**Meet the editor**

Dr Martin Molina is the Principal investigator of the Gene and Cell Therapy group at the department of human variability in the Centre for Genomics and Oncological Research (GENYO). He is PhD in Biology (extraordinary Price) by the CSIC-University of Granada in 1995. He worked in London from 1997 to 2003, first at the ICR Chester Beaty and then at the Windeyer Institute

of Medical Sciences (UCL) where he worked on retroviral vectors targeting to tumor cells for cancer immunotherapy. From 2003 to 2009 Dr Martin Molina consolidates his own research group thanks to a Miguel Servet and a Ramon y Cajal contracts. From 2009 he is Principal Investigator from the Fundación Progreso y Sadud, first at the Andalusian Stem Cell Bank (BACM) and then at GENYO. His group main interest is the development of safer and more efficient gene delivery tools for the treatment of immune-related diseases: Immunodeficiencies (Wiskott-Aldrich Syndrome) and autoimmune diseases (Multiple sclerosis). He has published over 35 international publications in high impact journals. His publications have been cited over 710 times and have an accumulate impact factor of 202.

Contents

**Preface IX**

Ángeles Solinís

**Variables 35**

**Perspective 49**

**Section 2 Gene Therapy Tools: Synthetic 69**

**Gene Transfer 91** Yahan Fan and Jian Wu

Chapter 1 **Non-Viral Delivery Systems in Gene Therapy 3**

Gerald C. O'Sullivan and Mark Tangney

Shengnan Xiang and Xiaoling Zhang

Chapter 3 **Silencing of Transgene Expression: A Gene Therapy**

Alicia Rodríguez Gascón, Ana del Pozo-Rodríguez and María

David Morrissey, Sara A. Collins, Simon Rajenderan, Garrett Casey,

Oleg E. Tolmachov, Tatiana Subkhankulova and Tanya Tolmachova

Chapter 2 **Plasmid Transgene Expression in vivo: Promoter and Tissue**

Chapter 4 **Cellular Uptake Mechanism of Non-Viral Gene Delivery and Means for Improving Transfection Efficiency 71**

Chapter 5 **Polylipid Nanoparticle, a Novel Lipid-Based Vector for Liver**

Chapter 6 **DNA Electrotransfer: An Effective Tool for Gene Therapy 109**

Chapter 7 **siRNA and Gene Formulation for Efficient Gene Therapy 135** Ian S. Blagbrough and Abdelkader A. Metwally

Aurore Burgain-Chain and Daniel Scherman

**Section 1 Introduction 1**

### Contents

#### **Preface XIII**


Chapter 7 **siRNA and Gene Formulation for Efficient Gene Therapy 135** Ian S. Blagbrough and Abdelkader A. Metwally

#### **X** Contents


Chapter 17 **Efficient AAV Vector Production System: Towards Gene Therapy For Duchenne Muscular Dystrophy 429**

Chapter 18 **Gene Therapy for Primary Immunodeficiencies 453**

Chapter 19 **Gene Therapy for Diabetic Retinopathy – Targeting the**

**Renin-Angiotensin System 467**

Chapter 20 **Gene Therapy for Retinitis Pigmentosa 493**

Francisco Martin, Alejandra Gutierrez-Guerrero and Karim

Nakagawa, Mohan K Raizada and William W Hauswirth

Chapter 21 **Gene Therapy for Erythroid Metabolic Inherited Diseases 511**

Bravo, S. Navarro, Zita Garate and Jose C. Segovia

Chapter 22 **Targeting the Lung: Challenges in Gene Therapy for Cystic**

Chapter 24 **Molecular Therapy for Lysosomal Storage Diseases 591**

Chapter 25 **Gene Therapy Perspectives Against Diseases of the**

Dimosthenis Lykouras, Kiriakos Karkoulias, Christos

Tourmousoglou, Efstratios Koletsis, Kostas Spiropoulos and

Qiuhong Li, Amrisha Verma, Ping Zhu, Bo Lei, Yiguo Qiu, Takahiko

Contents **VII**

Hiroshi Tomita, Eriko Sugano, Hitomi Isago, Namie Murayama and

Maria Garcia-Gomez, Oscar Quintana-Bustamante, Maria Garcia-

George Kotzamanis, Athanassios Kotsinas, Apostolos Papalois and

Takashi Okada

Benabdellah

Makoto Tamai

**Fibrosis 539**

**Section 5 Applications: Others 609**

Vassilis G. Gorgoulis

Chapter 23 **Gene Therapy for the COL7A1 Gene 561** E. Mayr, U. Koller and J.W. Bauer

Daisuke Tsuji and Kohji Itoh

**Respiratory System 611**

Dimitrios Dougenis

**Section 4 Applications: Inhereted Diseases 451**


**Section 3 Gene Therapy Tools: Biological 175**

**VI** Contents

**Bedside Approach 249**

**Directions 287**

**Potential 343**

Coroadinha

Chapter 8 **Mesenchymal Stem Cells as Gene Delivery Vehicles 177** Christopher D. Porada and Graça Almeida-Porada

Cian M. McCrudden and Helen O. McCarthy

Ana C. Calvo, Pilar Zaragoza and Rosario Osta

Chapter 12 **Lentiviral Gene Therapy Vectors: Challenges and Future**

Kochan, Karine Breckpot and David Escors

Chapter 14 **Targeted Lentiviral Vectors: Current Applications and Future**

Chapter 15 **Vectors for Highly Efficient and Neuron-Specific Retrograde**

Hélio A. Tomás, Ana F. Rodrigues, Paula M. Alves and Ana S.

Ines Dufait, Therese Liechtenstein, Alessio Lanna, Roberta Laranga, Antonella Padella, Christopher Bricogne, Frederick Arce, Grazyna

Cleo Goyvaerts, Therese Liechtenstein, Christopher Bricogne, David

**Gene Transfer for Gene Therapy of Neurological Diseases 387** Shigeki Kato, Kenta Kobayashi, Ken-ichi Inoue, Masahiko Takada

Chapter 11 **Transposons for Non-Viral Gene Transfer 269** Sunandan Saha and Matthew H. Wilson

Chapter 13 **Lentiviral Vectors in Immunotherapy 319**

Escors and Karine Breckpot

and Kazuto Kobayashi

Dustin T. Rae and Grant D. Trobridge

Chapter 16 **Retroviral Genotoxicity 399**

Chapter 9 **Cancer Gene Therapy – Key Biological Concepts in the Design of Multifunctional Non-Viral Delivery Systems 213**

Chapter 10 **Gene Therapy Based on Fragment C of Tetanus Toxin in ALS: A Promising Neuroprotective Strategy for the Bench to the**


Chapter 26 **Gene Therapy in Critical Care Medicine 631** Gabriel J. Moreno-González and Angel Zarain-Herzberg

#### Chapter 27 **Clinical and Translational Challenges in Gene Therapy of Cardiovascular Diseases 651** Divya Pankajakshan and Devendra K. Agrawal


Ann M. Simpson, M. Anne Swan, Guo Jun Liu, Chang Tao, Bronwyn A O'Brien, Edwin Ch'ng, Leticia M. Castro, Julia Ting, Zehra Elgundi, Tony An, Mark Lutherborrow, Fraser Torpy, Donald K. Martin, Bernard E. Tuch and Graham M. Nicholson

Preface

different experts in the gene therapy field.

In the last 10 years gene therapy has experienced a renascence thanks to the development of safer and more efficient gene transfer vectors and to the advances in the cell therapy field. This book brings together a comprehensive collection of gene therapy tools and their thera‐ peutic applications. The first part of the book covers different gene therapy vectors focusing on their advantages and disadvantages. The second part of the book gets into gene therapy applications, from the latest successes on clinical trials to the new gene therapy targets that are still under development. This book allows the reader to come across with the opinions of

Pfizer - Universidad de Granada - Junta de Andalucía Centre for Genomics and

**Francisco Martín Molina** Principal Investigator

Gene and Cell Therapy group

Oncological Research (GENYO)

Chapter 30 **Feasibility of Gene Therapy for Tooth Regeneration by Stimulation of a Third Dentition 727**

Katsu Takahashi, Honoka Kiso, Kazuyuki Saito, Yumiko Togo, Hiroko Tsukamoto, Boyen Huang and Kazuhisa Bessho

### Preface

Chapter 26 **Gene Therapy in Critical Care Medicine 631**

**Cardiovascular Diseases 651**

**Channels 703**

**VIII** Contents

Gabriel J. Moreno-González and Angel Zarain-Herzberg

Chapter 27 **Clinical and Translational Challenges in Gene Therapy of**

Divya Pankajakshan and Devendra K. Agrawal

Chapter 29 **Insulin Trafficking in a Glucose Responsive Engineered Human**

**Potassium Channels and Voltage-Gated Calcium**

Bernard E. Tuch and Graham M. Nicholson

Chapter 30 **Feasibility of Gene Therapy for Tooth Regeneration by Stimulation of a Third Dentition 727**

**Liver Cell Line is Regulated by the Interaction of ATP-Sensitive**

Ann M. Simpson, M. Anne Swan, Guo Jun Liu, Chang Tao, Bronwyn A O'Brien, Edwin Ch'ng, Leticia M. Castro, Julia Ting, Zehra Elgundi, Tony An, Mark Lutherborrow, Fraser Torpy, Donald K. Martin,

Katsu Takahashi, Honoka Kiso, Kazuyuki Saito, Yumiko Togo, Hiroko Tsukamoto, Boyen Huang and Kazuhisa Bessho

Chapter 28 **Gene Therapy for Chronic Pain Management 685** Isaura Tavares and Isabel Martins

> In the last 10 years gene therapy has experienced a renascence thanks to the development of safer and more efficient gene transfer vectors and to the advances in the cell therapy field. This book brings together a comprehensive collection of gene therapy tools and their thera‐ peutic applications. The first part of the book covers different gene therapy vectors focusing on their advantages and disadvantages. The second part of the book gets into gene therapy applications, from the latest successes on clinical trials to the new gene therapy targets that are still under development. This book allows the reader to come across with the opinions of different experts in the gene therapy field.

> > **Francisco Martín Molina** Principal Investigator Gene and Cell Therapy group Pfizer - Universidad de Granada - Junta de Andalucía Centre for Genomics and Oncological Research (GENYO)

**Section 1**

**Introduction**

**Section 1**

### **Introduction**

**Chapter 1**

**Non-Viral Delivery Systems in Gene Therapy**

Recent advances in molecular biology combined with the culmination of the Human Ge‐ nome Project [1] have provided a genetic understanding of cellular processes and disease pathogenesis; numerous genes involved in disease and cellular processes have been identi‐ fied as targets for therapeutic approaches. In addition, the development of high-throughput screening techniques (e.g., cDNA microarrays, differential display and database meaning) may drastically increase the rate at which these targets are identified [2,3]. Over the past years there has been a remarkable expansion of both the number of human genes directly associated with disease states and the number of vector systems available to express those genes for therapeutic purposes. However, the development of novel therapeutic strategies using these targets is dependent on the ability to manipulate the expression of these target genes in the desired cell population. In this chapter we explain the concept and aim of gene therapy, the different gene delivery systems and therapeutic strategies, how genes are deliv‐

A gene therapy medicinal product is a biological product which has the following character‐ istics: (a) it contains an active substance which contains or consists of a recombinant nucleic acid used in administered to human beings with a view to regulating, repairing, replacing, adding or deleting a genetic sequence; (b) its therapeutic, prophylactic or diagnostic effect relates directly to the recombinant nucleic acid sequence it contains, or to the product of ge‐

> © 2013 Gascón et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**2. Aim and concept of gene therapy with non-viral vectors**

Ana del Pozo-Rodríguez and María Ángeles Solinís

Additional information is available at the end of the chapter

Alicia Rodríguez Gascón,

http://dx.doi.org/10.5772/52704

ered and how they reach the target.

netic expression of this sequence [4].

**1. Introduction**

### **Non-Viral Delivery Systems in Gene Therapy**

Alicia Rodríguez Gascón, Ana del Pozo-Rodríguez and María Ángeles Solinís

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52704

#### **1. Introduction**

Recent advances in molecular biology combined with the culmination of the Human Ge‐ nome Project [1] have provided a genetic understanding of cellular processes and disease pathogenesis; numerous genes involved in disease and cellular processes have been identi‐ fied as targets for therapeutic approaches. In addition, the development of high-throughput screening techniques (e.g., cDNA microarrays, differential display and database meaning) may drastically increase the rate at which these targets are identified [2,3]. Over the past years there has been a remarkable expansion of both the number of human genes directly associated with disease states and the number of vector systems available to express those genes for therapeutic purposes. However, the development of novel therapeutic strategies using these targets is dependent on the ability to manipulate the expression of these target genes in the desired cell population. In this chapter we explain the concept and aim of gene therapy, the different gene delivery systems and therapeutic strategies, how genes are deliv‐ ered and how they reach the target.

#### **2. Aim and concept of gene therapy with non-viral vectors**

A gene therapy medicinal product is a biological product which has the following character‐ istics: (a) it contains an active substance which contains or consists of a recombinant nucleic acid used in administered to human beings with a view to regulating, repairing, replacing, adding or deleting a genetic sequence; (b) its therapeutic, prophylactic or diagnostic effect relates directly to the recombinant nucleic acid sequence it contains, or to the product of ge‐ netic expression of this sequence [4].

© 2013 Gascón et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The most important, and most difficult, challenge in gene therapy is the issue of delivery. The tools used to achieve gene modification are called gene therapy vectors and they are the "key" for an efficient and safe strategy. Therefore, there is a need for a delivery system, which must first overcome the extracellular barriers (such as avoiding particle clearance mechanisms, targeting specific cells or tissues and protecting the nucleic acid from degrada‐ tion) and, subsequently, the cellular barriers (cellular uptake, endosomal escape, nuclear en‐ try and nucleic release) [5]. An ideal gene delivery vector should be effective, specific, long lasting and safe.

**Gene types trasnferred in gene therapy clinical trials** Adhesion molecule

**Figure 2.** Gene types transferred in gene therapy clinical trials (adapted from http://www.wiley.co.k/genmed/clinical).

**Figure 3.** Vector systems used in gene therapy clinical trials (adapted from http://www.wiley.co.k/genmed/clinical).

**Vectors used for gene therapy clinical trials**

Antigen Antisense Cell cycle

Cytokine Deficiency Grow th factor Hormone Marker Oncogene regulator Oncolytic virus

Receptor Replication inhibitor Ribozyme siRNA Suicide Transcription factor Tumor suppressor Viral vaccine Others Unknow n

Cell protection/Drug resistance

Non-Viral Delivery Systems in Gene Therapy http://dx.doi.org/10.5772/52704 5

Porins, ion channels, transporters

Adennovirus Retrovirus

Lentivirus Other categories Unknown

Naked/plasmid DNA Vaccinia virus Lipofection Poxvirus

Adeno-associated virus Herpex simplex virus

Gene therapy has long been regarded a promising treatment for many diseases, including inherited through a genetic disorder (such as hemophilia, human severe combined immuno‐ deficiency, cystic fibrosis, etc) or acquired (such as AIDS or cancer). Figures 1 and 2 show the indications addressed and the gene types transferred in gene therapy clinical trials, re‐ spectively [6].

**Figure 1.** Indications addressed by gene therapy clinical trials (adapted from http://www.wiley.co.k/genmed/clinical).

Gene delivery systems include viral vectors and non-viral vectors. Viral vectors are the most effective, but their application is limited by their immunogenicity, oncogenicity and the small size of the DNA they can transport. Non-viral vectors are safer, of low cost, more re‐ producible and do not present DNA size limit. The main limitation of non-viral systems is their low transfection efficiency, although it has been improved by different strategies and the efforts are still ongoing [6]; actually, advances of non-viral delivery have lead to an in‐ creased number of products entering into clinical trials. However, viral vector has dominat‐ ed the clinical trials in gene therapy for its relatively high delivery efficiency. Figure 3 shows the proportion of vector systems currently in human trials [7].

The most important, and most difficult, challenge in gene therapy is the issue of delivery. The tools used to achieve gene modification are called gene therapy vectors and they are the "key" for an efficient and safe strategy. Therefore, there is a need for a delivery system, which must first overcome the extracellular barriers (such as avoiding particle clearance mechanisms, targeting specific cells or tissues and protecting the nucleic acid from degrada‐ tion) and, subsequently, the cellular barriers (cellular uptake, endosomal escape, nuclear en‐ try and nucleic release) [5]. An ideal gene delivery vector should be effective, specific, long

Gene therapy has long been regarded a promising treatment for many diseases, including inherited through a genetic disorder (such as hemophilia, human severe combined immuno‐ deficiency, cystic fibrosis, etc) or acquired (such as AIDS or cancer). Figures 1 and 2 show the indications addressed and the gene types transferred in gene therapy clinical trials, re‐

**Indications addressed by gene therapy clinical trials**

**Figure 1.** Indications addressed by gene therapy clinical trials (adapted from http://www.wiley.co.k/genmed/clinical).

Gene delivery systems include viral vectors and non-viral vectors. Viral vectors are the most effective, but their application is limited by their immunogenicity, oncogenicity and the small size of the DNA they can transport. Non-viral vectors are safer, of low cost, more re‐ producible and do not present DNA size limit. The main limitation of non-viral systems is their low transfection efficiency, although it has been improved by different strategies and the efforts are still ongoing [6]; actually, advances of non-viral delivery have lead to an in‐ creased number of products entering into clinical trials. However, viral vector has dominat‐ ed the clinical trials in gene therapy for its relatively high delivery efficiency. Figure 3 shows

the proportion of vector systems currently in human trials [7].

Cancer diseases Cardiovascular diseases

Gene marking Healthy volunteers Infectious diseases Inflammatory diseases Monogenic diseases Neurological diseases Ocular diseases Others

lasting and safe.

4 Gene Therapy - Tools and Potential Applications

spectively [6].

**Figure 2.** Gene types transferred in gene therapy clinical trials (adapted from http://www.wiley.co.k/genmed/clinical).

**Figure 3.** Vector systems used in gene therapy clinical trials (adapted from http://www.wiley.co.k/genmed/clinical).

#### **3. Non-viral methods for transfection**

Currently, three categories of non-viral systems are available:


Table 1 summarizes the most utilized non-viral vectors.


**3.1. Inorganic particles**

thermal effect.

ied as an efficient gene delivery and imaging agent [13].

**3.2. Synthetic or natural biodegradable particles**

*3.2.1. Polymer-based non-viral vectors*

Inorganic nanoparticles are nanostructures varying in size, shape and porosity, which can be engineered to evade the reticuloendothelial system or to protect an entrapped molecular payload from degradation or denaturation [8]. Calcium phosphate, silica, gold, and several magnetic compounds are the most studied [9-11]. Silica-coated nanoparticles are biocompat‐ ible structures that have been used for various biological applications including gene thera‐ py due to its biocompatibility [8]. Mesoporous silica nanoparticles have shown gene transfection efficiency "in vitro" in glial cells [12]. Magnetic inorganic nanoparticles (such as Fe3O4, MnO2) have been applied for cancer-targeted delivery of nucleic acids and simultane‐ ous diagnosis via magnetic resonance imaging [13,14]. Silica nanotubes have been also stud‐

Non-Viral Delivery Systems in Gene Therapy http://dx.doi.org/10.5772/52704 7

Inorganic particles can be easily prepared and surface-functionalized. They exhibit good storage stability and are not subject to microbial attack [13]. Bhattarai et al. [15] modified mesoporous silica nanoparticles with poly(ethylene glycol) and methacrylate derivatives

Gold nanoparticles have been lately investigated for gene therapy. They can be easily pre‐ pared, display low toxicity and the surface can be modified using various chemical techni‐ ques [16]. For instance, gold nanorods have been proposed to deliver nucleic acids to tumors [13]. They have strong absorption bands in the near-infrared region, and the absorbed light energy is then converted into heat by gold nanorods (photohermal effect). The near-infrared light can penetrate deeply into tissues; therefore, the surface of the gold could be modified with double-stranded DNA for controlled release [17]. After irradiation with near-infrared light, single stranded DNA is released due to thermal denaturation induced by the photo‐

Synthetic or natural biocompatible particles may be composed by cationic polymers, cationic lipids or cationic peptides, and also the combination of these components [18-21]. The poten‐ tial advantages of biodegradable carriers are their reduced toxicity (degradation leads to

Cationic polymers condense DNA into small particles (polyplexes) and prevent DNA from degradation. Polymeric nanoparticles are the most commonly used type of nano-scale deliv‐ ery systems. They are mostly spherical particles, in the size range of 1-1000 nm, carrying the nucleic acids of interest. DNA can be entrapped into the polymeric matrix or can be adsor‐ bed or conjugated on the surface of the nanoparticles. Moreover, the degradation of the pol‐ ymer can be used as a tool to release the plasmid DNA into the cytosol [22]. Table 1 shows

non-toxic products) and avoidance of accumulation of the polymer in the cells.

several commonly used polymers used for gene delivery [16].

and used them to deliver DNA or small interfering RNA (siRNA) "in vitro".

**Table 1.** Delivery systems for gene therapy.

#### **3.1. Inorganic particles**

**3. Non-viral methods for transfection**

6 Gene Therapy - Tools and Potential Applications

**•** Synthetic or natural biodegradable particles

Table 1 summarizes the most utilized non-viral vectors.

Inorganic particles Calcium phosphate

Physical methods Needle injection

**Table 1.** Delivery systems for gene therapy.

Synthetic or natural biodegradable particles 1. Polymeric-based non-viral vectors:

**Category System for gene delivery**

Poly(lactic-co-glycolic acid) (PLGA)

2. Cationic lipid-based non-viral vectors:

3. Peptide-based non-viral vectors:

Other peptides to functionalize other delivery systems: SAP, protamine

Poly lactic acid (PLA) Poly(ethylene imine) (PEI)

Cationic liposomes Cationic emulsions Solid lipid nanoparticles

Poly-L-lysine

Balistic DNA injection Electroporation Sonoporation Photoporation Magnetofection Hydroporation

Chitosan Dendrimers Polymethacrylates

Silica Gold Magnetic

**•** Inorganic particles

**•** Physical methods

Currently, three categories of non-viral systems are available:

Inorganic nanoparticles are nanostructures varying in size, shape and porosity, which can be engineered to evade the reticuloendothelial system or to protect an entrapped molecular payload from degradation or denaturation [8]. Calcium phosphate, silica, gold, and several magnetic compounds are the most studied [9-11]. Silica-coated nanoparticles are biocompat‐ ible structures that have been used for various biological applications including gene thera‐ py due to its biocompatibility [8]. Mesoporous silica nanoparticles have shown gene transfection efficiency "in vitro" in glial cells [12]. Magnetic inorganic nanoparticles (such as Fe3O4, MnO2) have been applied for cancer-targeted delivery of nucleic acids and simultane‐ ous diagnosis via magnetic resonance imaging [13,14]. Silica nanotubes have been also stud‐ ied as an efficient gene delivery and imaging agent [13].

Inorganic particles can be easily prepared and surface-functionalized. They exhibit good storage stability and are not subject to microbial attack [13]. Bhattarai et al. [15] modified mesoporous silica nanoparticles with poly(ethylene glycol) and methacrylate derivatives and used them to deliver DNA or small interfering RNA (siRNA) "in vitro".

Gold nanoparticles have been lately investigated for gene therapy. They can be easily pre‐ pared, display low toxicity and the surface can be modified using various chemical techni‐ ques [16]. For instance, gold nanorods have been proposed to deliver nucleic acids to tumors [13]. They have strong absorption bands in the near-infrared region, and the absorbed light energy is then converted into heat by gold nanorods (photohermal effect). The near-infrared light can penetrate deeply into tissues; therefore, the surface of the gold could be modified with double-stranded DNA for controlled release [17]. After irradiation with near-infrared light, single stranded DNA is released due to thermal denaturation induced by the photo‐ thermal effect.

#### **3.2. Synthetic or natural biodegradable particles**

Synthetic or natural biocompatible particles may be composed by cationic polymers, cationic lipids or cationic peptides, and also the combination of these components [18-21]. The poten‐ tial advantages of biodegradable carriers are their reduced toxicity (degradation leads to non-toxic products) and avoidance of accumulation of the polymer in the cells.

#### *3.2.1. Polymer-based non-viral vectors*

Cationic polymers condense DNA into small particles (polyplexes) and prevent DNA from degradation. Polymeric nanoparticles are the most commonly used type of nano-scale deliv‐ ery systems. They are mostly spherical particles, in the size range of 1-1000 nm, carrying the nucleic acids of interest. DNA can be entrapped into the polymeric matrix or can be adsor‐ bed or conjugated on the surface of the nanoparticles. Moreover, the degradation of the pol‐ ymer can be used as a tool to release the plasmid DNA into the cytosol [22]. Table 1 shows several commonly used polymers used for gene delivery [16].

#### *3.2.1.1. Poly(lactic-co-glycolic acid) (PLGA) and poly lactic acid (PLA)*

Biodegradable polyesters, PLGA and PLA, are the most commonly used polymers for deliv‐ ering drugs and biomolecules, including nucleic acids. They consist of units of lactic acid and glycolic acid connected through ester linkage. These biodegradable polymers undergo bulk hydrolysis thereby providing sustained delivery of the therapeutic agent. The degrada‐ tion products, lactic acid and glycolic acid, are removed from the body through citric acid cycle. The release of therapeutic agent from these polymers occurs by diffusion and polymer degradation [16].

ered using PLGA microparticles, on proliferation and differentiation capabilities of human

Non-Viral Delivery Systems in Gene Therapy http://dx.doi.org/10.5772/52704 9

Chitosan [b(1-4)2-amino-2-deoxy-D-glucose] is a biodegradable polysaccharide copolymer of N-acetyl-D-glucosamine and D-glucosamine obtained by the alkaline deacetylation of chi‐ tin, which is a polysaccharide found in the exoskeleton of crustaceans of marine arthropods and insects [37]. Chitosans differ in the degree of N-acetylation (40 to 98%) and molecular weight (50 to 2000 kDa) [38]. As the only natural polysaccharide with a positive charge, chi‐

**•** it is potentially safe and non-toxic, both in experimental animals [39] and humans [40]

**•** it has biocompatibility to the human body and does not elicit stimulation of the mucosa

**•** its cationic polyelectrolyte nature provides a strong electrostatic interaction with nega‐

**•** the mucoadhesive property of chitosan potentially leads to a sustained interaction be‐ tween the macromolecule being "delivered" and the membrane epithelia, promoting

**•** it has the ability to open intercellular tight junctions, facilitating its transport into the cells

Currently, there is a commercial transfection reagent based on chitosan (Novafect, NovaMa‐ trix, FMC, US), and many other prototypes are under development. Most of the chitosanbased nanocarriers for gene delivery have been based on direct complexation of chitosan and the nucleic acid [45], whereas in some instances additional polyelectrolytes, polymers and lipids have been used in order to form composite nanoparticles [46-49] or chitosan-coat‐

Many studies using cell cultures have shown that pDNA-loaded chitosan nanocarriers are able to achieve high transfection levels in most cell lines [50]. Chitosan nanocarriers loaded with siRNA have provided gene suppression values similar to the commercial reagent lipo‐

Chitosan of low molecular weight is more efficient for transfection than chitosan with high molecular weight. This enhancement in transfection efficacy observed with low molecular weight chitosan can be attributed to the easier release of pDNA from the nanocarrier upon cell internalization. Moreover, the presence of free low molecular chitosan has been deemed to be very important for the endosomal escape of the nanocarriers [50]. Concerning deacety‐ lation degree, its influence on transfection is not still clear. "In vitro" studies have shown that the best transfection is achieved with highly deacetylated chitosan [54,55]. However, "in

tively charged DNA [41], and protects the DNA from nuclease degradation [42]

**•** it can be degraded into H2O and CO2 in the body, which ensures its biosafety

tosan has the following unique properties as carrier for gene therapy:

mesenchymal stromal cells.

*3.2.1.2. Chitosan*

and the derma

[44]

more efficient uptake [43]

ed hydrophobic nanocarriers.

fectamine [51,52,18,53].

PLGA has a demonstrated FDA approved track record as a vehicle for drug and protein de‐ livery [23,24]. Biodegradable PLA and PLGA particles are biocompatible and have the ca‐ pacity to protect pDNA from nuclease degradation and increase pDNA stability [25,26].

PLGA particles typically less than 10 µm in size are efficiently phagocytosed by professional antigen presenting cells; therefore, they have significant potential for immunization applica‐ tions [27,28]. For example, intramuscular immunization of p55 Gag plasmid adsorbed on PLGA/cetyl trimethyl ammonium bromide (CTAB) particles induced potent antibody and cytotoxic T lymphocyte responses. These particles showed a 250-fold increase in antibody response at higher DNA doses and more rapid and complete seroconversion, at the lower doses, compared to other adjuvants, including cationic liposomes [29].

The encapsulation efficiency of DNA in PLGA nanoparticles is not very high, and it de‐ pends on the molecular weight of the PLGA and on the hydrophobicity of the polymer, be‐ ing the hydrophilic polymers those that provide higher loading efficiency [30]. To enhance the DNA loading, several strategies have been proposed. Kusonowiriyawong et al. [31] pre‐ pared cationic PLGA microparticles by dissolving cationic surfactants (like water insoluble stearylamine) in the organic solvent in which the PLGA was dissolved to prepare the micro‐ particles. Another strategy was to reduce the negative charge of plasmid DNA by condens‐ ing it with poly(aminoacids) (like poly-L-lysine) before encapsulation in PLGA microparticles [32,33].

Normally, after an initial burst release, plasmid DNA release from PLGA particles occurs slowly during several days/weeks [22]. The degradation of the PLGA nanoparticles, through a bulk homogeneous hydrolytic process, determines the release of plasmid DNA. Conse‐ quently, it can be expected that the use of more hydrophilic PLGA not only improves the encapsulation efficiency of DNA, but also results in a faster release of plasmid DNA. Deliv‐ ery of the plasmid DNA depends on the copolymer composition of the PLGA (lactic acid versus glycolic acid), molecular weight, particle size and morphology [22]. DNA release ki‐ netics depends also on the plasmid incorporation technique; Pérea et al. [34] reported that nanoparticles prepared by the water-oil emulsion/diffusion technique released their content rapidly, whereas those obtained by the water-oil-emulsion method showed an initial burst followed by a slow release during at least 28 days.

PLGA and PLA based nanoparticles have also been used for "in vitro" RNAi delivery [35]. For instance, Hong et al. [36] have shown the effects of glucocorticoid receptor siRNA deliv‐ ered using PLGA microparticles, on proliferation and differentiation capabilities of human mesenchymal stromal cells.

#### *3.2.1.2. Chitosan*

*3.2.1.1. Poly(lactic-co-glycolic acid) (PLGA) and poly lactic acid (PLA)*

doses, compared to other adjuvants, including cationic liposomes [29].

degradation [16].

8 Gene Therapy - Tools and Potential Applications

microparticles [32,33].

followed by a slow release during at least 28 days.

Biodegradable polyesters, PLGA and PLA, are the most commonly used polymers for deliv‐ ering drugs and biomolecules, including nucleic acids. They consist of units of lactic acid and glycolic acid connected through ester linkage. These biodegradable polymers undergo bulk hydrolysis thereby providing sustained delivery of the therapeutic agent. The degrada‐ tion products, lactic acid and glycolic acid, are removed from the body through citric acid cycle. The release of therapeutic agent from these polymers occurs by diffusion and polymer

PLGA has a demonstrated FDA approved track record as a vehicle for drug and protein de‐ livery [23,24]. Biodegradable PLA and PLGA particles are biocompatible and have the ca‐ pacity to protect pDNA from nuclease degradation and increase pDNA stability [25,26].

PLGA particles typically less than 10 µm in size are efficiently phagocytosed by professional antigen presenting cells; therefore, they have significant potential for immunization applica‐ tions [27,28]. For example, intramuscular immunization of p55 Gag plasmid adsorbed on PLGA/cetyl trimethyl ammonium bromide (CTAB) particles induced potent antibody and cytotoxic T lymphocyte responses. These particles showed a 250-fold increase in antibody response at higher DNA doses and more rapid and complete seroconversion, at the lower

The encapsulation efficiency of DNA in PLGA nanoparticles is not very high, and it de‐ pends on the molecular weight of the PLGA and on the hydrophobicity of the polymer, be‐ ing the hydrophilic polymers those that provide higher loading efficiency [30]. To enhance the DNA loading, several strategies have been proposed. Kusonowiriyawong et al. [31] pre‐ pared cationic PLGA microparticles by dissolving cationic surfactants (like water insoluble stearylamine) in the organic solvent in which the PLGA was dissolved to prepare the micro‐ particles. Another strategy was to reduce the negative charge of plasmid DNA by condens‐ ing it with poly(aminoacids) (like poly-L-lysine) before encapsulation in PLGA

Normally, after an initial burst release, plasmid DNA release from PLGA particles occurs slowly during several days/weeks [22]. The degradation of the PLGA nanoparticles, through a bulk homogeneous hydrolytic process, determines the release of plasmid DNA. Conse‐ quently, it can be expected that the use of more hydrophilic PLGA not only improves the encapsulation efficiency of DNA, but also results in a faster release of plasmid DNA. Deliv‐ ery of the plasmid DNA depends on the copolymer composition of the PLGA (lactic acid versus glycolic acid), molecular weight, particle size and morphology [22]. DNA release ki‐ netics depends also on the plasmid incorporation technique; Pérea et al. [34] reported that nanoparticles prepared by the water-oil emulsion/diffusion technique released their content rapidly, whereas those obtained by the water-oil-emulsion method showed an initial burst

PLGA and PLA based nanoparticles have also been used for "in vitro" RNAi delivery [35]. For instance, Hong et al. [36] have shown the effects of glucocorticoid receptor siRNA deliv‐ Chitosan [b(1-4)2-amino-2-deoxy-D-glucose] is a biodegradable polysaccharide copolymer of N-acetyl-D-glucosamine and D-glucosamine obtained by the alkaline deacetylation of chi‐ tin, which is a polysaccharide found in the exoskeleton of crustaceans of marine arthropods and insects [37]. Chitosans differ in the degree of N-acetylation (40 to 98%) and molecular weight (50 to 2000 kDa) [38]. As the only natural polysaccharide with a positive charge, chi‐ tosan has the following unique properties as carrier for gene therapy:


Currently, there is a commercial transfection reagent based on chitosan (Novafect, NovaMa‐ trix, FMC, US), and many other prototypes are under development. Most of the chitosanbased nanocarriers for gene delivery have been based on direct complexation of chitosan and the nucleic acid [45], whereas in some instances additional polyelectrolytes, polymers and lipids have been used in order to form composite nanoparticles [46-49] or chitosan-coat‐ ed hydrophobic nanocarriers.

Many studies using cell cultures have shown that pDNA-loaded chitosan nanocarriers are able to achieve high transfection levels in most cell lines [50]. Chitosan nanocarriers loaded with siRNA have provided gene suppression values similar to the commercial reagent lipo‐ fectamine [51,52,18,53].

Chitosan of low molecular weight is more efficient for transfection than chitosan with high molecular weight. This enhancement in transfection efficacy observed with low molecular weight chitosan can be attributed to the easier release of pDNA from the nanocarrier upon cell internalization. Moreover, the presence of free low molecular chitosan has been deemed to be very important for the endosomal escape of the nanocarriers [50]. Concerning deacety‐ lation degree, its influence on transfection is not still clear. "In vitro" studies have shown that the best transfection is achieved with highly deacetylated chitosan [54,55]. However, "in vivo", higher transfection was achieved after intramuscular administration of chitosan com‐ plexes with a low deacetylation degree [55].

tems [71-73]. Recent studies showed that the dendrimer-mediated siRNA delivery and gene silencing depends on the stoichiometry, concentration of siRNA and the dendrimer genera‐ tion [71]. In a recent study, a PAMAM dendrimer-delivered short hairpin RNA (shRNA) showed the ability to deplect a human telomerase reverse transcriptase, the catalytic subunit of telomerase complex, resulting in partial cellular apoptosis, and inhibition of tumor out‐

Non-Viral Delivery Systems in Gene Therapy http://dx.doi.org/10.5772/52704 11

The toxicity profile of dendrimers is good, although it depends on the number of terminal amino groups and positive charge density. Moreover, toxicity is concentration and genera‐ tion dependent with higher generations being more toxic as the number of surface groups

Polymethacrylates are cationic vinyl-based polymers that possess the ability to condense polynucleotides into nanometer size particles. They efficiently condense DNA by forming inter-polyelectrolyte complexes. A range of polymethacrylates, differing in molecular weights and structures, have been evaluated for their potential as gene delivery vector, such us poly[2-dimethylamino) ethyl methacrylate] (DMAEMA) and its co-polymers [16]. The use of polymethactrtylates for DNA transfection is, however, limited due to their low ability

In order to optimise the use of these compounds for gene transfer, Christiaens et al. [77] combined polymethacrylates with penetratin, a 16-residue water-soluble peptide that inter‐ nalises into cells through membrane translocation. Penetratin mainly enhanced the endoly‐ sosomal escape of the polymethacrylate–DNA complexes and increased their cellular uptake using COS-1 (kidney cells of the African green monkey). Nanoparticles with a methacrylate core and PEI shell prepared via graft copolymerization have also been employed lately for gene delivery [78,79]. This conjugation resulted in nanoparticles with a higher transfection

Cationic lipids have been among the more efficient synthetic gene delivery reagents "in vi‐ tro" since the landmark publications in the late 1980s [80]. Cationic lipids can condense nu‐ cleic acids into cationic particles when the components are mixed together. This cationic lipid/nucleic acid complex (lipoplex) can protect nucleic acids from enzymatic degradation and deliver the nucleic acids into cells by interacting with the negatively charged cell mem‐ brane [81]. Lipoplexes are not an ordered DNA phase surrounded by a lipid bilayer; rather, they are a partially condensed DNA complex with an ordered substructure and an irregular morphology [82,83]. Since the initial studies, hundreds of cationic lipids have been synthe‐ sized as candidates for non-viral gene delivery [84] and a few made it to clinical trials

Cationic lipids can be used to form lipoplexes by directly mixing the positively charged lip‐ ids at the physiological pH with the negatively charged DNA. However, cationic lipids are

growth in xenotransplanted mice [74].

*3.2.1.5. Polymethacrylates*

to interact with membranes.

doubles with increasing generation number [75,76].

efficiency and lower toxicity as compared with PEI.

*3.2.2. Cationic-lipid based non-viral vectors*

[85,86].

#### *3.2.1.3. Poly(ethylene imine) (PEI)*

PEI is one of the most potent polymers for gene delivery. PEI is produced by the polymeri‐ zation of aziridine and has been used to deliver genetic material into various cell types both "in vitro" and "in vivo" [56,57]. There are two forms of this polymer: the linear form and the branched form, being the branched structure more efficient in condensing nucleic acids than the linear PEI [58].

PEI has a high density of protonable amino groups, every third atom being amino nitrogen, which imparts a high buffering ability at practically any pH [16]. Hence, once inside the en‐ dosome, PEI disrupts the vacuole releasing the genetic material in the cytoplasm. This abili‐ ty to escape from the endosome, as well as the ability to form stable complexes with nucleic acids, make this polymer very useful as a gene delivery vector [56].

Depending on the type of polymer (e.g. linear or branched PEI), as well as the molecular weight, the particle sizes of the polyplexes formed are more or less uniformly distributed [59]. Transfection efficiency of PEI has been found to be dependent on a multitude of factors such as molecular weight, degree of branching, N/P ratio, complex size, etc [60].

The use of PEI for gene delivery is limited due to the relatively low transfection efficiency, short duration of gene expression, and elevated toxicity [61,62]. Conjugation of poly(ethyl‐ ene glycol) to PEI to form diblock or triblock copolymers has been used by some authors to reduce the toxicity of PEI [63,64,65]. Poly(ethylene glycol) also shields the positive charge of the polyplexes, thereby providing steric stability to the complex. Such stabilization prevents non-specific interaction with blood components during systemic delivery [66].

#### *3.2.1.4. Dendrimes*

Dendrimers are polymer-based molecules with a symmetrical structure in precise size and shapes, as well as terminal group functionality [8]. Dendrimers contain three regions: i) a central core (a single atom or a group of atoms having two or more identical chemical fun‐ cionalities); ii) branches emanating from core, which are composed of repeating units with at least one branching junction, whose repetition is organized in a geometric progression that results in a series of radially concentric layers; and iii) terminal function groups. Den‐ drimers bind to genetic material when peripheral groups, that are positively-charged at physiological pH, interact with the negatively-charged phosphate groups of the nucleic acid [67,68]. Due to their nanometric size, dendrimers can interact effectively and specifically with cell components such as membranes, organelles, and proteins [69].

For instance, Qi et al. [70] showed the ability of generations 5 and 6 (G5 and G6) of poly(amidoamine) (PAMAM) dendrimers, conjugated with poly(ethylene glycol) to effi‐ ciently transfect both "in vitro" and "in vivo" after intramuscular administration to neonatal mice. PAMAM has also the ability to deliver siRNAs, especially "in vitro" in cell culture sys‐ tems [71-73]. Recent studies showed that the dendrimer-mediated siRNA delivery and gene silencing depends on the stoichiometry, concentration of siRNA and the dendrimer genera‐ tion [71]. In a recent study, a PAMAM dendrimer-delivered short hairpin RNA (shRNA) showed the ability to deplect a human telomerase reverse transcriptase, the catalytic subunit of telomerase complex, resulting in partial cellular apoptosis, and inhibition of tumor out‐ growth in xenotransplanted mice [74].

The toxicity profile of dendrimers is good, although it depends on the number of terminal amino groups and positive charge density. Moreover, toxicity is concentration and genera‐ tion dependent with higher generations being more toxic as the number of surface groups doubles with increasing generation number [75,76].

#### *3.2.1.5. Polymethacrylates*

vivo", higher transfection was achieved after intramuscular administration of chitosan com‐

PEI is one of the most potent polymers for gene delivery. PEI is produced by the polymeri‐ zation of aziridine and has been used to deliver genetic material into various cell types both "in vitro" and "in vivo" [56,57]. There are two forms of this polymer: the linear form and the branched form, being the branched structure more efficient in condensing nucleic acids than

PEI has a high density of protonable amino groups, every third atom being amino nitrogen, which imparts a high buffering ability at practically any pH [16]. Hence, once inside the en‐ dosome, PEI disrupts the vacuole releasing the genetic material in the cytoplasm. This abili‐ ty to escape from the endosome, as well as the ability to form stable complexes with nucleic

Depending on the type of polymer (e.g. linear or branched PEI), as well as the molecular weight, the particle sizes of the polyplexes formed are more or less uniformly distributed [59]. Transfection efficiency of PEI has been found to be dependent on a multitude of factors

The use of PEI for gene delivery is limited due to the relatively low transfection efficiency, short duration of gene expression, and elevated toxicity [61,62]. Conjugation of poly(ethyl‐ ene glycol) to PEI to form diblock or triblock copolymers has been used by some authors to reduce the toxicity of PEI [63,64,65]. Poly(ethylene glycol) also shields the positive charge of the polyplexes, thereby providing steric stability to the complex. Such stabilization prevents

Dendrimers are polymer-based molecules with a symmetrical structure in precise size and shapes, as well as terminal group functionality [8]. Dendrimers contain three regions: i) a central core (a single atom or a group of atoms having two or more identical chemical fun‐ cionalities); ii) branches emanating from core, which are composed of repeating units with at least one branching junction, whose repetition is organized in a geometric progression that results in a series of radially concentric layers; and iii) terminal function groups. Den‐ drimers bind to genetic material when peripheral groups, that are positively-charged at physiological pH, interact with the negatively-charged phosphate groups of the nucleic acid [67,68]. Due to their nanometric size, dendrimers can interact effectively and specifically

For instance, Qi et al. [70] showed the ability of generations 5 and 6 (G5 and G6) of poly(amidoamine) (PAMAM) dendrimers, conjugated with poly(ethylene glycol) to effi‐ ciently transfect both "in vitro" and "in vivo" after intramuscular administration to neonatal mice. PAMAM has also the ability to deliver siRNAs, especially "in vitro" in cell culture sys‐

such as molecular weight, degree of branching, N/P ratio, complex size, etc [60].

non-specific interaction with blood components during systemic delivery [66].

with cell components such as membranes, organelles, and proteins [69].

acids, make this polymer very useful as a gene delivery vector [56].

plexes with a low deacetylation degree [55].

*3.2.1.3. Poly(ethylene imine) (PEI)*

10 Gene Therapy - Tools and Potential Applications

the linear PEI [58].

*3.2.1.4. Dendrimes*

Polymethacrylates are cationic vinyl-based polymers that possess the ability to condense polynucleotides into nanometer size particles. They efficiently condense DNA by forming inter-polyelectrolyte complexes. A range of polymethacrylates, differing in molecular weights and structures, have been evaluated for their potential as gene delivery vector, such us poly[2-dimethylamino) ethyl methacrylate] (DMAEMA) and its co-polymers [16]. The use of polymethactrtylates for DNA transfection is, however, limited due to their low ability to interact with membranes.

In order to optimise the use of these compounds for gene transfer, Christiaens et al. [77] combined polymethacrylates with penetratin, a 16-residue water-soluble peptide that inter‐ nalises into cells through membrane translocation. Penetratin mainly enhanced the endoly‐ sosomal escape of the polymethacrylate–DNA complexes and increased their cellular uptake using COS-1 (kidney cells of the African green monkey). Nanoparticles with a methacrylate core and PEI shell prepared via graft copolymerization have also been employed lately for gene delivery [78,79]. This conjugation resulted in nanoparticles with a higher transfection efficiency and lower toxicity as compared with PEI.

#### *3.2.2. Cationic-lipid based non-viral vectors*

Cationic lipids have been among the more efficient synthetic gene delivery reagents "in vi‐ tro" since the landmark publications in the late 1980s [80]. Cationic lipids can condense nu‐ cleic acids into cationic particles when the components are mixed together. This cationic lipid/nucleic acid complex (lipoplex) can protect nucleic acids from enzymatic degradation and deliver the nucleic acids into cells by interacting with the negatively charged cell mem‐ brane [81]. Lipoplexes are not an ordered DNA phase surrounded by a lipid bilayer; rather, they are a partially condensed DNA complex with an ordered substructure and an irregular morphology [82,83]. Since the initial studies, hundreds of cationic lipids have been synthe‐ sized as candidates for non-viral gene delivery [84] and a few made it to clinical trials [85,86].

Cationic lipids can be used to form lipoplexes by directly mixing the positively charged lip‐ ids at the physiological pH with the negatively charged DNA. However, cationic lipids are more frequently used to prepare lipoplex structures such as liposomes, nanoemulsions or solid lipid nanoparticles [81].

hand, emulsions are stable during storage and are highly biocompatible [94]. In addition, the physical characteristics and serum-resistant properties of the DNA/nanoemulsion com‐ plexes suggest that cationic nanoemulsions could be a more efficient carrier system for gene and/or immunogene delivery than liposomes. One of the reasons for the serum-resistant properties of the cationic lipid nanoemulsions may be the stability of the nanoemulsion/DNA complex [98]. However, in spite of extensive research on emulsions, very few reports using cationic amino-based nanoemulsions in gene delivery have been published. After "in vivo" administration, cationic nanoemulsions have shown higher trans‐

Non-Viral Delivery Systems in Gene Therapy http://dx.doi.org/10.5772/52704 13

The incorporation of noninonic surfactant with a branched poly(ethylene glycol), such as Tween 80®, increments the stability of the nanoemulsion and prevent the formation of large nanoemulsion/DNA complexes, probably because of their stearic hindrance and the genera‐ tion of a hydrophilic surface that may enhance the stability by preventing physical aggrega‐ tion [94]. In addition, this strategy may prevent from enzymatic degradation in blood, and due to the hydrophilic surface, they are taken up slowly by phagocytic cells, resulting in

Solid lipid nanoparticles are particles made from a lipid being solid at room temperature and also at body temperature. They combine advantages of different colloidal systems. Like emulsions or liposomes, they are physiologically compatible and, like polymeric nanoparti‐ cles, it is possible to modulate drug release from the lipid matrix. In addition, SLN possess certain advantages. They can be produced without use of organic solvents, using high pres‐ sure homogenization (HPH) method that is already successfully implemented in pharma‐ ceutical industry [102]. From the point of view of application, SLN have very good stability [103] and are subject to be lyophilized [104], which facilitates the industrial production.

Cationic SLN, for instance, SLN containing at least one cationic lipid, have been proposed as non-viral vectors for gene delivery [105,20]. It has been shown that cationic SLN can effec‐ tively bind nucleic acids, protect them from DNase I degradation and deliver them into liv‐ ing cells. Cationic lipids are used in the preparation of SLN applied in gene therapy not only due to their positive surface charge, but also due to their surfactant activity, necessary to produce an initial emulsion, which is a common step in most preparation techniques. By means of electrostatic interactions, cationic SLN condense nucleic acids on their surface, leading generally to an excess of positive charges in the final complexes. This is beneficial for transfection because condensation facilitates the mobility of nucleic acids, protects them from environmental enzymes and the cationic character of the vectors allows the interaction with negatively charged cell surface. The characteristics of the resulting complexes depend on the ratio between particle and nucleic acid; there must be an equilibrium between the binding forces of the nucleic acids to SLN to achieve protection without hampering the pos‐ terior release in the site of action [106]. Release of DNA from the complexes may be one of the most crucial steps determining the optimal ratio for cationic lipid system-mediated

fection and lower toxicity than liposomes [99].

prolonged circulation in blood [100,101].

*3.2.2.3. Solid lipid nanoparticles (SLN)*

transfection [107].

#### *3.2.2.1. Cationic liposomes*

Liposomes are spherical vesicles made of phospholipids used to deliver drugs or genes. They can range in size from 20 nm to a few microns. Cationic liposomes and DNA interact spontaneously to form complexes with 100% loading efficiency; in other words, all of the DNA molecules are complexed with the liposomes, if enough cationic liposomes are availa‐ ble. It is believed that the negative charges of the DNA interact with the positively charged groups of the liposomes [87]. The lipid to DNA ratio, and overall lipid concentration used in forming these complexes, are very important for efficient gene delivery and vary with appli‐ cations [88].

Liposomes offer several advantages for gene delivery [87]:


Successful delivery of DNA and RNA to a variety of cell types has been reported, including tumour, airway epithelial cells, endothelial cells, hepatocytes, muscle cells and others, by in‐ tratissue or intravenous injection into animals [89,90].

Several liposome-based vectors have been assayed in a number of clinical trials for cancer treatment. For instance, Allovectin-7® (Vical, San Diego, CA, USA), a plasmid DNA carrying HLAB and ß2-microglobulin genes complexed with DMRIE/DOPE liposomes have been as‐ sessed for safety and efficacy in phase I and II clinical trials [91,92].

#### *3.2.2.2. Lipid nanoemulsions*

An emulsion is a dispersion of one immiscible liquid in another stabilized by a third compo‐ nent, the emulsifying agent [93]. The nanoemulsion consists of oil, water and surfactants, and presents a droplet size distribution of around 200 nm. Lipid-based carrier systems rep‐ resent drug vehicles composed of physiological lipids, such as cholesterol, cholesterol esters, phospholipids and tryglicerides, and offer a number of advantages, making them an ideal drug delivery carrier [94]. Adding cationic lipids as surfactants to these dispersed systems makes them suitable for gene delivery. The presence of cationic surfactants, like DOTAP, DOTMA or DC-Chol, causes the formation of positively charged droplets that promote strong electrostatic interactions between emulsion and the anionic nucleic acid phosphate groups [95,96]. For instance, Bruxel et al. [97] prepared a cationic nanoemulsion with DO‐ TAP as a delivery system for oligonucleotides targeting malarial topoisomerase II.

Lipid emulsions are considered to be superior to liposomes mainly in a scaling-up point of view. On the one hand, emulsions can be produced on an industrial scale; on the other hand, emulsions are stable during storage and are highly biocompatible [94]. In addition, the physical characteristics and serum-resistant properties of the DNA/nanoemulsion com‐ plexes suggest that cationic nanoemulsions could be a more efficient carrier system for gene and/or immunogene delivery than liposomes. One of the reasons for the serum-resistant properties of the cationic lipid nanoemulsions may be the stability of the nanoemulsion/DNA complex [98]. However, in spite of extensive research on emulsions, very few reports using cationic amino-based nanoemulsions in gene delivery have been published. After "in vivo" administration, cationic nanoemulsions have shown higher trans‐ fection and lower toxicity than liposomes [99].

The incorporation of noninonic surfactant with a branched poly(ethylene glycol), such as Tween 80®, increments the stability of the nanoemulsion and prevent the formation of large nanoemulsion/DNA complexes, probably because of their stearic hindrance and the genera‐ tion of a hydrophilic surface that may enhance the stability by preventing physical aggrega‐ tion [94]. In addition, this strategy may prevent from enzymatic degradation in blood, and due to the hydrophilic surface, they are taken up slowly by phagocytic cells, resulting in prolonged circulation in blood [100,101].

#### *3.2.2.3. Solid lipid nanoparticles (SLN)*

more frequently used to prepare lipoplex structures such as liposomes, nanoemulsions or

Liposomes are spherical vesicles made of phospholipids used to deliver drugs or genes. They can range in size from 20 nm to a few microns. Cationic liposomes and DNA interact spontaneously to form complexes with 100% loading efficiency; in other words, all of the DNA molecules are complexed with the liposomes, if enough cationic liposomes are availa‐ ble. It is believed that the negative charges of the DNA interact with the positively charged groups of the liposomes [87]. The lipid to DNA ratio, and overall lipid concentration used in forming these complexes, are very important for efficient gene delivery and vary with appli‐

Successful delivery of DNA and RNA to a variety of cell types has been reported, including tumour, airway epithelial cells, endothelial cells, hepatocytes, muscle cells and others, by in‐

Several liposome-based vectors have been assayed in a number of clinical trials for cancer treatment. For instance, Allovectin-7® (Vical, San Diego, CA, USA), a plasmid DNA carrying HLAB and ß2-microglobulin genes complexed with DMRIE/DOPE liposomes have been as‐

An emulsion is a dispersion of one immiscible liquid in another stabilized by a third compo‐ nent, the emulsifying agent [93]. The nanoemulsion consists of oil, water and surfactants, and presents a droplet size distribution of around 200 nm. Lipid-based carrier systems rep‐ resent drug vehicles composed of physiological lipids, such as cholesterol, cholesterol esters, phospholipids and tryglicerides, and offer a number of advantages, making them an ideal drug delivery carrier [94]. Adding cationic lipids as surfactants to these dispersed systems makes them suitable for gene delivery. The presence of cationic surfactants, like DOTAP, DOTMA or DC-Chol, causes the formation of positively charged droplets that promote strong electrostatic interactions between emulsion and the anionic nucleic acid phosphate groups [95,96]. For instance, Bruxel et al. [97] prepared a cationic nanoemulsion with DO‐

TAP as a delivery system for oligonucleotides targeting malarial topoisomerase II.

Lipid emulsions are considered to be superior to liposomes mainly in a scaling-up point of view. On the one hand, emulsions can be produced on an industrial scale; on the other

solid lipid nanoparticles [81].

12 Gene Therapy - Tools and Potential Applications

Liposomes offer several advantages for gene delivery [87]:

**•** they can transport large pieces of DNA

*3.2.2.2. Lipid nanoemulsions*

**•** they can be targeted to specific cells or tissues

tratissue or intravenous injection into animals [89,90].

**•** they are relatively cheap to produce and do not cause diseases

**•** protection of the DNA from degradation, mainly due to nucleases

sessed for safety and efficacy in phase I and II clinical trials [91,92].

*3.2.2.1. Cationic liposomes*

cations [88].

Solid lipid nanoparticles are particles made from a lipid being solid at room temperature and also at body temperature. They combine advantages of different colloidal systems. Like emulsions or liposomes, they are physiologically compatible and, like polymeric nanoparti‐ cles, it is possible to modulate drug release from the lipid matrix. In addition, SLN possess certain advantages. They can be produced without use of organic solvents, using high pres‐ sure homogenization (HPH) method that is already successfully implemented in pharma‐ ceutical industry [102]. From the point of view of application, SLN have very good stability [103] and are subject to be lyophilized [104], which facilitates the industrial production.

Cationic SLN, for instance, SLN containing at least one cationic lipid, have been proposed as non-viral vectors for gene delivery [105,20]. It has been shown that cationic SLN can effec‐ tively bind nucleic acids, protect them from DNase I degradation and deliver them into liv‐ ing cells. Cationic lipids are used in the preparation of SLN applied in gene therapy not only due to their positive surface charge, but also due to their surfactant activity, necessary to produce an initial emulsion, which is a common step in most preparation techniques. By means of electrostatic interactions, cationic SLN condense nucleic acids on their surface, leading generally to an excess of positive charges in the final complexes. This is beneficial for transfection because condensation facilitates the mobility of nucleic acids, protects them from environmental enzymes and the cationic character of the vectors allows the interaction with negatively charged cell surface. The characteristics of the resulting complexes depend on the ratio between particle and nucleic acid; there must be an equilibrium between the binding forces of the nucleic acids to SLN to achieve protection without hampering the pos‐ terior release in the site of action [106]. Release of DNA from the complexes may be one of the most crucial steps determining the optimal ratio for cationic lipid system-mediated transfection [107].

Our research group showed for the first time the expression of a foreign protein with SLNs in an "in vivo" study [108]. After intravenous administration of SLN containing the EGFP plasmid to BALB/c mice, protein expression was detected in the liver and spleen from the third day after administration, and it was maintained for at least 1 week. In a later study [109], we incorporated dextran and protamine in the SLN and the transfection was im‐ proved, being detected also in lung. The improvement in the transfection was related to a longer circulation in the bloodstream due to the presence of dextran on the nanoparticle sur‐ face. The surface features of this new vector may also induce a lower opsonization and a slower uptake by the RES. Moreover, the high DNA condensation of protamine that contrib‐ utes to the nuclease resistance may result in an extended stay of plasmid in the organism. The presence of nuclear localization signals in protamine, which improves the nuclear enve‐ lope translocation, and its capacity to facilitate transcription [110] may also explain the im‐ provement of the transfection efficacy "in vivo".

from longer viral proteins can provide nuclear localization signals that help the transport of

Non-Viral Delivery Systems in Gene Therapy http://dx.doi.org/10.5772/52704 15

Poly-L-lysine is a biodegradable peptide synthesized by polimerization on N-carboxy-anhy‐ dride of lysine [123]. It is able to form nanometer size complexes with polynucleotides ow‐ ing to the presence of protonable amine groups on the lysine moiety [16]. The most commonly used poly-L-lysine has a polymerization degree of 90 to 450 [124]. This character‐ istic makes this peptide suitable for "in vivo" use because it is readily biodegradable [116]. However, as the length of the poly-L-lysine increases, so does the cytotoxicity. Moreover, poly-L-lysine exhibits modest transfection when used alone and requires the addition of an edosomolytic agent such as chloroquine or a fusogenic peptide to allow for release into the cytoplasm. An strategy to prevent plasma protein binding and increase circulation half-life

Due to the advantages of peptides for gene delivery, they are frequently used to funtionalize cationic lipoplexes or polyplexes. Since these vectors undergo endocytosis, decorating them with endosomolytic peptides for enhanced cytosolic release may be helpful. Moreover, com‐ bination with peptides endowed with the ability to target a specific tissue of interest is high‐ ly beneficial, since this allows for reduced dose and, therefore, reduced side effects following systemic administration [127]. In a study carried out by our group [19], we im‐ proved cell transfection of ARPE-19 cells by using a cell penetration peptide (SAP) with sol‐ id lipid nanoparticles. Kwon et al. [128] covalently attached a truncated endosomolytic peptide derived from the carboxy-terminus of the HIV cell entry protein gp41 to a PEI scaf‐ fold, obtaining improved gene transfection results compared with unmodified PEI. In other study [20], protamine induced a 6-fold increase in the transfection capacity of SLN in retinal cells due to a shift in the internalization mechanism from caveolae/raft-mediated to clathrinmediated endocytosis, which promotes the release of the protamine-DNA complexes from the solid lipid nanoparticles; afterwards the transport of the complexes into the nucleus is

Gene delivery using physical principles has attracted increasing attention. These methods usually employ a physical force to overcome the membrane barrier of the cells and facilitate intracellular gene transfer. The simplicity is one of the characteristics of these methods. The genetic material is introduced into cells without formulating in any particulate or viral sys‐ tem. In a recent publication, Kamimura et al. [87] revised the different physical methods for

is the attachment of poly(ethylene glycol) to the poly-L-lysine [125,126].

favoured by the nuclear localization signals of the protamine.

gene delivery. These methods include the following:

**3.3. Physical methods for gene delivery**

*3.2.3.2. Peptides in multifunctional delivery systems*

the nucleic acids to the nucleus [121,122].

*3.2.3.1. Poly-L-lysine*

SLN have also been applied for the treatment of ocular diseases by gene therapy. After ocu‐ lar injection of a SLN based vector to rat eyes, the expression of EGFP was detected in vari‐ ous types of cells depending on the administration route: intravitreal or subretinal. In addition, this vector was also able to transfect corneal cells after topical application [111].

SLN may also be used as delivery systems for siRNA or oligonucleotides. Apolipoproteinfree low-density lipoprotein (LDL) mimicking SLN [112] formed stable complexes with siR‐ NA and exhibited comparable gene silencing efficiency to siRNA complexed with the polymer PEI, and lower citotoxicity. Afterwards, Tao et al. [113] showed that lipid nanopar‐ ticles caused 90% reduction of luciferase expression for at least 10 days, after a single sys‐ temic administration of 3 mg/kg luciferase siRNA to a liver-luciferase mouse model. CTAB stabilized SLN bearing an antisense oligonucleotide against glucosylceramide synthase (asGCS) reduced the viability of the drug resistant NCI/ADR-RES human ovary cancer cells in the presence of the chemotherapeutic doxorubicin [114].

#### *3.2.3. Peptide-based gene non-viral vectors*

Many types of peptides, which are generally cationic in nature and able to interact with plasmid DNA through electrostatic interaction, are under intense investigation as a safe al‐ ternative for gene therapy [115]. There are mainly four barriers that must be overcome by non-viral vectors to achieve successful gene delivery. The vector must be able to tightly compact and protect DNA, target specific cell-surface receptors, disrupt the endosomal membrane, and deliver the DNA cargo to the nucleus [115]. Peptide-based vectors are ad‐ vantageous over other non-viral systems because they are able to achieve all of these goals [116]. Cationic peptides rich in basic residues such as lysine and/or arginine are able to effi‐ ciently condense DNA into small, compact particles that can be stabilized in serum [117,118]. Attachment of a peptide ligand to a polyplex or lipoplex allows targeting to spe‐ cific receptors and/or specific cell types. Peptide sequence derived from protein transduction domains are able to selectively lyse the endosomal membrane in its acidic environment lead‐ ing to cytoplasmic release of the particle [119,120]. Finally, short peptide sequences taken from longer viral proteins can provide nuclear localization signals that help the transport of the nucleic acids to the nucleus [121,122].

#### *3.2.3.1. Poly-L-lysine*

Our research group showed for the first time the expression of a foreign protein with SLNs in an "in vivo" study [108]. After intravenous administration of SLN containing the EGFP plasmid to BALB/c mice, protein expression was detected in the liver and spleen from the third day after administration, and it was maintained for at least 1 week. In a later study [109], we incorporated dextran and protamine in the SLN and the transfection was im‐ proved, being detected also in lung. The improvement in the transfection was related to a longer circulation in the bloodstream due to the presence of dextran on the nanoparticle sur‐ face. The surface features of this new vector may also induce a lower opsonization and a slower uptake by the RES. Moreover, the high DNA condensation of protamine that contrib‐ utes to the nuclease resistance may result in an extended stay of plasmid in the organism. The presence of nuclear localization signals in protamine, which improves the nuclear enve‐ lope translocation, and its capacity to facilitate transcription [110] may also explain the im‐

SLN have also been applied for the treatment of ocular diseases by gene therapy. After ocu‐ lar injection of a SLN based vector to rat eyes, the expression of EGFP was detected in vari‐ ous types of cells depending on the administration route: intravitreal or subretinal. In addition, this vector was also able to transfect corneal cells after topical application [111].

SLN may also be used as delivery systems for siRNA or oligonucleotides. Apolipoproteinfree low-density lipoprotein (LDL) mimicking SLN [112] formed stable complexes with siR‐ NA and exhibited comparable gene silencing efficiency to siRNA complexed with the polymer PEI, and lower citotoxicity. Afterwards, Tao et al. [113] showed that lipid nanopar‐ ticles caused 90% reduction of luciferase expression for at least 10 days, after a single sys‐ temic administration of 3 mg/kg luciferase siRNA to a liver-luciferase mouse model. CTAB stabilized SLN bearing an antisense oligonucleotide against glucosylceramide synthase (asGCS) reduced the viability of the drug resistant NCI/ADR-RES human ovary cancer cells

Many types of peptides, which are generally cationic in nature and able to interact with plasmid DNA through electrostatic interaction, are under intense investigation as a safe al‐ ternative for gene therapy [115]. There are mainly four barriers that must be overcome by non-viral vectors to achieve successful gene delivery. The vector must be able to tightly compact and protect DNA, target specific cell-surface receptors, disrupt the endosomal membrane, and deliver the DNA cargo to the nucleus [115]. Peptide-based vectors are ad‐ vantageous over other non-viral systems because they are able to achieve all of these goals [116]. Cationic peptides rich in basic residues such as lysine and/or arginine are able to effi‐ ciently condense DNA into small, compact particles that can be stabilized in serum [117,118]. Attachment of a peptide ligand to a polyplex or lipoplex allows targeting to spe‐ cific receptors and/or specific cell types. Peptide sequence derived from protein transduction domains are able to selectively lyse the endosomal membrane in its acidic environment lead‐ ing to cytoplasmic release of the particle [119,120]. Finally, short peptide sequences taken

provement of the transfection efficacy "in vivo".

14 Gene Therapy - Tools and Potential Applications

in the presence of the chemotherapeutic doxorubicin [114].

*3.2.3. Peptide-based gene non-viral vectors*

Poly-L-lysine is a biodegradable peptide synthesized by polimerization on N-carboxy-anhy‐ dride of lysine [123]. It is able to form nanometer size complexes with polynucleotides ow‐ ing to the presence of protonable amine groups on the lysine moiety [16]. The most commonly used poly-L-lysine has a polymerization degree of 90 to 450 [124]. This character‐ istic makes this peptide suitable for "in vivo" use because it is readily biodegradable [116]. However, as the length of the poly-L-lysine increases, so does the cytotoxicity. Moreover, poly-L-lysine exhibits modest transfection when used alone and requires the addition of an edosomolytic agent such as chloroquine or a fusogenic peptide to allow for release into the cytoplasm. An strategy to prevent plasma protein binding and increase circulation half-life is the attachment of poly(ethylene glycol) to the poly-L-lysine [125,126].

#### *3.2.3.2. Peptides in multifunctional delivery systems*

Due to the advantages of peptides for gene delivery, they are frequently used to funtionalize cationic lipoplexes or polyplexes. Since these vectors undergo endocytosis, decorating them with endosomolytic peptides for enhanced cytosolic release may be helpful. Moreover, com‐ bination with peptides endowed with the ability to target a specific tissue of interest is high‐ ly beneficial, since this allows for reduced dose and, therefore, reduced side effects following systemic administration [127]. In a study carried out by our group [19], we im‐ proved cell transfection of ARPE-19 cells by using a cell penetration peptide (SAP) with sol‐ id lipid nanoparticles. Kwon et al. [128] covalently attached a truncated endosomolytic peptide derived from the carboxy-terminus of the HIV cell entry protein gp41 to a PEI scaf‐ fold, obtaining improved gene transfection results compared with unmodified PEI. In other study [20], protamine induced a 6-fold increase in the transfection capacity of SLN in retinal cells due to a shift in the internalization mechanism from caveolae/raft-mediated to clathrinmediated endocytosis, which promotes the release of the protamine-DNA complexes from the solid lipid nanoparticles; afterwards the transport of the complexes into the nucleus is favoured by the nuclear localization signals of the protamine.

#### **3.3. Physical methods for gene delivery**

Gene delivery using physical principles has attracted increasing attention. These methods usually employ a physical force to overcome the membrane barrier of the cells and facilitate intracellular gene transfer. The simplicity is one of the characteristics of these methods. The genetic material is introduced into cells without formulating in any particulate or viral sys‐ tem. In a recent publication, Kamimura et al. [87] revised the different physical methods for gene delivery. These methods include the following:

#### *3.3.1. Needle injection*

The DNA is directly injected through a needle-carrying syringe into tissues. Several tissues have been transfected by this method [87]: muscle, skin, liver, cardiac muscle, and solid tu‐ mors. DNA vaccination is the major application of this gene delivery system [129]. The effi‐ ciency of needle injection of DNA is low; moreover, transfection is limited to the needle surroundings.

*3.3.5. Photoporation*

*3.3.6. Magnetofection*

*3.3.7. Hydroporation*

tissue damage.

vivo" administration.

**4.1. Target cell uptake and intracellular trafficking**

The photoporation method utilizes a single laser pulse as the physical force to generate tran‐ sient pores on a cell membrane to allow DNA to enter [87]. Efficiency seems to be controlled by the size of the focal point and pulse frequency of the laser. The level of transgene expres‐ sion reported is similar to that of electroporation. Further studies are needed before this

Non-Viral Delivery Systems in Gene Therapy http://dx.doi.org/10.5772/52704 17

This method employs a magnetic field to promote transfection. DNA is complexed with magnetic nanoparticles made of iron oxide and coated with cationic lipids or polymers through electrostatic interaction. The magnetic particles are then concentrated on the target cells by the influence of an external magnetic field. Similar to the mechanism of non-viral vector-based gene delivery, the cellular uptake of DNA is due to endocytosis and pinocyto‐ sis [136]. This method has been successfully applied to a wide range of primary cells, and

Hydroporation, also called hydrodynamic gene delivery method, is the most commonly method used for gene delivery to hepatocytes in rodents. Intrahepatic gene delivery is ach‐ ieved by a rapid injection of a large volume of DNA solution via the tail vein in rodents, that results in a transient enlargement of fenestrae, generation of a transient membrane defect on the plasma membrane and gene transfer to hepatocytes [87]. This method has been frequent‐ ly employed in gene therapy research. In order to apply this simple method of gene admin‐ istration to the clinic, efforts have been made to reduce the injection volume and avoid

**4. Strategies to improve transfection mediated by non-viral vectors**

The successful delivery of therapeutic genes to the desired target cells and their availability at the intracellular site of action are crucial requirements for efficient gene therapy. The de‐ sign of safe and efficient non-viral vectors depends mainly on our understanding of the mechanisms involved in the cellular uptake and intracellular disposition of the therapeutic genes as well as their carriers. Moreover, they have to overcome the difficulties after "in

Nucleic acid must be internalized to interact with the intracellular machinery to execute their effect. The positive surface charge of unshielded complexes facilitates cellular internali‐ zation. The non-viral vector can be functionalized with compounds that are recognized by the desire specific target cell type. Peptides, proteins, carbohydrates and small molecules

highly sophisticted procedure becomes a practical technique for gene delivery.

cells that are difficult to transfect by other non-viral vectors [137].

#### *3.3.2. Ballistic DNA injection*

This method is also called particle bombardment, microprojectile gene transfer or gene gun. DNA-coated gold particles are propelled against cells, forcing intracellular DNA transfer. The accelerating force for DNA-containing particles can be high-voltage electronic dis‐ charge, spark discharge or helium pressure discharge. One advantage of this method is that it allows delivering precise DNA doses. However, genes express transiently, and considera‐ ble cell damage occurs at the centre of the discharge site. This method has been used in vac‐ cination against the influenza virus [130] and in gene therapy for treatment of ovarian cancer [131].

#### *3.3.3. Electroporation*

Gene delivery is achieved by generating pores on a cell membrane through electric pulses. The efficiency is determined by the intensity of the pulses, frequency and duration [132]. Electroporation creates transient permeability of the cell membrane and induces a low level of inflammation at the injection site, facilitating DNA uptake by parenchyma cells and anti‐ gen-presenting cells [133]. As drawbacks, the number of cells transfected is low, and surgery is required to reach internal organs. This method has been clinically tested for DNA-based vaccination [134] and for cancer treatment [135].

#### *3.3.4. Sonoporation*

Sonoporation utilizes ultrasound to temporally permeabilize the cell membrane to allow cel‐ lular uptake of DNA. It is non-invasive and site-specific and could make it possible to destroy tumor cells after systemic delivery, while leave non-targeted organs unaffected [13]. Gene de‐ livery efficiency seems to be dependent on the intensisty of the pulses, frequency and dura‐ tion [87]. This method has been applied in the brain, cornea, kidney, peritoneal cavity, muscle, and heart, among others. Low-intensity ultrasonund in combination with microbubbles has recently acquired much attention as a safe method of gene delivery [13]. The use of microbub‐ bles as gene vectors is based on the hypothesis that destruction of DNA-loaded microbubbles by a focused ultrasound beam during their microvascular transit through the target area will result in localized transduction upon disruption of the microbubble shell while sparing non‐ targeted areas. The therapeutic effect of ultrasound-targeted microbubble destruction is rela‐ tive to the size, stability, and targeting function of microbubbles.

#### *3.3.5. Photoporation*

*3.3.1. Needle injection*

16 Gene Therapy - Tools and Potential Applications

surroundings.

cancer [131].

*3.3.3. Electroporation*

*3.3.4. Sonoporation*

vaccination [134] and for cancer treatment [135].

tive to the size, stability, and targeting function of microbubbles.

*3.3.2. Ballistic DNA injection*

The DNA is directly injected through a needle-carrying syringe into tissues. Several tissues have been transfected by this method [87]: muscle, skin, liver, cardiac muscle, and solid tu‐ mors. DNA vaccination is the major application of this gene delivery system [129]. The effi‐ ciency of needle injection of DNA is low; moreover, transfection is limited to the needle

This method is also called particle bombardment, microprojectile gene transfer or gene gun. DNA-coated gold particles are propelled against cells, forcing intracellular DNA transfer. The accelerating force for DNA-containing particles can be high-voltage electronic dis‐ charge, spark discharge or helium pressure discharge. One advantage of this method is that it allows delivering precise DNA doses. However, genes express transiently, and considera‐ ble cell damage occurs at the centre of the discharge site. This method has been used in vac‐ cination against the influenza virus [130] and in gene therapy for treatment of ovarian

Gene delivery is achieved by generating pores on a cell membrane through electric pulses. The efficiency is determined by the intensity of the pulses, frequency and duration [132]. Electroporation creates transient permeability of the cell membrane and induces a low level of inflammation at the injection site, facilitating DNA uptake by parenchyma cells and anti‐ gen-presenting cells [133]. As drawbacks, the number of cells transfected is low, and surgery is required to reach internal organs. This method has been clinically tested for DNA-based

Sonoporation utilizes ultrasound to temporally permeabilize the cell membrane to allow cel‐ lular uptake of DNA. It is non-invasive and site-specific and could make it possible to destroy tumor cells after systemic delivery, while leave non-targeted organs unaffected [13]. Gene de‐ livery efficiency seems to be dependent on the intensisty of the pulses, frequency and dura‐ tion [87]. This method has been applied in the brain, cornea, kidney, peritoneal cavity, muscle, and heart, among others. Low-intensity ultrasonund in combination with microbubbles has recently acquired much attention as a safe method of gene delivery [13]. The use of microbub‐ bles as gene vectors is based on the hypothesis that destruction of DNA-loaded microbubbles by a focused ultrasound beam during their microvascular transit through the target area will result in localized transduction upon disruption of the microbubble shell while sparing non‐ targeted areas. The therapeutic effect of ultrasound-targeted microbubble destruction is rela‐ The photoporation method utilizes a single laser pulse as the physical force to generate tran‐ sient pores on a cell membrane to allow DNA to enter [87]. Efficiency seems to be controlled by the size of the focal point and pulse frequency of the laser. The level of transgene expres‐ sion reported is similar to that of electroporation. Further studies are needed before this highly sophisticted procedure becomes a practical technique for gene delivery.

#### *3.3.6. Magnetofection*

This method employs a magnetic field to promote transfection. DNA is complexed with magnetic nanoparticles made of iron oxide and coated with cationic lipids or polymers through electrostatic interaction. The magnetic particles are then concentrated on the target cells by the influence of an external magnetic field. Similar to the mechanism of non-viral vector-based gene delivery, the cellular uptake of DNA is due to endocytosis and pinocyto‐ sis [136]. This method has been successfully applied to a wide range of primary cells, and cells that are difficult to transfect by other non-viral vectors [137].

#### *3.3.7. Hydroporation*

Hydroporation, also called hydrodynamic gene delivery method, is the most commonly method used for gene delivery to hepatocytes in rodents. Intrahepatic gene delivery is ach‐ ieved by a rapid injection of a large volume of DNA solution via the tail vein in rodents, that results in a transient enlargement of fenestrae, generation of a transient membrane defect on the plasma membrane and gene transfer to hepatocytes [87]. This method has been frequent‐ ly employed in gene therapy research. In order to apply this simple method of gene admin‐ istration to the clinic, efforts have been made to reduce the injection volume and avoid tissue damage.

#### **4. Strategies to improve transfection mediated by non-viral vectors**

The successful delivery of therapeutic genes to the desired target cells and their availability at the intracellular site of action are crucial requirements for efficient gene therapy. The de‐ sign of safe and efficient non-viral vectors depends mainly on our understanding of the mechanisms involved in the cellular uptake and intracellular disposition of the therapeutic genes as well as their carriers. Moreover, they have to overcome the difficulties after "in vivo" administration.

#### **4.1. Target cell uptake and intracellular trafficking**

Nucleic acid must be internalized to interact with the intracellular machinery to execute their effect. The positive surface charge of unshielded complexes facilitates cellular internali‐ zation. The non-viral vector can be functionalized with compounds that are recognized by the desire specific target cell type. Peptides, proteins, carbohydrates and small molecules have been used to induce target cell-specific internalization [138]. For instance, SLN have been combined with peptides that show penetrating properties, such as the dimeric HIV-1 TAT (Trans-Activator of Transcription) peptide [139] or the synthetic SAP (Sweet Arrow Peptide) [19].

depends on the type of vectors [145]. Comparisons between different delivery vehicles showed that higher copy numbers of DNA molecules in the nucleus do not necessarily cor‐ relate with higher transfection efficiency. At similar plasmid/nucleus copies, lipofectamine mediated 10-fold higher transfection efficiency than PEI. This suggests that the DNA deliv‐ ered by PEI is biologically less active than the DNA delivered by lipofectamine. It also em‐ phasizes that a deeper understanding of the nuclear events in gene delivery is required for

Non-Viral Delivery Systems in Gene Therapy http://dx.doi.org/10.5772/52704 19

Vectors mediating high transfection efficiency "in vitro" often fail to achieve similar results "in vivo". One possible reason is that lipidic and polymeric vectors are optimized "in vitro" using two-dimensional (2D) cultures that lack extracellular "in vivo" barriers and do not re‐ alistically reflect "in vivo" conditions. While cells "in vitro" grow in monolayers, cells "in vivo" grow in 3D tissue layers held together by the extracellular matrix [145]. This results in cells with reduced thicknesses but larger widths and lengths. Particles that are taken up di‐ rectly above the nucleus (supranuclear region) have the shortest transport distance to the nucleus and hence a greater chance of delivery success. The spatiotemporal distribution of carriers, however, determines the optimal time for endosomal escape and the optimal intra‐ cellular pathway [151]. This highlights the need to develop adequate "in vitro" models that mimics as much as possible the "in vivo" conditions to optimize carriers for gene therapy. After intravenous administration, plasma nuclease degradation of the nucleic acid is the first barrier that needs to be overcome for therapeutic nucleic acid action. Nucleic acids can be degraded by hydrolytic endo- and exo-nucleases. Both types of nucleases are present in blood. Therefore, increasing nuclease resistance is crucial for achieving therapeutic effects. Naked nucleic acids are not only rapidly degraded upon intravenous injection, they are also cleared from the circulation rapidly, further limiting target tissue localization [138]. To im‐ prove nuclease resistance and colloidal stability, complexation strength is an important fac‐ tor. Shielding the non-viral vectors with poly-L-lysine or poly(ethylene glycol), as

mentioned previously, prolongs the circulation time in blood of the vectors.

Vectors delivered "in vivo" by systemic administration not only have to withstand the bloodstream, but also have to overcome the cellular matrix to reach all cell layers of the tis‐ sue. While large particles seem to have an advantage "in vitro" due to a sedimentation effect on cells, efficient delivery of particles deep into organs requires particles <100 nm. Small particles (40 nm) diffuse faster and more effectively in the extracellular matrix and inner lay‐ ers of tissues, whereas larger particles (>100 nm) are restricted by steric hindrance [152].

The net cationic charge of the synthetic vector is a determinant of circulation time, tissue dis‐ tribution and cellular uptake of synthetic vectors by inducing interactions with negatively blood constituents, such as erythrocytes and proteins. The opsonisation of foreign particles by plasma proteins actually represents one of the steps in the natural process of removal of foreign particles by the innate immune system [153]. This may result in obstruction of small capillaries, possibly leading to serious complication, such as pulmonary embolism [154]. Part of the complexes end up in the reticuoloendothelial system (RES), where they are re‐

future progress.

**4.2. "In vivo" optimization**

Endocytosis has been postulated as the main entry mechanism for non-viral systems. Vari‐ ous endocytosis mechanisms have been described to date: phagocytosis, pinocytosis, cla‐ thrin-mediated endocytosis, caveolae/raft-mediated endocytosis and chathrin and caveolae independent endocytosis. Clathrin-mediated endocytosis leads to an intracellular pathway in which endosomes fuse with lysosomes, which degrade their content, whereas caveolae/ raft-mediated endocytosis avoids the lysosomal pathway and its consequent vector degra‐ dation [20]. Cytosolic delivery from either endosomes or lisosomes has been reported a ma‐ jor limitation in transfection [140]. In consequence, some research groups have used substances that facilitate endosomal escape before lysosomal degradation. For clathrinmediated endocytosis, the drop in pH is a useful strategy for endosomal scape via proton destabilization conferred by the cationic carrier, or by pH-dependent activation of mem‐ brane disruptive helper molecules, such as DOPE or fusogenic peptides [141-143]. More re‐ cently, Leung et al. [144] have patented lipids with 4-amino-butiric acid (FAB) as headgroup to form lipid nanoparticles able to introduce nucleic acids, specifically siRNA, into mamma‐ lian cells. FAB lipids also demonstrated membrane destabilizing properties.

Once genes are delivered in the cytoplasm they have to diffuse toward the nuclear region. DNA plasmids have difficulties to diffuse in the cytoplasm because they are large in size. Therefore, packaging and complexing them into small particles facilitates its displacement intracellularly. Diffusion is a function of diameter; hence, smaller particles move faster than larger ones. Thus, another way to optimize gene delivery to the nucleus would be to de‐ crease the size of the particles to increase the velocity of passive diffusion through the cyto‐ plasm [145].

The pass through the nuclear membrane is the next step, and it is in general, quite difficult. There are two mechanisms large molecules can use to overcome that barrier: disruption of the nuclear membrane during mitosis, which is conditioned by the division rate of targeted cells, or import through the nuclear pore complex (NPC). This latter mechanism requires nuclear localization signals, which can be used to improve transfection by non-viral vectors [146]. In this regard, protamine is a peptide that condenses DNA and presents sequences of 6 consecutive arginine residues [147], which make this peptide able to translocate molecules such as DNA from the cytoplasm to the nucleus of living cells. Although protamine/DNA polyplexes are not effective gene vectors [148], the combination of protamine with SLN pro‐ duced good results in both COS-1 and Na 1330 (murine neuroblastoma) culture cells [149,150]. Precondensation of plasmids with this peptide, to form protamine-DNA com‐ plexes that are later bound to cationic SLN, is another alternative that has shown higher transfection capacity in retinal cells compared to SLN prepared without protamine [20].

Once inside the nucleus, level of transgene expression depends on the copy number of DNA and its accessibility for the transcription machinery. Studies have shown that the minimum number of plasmids delivered to the nucleus required for measurable transgene expression depends on the type of vectors [145]. Comparisons between different delivery vehicles showed that higher copy numbers of DNA molecules in the nucleus do not necessarily cor‐ relate with higher transfection efficiency. At similar plasmid/nucleus copies, lipofectamine mediated 10-fold higher transfection efficiency than PEI. This suggests that the DNA deliv‐ ered by PEI is biologically less active than the DNA delivered by lipofectamine. It also em‐ phasizes that a deeper understanding of the nuclear events in gene delivery is required for future progress.

#### **4.2. "In vivo" optimization**

have been used to induce target cell-specific internalization [138]. For instance, SLN have been combined with peptides that show penetrating properties, such as the dimeric HIV-1 TAT (Trans-Activator of Transcription) peptide [139] or the synthetic SAP (Sweet Arrow

Endocytosis has been postulated as the main entry mechanism for non-viral systems. Vari‐ ous endocytosis mechanisms have been described to date: phagocytosis, pinocytosis, cla‐ thrin-mediated endocytosis, caveolae/raft-mediated endocytosis and chathrin and caveolae independent endocytosis. Clathrin-mediated endocytosis leads to an intracellular pathway in which endosomes fuse with lysosomes, which degrade their content, whereas caveolae/ raft-mediated endocytosis avoids the lysosomal pathway and its consequent vector degra‐ dation [20]. Cytosolic delivery from either endosomes or lisosomes has been reported a ma‐ jor limitation in transfection [140]. In consequence, some research groups have used substances that facilitate endosomal escape before lysosomal degradation. For clathrinmediated endocytosis, the drop in pH is a useful strategy for endosomal scape via proton destabilization conferred by the cationic carrier, or by pH-dependent activation of mem‐ brane disruptive helper molecules, such as DOPE or fusogenic peptides [141-143]. More re‐ cently, Leung et al. [144] have patented lipids with 4-amino-butiric acid (FAB) as headgroup to form lipid nanoparticles able to introduce nucleic acids, specifically siRNA, into mamma‐

lian cells. FAB lipids also demonstrated membrane destabilizing properties.

Once genes are delivered in the cytoplasm they have to diffuse toward the nuclear region. DNA plasmids have difficulties to diffuse in the cytoplasm because they are large in size. Therefore, packaging and complexing them into small particles facilitates its displacement intracellularly. Diffusion is a function of diameter; hence, smaller particles move faster than larger ones. Thus, another way to optimize gene delivery to the nucleus would be to de‐ crease the size of the particles to increase the velocity of passive diffusion through the cyto‐

The pass through the nuclear membrane is the next step, and it is in general, quite difficult. There are two mechanisms large molecules can use to overcome that barrier: disruption of the nuclear membrane during mitosis, which is conditioned by the division rate of targeted cells, or import through the nuclear pore complex (NPC). This latter mechanism requires nuclear localization signals, which can be used to improve transfection by non-viral vectors [146]. In this regard, protamine is a peptide that condenses DNA and presents sequences of 6 consecutive arginine residues [147], which make this peptide able to translocate molecules such as DNA from the cytoplasm to the nucleus of living cells. Although protamine/DNA polyplexes are not effective gene vectors [148], the combination of protamine with SLN pro‐ duced good results in both COS-1 and Na 1330 (murine neuroblastoma) culture cells [149,150]. Precondensation of plasmids with this peptide, to form protamine-DNA com‐ plexes that are later bound to cationic SLN, is another alternative that has shown higher transfection capacity in retinal cells compared to SLN prepared without protamine [20].

Once inside the nucleus, level of transgene expression depends on the copy number of DNA and its accessibility for the transcription machinery. Studies have shown that the minimum number of plasmids delivered to the nucleus required for measurable transgene expression

Peptide) [19].

18 Gene Therapy - Tools and Potential Applications

plasm [145].

Vectors mediating high transfection efficiency "in vitro" often fail to achieve similar results "in vivo". One possible reason is that lipidic and polymeric vectors are optimized "in vitro" using two-dimensional (2D) cultures that lack extracellular "in vivo" barriers and do not re‐ alistically reflect "in vivo" conditions. While cells "in vitro" grow in monolayers, cells "in vivo" grow in 3D tissue layers held together by the extracellular matrix [145]. This results in cells with reduced thicknesses but larger widths and lengths. Particles that are taken up di‐ rectly above the nucleus (supranuclear region) have the shortest transport distance to the nucleus and hence a greater chance of delivery success. The spatiotemporal distribution of carriers, however, determines the optimal time for endosomal escape and the optimal intra‐ cellular pathway [151]. This highlights the need to develop adequate "in vitro" models that mimics as much as possible the "in vivo" conditions to optimize carriers for gene therapy.

After intravenous administration, plasma nuclease degradation of the nucleic acid is the first barrier that needs to be overcome for therapeutic nucleic acid action. Nucleic acids can be degraded by hydrolytic endo- and exo-nucleases. Both types of nucleases are present in blood. Therefore, increasing nuclease resistance is crucial for achieving therapeutic effects. Naked nucleic acids are not only rapidly degraded upon intravenous injection, they are also cleared from the circulation rapidly, further limiting target tissue localization [138]. To im‐ prove nuclease resistance and colloidal stability, complexation strength is an important fac‐ tor. Shielding the non-viral vectors with poly-L-lysine or poly(ethylene glycol), as mentioned previously, prolongs the circulation time in blood of the vectors.

Vectors delivered "in vivo" by systemic administration not only have to withstand the bloodstream, but also have to overcome the cellular matrix to reach all cell layers of the tis‐ sue. While large particles seem to have an advantage "in vitro" due to a sedimentation effect on cells, efficient delivery of particles deep into organs requires particles <100 nm. Small particles (40 nm) diffuse faster and more effectively in the extracellular matrix and inner lay‐ ers of tissues, whereas larger particles (>100 nm) are restricted by steric hindrance [152].

The net cationic charge of the synthetic vector is a determinant of circulation time, tissue dis‐ tribution and cellular uptake of synthetic vectors by inducing interactions with negatively blood constituents, such as erythrocytes and proteins. The opsonisation of foreign particles by plasma proteins actually represents one of the steps in the natural process of removal of foreign particles by the innate immune system [153]. This may result in obstruction of small capillaries, possibly leading to serious complication, such as pulmonary embolism [154]. Part of the complexes end up in the reticuoloendothelial system (RES), where they are re‐ moved rapidly by phagocytosis or by trapping in fine capillary beds [155]. The nanocarriers, when circulating in blood, can activate the complement system and it seems that the com‐ plement activation is higher as the surface charge increases [156,157].

tions. A number of potentially rate-limiting steps in the processes of non-viral-mediated gene delivery have been identified, which include the efficiency of cell surface association, internalization, release of gene from intracellular compartments such as endosomes, transfer via the cytosol and translocation into the nucleus and transcription efficacy. Insight into mo‐ lecular features of each of these steps is essential in order to determine their effectiveness as a barrier and to identify means of overcoming these hurdles. Although non-viral vectors may work reasonably well "in vitro", clinical success is still far from ideal. Considering the number of research groups that focus their investigations on the development of new vec‐ tors for gene therapy, together with the advances in the development of new technologies to better understand their "in vitro" and "in vivo" behavior, the present limitations of non-vi‐

Non-Viral Delivery Systems in Gene Therapy http://dx.doi.org/10.5772/52704 21

Alicia Rodríguez Gascón, Ana del Pozo-Rodríguez and María Ángeles Solinís

Pharmacokinetics, Nanotechnology and Gene Therapy Group, Faculty of Phamacy, Univer‐

[1] Venter JC, Adams MD, Myers EW, et al. The Sequence of the Human Genome. Sci‐

[2] Kassner PD. Discovery of Novel Targets with High Throughput RNA Interference Screening. Combinatorial Chemistry & High Throughput Screen 2008;11(3) 175-184.

[3] Wiltgen M, Tilz GP. DNA Microarray Analysis: Principles and Clinical Impact. Hem‐

[4] Directive 2009/120/EC of the European Parliament. http://eur-lex.europa.eu/LexUri‐ Serv/LexUriServ.do?uri=OJ:L:2009:242:0003:0012:EN:PDF (accessed 07 August 2012).

[5] Zhang Y, Satterlee A, Huang L. In Vivo Gene Delivery by Nonviral Vectors: Over‐

[6] Gene Therapy Clinical Trials Worldwide.Provided by the Journal of Gene Medicine. Jon Wiley and Sons Ltd, 2012; http://www.wiley.co.k/genmed/clinical (accessed 01

[7] Li SD, Huang L. Non-viral is Superior to Viral Gene Delivery. Journal of Controlled

coming Hurdles?. Molecular Therapy 2012; 20(7) 1298-1304.

\*Address all correspondence to: alicia.rodriguez@ehu.es

sity of the Basque Country UPV/EHU, Spain

ence 2001;291(5507) 1304-51.

atology 2007;12(4) 27-87.

Release 2007;123(3) 181-183.

August 2012).

ral vectors will be resolved rationally.

**Author details**

**References**

The interaction with blood components is related to the intrinsic properties of the cationic compound (side chain end groups, its spatial conformation and molecular weight), as well as the applied Nitrogen:Phosphate (N:P) ratio [138]. Shielding of the positive surface charge of complexes is currently an important strategy to circumvent the aforementioned problems. The most popular strategy is based on the attachment of water-soluble, neutral, flexible pol‐ ymers, as poly(ethylene glycol), poly(vinylpyrrolidone) and poly(hydroxyethyl-L-aspara‐ gine). The efficacy of the shielding effect of these polymers is determined by the molecular weight and grafting density of the shielding polymer [158]. Longer chains are usually more effective in protecting the particle (surface) from aggregation and opsonisation.

The nanocarriers must arrive to the target tissue to exert their action. Although most common‐ ly used targeting strategies consist of proteins and peptides, carbohydrates have also been uti‐ lized [159]. The access of non-viral vector to tumors has been investigated extensively. The discontinuous endothelial cell layer has gaps that give the nanocarriers the opportunity to es‐ cape the vascular bed and migrate into the tumoral mass. The most common entities used for tumor targeting include transferrin, epidermal growth factor, and the integrin-binding tripeptide arginine-glycine-aspartic acid (RGD) [159]. Brain targeting has also a great interest; most gene vector do not cross the blood-brain barrier (BBB) after intravenous administration and must be administered through intracerebral injection, which is highly invasive and does not allow for delivery of the gene to other areas of the brain. Injection in the cerebrospinal flu‐ id is also another strategy. Commonly used ligands for mediated uptake are insuline-like growth factors, transferrin or low-density lipid protein [159]. Targeting to the liver has been also investigated in a great extension by many researchers. Carbohydrate-related molecules, such as galactose, asialofetuin, N-acetylgalactosamine and folic acid are the most commonly molecules used for liver targeting [159]. Targeting to endothelial cells provides avenues for improvement of specificity and effectiveness of treatment of many diseases, such as cardio‐ vascular or metabolic diseases [160]. Among other endothelial cell surface determinants, in‐ tercellular adhesion molecule-1 (CD54 or ICA-1, a 110-KDa Ig-like transmembrane constitutive endothelial adhesion molecule) is a good candidate target for this goal. ICAM-1 targeting can be achieved by coupling Anti-ICAM-1 antibodies to carriers [161].

#### **5. Conclusion**

The success of gene therapy is highly dependent on the delivery vector. Viral vectors have dominated the clinical trials in gene therapy for its relatively high delivery efficiency. How‐ ever, the improvement of efficacy of non-viral vectors has lead to an increased number of products entering into clinical trials. A better understanding of the mechanisms governing the efficiency of transfection, from the formation of the complexes to their intracellular de‐ livery, will lead to the design of better adapted non-viral vectors for gene therapy applica‐ tions. A number of potentially rate-limiting steps in the processes of non-viral-mediated gene delivery have been identified, which include the efficiency of cell surface association, internalization, release of gene from intracellular compartments such as endosomes, transfer via the cytosol and translocation into the nucleus and transcription efficacy. Insight into mo‐ lecular features of each of these steps is essential in order to determine their effectiveness as a barrier and to identify means of overcoming these hurdles. Although non-viral vectors may work reasonably well "in vitro", clinical success is still far from ideal. Considering the number of research groups that focus their investigations on the development of new vec‐ tors for gene therapy, together with the advances in the development of new technologies to better understand their "in vitro" and "in vivo" behavior, the present limitations of non-vi‐ ral vectors will be resolved rationally.

#### **Author details**

moved rapidly by phagocytosis or by trapping in fine capillary beds [155]. The nanocarriers, when circulating in blood, can activate the complement system and it seems that the com‐

The interaction with blood components is related to the intrinsic properties of the cationic compound (side chain end groups, its spatial conformation and molecular weight), as well as the applied Nitrogen:Phosphate (N:P) ratio [138]. Shielding of the positive surface charge of complexes is currently an important strategy to circumvent the aforementioned problems. The most popular strategy is based on the attachment of water-soluble, neutral, flexible pol‐ ymers, as poly(ethylene glycol), poly(vinylpyrrolidone) and poly(hydroxyethyl-L-aspara‐ gine). The efficacy of the shielding effect of these polymers is determined by the molecular weight and grafting density of the shielding polymer [158]. Longer chains are usually more

The nanocarriers must arrive to the target tissue to exert their action. Although most common‐ ly used targeting strategies consist of proteins and peptides, carbohydrates have also been uti‐ lized [159]. The access of non-viral vector to tumors has been investigated extensively. The discontinuous endothelial cell layer has gaps that give the nanocarriers the opportunity to es‐ cape the vascular bed and migrate into the tumoral mass. The most common entities used for tumor targeting include transferrin, epidermal growth factor, and the integrin-binding tripeptide arginine-glycine-aspartic acid (RGD) [159]. Brain targeting has also a great interest; most gene vector do not cross the blood-brain barrier (BBB) after intravenous administration and must be administered through intracerebral injection, which is highly invasive and does not allow for delivery of the gene to other areas of the brain. Injection in the cerebrospinal flu‐ id is also another strategy. Commonly used ligands for mediated uptake are insuline-like growth factors, transferrin or low-density lipid protein [159]. Targeting to the liver has been also investigated in a great extension by many researchers. Carbohydrate-related molecules, such as galactose, asialofetuin, N-acetylgalactosamine and folic acid are the most commonly molecules used for liver targeting [159]. Targeting to endothelial cells provides avenues for improvement of specificity and effectiveness of treatment of many diseases, such as cardio‐ vascular or metabolic diseases [160]. Among other endothelial cell surface determinants, in‐ tercellular adhesion molecule-1 (CD54 or ICA-1, a 110-KDa Ig-like transmembrane constitutive endothelial adhesion molecule) is a good candidate target for this goal. ICAM-1

plement activation is higher as the surface charge increases [156,157].

20 Gene Therapy - Tools and Potential Applications

effective in protecting the particle (surface) from aggregation and opsonisation.

targeting can be achieved by coupling Anti-ICAM-1 antibodies to carriers [161].

The success of gene therapy is highly dependent on the delivery vector. Viral vectors have dominated the clinical trials in gene therapy for its relatively high delivery efficiency. How‐ ever, the improvement of efficacy of non-viral vectors has lead to an increased number of products entering into clinical trials. A better understanding of the mechanisms governing the efficiency of transfection, from the formation of the complexes to their intracellular de‐ livery, will lead to the design of better adapted non-viral vectors for gene therapy applica‐

**5. Conclusion**

Alicia Rodríguez Gascón, Ana del Pozo-Rodríguez and María Ángeles Solinís

\*Address all correspondence to: alicia.rodriguez@ehu.es

Pharmacokinetics, Nanotechnology and Gene Therapy Group, Faculty of Phamacy, Univer‐ sity of the Basque Country UPV/EHU, Spain

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**Chapter 2**

**Plasmid Transgene Expression** *in vivo***:**

David Morrissey, Sara A. Collins, Simon Rajenderan,

Ensuring an appropriate level and duration of expression is essential in achieving an effi‐ cient and safe gene therapy. While the length of time a gene must be expressed for efficacy depends on both the therapeutic strategy and the disease, many gene therapy approaches prove ineffective as the therapeutic is expressed for a limited duration (Frank et al. 2004). Proposed causes of transient expression include loss of DNA due to cell turnover, immune responses against transfected cells and/or expressed proteins, and inhibition of transcription through host cell methylation of microbial DNA sequences (Prosch et al. 1996; Scheule 2000; Greenland et al. 2007). Vector related elements or activity also contribute to duration of gene expression post administration. Adenovirus is known to stimulate severe innate and adap‐ tive immune responses, and can induce cellular and humoural responses to the transgene product and its capsid proteins resulting in failure to provide long-term gene expression

Plasmid electroporation, on the other hand, has been shown not to elicit such transgene gene silencing immune responses (Jooss et al. 1998; Mir et al. 1999), and presents an attrac‐ tive option in achieving long-term gene expression, especially in light of recent improve‐ ments in plasmid vectors (Gill et al. 2009). Although plasmid based systems offer certain advantages, they do, however, have drawbacks. The magnitude of transgene expression is generally lower with plasmid vectors than that with viruses. In addition, most plasmids are not passed on to daughter cells following cell division leading to eventual loss of expression in rapidly dividing tissues. This can result in sub-therapeutic effects, a significant problem

> © 2013 Morrissey et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

(Jooss et al. 1998; Yuasa et al. 2002; Louboutin et al. 2005; Wang et al. 2005).

**Promoter and Tissue Variables**

Garrett Casey, Gerald C. O'Sullivan and

Additional information is available at the end of the chapter

Mark Tangney

**1. Introduction**

http://dx.doi.org/10.5772/52658

#### **Chapter 2**

### **Plasmid Transgene Expression** *in vivo***: Promoter and Tissue Variables**

David Morrissey, Sara A. Collins, Simon Rajenderan, Garrett Casey, Gerald C. O'Sullivan and Mark Tangney

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52658

#### **1. Introduction**

Ensuring an appropriate level and duration of expression is essential in achieving an effi‐ cient and safe gene therapy. While the length of time a gene must be expressed for efficacy depends on both the therapeutic strategy and the disease, many gene therapy approaches prove ineffective as the therapeutic is expressed for a limited duration (Frank et al. 2004). Proposed causes of transient expression include loss of DNA due to cell turnover, immune responses against transfected cells and/or expressed proteins, and inhibition of transcription through host cell methylation of microbial DNA sequences (Prosch et al. 1996; Scheule 2000; Greenland et al. 2007). Vector related elements or activity also contribute to duration of gene expression post administration. Adenovirus is known to stimulate severe innate and adap‐ tive immune responses, and can induce cellular and humoural responses to the transgene product and its capsid proteins resulting in failure to provide long-term gene expression (Jooss et al. 1998; Yuasa et al. 2002; Louboutin et al. 2005; Wang et al. 2005).

Plasmid electroporation, on the other hand, has been shown not to elicit such transgene gene silencing immune responses (Jooss et al. 1998; Mir et al. 1999), and presents an attrac‐ tive option in achieving long-term gene expression, especially in light of recent improve‐ ments in plasmid vectors (Gill et al. 2009). Although plasmid based systems offer certain advantages, they do, however, have drawbacks. The magnitude of transgene expression is generally lower with plasmid vectors than that with viruses. In addition, most plasmids are not passed on to daughter cells following cell division leading to eventual loss of expression in rapidly dividing tissues. This can result in sub-therapeutic effects, a significant problem

© 2013 Morrissey et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. with gene therapy. Efforts have been made to ensure that therapeutic protein production is active for an appropriate length of time to address some of these failings. To counteract the effects of episomal DNA loss, the use of integrating DNA in the form of retroviruses or transposon containing plasmids has been examined and shown some efficacy(Sandrin et al. 2003; Ohlfest et al. 2005). Delivery in this fashion would lead to long lasting, possibly indefi‐ nite gene expression. Although this addresses one failing of plasmid delivery, the potential of indefinite and uncontrollable protein production to cause unexpected side effects is an is‐ sue. Unlike the current situation, where therapy related complications results in withdrawal of the medication, the "offending gene" cannot easily be removed, and may continue to cause significant side effects. In addition, integration of foreign DNA is not ideal as it can lead to mutagenesis, with subsequent alteration in the patient's protein expression profile and potentially carcinogenesis. With this in mind, methods of prolonging and/or controlling episomal gene expression are preferred, provided this expression is of sufficient magnitude.

plasmid, designated pUb-luc, containing the firefly *luciferase* gene transcriptionally control‐ led from the human Ubiquitin-B promoter was constructed, by excising the firefly *luciferase* gene from pGL3-Control using restriction enzymes Nco1 and Xba1 (New England Biolabs, USA) and cloning it in the Nhe1 (site 2) and Nco1 sites of pDRIVE03-UbiquitinB(h) down‐ stream of the ubiquitin promoter. Plasmid copy number was calculated using the formula

luc = 5.9 x 103 and pGL3 = 5.2 x 103 bp respectively. Endotoxin-free plasmid DNA was isolat‐ ed from TOP10F *E.coli* (Invitrogen) using the MegaPrep kit (Qiagen, West Sussex, England).

Murine JBS fibrosarcoma tumour cells were maintained in culture in Dulbecco's Modified Essential Medium (DMEM) (GIBCO, Invitrogen Corp., Paisley, Scotland) as previously de‐ scribed (Collins, C. G. et al. 2006; Collins, S. A. et al. 2010). Female Balb/C and MF1nu/nu mice of 6–8 weeks of age were obtained from Harlan Laboratories (Oxfordshire, England).

For tumour experiments, mice were treated at a tumour volume of approximately 100 mm3 in volume (5-7 mm major diameter). Mice were anaesthetized during all treatments by intra‐ peritoneal (i.p.) administration of 200 µg xylazine and 2 mg ketamine. For liver transfection, a 1 cm subcostal incision was made over the liver and the peritoneum opened. The right lobe of the exposed liver was administered plasmid by electroporation as described below (Casey et al. 2010; Collins, S. A. et al. 2011). The wound was closed in two layers, peritoneal and skin, using 4/0 prolene sutures (Promed, Killorglin, Ireland). For plasmid delivery by electroporation, a custom-designed applicator with 2 needles 4 mm apart was used, with both needles placed through the skin central to the tissue. Tissue was injected between elec‐ trode needles with 8 x 1011 copies of plasmid DNA in sterile injectable saline in an injection volume of 50 µl. After 80 seconds, square-wave pulses (1200 V/cm 100 µsec x 1 and 120 V/cm 20 msec, 8 pulses) were administered in sequence using a custom designed pulse gen‐

Individual animals were weighed and dosed by i.p. injection of trichostatin A (TSA) (Sigma) at 10 mg/kg in 60 µl 10% (v/v) dimethyl sulfoxide in filtered peanut oil, daily for the dura‐ tion of the experiment. *In vivo* luciferase activity was assessed 4 hours after administration

*In vivo* luciferase activity from tissues was analysed at set time points post-transfection as follows: 80 µl of 30 mg/ml firefly luciferin (Biosynth, Basil, Switzerland) was injected i.p.

JBS cells suspended in 200 µl serum free DMEM were

Plasmid Transgene Expression *in vivo*; Promoter and Tissue Variables

, pCMV-

37

http://dx.doi.org/10.5772/52658

number DNA copies = weight/(Plasmid size x 1.096 x 10-21) with pUb-luc = 4.3 x103

**2.2. Animals and tumour induction**

For routine tumour induction, 2 × 106

injected subcutaneously into the flank.

erator (Cliniporator (IGEA, Carpi, Italy).

of TSA.

**2.5. Whole body imaging**

**2.4. Inhibition of DNA acetylation** *in vivo*

**2.3.** *In-vivo* **gene delivery**

Plasmid loss alone may not fully account for the temporal loss of expression seen with these vectors. Epigenetic modification of the therapeutic has also been implicated in gene silencing, but the exact mechanisms by which this occurs have not yet been fully elucidated. It has been demonstrated that duration of transgene expression may by increased by use of 'native' pro‐ moters of mammalian origin rather than viral promoters (Gazdhar et al. 2006). The postulated mechanism behind this difference of expression relates to the presence and subsequent meth‐ ylation of CpG sequences on promoters. This methylation is a naturally occurring phenomen‐ on and reports have correlated methylation of CpG-rich sequences with silencing of gene expression (Gazdhar, Bilici et al. 2006). Native mammalian promoters possess fewer CpG se‐ quences than their viral counterparts and are theoretically less prone to silencing. By employ‐ ing mammalian promoters, the duration of gene expression may be extended, allowing for sustained therapeutic production. Anecdotal evidence suggests that the degree of viral pro‐ moter silencing varies between tissue types, and that the duration of gene expression in tu‐ mour tissue in particular may be short-lived (Jaenisch et al. 1985; Momparler & Bovenzi 2000; Bartoli et al. 2003). This may, in part, be due to abnormal cell turnover in tumour tissue, but the disorganised methylation pattern in tumour tissue could also play a role.

In this chapter, we assess the influence of promoter type on electroporated plasmid transgene expression in murine models. Expression is examined by utilising the reporter gene lucifer‐ ase. The activity of luciferase can then be measured *in vivo*, allowing for repeated assessment of gene expression in the same test subjects over time. The pattern of expression is also exam‐ ined in different tissue types as is the role of epigenetic modification in gene silencing.

#### **2. Materials and methods**

#### **2.1. DNA constructs**

pGL3-Control and pCMV-luc were purchased from Stratagene (Techno-Path, Limerick, Ire‐ land) and Promega (Medical Supply Co., Dublin, Ireland) respectively. pDRIVE03-Ubiqui‐ tinB(h) v02 was purchased from Invivogen (Cayla SAS, Toulouse, France). A version of this plasmid, designated pUb-luc, containing the firefly *luciferase* gene transcriptionally control‐ led from the human Ubiquitin-B promoter was constructed, by excising the firefly *luciferase* gene from pGL3-Control using restriction enzymes Nco1 and Xba1 (New England Biolabs, USA) and cloning it in the Nhe1 (site 2) and Nco1 sites of pDRIVE03-UbiquitinB(h) down‐ stream of the ubiquitin promoter. Plasmid copy number was calculated using the formula number DNA copies = weight/(Plasmid size x 1.096 x 10-21) with pUb-luc = 4.3 x103 , pCMVluc = 5.9 x 103 and pGL3 = 5.2 x 103 bp respectively. Endotoxin-free plasmid DNA was isolat‐ ed from TOP10F *E.coli* (Invitrogen) using the MegaPrep kit (Qiagen, West Sussex, England).

#### **2.2. Animals and tumour induction**

with gene therapy. Efforts have been made to ensure that therapeutic protein production is active for an appropriate length of time to address some of these failings. To counteract the effects of episomal DNA loss, the use of integrating DNA in the form of retroviruses or transposon containing plasmids has been examined and shown some efficacy(Sandrin et al. 2003; Ohlfest et al. 2005). Delivery in this fashion would lead to long lasting, possibly indefi‐ nite gene expression. Although this addresses one failing of plasmid delivery, the potential of indefinite and uncontrollable protein production to cause unexpected side effects is an is‐ sue. Unlike the current situation, where therapy related complications results in withdrawal of the medication, the "offending gene" cannot easily be removed, and may continue to cause significant side effects. In addition, integration of foreign DNA is not ideal as it can lead to mutagenesis, with subsequent alteration in the patient's protein expression profile and potentially carcinogenesis. With this in mind, methods of prolonging and/or controlling episomal gene expression are preferred, provided this expression is of sufficient magnitude. Plasmid loss alone may not fully account for the temporal loss of expression seen with these vectors. Epigenetic modification of the therapeutic has also been implicated in gene silencing, but the exact mechanisms by which this occurs have not yet been fully elucidated. It has been demonstrated that duration of transgene expression may by increased by use of 'native' pro‐ moters of mammalian origin rather than viral promoters (Gazdhar et al. 2006). The postulated mechanism behind this difference of expression relates to the presence and subsequent meth‐ ylation of CpG sequences on promoters. This methylation is a naturally occurring phenomen‐ on and reports have correlated methylation of CpG-rich sequences with silencing of gene expression (Gazdhar, Bilici et al. 2006). Native mammalian promoters possess fewer CpG se‐ quences than their viral counterparts and are theoretically less prone to silencing. By employ‐ ing mammalian promoters, the duration of gene expression may be extended, allowing for sustained therapeutic production. Anecdotal evidence suggests that the degree of viral pro‐ moter silencing varies between tissue types, and that the duration of gene expression in tu‐ mour tissue in particular may be short-lived (Jaenisch et al. 1985; Momparler & Bovenzi 2000; Bartoli et al. 2003). This may, in part, be due to abnormal cell turnover in tumour tissue, but

the disorganised methylation pattern in tumour tissue could also play a role.

**2. Materials and methods**

36 Gene Therapy - Tools and Potential Applications

**2.1. DNA constructs**

In this chapter, we assess the influence of promoter type on electroporated plasmid transgene expression in murine models. Expression is examined by utilising the reporter gene lucifer‐ ase. The activity of luciferase can then be measured *in vivo*, allowing for repeated assessment of gene expression in the same test subjects over time. The pattern of expression is also exam‐

pGL3-Control and pCMV-luc were purchased from Stratagene (Techno-Path, Limerick, Ire‐ land) and Promega (Medical Supply Co., Dublin, Ireland) respectively. pDRIVE03-Ubiqui‐ tinB(h) v02 was purchased from Invivogen (Cayla SAS, Toulouse, France). A version of this

ined in different tissue types as is the role of epigenetic modification in gene silencing.

Murine JBS fibrosarcoma tumour cells were maintained in culture in Dulbecco's Modified Essential Medium (DMEM) (GIBCO, Invitrogen Corp., Paisley, Scotland) as previously de‐ scribed (Collins, C. G. et al. 2006; Collins, S. A. et al. 2010). Female Balb/C and MF1nu/nu mice of 6–8 weeks of age were obtained from Harlan Laboratories (Oxfordshire, England). For routine tumour induction, 2 × 106 JBS cells suspended in 200 µl serum free DMEM were injected subcutaneously into the flank.

#### **2.3.** *In-vivo* **gene delivery**

For tumour experiments, mice were treated at a tumour volume of approximately 100 mm3 in volume (5-7 mm major diameter). Mice were anaesthetized during all treatments by intra‐ peritoneal (i.p.) administration of 200 µg xylazine and 2 mg ketamine. For liver transfection, a 1 cm subcostal incision was made over the liver and the peritoneum opened. The right lobe of the exposed liver was administered plasmid by electroporation as described below (Casey et al. 2010; Collins, S. A. et al. 2011). The wound was closed in two layers, peritoneal and skin, using 4/0 prolene sutures (Promed, Killorglin, Ireland). For plasmid delivery by electroporation, a custom-designed applicator with 2 needles 4 mm apart was used, with both needles placed through the skin central to the tissue. Tissue was injected between elec‐ trode needles with 8 x 1011 copies of plasmid DNA in sterile injectable saline in an injection volume of 50 µl. After 80 seconds, square-wave pulses (1200 V/cm 100 µsec x 1 and 120 V/cm 20 msec, 8 pulses) were administered in sequence using a custom designed pulse gen‐ erator (Cliniporator (IGEA, Carpi, Italy).

#### **2.4. Inhibition of DNA acetylation** *in vivo*

Individual animals were weighed and dosed by i.p. injection of trichostatin A (TSA) (Sigma) at 10 mg/kg in 60 µl 10% (v/v) dimethyl sulfoxide in filtered peanut oil, daily for the dura‐ tion of the experiment. *In vivo* luciferase activity was assessed 4 hours after administration of TSA.

#### **2.5. Whole body imaging**

*In vivo* luciferase activity from tissues was analysed at set time points post-transfection as follows: 80 µl of 30 mg/ml firefly luciferin (Biosynth, Basil, Switzerland) was injected i.p. and intratumourally where appropriate. Mice were anaesthetised as before. Ten minutes post-luciferin injection, live anaesthetised mice were imaged for 3 min at high sensitivity us‐ ing an intensified CCD camera (IVIS Imaging System, Xenogen, Caliper Life Sciences, Eng‐ land). Exposure conditions were maintained at identical levels so that all measurements would be comparable. All data analysis was carried out on Living Image 2.5 software (Xeno‐ gen). Luminescence levels were calculated using standardised regions of interest (ROI) for all three anatomical areas. Actual levels were obtained by subtracting the corresponding ROI of an untransfected mouse to account for background luminescence. For comparison between plasmids, luminescence was represented as p/sec/cm2 /sr/plasmid copy.

the Ub promoter plasmid was examined in livers (figure 1b), luminescence was initially low but increased during the first week post transfection, before decreasing slowly, and re‐ mained higher than the CMV levels up to day 25. To examine other viral promoter activity in liver, pGL3 (SV40 promoter) was assessed (figure 1c). Like pCMV-luc, pGL3 displayed significantly faster reduction in expression than pUb-luc. A different temporal pattern of ex‐ pression was observed in muscle for both the CMV and Ub promoters. Although promoter activity fluctuated over the period examined, a gross reduction in expression over time was

> 0.00E+00 5.00E-08 1.00E-07 1.50E-07 2.00E-07 2.50E-07 3.00E-07

Average Luminescence

Average Luminescence

Average Luminescence

**Figure 1. Duration of CMV and Ub promoter activity** *in vivo* **in liver and muscle** pCMV-luc, pUb-luc or pGL3 was delivered to liver and quadriceps muscle (n=8) by electroporation and luminescence analysed *in vivo* over time using IVIS imaging. **(a)** CMV activity in liver was initially high, reducing to background levels by day 7. **(b)** Initial Ub activity levels were lower than those detected for the CMV promoter but increased and remained higher than that detected for CMV at later time points up to day 25. A gross reduction in expression over time was not apparent in muscle with either CMV or Ub promoters. **(c)** SV40 expression in liver decreased to background levels by Day 7. **(d)** Expression lev‐

The kinetics of CMV, Ub and SV40 promoter activity were also analysed in tumour bearing mice. pCMV-luc, pUb-luc or pGL3 DNA was electroporated to subcutaneous (s.c.) JBS fi‐

els from both viral promoters (CMV and SV40) decline significantly faster than Ub in liver.

p/sec/cm^2/sr/Gene Copy

p/sec/cm^2/sr/Gene Copy

p/sec/cm^2/sr/Gene Copy

0.00E+00 5.00E-08 1.00E-07 1.50E-07 2.00E-07 2.50E-07 3.00E-07

0.00E+00 5.00E-08 1.00E-07 1.50E-07 2.00E-07 2.50E-07 3.00E-07 LIVER MUSCLE

> pUb pCMV pGL3

0 10 20 30 88 189 374

0 10 20 30 88 189 374

0 10 20 30 88 189 374

**Time (Days)**

in volume. IVIS imaging over 18 days (the lim‐

**Time (Days)**

**pCMV-luc, pUb-luc, pGL3 Liver** 

**Time (Days)**

 **pUb-luc Liver & Muscle** 

 **pCMV-luc Liver & Muscle** 

Plasmid Transgene Expression *in vivo*; Promoter and Tissue Variables

http://dx.doi.org/10.5772/52658

LIVER MUSCLE 39

not observed in this tissue with either CMV or Ub promoters (figure 1).

pCMV-luc

Day 1 Day 4 Day 7 Day 12 Day 32 Day 370

pUb-luc

Day 1 Day 4 Day 7 Day 12 Day 32 Day 370

**(c) (d)**

brosarcoma tumours upon reaching 80 mm3

pGL3

**(a)**

**(b)**

Day 1 Day 4 Day 7 Day 12

#### **2.6. Assessment of plasmid DNA in liver tissue using PCR analysis**

To determine the presence of plasmid DNA in liver tissue, pCMV-Luc was delivered to the livers of 9 mice using electroporation as previously described. Luciferase expression was as‐ sessed by IVIS imaging at the time of sampling, 24 hr, 3 days and 10 days post treatment. Livers from three mice were excised at each time-point and snap frozen in liquid nitrogen. Livers were homogenized in TRIZOL Reagent (Invitrogen) using an Ultra Turrax T25 ho‐ mogeniser (IKA Werke GmbH & Co. KG, Staufen, Germany) and total DNA was extracted as per the manufacturer's protocol. The presence of the plasmid DNA in the total DNA was determined by PCR using *luciferase* specific primers (For- 5'-AATCCATCTTGCTCCAA‐ CAC-3' Rev- 5'ATCTCTTTTTCCGTCATCGTC-3'). PCR conditions were: Initial denatura‐ tion at 95 ºC for 15mins followed by 35 cycles (95 ºC for 1 min, 60 ºC for 1 min, 72 ºC for 1 min) and a final extension of 10 m at 72 ºC. The resulting PCR products were analyzed on a 1 % agarose gel.

#### **2.7. Statistical analysis**

The primary outcome variable of the statistical analyses was luminescence per cell per gene copy administered in each cell line or luminescence per gene copy administered in each or‐ gan measured at each time point. The principal explanatory variables were the delivery mo‐ dalites used. *In vivo* luminescence was analysed as continuous. At specified time points, a two-sampled t-test was used to compare mean luminescence per gene copy administered for each delivery modality. Microsoft Excel 11.0 (Microsoft) and GraphPad Prism Version 4.0 (GraphPad Prism Software Inc, San Diego, CA, USA) were used to manage and analyze da‐ ta. Statistical significance was defined at the standard 5 % level.

#### **3. Results**

Plasmid DNA encoding the luciferase gene transcribed from either the CMV (pCMV-luc) or Ubiquitin-B (pUb-luc) promoter was delivered to murine liver or quadriceps muscle by *in vivo* electroporation. Live whole body imaging (IVIS) was performed at various times over 370 days to determine luciferase expression. Expression mediated by the CMV promoter in liver, while initially high, reduced rapidly to background level by day 7 (figure 1a). When the Ub promoter plasmid was examined in livers (figure 1b), luminescence was initially low but increased during the first week post transfection, before decreasing slowly, and re‐ mained higher than the CMV levels up to day 25. To examine other viral promoter activity in liver, pGL3 (SV40 promoter) was assessed (figure 1c). Like pCMV-luc, pGL3 displayed significantly faster reduction in expression than pUb-luc. A different temporal pattern of ex‐ pression was observed in muscle for both the CMV and Ub promoters. Although promoter activity fluctuated over the period examined, a gross reduction in expression over time was not observed in this tissue with either CMV or Ub promoters (figure 1).

and intratumourally where appropriate. Mice were anaesthetised as before. Ten minutes post-luciferin injection, live anaesthetised mice were imaged for 3 min at high sensitivity us‐ ing an intensified CCD camera (IVIS Imaging System, Xenogen, Caliper Life Sciences, Eng‐ land). Exposure conditions were maintained at identical levels so that all measurements would be comparable. All data analysis was carried out on Living Image 2.5 software (Xeno‐ gen). Luminescence levels were calculated using standardised regions of interest (ROI) for all three anatomical areas. Actual levels were obtained by subtracting the corresponding ROI of an untransfected mouse to account for background luminescence. For comparison

To determine the presence of plasmid DNA in liver tissue, pCMV-Luc was delivered to the livers of 9 mice using electroporation as previously described. Luciferase expression was as‐ sessed by IVIS imaging at the time of sampling, 24 hr, 3 days and 10 days post treatment. Livers from three mice were excised at each time-point and snap frozen in liquid nitrogen. Livers were homogenized in TRIZOL Reagent (Invitrogen) using an Ultra Turrax T25 ho‐ mogeniser (IKA Werke GmbH & Co. KG, Staufen, Germany) and total DNA was extracted as per the manufacturer's protocol. The presence of the plasmid DNA in the total DNA was determined by PCR using *luciferase* specific primers (For- 5'-AATCCATCTTGCTCCAA‐ CAC-3' Rev- 5'ATCTCTTTTTCCGTCATCGTC-3'). PCR conditions were: Initial denatura‐ tion at 95 ºC for 15mins followed by 35 cycles (95 ºC for 1 min, 60 ºC for 1 min, 72 ºC for 1 min) and a final extension of 10 m at 72 ºC. The resulting PCR products were analyzed on a

The primary outcome variable of the statistical analyses was luminescence per cell per gene copy administered in each cell line or luminescence per gene copy administered in each or‐ gan measured at each time point. The principal explanatory variables were the delivery mo‐ dalites used. *In vivo* luminescence was analysed as continuous. At specified time points, a two-sampled t-test was used to compare mean luminescence per gene copy administered for each delivery modality. Microsoft Excel 11.0 (Microsoft) and GraphPad Prism Version 4.0 (GraphPad Prism Software Inc, San Diego, CA, USA) were used to manage and analyze da‐

Plasmid DNA encoding the luciferase gene transcribed from either the CMV (pCMV-luc) or Ubiquitin-B (pUb-luc) promoter was delivered to murine liver or quadriceps muscle by *in vivo* electroporation. Live whole body imaging (IVIS) was performed at various times over 370 days to determine luciferase expression. Expression mediated by the CMV promoter in liver, while initially high, reduced rapidly to background level by day 7 (figure 1a). When

/sr/plasmid copy.

between plasmids, luminescence was represented as p/sec/cm2

38 Gene Therapy - Tools and Potential Applications

ta. Statistical significance was defined at the standard 5 % level.

1 % agarose gel.

**3. Results**

**2.7. Statistical analysis**

**2.6. Assessment of plasmid DNA in liver tissue using PCR analysis**

**Figure 1. Duration of CMV and Ub promoter activity** *in vivo* **in liver and muscle** pCMV-luc, pUb-luc or pGL3 was delivered to liver and quadriceps muscle (n=8) by electroporation and luminescence analysed *in vivo* over time using IVIS imaging. **(a)** CMV activity in liver was initially high, reducing to background levels by day 7. **(b)** Initial Ub activity levels were lower than those detected for the CMV promoter but increased and remained higher than that detected for CMV at later time points up to day 25. A gross reduction in expression over time was not apparent in muscle with either CMV or Ub promoters. **(c)** SV40 expression in liver decreased to background levels by Day 7. **(d)** Expression lev‐ els from both viral promoters (CMV and SV40) decline significantly faster than Ub in liver.

The kinetics of CMV, Ub and SV40 promoter activity were also analysed in tumour bearing mice. pCMV-luc, pUb-luc or pGL3 DNA was electroporated to subcutaneous (s.c.) JBS fi‐ brosarcoma tumours upon reaching 80 mm3 in volume. IVIS imaging over 18 days (the lim‐ it of tumour monitoring before animals required culling) demonstrated that the initially high expression driven by the CMV promoter was rapidly reduced to background level by day 4-post transfection (figure 2). Reduction was also observed with SV40 promoter, albeit with a heterologous temporal expression pattern to CMV, with pGL3 expression peaking at day 4 before rapidly reducing to background levels. Ub promoter activity was still evident at the final time point. pCMV-luc and pGL3 displayed statistically similar (p = 0.98) maxi‐ mum to minimum rates of silencing (2.9 x 10-7 p/sec/cm2 /sr/gene copy per day), higher than that of pUb-luc (6.8 x 10-8 p/sec/cm2 /sr/gene copy per day). pCMV-luc expression was also found to rapidly reduce in s.c. human MCF7 breast carcinoma tumours growing in athy‐ mic mice (data not shown). Ubiquitin-B promoter transcriptional activity may be related to the normal functions of ubiquitin in cells, which is expressed constitutively for removing abnormal proteins and for modification of histones leading to gene activation, and so may not be subject to the down-regulation observed with many viral promoters (Ciechanover et al. 2000; Yew et al. 2001). Ubiquitin is also induced in response to cell stress, and expres‐ sion might be up-regulated in response to cellular necrosis and apoptosis, which is espe‐ cially relevant in growing tumours. Given that pUb-luc expression is evident long after viral promoter activity diminishes (up to day 25 for pUb-luc as opposed to day 7 for pCMV-luc and pGL3; figure 2), it is plausible that viral promoter plasmids remain present in liver post cessation of expression.

To test for the presence of plasmid, DNA was extracted from murine livers at various times post transfection with pCMV-luc and PCR analysis performed. DNA PCR results from days 1, 3 and 10 confirmed the presence of *luciferase* DNA in tissue after cessation of expression, suggesting that inhibition of transgene expression occurred at the level of or post transcrip‐ tion (figure 3). Our findings indicate that both viral promoters examined provided shortlived expression in tumours and liver, whereas use of Ub promoter significantly prolonged transgene expression. Importantly, we also found that viral promoter activity was depend‐ ent on target tissue, since no reduction in expression was observed in plasmid electroporat‐

Plasmid Transgene Expression *in vivo*; Promoter and Tissue Variables

http://dx.doi.org/10.5772/52658

41

**Figure 3. Plasmid DNA persists in liver after cessation of expression** PCR analysis of DNA extracted from murine livers (n=3) on days 1, 3 or 10-post electroporation with pCMV-luc. A representative mouse from which DNA was ex‐ tracted at each time-point is shown. PCR using primers specific for the *luciferase* gene indicates presence of plasmid.

In order to examine any effects of T-cell mediated immune activity on viral promoter con‐ struct expression, pCMV-luc expression in livers of athymic mice was examined. No differ‐ ence in the magnitude or duration of expression was observed between immune competent Balb/C and T-cell deficient mice, suggesting that cellular immune responses were not in‐ volved in the observed reduction in hepatic expression of pCMV-luc (figure 4a). Other stud‐ ies have indicated that luciferase protein has low immunogenicity, and immune-mediated destruction of luciferase-producing cells does not occur in mice (Davis et al. 1997), while the persistence of expression in muscle here also makes this unlikely as a cause for silencing in other tissues. The observation of indefinite expression in plasmid electroporated muscle is in direct contrast to Ad expression in quadriceps muscle, which has been shown to be eliminat‐ ed through T cell and antibody immune activities and/or CMV promoter methylation (Jooss,

ed muscle with both viral and mammalian promoters.

Untransfected liver samples did not yield PCR product.

Yang et al. 1998; Brooks et al. 2004).

**Figure 2. Duration of viral and native promoter activity in tumour** pCMV-luc, pGL3 or pUb-luc was delivered *in vivo* to growing tumours (n=6) by electroporation and luminescence analysed *in vivo* over time using IVIS imaging. Expression from both viral promoters (CMV and SV40) rapidly diminished, whereas Ub promoter activity was still evi‐ dent at the final time point (day 18) when mice required culling due to tumour size. Ub mediated expression levels were at 39.4 % of maximal level on final time-point, compared with 2.4 % and 3.5 % for CMV and SV40 respectively.

To test for the presence of plasmid, DNA was extracted from murine livers at various times post transfection with pCMV-luc and PCR analysis performed. DNA PCR results from days 1, 3 and 10 confirmed the presence of *luciferase* DNA in tissue after cessation of expression, suggesting that inhibition of transgene expression occurred at the level of or post transcrip‐ tion (figure 3). Our findings indicate that both viral promoters examined provided shortlived expression in tumours and liver, whereas use of Ub promoter significantly prolonged transgene expression. Importantly, we also found that viral promoter activity was depend‐ ent on target tissue, since no reduction in expression was observed in plasmid electroporat‐ ed muscle with both viral and mammalian promoters.

it of tumour monitoring before animals required culling) demonstrated that the initially high expression driven by the CMV promoter was rapidly reduced to background level by day 4-post transfection (figure 2). Reduction was also observed with SV40 promoter, albeit with a heterologous temporal expression pattern to CMV, with pGL3 expression peaking at day 4 before rapidly reducing to background levels. Ub promoter activity was still evident at the final time point. pCMV-luc and pGL3 displayed statistically similar (p = 0.98) maxi‐

found to rapidly reduce in s.c. human MCF7 breast carcinoma tumours growing in athy‐ mic mice (data not shown). Ubiquitin-B promoter transcriptional activity may be related to the normal functions of ubiquitin in cells, which is expressed constitutively for removing abnormal proteins and for modification of histones leading to gene activation, and so may not be subject to the down-regulation observed with many viral promoters (Ciechanover et al. 2000; Yew et al. 2001). Ubiquitin is also induced in response to cell stress, and expres‐ sion might be up-regulated in response to cellular necrosis and apoptosis, which is espe‐ cially relevant in growing tumours. Given that pUb-luc expression is evident long after viral promoter activity diminishes (up to day 25 for pUb-luc as opposed to day 7 for pCMV-luc and pGL3; figure 2), it is plausible that viral promoter plasmids remain present

/sr/gene copy per day), higher than

/sr/gene copy per day). pCMV-luc expression was also

mum to minimum rates of silencing (2.9 x 10-7 p/sec/cm2

that of pUb-luc (6.8 x 10-8 p/sec/cm2

40 Gene Therapy - Tools and Potential Applications

in liver post cessation of expression.

Day 1

Day 4

Day 6

Day 8

Day 11

Day 15

Day 18

Day 18

**Figure 2. Duration of viral and native promoter activity in tumour** pCMV-luc, pGL3 or pUb-luc was delivered *in vivo* to growing tumours (n=6) by electroporation and luminescence analysed *in vivo* over time using IVIS imaging. Expression from both viral promoters (CMV and SV40) rapidly diminished, whereas Ub promoter activity was still evi‐ dent at the final time point (day 18) when mice required culling due to tumour size. Ub mediated expression levels were at 39.4 % of maximal level on final time-point, compared with 2.4 % and 3.5 % for CMV and SV40 respectively.

Average Luminescence

p/sec/cm^2/sr/Gene Copy

0.00E+00 2.00E-07 4.00E-07 6.00E-07 8.00E-07 1.00E-06 1.20E-06 1.40E-06

> 0 5 10 15 **Time (Days)**

Ub CMV SV40

Day 15

Day 11

Day 8

Day 1 Day 4 Day 6 Day 8 Day 11 Day 15 Day 18

Day 6

Day 4

Day 1

**SV 40**

**CMV**

**UB**

**Figure 3. Plasmid DNA persists in liver after cessation of expression** PCR analysis of DNA extracted from murine livers (n=3) on days 1, 3 or 10-post electroporation with pCMV-luc. A representative mouse from which DNA was ex‐ tracted at each time-point is shown. PCR using primers specific for the *luciferase* gene indicates presence of plasmid. Untransfected liver samples did not yield PCR product.

In order to examine any effects of T-cell mediated immune activity on viral promoter con‐ struct expression, pCMV-luc expression in livers of athymic mice was examined. No differ‐ ence in the magnitude or duration of expression was observed between immune competent Balb/C and T-cell deficient mice, suggesting that cellular immune responses were not in‐ volved in the observed reduction in hepatic expression of pCMV-luc (figure 4a). Other stud‐ ies have indicated that luciferase protein has low immunogenicity, and immune-mediated destruction of luciferase-producing cells does not occur in mice (Davis et al. 1997), while the persistence of expression in muscle here also makes this unlikely as a cause for silencing in other tissues. The observation of indefinite expression in plasmid electroporated muscle is in direct contrast to Ad expression in quadriceps muscle, which has been shown to be eliminat‐ ed through T cell and antibody immune activities and/or CMV promoter methylation (Jooss, Yang et al. 1998; Brooks et al. 2004).

diluted rapidly in tissues with a high mitotic index. Liver hepatocytes and skeletal myocytes are fully differentiated and have a low turnover, unlike tumour cells. (Ayers & Jeffery 1988) It may be hypothesised that the static nature of cell turnover in muscle compared with tu‐ mour is relevant in this context. However, this cannot fully account for the observed loss of expression, since in our study, the rate of reduction of expression for plasmids with promot‐ ers of mammalian and viral origin was different. Also, previous studies have shown no al‐ teration in longevity of transgene expression when cell turnover was inhibited (Herweijer et al. 2001). Furthermore, we demonstrated by PCR that pCMV-luc persisted in liver cells after expression ceased. We think it is unlikely that the reduction in pCMV-luc expression was due to a parallel reduction in the plasmid DNA as this was not seen for the Ub promoter

Plasmid Transgene Expression *in vivo*; Promoter and Tissue Variables

http://dx.doi.org/10.5772/52658

43

It has previously been demonstrated that plasmid transgene expression can be modulated with chromatin remodelling agents (Bartoli, Fettucciari et al. 2003). To this end, murine liv‐ ers were electroporated with pCMV-luc and mice systemically administered the histone de‐ acetylase inhibitor trichostatin-A (TSA) daily for the duration of experiment. TSA is a specific inhibitor for histone deacetylase (HDAC) and is known to enhance gene expression in viral and plasmid-transfected cells *in vitro* and *in vivo* (Vanniasinkam et al. 2006). It has been shown that HDAC binds to the CMV promoter, and TSA may act to overcome such transcriptional repression (Tang & Maul 2003). In our experiments, TSA administration sig‐ nificantly increased levels of expression at later time points, compared with control (p < 0.002 on day 7; figure 4b). Interestingly, a further increase was noted when 5′ azacytidine (aza-C), a non-specific methylation inhibitor, and trichostatin were used in combination,

While this study did not generate data to correlate RNA levels with luminescence, differen‐ ces in transcription appears to be the key element in observed expression levels. Firefly luci‐ ferase protein is known to have a short half-life *in vivo*, in the region of 1 - 4 hours (Baggett et al. 2004; Tangney & Francis 2012), and any luminescence detected in our experiments was due to recently transcribed gene. Furthermore, given that pUb-luc expression is evident long after viral promoter activity diminishes (up to day 25 for pUb-luc as opposed to day 7 for pCMV-luc and pGL3; figure 1), it is likely that viral promoter plasmids remain present in liver post cessation of expression, and we demonstrated by PCR that pCMV-luc DNA was present in liver 10 days post transfection.There exist numerous reports linking viral promot‐ er DNA methylation with transcriptional silencing in gene therapy settings *in vitro* and *in*

Our findings are consistent with previous studies in lung tissue where the levels and dura‐ tion of transgene expression *in vivo* were compared using plasmid vectors coding for the CMV or Ubiquitin promoters (Gill et al. 2001; Yew, Przybylska et al. 2001; Gazdhar, Bilici et al. 2006). Further specific methylation assays may elucidate the precise mechanism of viral promoter silencing here. Given that many tumour types have been shown to have abnormal methylation, this phenomenon may represent a serious hindrance to cancer gene therapy which use of native promoters may abrogate as demonstrated here (Kanai 2008). Further‐ more, the finding of indefinite high-level expression in plasmid electroporated muscle irre‐

*vivo* (Di Ianni et al. 1999; Brooks, Harkins et al. 2004; Al-Dosari et al. 2006).

where similar plasmid copy numbers would be expected.

while aza-C in isolation had no effect (data not shown).

**Figure 4. (a) pCMV-luc is silenced in livers in absence of T cells** pCMV-luc was electroporated *in vivo* to livers of athymic mice and IVIS imaged (n=4). No difference was observed in plasmid expression at any time point when com‐ pared with expression in immunocompetent Balb/C mice (p > 0.25). **(b) Effect of deacetylation agent on pCMV-luc expression** *in vivo* pCMV-luc was delivered to livers by electroporation (n=4). TSA or PBS was i.p. administered daily. Gene expression was analysed using the IVIS imaging system. The magnitude and duration of gene expression in ani‐ mals treated with TSA was significantly increased. \* denotes statistically significant difference between groups, p < 0.05.

#### **4. Discussion**

We did not determine the reasons for the observed tissue-specific nature of viral promoter silencing, and it remains unclear as to why liver and tumour, but not muscle, affected plas‐ mid expression. Plasmids function predominantly in an episomal fashion and copy number per cell is reduced proportional to cell replication. As such, genes would be expected to be diluted rapidly in tissues with a high mitotic index. Liver hepatocytes and skeletal myocytes are fully differentiated and have a low turnover, unlike tumour cells. (Ayers & Jeffery 1988) It may be hypothesised that the static nature of cell turnover in muscle compared with tu‐ mour is relevant in this context. However, this cannot fully account for the observed loss of expression, since in our study, the rate of reduction of expression for plasmids with promot‐ ers of mammalian and viral origin was different. Also, previous studies have shown no al‐ teration in longevity of transgene expression when cell turnover was inhibited (Herweijer et al. 2001). Furthermore, we demonstrated by PCR that pCMV-luc persisted in liver cells after expression ceased. We think it is unlikely that the reduction in pCMV-luc expression was due to a parallel reduction in the plasmid DNA as this was not seen for the Ub promoter where similar plasmid copy numbers would be expected.

It has previously been demonstrated that plasmid transgene expression can be modulated with chromatin remodelling agents (Bartoli, Fettucciari et al. 2003). To this end, murine liv‐ ers were electroporated with pCMV-luc and mice systemically administered the histone de‐ acetylase inhibitor trichostatin-A (TSA) daily for the duration of experiment. TSA is a specific inhibitor for histone deacetylase (HDAC) and is known to enhance gene expression in viral and plasmid-transfected cells *in vitro* and *in vivo* (Vanniasinkam et al. 2006). It has been shown that HDAC binds to the CMV promoter, and TSA may act to overcome such transcriptional repression (Tang & Maul 2003). In our experiments, TSA administration sig‐ nificantly increased levels of expression at later time points, compared with control (p < 0.002 on day 7; figure 4b). Interestingly, a further increase was noted when 5′ azacytidine (aza-C), a non-specific methylation inhibitor, and trichostatin were used in combination, while aza-C in isolation had no effect (data not shown).

**(b)**

0.00E+00

1.00E-06

5.00E-08

0 5 10 15 **Time (Days)**

1 2 3 4 5 6 7 **Time (Days)**

**Figure 4. (a) pCMV-luc is silenced in livers in absence of T cells** pCMV-luc was electroporated *in vivo* to livers of athymic mice and IVIS imaged (n=4). No difference was observed in plasmid expression at any time point when com‐ pared with expression in immunocompetent Balb/C mice (p > 0.25). **(b) Effect of deacetylation agent on pCMV-luc expression** *in vivo* pCMV-luc was delivered to livers by electroporation (n=4). TSA or PBS was i.p. administered daily. Gene expression was analysed using the IVIS imaging system. The magnitude and duration of gene expression in ani‐ mals treated with TSA was significantly increased. \* denotes statistically significant difference between groups, p <

We did not determine the reasons for the observed tissue-specific nature of viral promoter silencing, and it remains unclear as to why liver and tumour, but not muscle, affected plas‐ mid expression. Plasmids function predominantly in an episomal fashion and copy number per cell is reduced proportional to cell replication. As such, genes would be expected to be

\*

Athymic Balb/C

> TSA PBS

> > \*

1.00E-07

1.50E-07

2.00E-07

2.50E-07

Average Luminescence

0.05.

**4. Discussion**

p/sec/cm^2/sr/Gene Copy

1.00E-08

p/sec/cm^2/sr/Gene Copy

1.00E-07

**(a)**

42 Gene Therapy - Tools and Potential Applications

Average Luminescence

**av**

p/sec/cm^2/sr/Gene Copy

While this study did not generate data to correlate RNA levels with luminescence, differen‐ ces in transcription appears to be the key element in observed expression levels. Firefly luci‐ ferase protein is known to have a short half-life *in vivo*, in the region of 1 - 4 hours (Baggett et al. 2004; Tangney & Francis 2012), and any luminescence detected in our experiments was due to recently transcribed gene. Furthermore, given that pUb-luc expression is evident long after viral promoter activity diminishes (up to day 25 for pUb-luc as opposed to day 7 for pCMV-luc and pGL3; figure 1), it is likely that viral promoter plasmids remain present in liver post cessation of expression, and we demonstrated by PCR that pCMV-luc DNA was present in liver 10 days post transfection.There exist numerous reports linking viral promot‐ er DNA methylation with transcriptional silencing in gene therapy settings *in vitro* and *in vivo* (Di Ianni et al. 1999; Brooks, Harkins et al. 2004; Al-Dosari et al. 2006).

Our findings are consistent with previous studies in lung tissue where the levels and dura‐ tion of transgene expression *in vivo* were compared using plasmid vectors coding for the CMV or Ubiquitin promoters (Gill et al. 2001; Yew, Przybylska et al. 2001; Gazdhar, Bilici et al. 2006). Further specific methylation assays may elucidate the precise mechanism of viral promoter silencing here. Given that many tumour types have been shown to have abnormal methylation, this phenomenon may represent a serious hindrance to cancer gene therapy which use of native promoters may abrogate as demonstrated here (Kanai 2008). Further‐ more, the finding of indefinite high-level expression in plasmid electroporated muscle irre‐ spective of the promoter type has important therapeutic implications. Skeletal muscle is a large and accessible tissue, within which a plasmid-based gene therapy might be a safe and efficient method for systemic protein production, particularly when combined with either endogenous or exogenous regulatable systems. We have previously demonstrated the appli‐ cation of an inducible plasmid based system in *in vivo* murine tissue (Morrissey et al. 2012). In addition to providing an "off switch" to safeguard against side effects, this also allows optimal temporal delivery of therapeutic, tailored to when it can most efficiently achieve a biological response.

[4] Bartoli, A., Fettucciari, K., Fetriconi, I., Rosati, E., Di Ianni, M., Tabilio, A., Delfino, D. V., Rossi, R., & Marconi, P. (2003). Effect of trichostatin a and 5'-azacytidine on trans‐

Plasmid Transgene Expression *in vivo*; Promoter and Tissue Variables

http://dx.doi.org/10.5772/52658

45

[5] Brooks, A. R., Harkins, R. N., Wang, P., Qian, H. S., Liu, P., & Rubanyi, G. M. (2004). Transcriptional silencing is associated with extensive methylation of the CMV pro‐ moter following adenoviral gene delivery to muscle. J Gene Med , 6(4), 395-404.

[6] Casey, G., Cashman, J. P., Morrissey, D., Whelan, M. C., Larkin, J. O., Soden, D. M., Tangney, M., & O'Sullivan, G. C. (2010). Sonoporation mediated immunogene thera‐ py of solid tumors. Ultrasound Med Biol 36(3): X (Electronic)0301-5629 (Linking), 430

[7] Ciechanover, A., Orian, A., & Schwartz, A. L. (2000). Ubiquitin-mediated proteolysis:

[8] Collins, C. G., Tangney, M., Larkin, J. O., Casey, G., Whelan, M. C., Cashman, J., Murphy, J., Soden, D., Vejda, S., Mc Kenna, S., Kiely, B., Collins, J. K., Barrett, J., Aar‐ ons, S., & O'Sullivan, G. C. (2006). Local gene therapy of solid tumors with GM-CSF

[9] Collins, S. A., Buhles, A., Scallan, M. F., Harrison, P. T., O'Hanlon, D. M., O'Sullivan, G. C., & Tangney, M. (2010). AAV2 -mediated in vivo immune gene therapy of solid

[10] Collins, S. A., Morrissey, D., Rajendran, S., Casey, G., Scallan, M. F., Harrison, P. T., O'Sullivan, G. C., & Tangney, M. (2011). Comparison of DNA Delivery and Expres‐ sion Using Frequently Used Delivery Methods. Gene Therapy- developments and fu‐

[11] Davis, H. L., Millan, C. L., & Watkins, S. C. (1997). Immune-mediated destruction of transfected muscle fibers after direct gene transfer with antigen-expressing plasmid

[12] Di Ianni, M., Terenzi, A., Perruccio, K., Ciurnelli, R., Lucheroni, F., Benedetti, R., Martelli, M. F., & Tabilio, A. (1999). 5 -Azacytidine prevents transgene methylation in

[13] Frank, O., Rudolph, C., Heberlein, C., von, Neuhoff. N., Schrock, E., Schambach, A., Schlegelberger, B., Fehse, B., Ostertag, W., Stocking, C., & Baum, C. (2004). Tumor cells escape suicide gene therapy by genetic and epigenetic instability. *Blood*, 104(12),

[14] Gazdhar, A., Bilici, M., Pierog, J., Ayuni, E. L., Gugger, M., Wetterwald, A., Cecchini, M., & Schmid, R. A. (2006). In vivo electroporation and ubiquitin promoter- a proto‐

[15] Gill, D. R., Pringle, I. A., & Hyde, S. C. (2009). Progress and prospects: the design and

col for sustained gene expression in the lung." J Gene Med , 8(7), 910-918.

production of plasmid vectors. Gene Ther , 16(2), 165-71.

tumours. Genet Vaccines Ther 8: 81479-0556 (Electronic)1479-0556 (Linking)

biological regulation via destruction. Bioessays , 22(5), 442-51.

and B71 eradicates both treated and distal tumors. Cancer Gene Ther

gene reactivation in U937 transduced cells. Pharmacol Res , 48(1), 111-8.

EOF-440 EOF.

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vivo. Gene Ther 6(4): 703-70969-7128 (Print)

DNA. Gene Ther , 4(3), 181-8.

3543-9.

#### **5. Conclusion**

In summary these results highlight the importance of promoter,tissue and vector variables in achieving appropriate transgene expression for DNA therapeutic strategies.

#### **Acknowledgment**

This work was funded by Cancer Research Ireland (CRI07TAN) and the Cork Cancer Re‐ search Centre.

#### **Author details**

David Morrissey, Sara A. Collins, Simon Rajenderan, Garrett Casey, Gerald C. O'Sullivan and Mark Tangney

Cork Cancer Research Centre, Mercy University Hospital and Leslie C. Quick Jnr. Laborato‐ ry, University College Cork, Cork, Ireland

#### **References**


[4] Bartoli, A., Fettucciari, K., Fetriconi, I., Rosati, E., Di Ianni, M., Tabilio, A., Delfino, D. V., Rossi, R., & Marconi, P. (2003). Effect of trichostatin a and 5'-azacytidine on trans‐ gene reactivation in U937 transduced cells. Pharmacol Res , 48(1), 111-8.

spective of the promoter type has important therapeutic implications. Skeletal muscle is a large and accessible tissue, within which a plasmid-based gene therapy might be a safe and efficient method for systemic protein production, particularly when combined with either endogenous or exogenous regulatable systems. We have previously demonstrated the appli‐ cation of an inducible plasmid based system in *in vivo* murine tissue (Morrissey et al. 2012). In addition to providing an "off switch" to safeguard against side effects, this also allows optimal temporal delivery of therapeutic, tailored to when it can most efficiently achieve a

In summary these results highlight the importance of promoter,tissue and vector variables

This work was funded by Cancer Research Ireland (CRI07TAN) and the Cork Cancer Re‐

Cork Cancer Research Centre, Mercy University Hospital and Leslie C. Quick Jnr. Laborato‐

[1] Al-Dosari, M., Zhang, G., Knapp, J. E., & Liu, D. (2006). Evaluation of viral and mam‐ malian promoters for driving transgene expression in mouse liver. Biochem Biophys

[2] Ayers, M. M., & Jeffery, P. K. (1988). Proliferation and differentiation in mammalian

[3] Baggett, B., Roy, R., Momen, S., Morgan, S., Tisi, L., Morse, D., & Gillies, R. J. (2004). Thermostability of firefly luciferases affects efficiency of detection by in vivo biolu‐

in achieving appropriate transgene expression for DNA therapeutic strategies.

David Morrissey, Sara A. Collins, Simon Rajenderan, Garrett Casey,

biological response.

44 Gene Therapy - Tools and Potential Applications

**5. Conclusion**

**Acknowledgment**

search Centre.

**Author details**

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ry, University College Cork, Cork, Ireland

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airway epithelium. Eur Respir J , 1(1), 58-80.

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[16] Gill, D. R., Smyth, S. E., Goddard, C. A., Pringle, I. A., Higgins, C. F., Colledge, W. H., & Hyde, S. C. (2001). Increased persistence of lung gene expression using plas‐ mids containing the ubiquitin C or elongation factor 1alpha promoter. Gene Ther , 8(20), 1539-46.

[28] Prosch, S., Stein, J., Staak, K., Liebenthal, C., Volk, H. D., & Kruger, D. H. (1996). Inac‐ tivation of the very strong HCMV immediate early promoter by DNA CpG methyla‐

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[29] Sandrin, V., Russell, S. J., & Cosset, F. L. (2003). Targeting retroviral and lentiviral

[30] Scheule, R. K. (2000). The role of CpG motifs in immunostimulation and gene thera‐

[31] Tang, Q., & Maul, G. G. (2003). Mouse cytomegalovirus immediate-early protein 1 binds with host cell repressors to relieve suppressive effects on viral transcription

[32] Tangney, M., & Francis, K. P. (2012). In vivo optical imaging in gene & cell therapy."

[33] Vanniasinkam, T., Ertl, H., & Tang, Q. (2006). Trichostatin-A enhances adaptive im‐

[34] Wang, L., Dobrzynski, E., Schlachterman, A., Cao, O., & Herzog, R. W. (2005). Sys‐ temic protein delivery by muscle-gene transfer is limited by a local immune re‐

[35] Yew, N. S., Przybylska, M., Ziegler, R. J., Liu, D., & Cheng, S. H. (2001). High and sustained transgene expression in vivo from plasmid vectors containing a hybrid

[36] Yuasa, K., Sakamoto, M., Miyagoe-Suzuki, Y., Tanouchi, A., Yamamoto, H., Li, J., Chamberlain, J. S., Xiao, X., & Takeda, S. (2002). Adeno-associated virus vector-medi‐ ated gene transfer into dystrophin-deficient skeletal muscles evokes enhanced im‐

mune response against the transgene product. Gene Ther , 9(23), 1576-88.

tion in vitro. Biol Chem Hoppe Seyler , 377(3), 195-201.

vectors." Curr Top Microbiol Immunol , 281, 137-78.

py. Adv Drug Deliv Rev 44(2-3): , 119 EOF-34 EOF.

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ubiquitin promoter. Mol Ther , 4(1), 75-82.

and replication during lytic infection. J Virol , 77(2), 1357-67.

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mune responses to DNA vaccination. J Clin Virol , 36(4), 292-7.


[28] Prosch, S., Stein, J., Staak, K., Liebenthal, C., Volk, H. D., & Kruger, D. H. (1996). Inac‐ tivation of the very strong HCMV immediate early promoter by DNA CpG methyla‐ tion in vitro. Biol Chem Hoppe Seyler , 377(3), 195-201.

[16] Gill, D. R., Smyth, S. E., Goddard, C. A., Pringle, I. A., Higgins, C. F., Colledge, W. H., & Hyde, S. C. (2001). Increased persistence of lung gene expression using plas‐ mids containing the ubiquitin C or elongation factor 1alpha promoter. Gene Ther ,

[17] Greenland, J. R., Geiben, R., Ghosh, S., Pastor, W. A., & Letvin, N. L. (2007). Plasmid DNA vaccine-elicited cellular immune responses limit in vivo vaccine antigen ex‐

[18] Herweijer, H., Zhang, G., Subbotin, V. M., Budker, V., Williams, P., & Wolff, J. A. (2001). Time course of gene expression after plasmid DNA gene transfer to the liver. J

[19] Jaenisch, R., Schnieke, A., & Harbers, K. (1985). Treatment of mice with 5-azacytidine efficiently activates silent retroviral genomes in different tissues. Proc Natl Acad Sci

[20] Jooss, K., Ertl, H. C., & Wilson, J. M. (1998). Cytotoxic T-lymphocyte target proteins and their major histocompatibility complex class I restriction in response to adenovi‐

[21] Jooss, K., Yang, Y., Fisher, K. J., & Wilson, J. M. (1998). Transduction of dendritic cells by DNA viral vectors directs the immune response to transgene products in muscle

[22] Kanai, Y. (2008). Alterations of DNA methylation and clinicopathological diversity of

[23] Louboutin, J. P., Wang, L., & Wilson, J. M. (2005). Gene transfer into skeletal muscle

[24] Mir, L. M., Bureau, M. F., Gehl, J., Rangara, R., Rouy, D., Caillaud, J. M., Delaere, P., Branellec, D., Schwartz, B., & Scherman, D. (1999). High-efficiency gene transfer into skeletal muscle mediated by electric pulses. Proc Natl Acad Sci U S A , 96(8), 4262-7.

[25] Momparler, R. L., & Bovenzi, V. (2000). DNA methylation and cancer. J Cell Physiol ,

[26] Morrissey, D., van Pijkeren, J. P., Rajendran, S., Collins, S. A., Casey, G., O'Sullivan, G. C., & Tangney, M. (2012). Control and augmentation of long-term plasmid trans‐ gene expression in vivo in murine muscle tissue and ex vivo in patient mesenchymal tissue. J Biomed Biotechnol 2012: 3798451110-7251 (Electronic)1110-7243 (Linking)

[27] Ohlfest, J. R., Demorest, Z. L., Motooka, Y., Vengco, I., Oh, S., Chen, E., Scappaticci, F. A., Saplis, R. J., Ekker, S. C., Low, W. C., Freese, A. B., & Largaespada, D. A. (2005). Combinatorial antiangiogenic gene therapy by nonviral gene transfer using the sleeping beauty transposon causes tumor regression and improves survival in mice

bearing intracranial human glioblastoma. Mol Ther , 12(5), 778-88.

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46 Gene Therapy - Tools and Potential Applications

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human cancers. Pathol Int , 58(9), 544-58.


**Chapter 3**

**Silencing of Transgene Expression:**

Oleg E. Tolmachov, Tatiana Subkhankulova and

The treatment of a number of diseases can be achieved through gene addition therapy, where curative transgenes are established within the patient's cells after delivery with viral or non-viral vectors. The defective cells requiring treatment are typically differentiated; these cells or their progenitors can be targeted for therapeutic gene transfer. However, as the abundance of progenitor cells varies between different tissues and in the same tissue during the fetal, neonatal and adult stages of development, the scarcity of a particular progenitor cell pool, the paucity of spontaneous departures of progenitor cells down differentiation pathways and unclear differentiation induction conditions can complicate genetic therapeu‐ tic intervention via these cells. Nevertheless, gene transfer to progenitor cells can be a pre‐ ferred option when differentiated cells are either poorly accessible for the vector or, once differentiated, are defective beyond repair by gene therapy. Genetic conditions with consid‐ erable value in therapeutic gene transfer to progenitor cells include cystic fibrosis (CF) and

The delivered transgenes can integrate into the chromosomal DNA, replicate episomally or persist as non-replicating episomal elements in non-dividing cells. Depending on the prop‐ erties of the transgene expression cassette, particular features of specific transgene integra‐ tion sites and the state of the individual recipient cells, the transgenes are expressed with varying degree of efficiency. On some occasions, the transgenes are permanently silenced immediately after introduction, on other occasions transgene silencing occurs only after a certain period of adequate expression and on still other occasions transgene expression var‐ ies dramatically among the individual clones of transgene-harbouring cells. Such variation is thought to be mainly due to the transgene's interaction with its immediate genetic neigh‐

> © 2013 Tolmachov et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

severe combined immunodeficiency (SCID).

**A Gene Therapy Perspective**

Tanya Tolmachova

**1. Introduction**

http://dx.doi.org/10.5772/53379

**Chapter 3**

### **Silencing of Transgene Expression: A Gene Therapy Perspective**

Oleg E. Tolmachov, Tatiana Subkhankulova and Tanya Tolmachova

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53379

#### **1. Introduction**

The treatment of a number of diseases can be achieved through gene addition therapy, where curative transgenes are established within the patient's cells after delivery with viral or non-viral vectors. The defective cells requiring treatment are typically differentiated; these cells or their progenitors can be targeted for therapeutic gene transfer. However, as the abundance of progenitor cells varies between different tissues and in the same tissue during the fetal, neonatal and adult stages of development, the scarcity of a particular progenitor cell pool, the paucity of spontaneous departures of progenitor cells down differentiation pathways and unclear differentiation induction conditions can complicate genetic therapeu‐ tic intervention via these cells. Nevertheless, gene transfer to progenitor cells can be a pre‐ ferred option when differentiated cells are either poorly accessible for the vector or, once differentiated, are defective beyond repair by gene therapy. Genetic conditions with consid‐ erable value in therapeutic gene transfer to progenitor cells include cystic fibrosis (CF) and severe combined immunodeficiency (SCID).

The delivered transgenes can integrate into the chromosomal DNA, replicate episomally or persist as non-replicating episomal elements in non-dividing cells. Depending on the prop‐ erties of the transgene expression cassette, particular features of specific transgene integra‐ tion sites and the state of the individual recipient cells, the transgenes are expressed with varying degree of efficiency. On some occasions, the transgenes are permanently silenced immediately after introduction, on other occasions transgene silencing occurs only after a certain period of adequate expression and on still other occasions transgene expression var‐ ies dramatically among the individual clones of transgene-harbouring cells. Such variation is thought to be mainly due to the transgene's interaction with its immediate genetic neigh‐

© 2013 Tolmachov et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. bourhood within the host genome; a phenomenon, which is similar to 'position effect varie‐ gation' in normal development caused by spontaneous clone-wise silencing of some resident genes [1]. Typical position effect variegation is epigenetic instability and should be distinguished from variegation due to somatic mutations, e.g. due to variations in the length of polynucleotide repeat expansions [2] or due to the sorting of mitochondrial genomes in mitochondrial heteroplasmia [3]. The element of randomness, which is inherently present in position effect variegation, should not come as a surprise. In fact, stochastic fluctuations of gene expression are typical both at the level of variation between different cells of tissue and at the level of temporal variation within one cell. Both of these modes of variation are essen‐ tial for normal differentiation and tissue-patterning with the input of stochastic variation be‐ ing decisive when a developmental signal is present at a near-critical level. For the gene therapist, it is important that the permanent silencing of transgene expression can occur both in postmitotic target cells and target cells undergoing clonal expansion, while variega‐ tion is typically associated with clones of dividing cells. Stable long-term transgene expres‐ sion in differentiating cells is particularly challenging. In fact, the introduced genes are subject to the pre-existing and developing gene expression patterns in the target cells, which can override the signals from the transgenes' own regulatory elements and, thus, can cause transgene expression shutdown. Indeed, at a transcriptional level, the changing scenery of transcription initiation factor pools, chromatin re-modelling and DNA methylation events during differentiation contribute to the transiency of transgene expression.

which should be taken into account where choosing or designing effective gene therapy vec‐

Silencing of Transgene Expression: A Gene Therapy Perspective

http://dx.doi.org/10.5772/53379

51

Patterns for maintaining gene repression or activation are governed by regulatory machi‐ nery acting at multiple levels: 1) transcription; 2) mRNA processing, export from the nu‐ cleus, translation and degradation; 3) protein folding, modification, transport and degradation. Control of gene expression is well-coordinated and highly hierarchical, with the control of transcription initiation situated at the top of the regulatory ladder. A number of interacting instruments of transcriptional gene activation and silencing in mammals are known: DNA methylation (e.g. methylation within CpG-islands of promoters), amino acid sequence variants of histones, covalent modifications of histones, histone-binding proteins (e.g. powerful inhibitors of gene activity from the Polycomb Group) and combinations of transcription initiation factors specific for particular tissues and developmental stages. The pivotal point is the access of the transcription machinery to DNA, which is regulated via DNA methylation and chromatin remodelling. With some simplification, it can be general‐ ized that 'coarse tuning' of gene expression (e.g. long-term silencing) is provided by DNA methylation, 'medium tuning' is provided by chromatin remodelling and 'fine tuning' is achieved via various transcription factors and a multitude of other regulatory devices.

The various branches of the regulatory machinery play their own particular roles and yet are inherently interconnected. As detailed below, a prime example of this is the deep in‐ volvement of the miRNA pathway both in mRNA degradation and in the establishment of

DNA methylation is an important epigenetic mark involved in cell differentiation and or‐ gan and tissue development, which plays a crucial role in the establishment of genomic imprinting (parent-dependent silencing of alternative alleles) in both male and female germ lines. However, in gene transfer experiments, the methylation of transgenes was shown to be just one ingredient in the dynamic interplay of various factors responsible for

*De-novo* methylation patterns in humans are established mainly on implantation and in ga‐ metogenesis. Two DNA (cytosine-5)-methyltransferases, DNMT3A and DNMT3B, play an essential role in *de-novo* methylation while DNMT3A in cooperation with the auxiliary pro‐ tein DNMT3L is responsible for imprinting. There is still much we do not know about the manner in which the inactive state of the imprinted chromosomal domains is achieved and what factors trigger this type of silencing. The available evidence indicates that 'Smc hinge proteins' can be particularly important in epigenetic silencing [7]. Thus, in studies based on X-linked GFP transgene silencing, the SmcHD1 gene was shown to play a critical role in Xchromosome inactivation in mammals [8,9]. The recruitment of SmcHD1 to the X-chromo‐

**2. Host genetic factors of silencing and position effect variegation**

tors and strategies for their administration.

chromatin methylation patterns [5].

silencing and variegation [6].

**2.1. The role of DNA methylation in silencing**

Genomes in general and, in particular, mammalian genomes have a mosaic organisation with functionally related genetic elements often being in close physical proximity. There are three teleological reasons for this: 1) expediency of genetic exchange; 2) straightforward temporal control of gene expression; 3) economy of energy, enzymes and other factors serv‐ ing the genetic elements. The second and the third of these reasons are also sufficient for the existence of a finely patterned 3D-arrangement of DNA in interphase nuclei, simplifying the functional interactions between distant genetic elements, e.g. interactions regulating gene expression. It is intriguing to propose that the need to orchestrate gene expression in time and the economy need are also driving the astonishing interconnectedness of all gene silenc‐ ing mechanisms, which we shall address in this chapter.

The gene therapist should take advantage of the pre-existing regulatory moduli present in the target cells and should also supply the transgenes with their own expression control ele‐ ments. The regulatory elements required for reliable, long-term and tissue-specific transgene expression include minimal promoters, enhancers, regulatory introns and locus control re‐ gions. The functional arrangement of all these elements is ultimately achieved in 3D. This should be borne in mind, when 2D assemblies of regulatory elements are called 'promoters'. Some 'promoters' are, in fact, motley artificial chimeras. For example, a fusion between a hu‐ man cytomegalovirus (CMV) immediate-early enhancer and chicken beta-actin promoter, exon1 and intron1 is called 'CBA promoter' or 'CAG promoter' [4].

In general, in the majority of situations in gene therapy, transgene silencing and variegation are undesirable. We review here different factors, both host-dependent and vector-depend‐ ent, which are known to contribute to silencing and variegation of transgene expression and which should be taken into account where choosing or designing effective gene therapy vec‐ tors and strategies for their administration.

#### **2. Host genetic factors of silencing and position effect variegation**

Patterns for maintaining gene repression or activation are governed by regulatory machi‐ nery acting at multiple levels: 1) transcription; 2) mRNA processing, export from the nu‐ cleus, translation and degradation; 3) protein folding, modification, transport and degradation. Control of gene expression is well-coordinated and highly hierarchical, with the control of transcription initiation situated at the top of the regulatory ladder. A number of interacting instruments of transcriptional gene activation and silencing in mammals are known: DNA methylation (e.g. methylation within CpG-islands of promoters), amino acid sequence variants of histones, covalent modifications of histones, histone-binding proteins (e.g. powerful inhibitors of gene activity from the Polycomb Group) and combinations of transcription initiation factors specific for particular tissues and developmental stages. The pivotal point is the access of the transcription machinery to DNA, which is regulated via DNA methylation and chromatin remodelling. With some simplification, it can be general‐ ized that 'coarse tuning' of gene expression (e.g. long-term silencing) is provided by DNA methylation, 'medium tuning' is provided by chromatin remodelling and 'fine tuning' is achieved via various transcription factors and a multitude of other regulatory devices.

The various branches of the regulatory machinery play their own particular roles and yet are inherently interconnected. As detailed below, a prime example of this is the deep in‐ volvement of the miRNA pathway both in mRNA degradation and in the establishment of chromatin methylation patterns [5].

#### **2.1. The role of DNA methylation in silencing**

bourhood within the host genome; a phenomenon, which is similar to 'position effect varie‐ gation' in normal development caused by spontaneous clone-wise silencing of some resident genes [1]. Typical position effect variegation is epigenetic instability and should be distinguished from variegation due to somatic mutations, e.g. due to variations in the length of polynucleotide repeat expansions [2] or due to the sorting of mitochondrial genomes in mitochondrial heteroplasmia [3]. The element of randomness, which is inherently present in position effect variegation, should not come as a surprise. In fact, stochastic fluctuations of gene expression are typical both at the level of variation between different cells of tissue and at the level of temporal variation within one cell. Both of these modes of variation are essen‐ tial for normal differentiation and tissue-patterning with the input of stochastic variation be‐ ing decisive when a developmental signal is present at a near-critical level. For the gene therapist, it is important that the permanent silencing of transgene expression can occur both in postmitotic target cells and target cells undergoing clonal expansion, while variega‐ tion is typically associated with clones of dividing cells. Stable long-term transgene expres‐ sion in differentiating cells is particularly challenging. In fact, the introduced genes are subject to the pre-existing and developing gene expression patterns in the target cells, which can override the signals from the transgenes' own regulatory elements and, thus, can cause transgene expression shutdown. Indeed, at a transcriptional level, the changing scenery of transcription initiation factor pools, chromatin re-modelling and DNA methylation events

50 Gene Therapy - Tools and Potential Applications

during differentiation contribute to the transiency of transgene expression.

ing mechanisms, which we shall address in this chapter.

exon1 and intron1 is called 'CBA promoter' or 'CAG promoter' [4].

Genomes in general and, in particular, mammalian genomes have a mosaic organisation with functionally related genetic elements often being in close physical proximity. There are three teleological reasons for this: 1) expediency of genetic exchange; 2) straightforward temporal control of gene expression; 3) economy of energy, enzymes and other factors serv‐ ing the genetic elements. The second and the third of these reasons are also sufficient for the existence of a finely patterned 3D-arrangement of DNA in interphase nuclei, simplifying the functional interactions between distant genetic elements, e.g. interactions regulating gene expression. It is intriguing to propose that the need to orchestrate gene expression in time and the economy need are also driving the astonishing interconnectedness of all gene silenc‐

The gene therapist should take advantage of the pre-existing regulatory moduli present in the target cells and should also supply the transgenes with their own expression control ele‐ ments. The regulatory elements required for reliable, long-term and tissue-specific transgene expression include minimal promoters, enhancers, regulatory introns and locus control re‐ gions. The functional arrangement of all these elements is ultimately achieved in 3D. This should be borne in mind, when 2D assemblies of regulatory elements are called 'promoters'. Some 'promoters' are, in fact, motley artificial chimeras. For example, a fusion between a hu‐ man cytomegalovirus (CMV) immediate-early enhancer and chicken beta-actin promoter,

In general, in the majority of situations in gene therapy, transgene silencing and variegation are undesirable. We review here different factors, both host-dependent and vector-depend‐ ent, which are known to contribute to silencing and variegation of transgene expression and DNA methylation is an important epigenetic mark involved in cell differentiation and or‐ gan and tissue development, which plays a crucial role in the establishment of genomic imprinting (parent-dependent silencing of alternative alleles) in both male and female germ lines. However, in gene transfer experiments, the methylation of transgenes was shown to be just one ingredient in the dynamic interplay of various factors responsible for silencing and variegation [6].

*De-novo* methylation patterns in humans are established mainly on implantation and in ga‐ metogenesis. Two DNA (cytosine-5)-methyltransferases, DNMT3A and DNMT3B, play an essential role in *de-novo* methylation while DNMT3A in cooperation with the auxiliary pro‐ tein DNMT3L is responsible for imprinting. There is still much we do not know about the manner in which the inactive state of the imprinted chromosomal domains is achieved and what factors trigger this type of silencing. The available evidence indicates that 'Smc hinge proteins' can be particularly important in epigenetic silencing [7]. Thus, in studies based on X-linked GFP transgene silencing, the SmcHD1 gene was shown to play a critical role in Xchromosome inactivation in mammals [8,9]. The recruitment of SmcHD1 to the X-chromo‐ some may involve the non-coding Xist RNA, proteins from the Polycomb group and DNA methyltransferases [7].

sides the normal requirement for the recruitment of transcription factors and co-activators, the genomic targets of PcG proteins require the activity of specific demethylases and meth‐

Silencing of Transgene Expression: A Gene Therapy Perspective

http://dx.doi.org/10.5772/53379

53

Importantly for gene therapy, PcG protein complexes have been recently demonstrated to be

There are two types of transcription factors: 1) auxiliary proteins, which bind other proteins in the transcription complex; 2) DNA-binding sequence-specific transcription factors. The latter type can straightforwardly be recognised *in silico* by the observation of some distinct patterns within the DNA-binding domains of transcription factors, e.g. the zinc-finger motif, the helix-loop-helix motif or the leucine-zipper motif. *In silico* analysis, e.g. using *Biobase* software (http://www.biobase-international.com), is currently also a method of choice for pinpointing transcription factor binding sites and, therefore, for predicting gene expression

It has become clear that non-coding RNAs have an important bearing on gene and transgene expression. In general, there are several mechanisms for the regulatory effects of non-coding RNAs in gene expression. The two most important control points appear to be the direct regulation of transcription initiation and the regulation of mRNA degradation through RNAi by miRNAs. Recent findings revealed that non-coding RNAs are critical factors in the recruitment of PcG members to the cell chromatin [20,21]. At the same time, the miRNA pathway turned out to be significant in establishing the DNA methylation and histone mod‐

In animals, small RNAs, namely piRNA species, which are typically 24-32 nucleotides in length, have been shown to mediate genomic DNA methylation. These non-coding RNAs associate with Piwi clade proteins from the Argonaut superfamily and act analogously to the well-documented RdMD complexes in plants. The primary role of piRNA in many ani‐ mals appears to be the silencing of retrotransposones via DNA methylation in germ lines. In fact, the lack of transposons' suppression in spermatogenesis often results in defects and the loss of germ cells with age. Although it is not clear whether the same mechanism is respon‐ sible for the protective silencing of viral genomes after viral infections of mammalian cells, the small RNAs are likely to be involved in *de-novo* methylation of viral DNA through a sim‐ ilar mechanism. Thus, small noncoding RNAs could potentially provide a flexible regulato‐ ry link between transgene recognition, PcG proteins recruitment and transgene silencing

It appears that, in general, regulation via RNAi has a smaller long-term influence on gene expression than histone modifications and DNA methylation, acting rather as a rapid re‐ sponse system. Indeed, it would be too energetically inconvenient for cells to synthesize

through DNA methylation, histone modifications and chromatin remodelling.

mRNA and then to destroy it on a permanent basis.

able to repress transcription activity in genomic repeats and some transgenes [19].

**2.4. Tissue specific and developmental stage specific transcription factors**

yltransferases for the gene expression to proceed [18].

**2.5. Silencing mediated by non-coding RNAs**

activation patterns.

ification patterns [5,22].

An area of intriguing research is the relationship between DNA hypermethylation and the function of locus control regions (LCRs), controlling the local state of chromatin [10,11].

#### **2.2. The role of histone variants and histone modifications in silencing**

There are two types of structural variations among histone molecules. Firstly, there are low abundance species of histones with unusual amino acid sequences, so-called histone var‐ iants. Secondly, histones are amenable to standard covalent protein modifications such as acetylations and methylations of specific amino acid residues. Both structural variations are known to play important roles in the regulation of gene expression activity.

Regions of constitutive heterochromatin are particularly prone to encroaching on the trans‐ gene in a variable pattern in different cells and, thus, to interfering with transgene expres‐ sion. Different loci in human chromosomes have a variable tendency to become involved in heterochromatin structures. For example, chromosomes' centromeres and telomeres are typ‐ ical regions of heterochromatin, which are known to expand occasionally, inducing steady or intermittent silencing. In the case of centromeres, the silencing machinery might involve the histone variant CENP-A, which is found exclusively in centromeres. Other histone var‐ iants could also play a role in silencing. Thus, the histone variant macroH2A appears to be important in gene silencing on the inactive X-chromosome. In contrast, the histone variants H2A.Z and H3.3 are known to be conducive for transcription.

DNA methylation and histone modifications are closely linked to chromatin remodelling and are often jointly implicated in gene silencing and position effect variegation. Using an *in vivo* mammalian model for position effect variegation, Hiragami-Hamada and co-workers [12] extensively investigated the molecular basis for the stability of heterochromatin-mediat‐ ed silencing in mammals. Comparison between two transgenic lines, containing different numbers of copies of human CD2 transgenes integrated within or close to a block of the per‐ icentric heterochromatin, revealed that the variegation of CD2 expression is indeed associat‐ ed with both genomic DNA methylation and histone modifications such as H3K9me3. However, DNA methylation was the key modification that accompanied the formation of an inaccessible chromatin structure and more stable gene silencing [12,13].

#### **2.3. Silencing mediated by Polycomb proteins**

Silencing can be mediated by proteins from the Polycomb group (PcG). These proteins can form giant complexes, which are tethered to histones and regulatory DNA sequences called Polycomb Response Elements (PREs). When the PcG proteins bind histones, they suppress all the gene expression activity in the respective area of chromatin. In mammals, PcG pro‐ teins are known to be involved in cell differentiation and tissue formation and also to con‐ tribute to tumorigenesis, genomic imprinting, stem cell maintenance and aging [14-16]. The emerging picture from fundamental research suggests that counteracting PcG repression can only be achieved by a combination of multiple inputs converging at chromatin [17]. Be‐ sides the normal requirement for the recruitment of transcription factors and co-activators, the genomic targets of PcG proteins require the activity of specific demethylases and meth‐ yltransferases for the gene expression to proceed [18].

Importantly for gene therapy, PcG protein complexes have been recently demonstrated to be able to repress transcription activity in genomic repeats and some transgenes [19].

#### **2.4. Tissue specific and developmental stage specific transcription factors**

There are two types of transcription factors: 1) auxiliary proteins, which bind other proteins in the transcription complex; 2) DNA-binding sequence-specific transcription factors. The latter type can straightforwardly be recognised *in silico* by the observation of some distinct patterns within the DNA-binding domains of transcription factors, e.g. the zinc-finger motif, the helix-loop-helix motif or the leucine-zipper motif. *In silico* analysis, e.g. using *Biobase* software (http://www.biobase-international.com), is currently also a method of choice for pinpointing transcription factor binding sites and, therefore, for predicting gene expression activation patterns.

#### **2.5. Silencing mediated by non-coding RNAs**

some may involve the non-coding Xist RNA, proteins from the Polycomb group and DNA

An area of intriguing research is the relationship between DNA hypermethylation and the function of locus control regions (LCRs), controlling the local state of chromatin [10,11].

There are two types of structural variations among histone molecules. Firstly, there are low abundance species of histones with unusual amino acid sequences, so-called histone var‐ iants. Secondly, histones are amenable to standard covalent protein modifications such as acetylations and methylations of specific amino acid residues. Both structural variations are

Regions of constitutive heterochromatin are particularly prone to encroaching on the trans‐ gene in a variable pattern in different cells and, thus, to interfering with transgene expres‐ sion. Different loci in human chromosomes have a variable tendency to become involved in heterochromatin structures. For example, chromosomes' centromeres and telomeres are typ‐ ical regions of heterochromatin, which are known to expand occasionally, inducing steady or intermittent silencing. In the case of centromeres, the silencing machinery might involve the histone variant CENP-A, which is found exclusively in centromeres. Other histone var‐ iants could also play a role in silencing. Thus, the histone variant macroH2A appears to be important in gene silencing on the inactive X-chromosome. In contrast, the histone variants

DNA methylation and histone modifications are closely linked to chromatin remodelling and are often jointly implicated in gene silencing and position effect variegation. Using an *in vivo* mammalian model for position effect variegation, Hiragami-Hamada and co-workers [12] extensively investigated the molecular basis for the stability of heterochromatin-mediat‐ ed silencing in mammals. Comparison between two transgenic lines, containing different numbers of copies of human CD2 transgenes integrated within or close to a block of the per‐ icentric heterochromatin, revealed that the variegation of CD2 expression is indeed associat‐ ed with both genomic DNA methylation and histone modifications such as H3K9me3. However, DNA methylation was the key modification that accompanied the formation of an

Silencing can be mediated by proteins from the Polycomb group (PcG). These proteins can form giant complexes, which are tethered to histones and regulatory DNA sequences called Polycomb Response Elements (PREs). When the PcG proteins bind histones, they suppress all the gene expression activity in the respective area of chromatin. In mammals, PcG pro‐ teins are known to be involved in cell differentiation and tissue formation and also to con‐ tribute to tumorigenesis, genomic imprinting, stem cell maintenance and aging [14-16]. The emerging picture from fundamental research suggests that counteracting PcG repression can only be achieved by a combination of multiple inputs converging at chromatin [17]. Be‐

**2.2. The role of histone variants and histone modifications in silencing**

known to play important roles in the regulation of gene expression activity.

H2A.Z and H3.3 are known to be conducive for transcription.

inaccessible chromatin structure and more stable gene silencing [12,13].

**2.3. Silencing mediated by Polycomb proteins**

methyltransferases [7].

52 Gene Therapy - Tools and Potential Applications

It has become clear that non-coding RNAs have an important bearing on gene and transgene expression. In general, there are several mechanisms for the regulatory effects of non-coding RNAs in gene expression. The two most important control points appear to be the direct regulation of transcription initiation and the regulation of mRNA degradation through RNAi by miRNAs. Recent findings revealed that non-coding RNAs are critical factors in the recruitment of PcG members to the cell chromatin [20,21]. At the same time, the miRNA pathway turned out to be significant in establishing the DNA methylation and histone mod‐ ification patterns [5,22].

In animals, small RNAs, namely piRNA species, which are typically 24-32 nucleotides in length, have been shown to mediate genomic DNA methylation. These non-coding RNAs associate with Piwi clade proteins from the Argonaut superfamily and act analogously to the well-documented RdMD complexes in plants. The primary role of piRNA in many ani‐ mals appears to be the silencing of retrotransposones via DNA methylation in germ lines. In fact, the lack of transposons' suppression in spermatogenesis often results in defects and the loss of germ cells with age. Although it is not clear whether the same mechanism is respon‐ sible for the protective silencing of viral genomes after viral infections of mammalian cells, the small RNAs are likely to be involved in *de-novo* methylation of viral DNA through a sim‐ ilar mechanism. Thus, small noncoding RNAs could potentially provide a flexible regulato‐ ry link between transgene recognition, PcG proteins recruitment and transgene silencing through DNA methylation, histone modifications and chromatin remodelling.

It appears that, in general, regulation via RNAi has a smaller long-term influence on gene expression than histone modifications and DNA methylation, acting rather as a rapid re‐ sponse system. Indeed, it would be too energetically inconvenient for cells to synthesize mRNA and then to destroy it on a permanent basis.

### **3. Gene vector properties, which are known to contribute to transgene silencing**

Some bacterial plasmid backbones are known to cause transgene silencing [27-29]. In addi‐ tion, bacterial plasmid backbones interfere with gene delivery into human cells after DNA administration *in vivo* because of the innate TLR9-receptor-mediated immune reaction to un‐ methylated bacterial 'CpG-motifs' within these backbones. In an attempt to alleviate the im‐ mune reaction, methylation of these sequences *in vitro* was attempted. Disappointingly, on some occasions the methylation of plasmid gene vector DNA resulted in increased silencing of transgene expression [30]. The depletion or ablation of CpG motifs from bacterial plasmid backbones is known to substantially reduce their immunogenicity. The effects of CpG-de‐ pletion and ablation on transgene silencing are expected, but the available data on this issue

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55

Bacterial lypopolysaccharides (LPS) often co-purify and contaminate plasmid gene vector DNA. These endotoxins can substantially reduce the efficiency of transfection *in vitro* [31,32] and *in vivo*, where LPS are known to induce a TLR4-receptor-mediated innate immune re‐ sponse. Bacterial endotoxins exhibit a profound effect on cellular regulatory networks [33]. Therefore, it is possible that tilting cells towards 'transgene-silencing mode' is an important

**4. Therapeutic gene vectors and the strategies for their use, which are**

Stable long-term transgene expression depends on the intertwined issues of reliable mainte‐ nance of transgenes in target cells and a robust policy to prevent undesired transgene silenc‐ ing. In general, these two issues are to a large extent under the control of the gene therapist, as both of them can be addressed through the gene vector design and the delivery mode. The regulation of gene expression in eukaryotic cells is exceptionally complex and multi-fac‐ eted. As a result, the strategies used to achieve sustainable transgene expression should ad‐

As most silencing mechanisms are nuclear-based, gene vectors with direct cytoplasmic ex‐ pression, which are not required to enter the nucleoplasm, are well-positioned to avoid si‐ lencing. Thus, non-viral mRNA vectors [34] or positive strand RNA-based viral vectors such as Sendai virus based vectors [35] can be employed. In addition to the escape from silencing, the advantages of extra-nuclear-delivery vectors include relatively fast transgene expression and the absence of potentially mutagenic genomic insertions. The downside is that trans‐ gene expression using such vectors is never long-term because of the eventual degradation of RNA in cells and because of RNA dilution in the dividing cells. Moreover, the fundamen‐ tally low fidelity of RNA replication undermines efforts to generate artificial vector systems with replicating RNA episomes. The key upside is that low immunogenicity and minimal

contributing factor in the endotoxin-mediated inhibition of transfection.

dress multiple possible reasons for the transgene expression shutdown.

toxicity of such vectors accommodate their repeated administration well.

**4.1. Employment of cytoplasmic-only (non-nuclear) vectors**

**employed to avoid transgene silencing**

are currently quite limited.

Long-term transgene expression is highly desirable for most gene therapy applications. However, it is a relatively common occurrence for transgene expression to die out both in terms of the decrease of the efficiency of expression in individual cells and in terms of the reduction of the fraction of expressing cells.

A wide variety of vectors can be used for the delivery and establishment of transgenes and their control elements. Some of the vectors, so called 'viral vectors', are generated using a top-down approach by piggy-backing on the natural gene transfer machinery of viruses. In contrast, 'non-viral' vectors are either pure nucleic acids or synthetic nano-particles, which are generated using a bottom-up strategy. A pivotal feature of any gene therapy vector (with the obvious exception of cytoplasmic-only vectors such as mRNA-based vectors) is the final localization of the delivered transgenes in the nuclei of the target cells. In general, transgenes can be integrated into random chromosomal sites, integrated into pre-selected chromosomal sites and/or left to exist episomally. Specialized molecular machinery for effi‐ cient random integration is born by retroviral vectors [23], lentiviral vectors and eukaryotic transposon vectors. Although the bulk of the DNA delivered with non-transposable plas‐ mid, minicircle and PCR-generated vectors stays episomally, some of the vector DNA also randomly integrates into the chromosomal DNA. The genetic neighbourhood at a transgene integration site has an important bearing on the temporal profile of transgene expression. Nevertheless, many factors that determine the susceptibility of transgene to silencing are de‐ fined by the properties of the employed vector, transgene and co-introduced expression con‐ trol elements.

Multimeric transgene inserts were reported to induce silencing [24]. Unfavourably, even if a gene vector delivers monomeric DNA, spontaneous chromosomal integrations often result in vector DNA multimers (it remains unclear whether the multimers are formed before or after the initial integration event). Silencing due to repetitive DNA was also demonstrated when the introduced DNA contained trinucleotide repeat expansions [25]. This result has an implication for the gene therapy of recessive polyglutamine diseases, as therapeutic trans‐ genes can contain triplet expansions of some minimal length. The precise mechanism for si‐ lencing through the recognition of multimeric transgenes and trinucleotide repeats in the host genomic DNA still remains unclear.

Transgene silencing is often blamed on the malfunction of foreign gene expression control elements. Indeed, this phenomenon is sometimes referred to as 'promoter shut down'. Cer‐ tainly, different promoters vary in their ability to maintain long-term transgene expression in specific cell populations. In particular, there is a clear tendency for some promoters to turn off in cells where they are not normally active. The mechanisms for such effects can be quite indirect. Thus, the ubiquitous CMV promoter can activate transgene expression in an‐ tigen-presenting cells with the ensuing immune response and elimination of all vulnerable transgene expressing cells [26].

Some bacterial plasmid backbones are known to cause transgene silencing [27-29]. In addi‐ tion, bacterial plasmid backbones interfere with gene delivery into human cells after DNA administration *in vivo* because of the innate TLR9-receptor-mediated immune reaction to un‐ methylated bacterial 'CpG-motifs' within these backbones. In an attempt to alleviate the im‐ mune reaction, methylation of these sequences *in vitro* was attempted. Disappointingly, on some occasions the methylation of plasmid gene vector DNA resulted in increased silencing of transgene expression [30]. The depletion or ablation of CpG motifs from bacterial plasmid backbones is known to substantially reduce their immunogenicity. The effects of CpG-de‐ pletion and ablation on transgene silencing are expected, but the available data on this issue are currently quite limited.

**3. Gene vector properties, which are known to contribute to transgene**

Long-term transgene expression is highly desirable for most gene therapy applications. However, it is a relatively common occurrence for transgene expression to die out both in terms of the decrease of the efficiency of expression in individual cells and in terms of the

A wide variety of vectors can be used for the delivery and establishment of transgenes and their control elements. Some of the vectors, so called 'viral vectors', are generated using a top-down approach by piggy-backing on the natural gene transfer machinery of viruses. In contrast, 'non-viral' vectors are either pure nucleic acids or synthetic nano-particles, which are generated using a bottom-up strategy. A pivotal feature of any gene therapy vector (with the obvious exception of cytoplasmic-only vectors such as mRNA-based vectors) is the final localization of the delivered transgenes in the nuclei of the target cells. In general, transgenes can be integrated into random chromosomal sites, integrated into pre-selected chromosomal sites and/or left to exist episomally. Specialized molecular machinery for effi‐ cient random integration is born by retroviral vectors [23], lentiviral vectors and eukaryotic transposon vectors. Although the bulk of the DNA delivered with non-transposable plas‐ mid, minicircle and PCR-generated vectors stays episomally, some of the vector DNA also randomly integrates into the chromosomal DNA. The genetic neighbourhood at a transgene integration site has an important bearing on the temporal profile of transgene expression. Nevertheless, many factors that determine the susceptibility of transgene to silencing are de‐ fined by the properties of the employed vector, transgene and co-introduced expression con‐

Multimeric transgene inserts were reported to induce silencing [24]. Unfavourably, even if a gene vector delivers monomeric DNA, spontaneous chromosomal integrations often result in vector DNA multimers (it remains unclear whether the multimers are formed before or after the initial integration event). Silencing due to repetitive DNA was also demonstrated when the introduced DNA contained trinucleotide repeat expansions [25]. This result has an implication for the gene therapy of recessive polyglutamine diseases, as therapeutic trans‐ genes can contain triplet expansions of some minimal length. The precise mechanism for si‐ lencing through the recognition of multimeric transgenes and trinucleotide repeats in the

Transgene silencing is often blamed on the malfunction of foreign gene expression control elements. Indeed, this phenomenon is sometimes referred to as 'promoter shut down'. Cer‐ tainly, different promoters vary in their ability to maintain long-term transgene expression in specific cell populations. In particular, there is a clear tendency for some promoters to turn off in cells where they are not normally active. The mechanisms for such effects can be quite indirect. Thus, the ubiquitous CMV promoter can activate transgene expression in an‐ tigen-presenting cells with the ensuing immune response and elimination of all vulnerable

**silencing**

trol elements.

reduction of the fraction of expressing cells.

54 Gene Therapy - Tools and Potential Applications

host genomic DNA still remains unclear.

transgene expressing cells [26].

Bacterial lypopolysaccharides (LPS) often co-purify and contaminate plasmid gene vector DNA. These endotoxins can substantially reduce the efficiency of transfection *in vitro* [31,32] and *in vivo*, where LPS are known to induce a TLR4-receptor-mediated innate immune re‐ sponse. Bacterial endotoxins exhibit a profound effect on cellular regulatory networks [33]. Therefore, it is possible that tilting cells towards 'transgene-silencing mode' is an important contributing factor in the endotoxin-mediated inhibition of transfection.

#### **4. Therapeutic gene vectors and the strategies for their use, which are employed to avoid transgene silencing**

Stable long-term transgene expression depends on the intertwined issues of reliable mainte‐ nance of transgenes in target cells and a robust policy to prevent undesired transgene silenc‐ ing. In general, these two issues are to a large extent under the control of the gene therapist, as both of them can be addressed through the gene vector design and the delivery mode. The regulation of gene expression in eukaryotic cells is exceptionally complex and multi-fac‐ eted. As a result, the strategies used to achieve sustainable transgene expression should ad‐ dress multiple possible reasons for the transgene expression shutdown.

#### **4.1. Employment of cytoplasmic-only (non-nuclear) vectors**

As most silencing mechanisms are nuclear-based, gene vectors with direct cytoplasmic ex‐ pression, which are not required to enter the nucleoplasm, are well-positioned to avoid si‐ lencing. Thus, non-viral mRNA vectors [34] or positive strand RNA-based viral vectors such as Sendai virus based vectors [35] can be employed. In addition to the escape from silencing, the advantages of extra-nuclear-delivery vectors include relatively fast transgene expression and the absence of potentially mutagenic genomic insertions. The downside is that trans‐ gene expression using such vectors is never long-term because of the eventual degradation of RNA in cells and because of RNA dilution in the dividing cells. Moreover, the fundamen‐ tally low fidelity of RNA replication undermines efforts to generate artificial vector systems with replicating RNA episomes. The key upside is that low immunogenicity and minimal toxicity of such vectors accommodate their repeated administration well.

#### **4.2. CpG ablation, CpG depletion and minimized DNA vectors**

The methylation of chromosomal DNA is one of the most powerful mechanisms for the shut-down of gene expression. Thus, the design of gene therapy vectors should take into ac‐ count the amenity of the vector sequences to methylation. Firstly, the purposeful exclusion of entire methylation-prone CpG islands should be considered. Secondly, CpG-depleted or CpG-ablated modules, produced through the point-wise replacement or removal of CpG di‐ nucleotides, should be taken advantage of. The generation of functionally active CpG-ablat‐ ed sequences is fairly laborious; the CpG-ablated gamma replicon from the bacterial plasmid R6K and some antibiotic-resistance genes are available from *Invivogen*.

**4.3. Judicious choice of tissue-specific, inducible and ubiquitous promoters to control**

**4.4. Multiple transgene insertions into random chromosomal sites**

Random integration of transgenes into chromosomes is typical for a number of gene deliv‐ ery systems. Spontaneous chromosomal integration of vector DNA within target cells is not efficient. Thus, enhanced random chromosomal integration of plasmid gene vectors can be attained using genetic elements of eukaryotic transposons, retroviruses or lentiviruses (lenti‐ viruses form a subgroup of retroviruses with a somewhat larger genome and the ability to infect non-dividing cells). However, many integration events occur in unfavourable genetic neighbourhoods resulting in the silencing of the respective copies of the transgenes. Hence, position-dependent silencing means that individual transfected or transduced cell clones differ in terms of the longevity of the transgene expression. Random chromosomal integra‐ tion of transgenes tend to occur in transcriptionally active areas of the genome where heter‐ ochromatin condensation and DNA methylation are unlikely to interfere with transgene expression. However, as cells differentiate, the pattern of heterochromatization and DNA methylation changes and some of the transgenes find themselves in transcriptionally silent areas of the genome. Therefore, the shutdown of transgene expression is particularly com‐ mon in cell populations undergoing differentiation. In these circumstances, it is certainly possible to increase the chances of long-term transgene expression by increasing the number of randomly chromosomally integrated transgenes through a higher concentration of vector and/or repeated rounds of vector administration. Thus, the gene therapist can aim to gener‐

Promoters are the gene expression control elements, which are typically co-introduced with therapeutic transgenes. In scientific literature, the word 'promoter' is often an umbrella term, which in addition to a minimal promoter also incorporates other linked genetic ele‐ ments such as enhancers, transcription factor binding sites and even regulatory introns. Pro‐ moter is a key element of the regulatory machinery required for long-term non-silenced transgene expression. Different promoters vary in their strength, tissue specificity, specifici‐ ty for particular developmental stages and ability to react to external stimuli (inducibility). Each therapeutic setting requires a thoughtful choice of a transgene promoter. Thus, some ubiquitous promoters are appropriate for consistent long-term transgene expression in dif‐ ferentiating stem cells passing through a number of developmental phases [43]. Ubiquitous promoters are also appropriate in situations where the resident homologue of the therapeu‐ tic gene is naturally expressed ubiquitously [44]. Tissue-specific promoters have been known for a long time to be instrumental for long-term transgene expression in terminally differentiated cells in the liver, vascular tissue, muscle and central nervous system [45]. In‐ ducible promoters are appropriate where the constitutive expression of the therapeutic transgene is undesired and/or where bespoke activation of the therapeutic transgene is re‐ quired. In addition to the heavily used tetracycline-sensing promoter systems, inducible promoters can be activated by heat, light and gas-born acetaldehyde [46]. Clearly, the con‐ struction and determined exploitation of new hybrid promoters can resolve many issues in

Silencing of Transgene Expression: A Gene Therapy Perspective

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57

**transgene expression**

transgene silencing.

Clearly, as repetitive sequences are known to induce silencing, their use in therapeutic gene vectors should be avoided as far as possible.

A common way to reduce the chances of transgene silencing is to shorten the auxiliary vector sequences outside of the therapeutic transgene expression cassette. For example, the plasmid selection markers can be very short indeed [36]. In fact, a plasmid replica‐ tion origin can be re-utilised as a plasmid marker using the 'plasmid addiction' phe‐ nomenon [37].

The trend to exclude unwanted sequences from gene transfer vectors led to the genera‐ tion of specialized minimized DNA vectors. The most tested versions of such vectors are DNA fragments amplified *in vitro* using polymerase chain reaction (PCR) [38], plas‐ mid-derived linear terminally looped 'midges' [39] or circular supercoiled 'minicircles' [40]. Minicircle vectors are produced by intramolecular site-specific recombination with‐ in bacterial plasmids. The superior efficiency of gene delivery and the longevity of transgene expression achieved with minicircle DNA was observed in multiple studies (e.g. [41]). The production of minimized DNA vectors is a biotechnological challenge. For example, advanced methods and bacterial strains were developed for efficient bacte‐ ria-based minicircle DNA production. The generation of PCR amplicons with Taq-poly‐ merase is relatively inexpensive. However, the load of Taq-polymerase-introduced mutations may make one consider alternative *in vitro* amplification methods for the large-scale synthesis of double-stranded DNA, e.g. ligase chain reaction (LCR), which is based on the ligation of preassembled oligonucleotides.

The usual aim in the production of minimized DNA vectors is the removal of sequences of bacterial origin, such as plasmid backbone sequences, as they can be immunogenic and some of them were reported to cause silencing [27,29]. It should be emphasized that trans‐ gene silencing through the co-delivery of specific plasmid sequences should not be general‐ ized to all plasmid sequences and each plasmid sequence or bacterial sequence needs to be tested individually. More research is required to identify the affected bacterial replicons and to pinpoint the mechanism for the induction of silencing by bacterial DNA sequences. An‐ other avenue is the development of novel specialized forms of minimized vectors, such as 'minivectors' for RNAi-based therapy [42].

#### **4.3. Judicious choice of tissue-specific, inducible and ubiquitous promoters to control transgene expression**

**4.2. CpG ablation, CpG depletion and minimized DNA vectors**

vectors should be avoided as far as possible.

56 Gene Therapy - Tools and Potential Applications

based on the ligation of preassembled oligonucleotides.

'minivectors' for RNAi-based therapy [42].

nomenon [37].

The methylation of chromosomal DNA is one of the most powerful mechanisms for the shut-down of gene expression. Thus, the design of gene therapy vectors should take into ac‐ count the amenity of the vector sequences to methylation. Firstly, the purposeful exclusion of entire methylation-prone CpG islands should be considered. Secondly, CpG-depleted or CpG-ablated modules, produced through the point-wise replacement or removal of CpG di‐ nucleotides, should be taken advantage of. The generation of functionally active CpG-ablat‐ ed sequences is fairly laborious; the CpG-ablated gamma replicon from the bacterial

Clearly, as repetitive sequences are known to induce silencing, their use in therapeutic gene

A common way to reduce the chances of transgene silencing is to shorten the auxiliary vector sequences outside of the therapeutic transgene expression cassette. For example, the plasmid selection markers can be very short indeed [36]. In fact, a plasmid replica‐ tion origin can be re-utilised as a plasmid marker using the 'plasmid addiction' phe‐

The trend to exclude unwanted sequences from gene transfer vectors led to the genera‐ tion of specialized minimized DNA vectors. The most tested versions of such vectors are DNA fragments amplified *in vitro* using polymerase chain reaction (PCR) [38], plas‐ mid-derived linear terminally looped 'midges' [39] or circular supercoiled 'minicircles' [40]. Minicircle vectors are produced by intramolecular site-specific recombination with‐ in bacterial plasmids. The superior efficiency of gene delivery and the longevity of transgene expression achieved with minicircle DNA was observed in multiple studies (e.g. [41]). The production of minimized DNA vectors is a biotechnological challenge. For example, advanced methods and bacterial strains were developed for efficient bacte‐ ria-based minicircle DNA production. The generation of PCR amplicons with Taq-poly‐ merase is relatively inexpensive. However, the load of Taq-polymerase-introduced mutations may make one consider alternative *in vitro* amplification methods for the large-scale synthesis of double-stranded DNA, e.g. ligase chain reaction (LCR), which is

The usual aim in the production of minimized DNA vectors is the removal of sequences of bacterial origin, such as plasmid backbone sequences, as they can be immunogenic and some of them were reported to cause silencing [27,29]. It should be emphasized that trans‐ gene silencing through the co-delivery of specific plasmid sequences should not be general‐ ized to all plasmid sequences and each plasmid sequence or bacterial sequence needs to be tested individually. More research is required to identify the affected bacterial replicons and to pinpoint the mechanism for the induction of silencing by bacterial DNA sequences. An‐ other avenue is the development of novel specialized forms of minimized vectors, such as

plasmid R6K and some antibiotic-resistance genes are available from *Invivogen*.

Promoters are the gene expression control elements, which are typically co-introduced with therapeutic transgenes. In scientific literature, the word 'promoter' is often an umbrella term, which in addition to a minimal promoter also incorporates other linked genetic ele‐ ments such as enhancers, transcription factor binding sites and even regulatory introns. Pro‐ moter is a key element of the regulatory machinery required for long-term non-silenced transgene expression. Different promoters vary in their strength, tissue specificity, specifici‐ ty for particular developmental stages and ability to react to external stimuli (inducibility). Each therapeutic setting requires a thoughtful choice of a transgene promoter. Thus, some ubiquitous promoters are appropriate for consistent long-term transgene expression in dif‐ ferentiating stem cells passing through a number of developmental phases [43]. Ubiquitous promoters are also appropriate in situations where the resident homologue of the therapeu‐ tic gene is naturally expressed ubiquitously [44]. Tissue-specific promoters have been known for a long time to be instrumental for long-term transgene expression in terminally differentiated cells in the liver, vascular tissue, muscle and central nervous system [45]. In‐ ducible promoters are appropriate where the constitutive expression of the therapeutic transgene is undesired and/or where bespoke activation of the therapeutic transgene is re‐ quired. In addition to the heavily used tetracycline-sensing promoter systems, inducible promoters can be activated by heat, light and gas-born acetaldehyde [46]. Clearly, the con‐ struction and determined exploitation of new hybrid promoters can resolve many issues in transgene silencing.

#### **4.4. Multiple transgene insertions into random chromosomal sites**

Random integration of transgenes into chromosomes is typical for a number of gene deliv‐ ery systems. Spontaneous chromosomal integration of vector DNA within target cells is not efficient. Thus, enhanced random chromosomal integration of plasmid gene vectors can be attained using genetic elements of eukaryotic transposons, retroviruses or lentiviruses (lenti‐ viruses form a subgroup of retroviruses with a somewhat larger genome and the ability to infect non-dividing cells). However, many integration events occur in unfavourable genetic neighbourhoods resulting in the silencing of the respective copies of the transgenes. Hence, position-dependent silencing means that individual transfected or transduced cell clones differ in terms of the longevity of the transgene expression. Random chromosomal integra‐ tion of transgenes tend to occur in transcriptionally active areas of the genome where heter‐ ochromatin condensation and DNA methylation are unlikely to interfere with transgene expression. However, as cells differentiate, the pattern of heterochromatization and DNA methylation changes and some of the transgenes find themselves in transcriptionally silent areas of the genome. Therefore, the shutdown of transgene expression is particularly com‐ mon in cell populations undergoing differentiation. In these circumstances, it is certainly possible to increase the chances of long-term transgene expression by increasing the number of randomly chromosomally integrated transgenes through a higher concentration of vector and/or repeated rounds of vector administration. Thus, the gene therapist can aim to gener‐ ate multiple copies of transgenes, indiscriminately integrated within the target genome, hoping that at least one of the copies will reside in a suitable chromosomal site that will be immune to silencing.

catalysing a one-off recombination event between the lambda's *attP* site and the chromoso‐ mal *attB* site. The reverse reaction, excision of prophage, is often possible; however, a sepa‐ rate enzyme or a separate subunit of bacteriophage integrase is normally required to catalyze the excision. The *attB* sites are typically shorter than the corresponding *attP* sites. Thus, in the recombination system from the *Streptomyces coelicolor* bacteriophage phiC31, *attP* is 39 bp long and *attB* is 34 bp long. Similarly, the recombination system from the *Lacto‐ coccus lactis* bacteriophage TP901-1 has 50 bp long *attP* and 31 bp long *attB*. Consequently, in artificial recombination systems within the mammalian setting, higher specificity of integra‐ tion is achieved with longer *attP* sites positioned within the chromosomal loci. It has turned out that the human genome contains a close analogue of the phiC31 *attP* site. Extensive mu‐ tagenesis of the phiC31 integrase gene has produced versions of the enzymes with very high specific activity towards this native human site [49]. Cell-permeable and nuclear targeted versions of phiC31 integrase were also created, these recombinant enzymes can be used to create transient, 'hit-and-run', recombinase activity in human cells that is required for the

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59

The typical original *in vivo* function of the reversible site-specific recombination systems is to preserve the monomeric status of a plasmid, prophage or episome via the resolution of circular DNA multimers to monomers; monomeric status is important for the maintenance stability of many plasmid replicons. Commonly used reversible systems include bacterio‐ phage's P1Cre recombinase with its cognate *loxP* sites and FLP recombinase (flipase) with its cognate *FTR* sites from the yeast *Saccharomyces cerevisiae* '2-micron circle' episome. Many re‐ versible systems were successfully used for the chromosomal integration of transgenes in pre-engineered cells. However, it should be noted that some site-specific recombination sys‐ tems are fundamentally unsuitable for chromosomal integration strategies. Thus, ParA re‐ solvase and *MRS* sites from the plasmid RK2 constitute a reversible system for intramolecular recombination; however, in this system there is no molecular recombination

Of course, the employed bacterial recombination systems have to be functional in eukaryotic cells [50]. A potential pitfall to be aware of is that some of the site-specific recombinases re‐ quire an additional co-factor; e.g., IHF (integration host factor) is an obligatory element for lambda Int/*attB*/*attP* system. Unexpectedly and encouragingly, at least on some occasions

The wild type human adeno-associated virus type 2 (AAV2) is the only known human virus capable of site-specific chromosomal integration. AAV2 uses the chromosome-tethering strategy for genomic insertions. Expression of the Rep gene is required for integration of the viral genome into a unique DNA sequence within specific chromosomal loci. The Rep pro‐ teins of this virus bind both several Rep Binding Sites (RBS) within the viral DNA and the RBS sites in the human genome (known as AAVS1, AAVS2 and AAVS3) leading to prefer‐

An important step forward in the exploitation of the site-specific integration system of AAV was achieved when the AAV Rep protein was used to direct the integration of integrase-de‐ fective retroviral vectors into human 19q13.42 locus [51]. The transfer of the locus-specific

ential integration of the viral DNA in the genomic loci 19q13.42, 5p13.3 and 3p24.3.

stable integration of therapeutic transgenes.

between *MRS* sites situated on separate DNA molecules.

mammalian cells are able to provide suitable co-factors [50].

The employment of transposable genetic elements for efficient random integration of thera‐ peutic transgenes was complicated by the fact that mammals do not have their own active or easily re-activatable transposons. Therefore, a number of heterologous transposons were adapted for use in human cells. Recombination machinery from Sleeping Beauty, PiggyBac, Tol2 and Mos1 transposons was shown to be capable of directing chromosomal integration of transgenes [47]. Genes for transposases were either included within the cargo gene vector plasmid or were delivered into human cells on a separate plasmid. Mutant transposases with enhanced activity for random DNA integration were developed.

A caveat of the anti-silencing strategy relying on multiple transgene insertions into random chromosome sites is a possibility of potentially deleterious or tumourigenic mutations due to insertional mutagenesis. However, this drawback is irrelevant for highly differentiated and non-dividing cells where, firstly, only a limited set of gene products is required for cell survival and functional competence and, secondly, only a minimal risk is present for the se‐ lection of malignancies. In fact, many terminally differentiated cells are either polyploid or polynucleated; both of these statuses can alleviate the impact of insertional mutagenesis.

#### **4.5. Site-specific chromosomal integration**

One of the ideal scenarios, where transgene silencing is avoided, involves the transgene DNA being site-specifically integrated into a 'benign', silencing-resistant chromosomal site where there is little chance of transgene consumption by heterochromatin. Thus, targeting transgenes to a continuously active chromosomal locus can resolve the transgene expression shutdown problem. In particular, sites could exist within chromosomal DNA, where an inte‐ grated transgene would be immune to chromatin re-arrangements and other regulatory events during differentiation. A possible candidate site is the human homologue of mouse Rosa 26 locus, which is being successfully used to express various transgenes in mouse transgenic studies.

In principle, both transposases and retroviral integrases can be re-engineered into site-di‐ rected recombination enzymes through their fusion with appropriate site-specific 'tethering' domains [48]. In addition to tethered transposases and retroviral integrases, the site-specific integration of transgenes into human chromosomes can be achieved via the modification of *bona fide* site-specific recombination systems.

Site-specific DNA recombination systems are comprised of recombinase enzymes, their cofactors and their cognate recombination sites. Site-specific recombination systems can be classified into two general types: irreversible and reversible ones.

Site-specific recombination machinery for irreversible recombination is typically borrowed from the chromosome integration systems of temperate bacteriophages. In integrative re‐ combination systems there are two types of recombination sites, which are normally refer‐ red to as *attP* and *attB*. An archetypical example is bacteriophage lambda integrase (Int) catalysing a one-off recombination event between the lambda's *attP* site and the chromoso‐ mal *attB* site. The reverse reaction, excision of prophage, is often possible; however, a sepa‐ rate enzyme or a separate subunit of bacteriophage integrase is normally required to catalyze the excision. The *attB* sites are typically shorter than the corresponding *attP* sites. Thus, in the recombination system from the *Streptomyces coelicolor* bacteriophage phiC31, *attP* is 39 bp long and *attB* is 34 bp long. Similarly, the recombination system from the *Lacto‐ coccus lactis* bacteriophage TP901-1 has 50 bp long *attP* and 31 bp long *attB*. Consequently, in artificial recombination systems within the mammalian setting, higher specificity of integra‐ tion is achieved with longer *attP* sites positioned within the chromosomal loci. It has turned out that the human genome contains a close analogue of the phiC31 *attP* site. Extensive mu‐ tagenesis of the phiC31 integrase gene has produced versions of the enzymes with very high specific activity towards this native human site [49]. Cell-permeable and nuclear targeted versions of phiC31 integrase were also created, these recombinant enzymes can be used to create transient, 'hit-and-run', recombinase activity in human cells that is required for the stable integration of therapeutic transgenes.

ate multiple copies of transgenes, indiscriminately integrated within the target genome, hoping that at least one of the copies will reside in a suitable chromosomal site that will be

The employment of transposable genetic elements for efficient random integration of thera‐ peutic transgenes was complicated by the fact that mammals do not have their own active or easily re-activatable transposons. Therefore, a number of heterologous transposons were adapted for use in human cells. Recombination machinery from Sleeping Beauty, PiggyBac, Tol2 and Mos1 transposons was shown to be capable of directing chromosomal integration of transgenes [47]. Genes for transposases were either included within the cargo gene vector plasmid or were delivered into human cells on a separate plasmid. Mutant transposases

A caveat of the anti-silencing strategy relying on multiple transgene insertions into random chromosome sites is a possibility of potentially deleterious or tumourigenic mutations due to insertional mutagenesis. However, this drawback is irrelevant for highly differentiated and non-dividing cells where, firstly, only a limited set of gene products is required for cell survival and functional competence and, secondly, only a minimal risk is present for the se‐ lection of malignancies. In fact, many terminally differentiated cells are either polyploid or polynucleated; both of these statuses can alleviate the impact of insertional mutagenesis.

One of the ideal scenarios, where transgene silencing is avoided, involves the transgene DNA being site-specifically integrated into a 'benign', silencing-resistant chromosomal site where there is little chance of transgene consumption by heterochromatin. Thus, targeting transgenes to a continuously active chromosomal locus can resolve the transgene expression shutdown problem. In particular, sites could exist within chromosomal DNA, where an inte‐ grated transgene would be immune to chromatin re-arrangements and other regulatory events during differentiation. A possible candidate site is the human homologue of mouse Rosa 26 locus, which is being successfully used to express various transgenes in mouse

In principle, both transposases and retroviral integrases can be re-engineered into site-di‐ rected recombination enzymes through their fusion with appropriate site-specific 'tethering' domains [48]. In addition to tethered transposases and retroviral integrases, the site-specific integration of transgenes into human chromosomes can be achieved via the modification of

Site-specific DNA recombination systems are comprised of recombinase enzymes, their cofactors and their cognate recombination sites. Site-specific recombination systems can be

Site-specific recombination machinery for irreversible recombination is typically borrowed from the chromosome integration systems of temperate bacteriophages. In integrative re‐ combination systems there are two types of recombination sites, which are normally refer‐ red to as *attP* and *attB*. An archetypical example is bacteriophage lambda integrase (Int)

with enhanced activity for random DNA integration were developed.

**4.5. Site-specific chromosomal integration**

*bona fide* site-specific recombination systems.

classified into two general types: irreversible and reversible ones.

immune to silencing.

58 Gene Therapy - Tools and Potential Applications

transgenic studies.

The typical original *in vivo* function of the reversible site-specific recombination systems is to preserve the monomeric status of a plasmid, prophage or episome via the resolution of circular DNA multimers to monomers; monomeric status is important for the maintenance stability of many plasmid replicons. Commonly used reversible systems include bacterio‐ phage's P1Cre recombinase with its cognate *loxP* sites and FLP recombinase (flipase) with its cognate *FTR* sites from the yeast *Saccharomyces cerevisiae* '2-micron circle' episome. Many re‐ versible systems were successfully used for the chromosomal integration of transgenes in pre-engineered cells. However, it should be noted that some site-specific recombination sys‐ tems are fundamentally unsuitable for chromosomal integration strategies. Thus, ParA re‐ solvase and *MRS* sites from the plasmid RK2 constitute a reversible system for intramolecular recombination; however, in this system there is no molecular recombination between *MRS* sites situated on separate DNA molecules.

Of course, the employed bacterial recombination systems have to be functional in eukaryotic cells [50]. A potential pitfall to be aware of is that some of the site-specific recombinases re‐ quire an additional co-factor; e.g., IHF (integration host factor) is an obligatory element for lambda Int/*attB*/*attP* system. Unexpectedly and encouragingly, at least on some occasions mammalian cells are able to provide suitable co-factors [50].

The wild type human adeno-associated virus type 2 (AAV2) is the only known human virus capable of site-specific chromosomal integration. AAV2 uses the chromosome-tethering strategy for genomic insertions. Expression of the Rep gene is required for integration of the viral genome into a unique DNA sequence within specific chromosomal loci. The Rep pro‐ teins of this virus bind both several Rep Binding Sites (RBS) within the viral DNA and the RBS sites in the human genome (known as AAVS1, AAVS2 and AAVS3) leading to prefer‐ ential integration of the viral DNA in the genomic loci 19q13.42, 5p13.3 and 3p24.3.

An important step forward in the exploitation of the site-specific integration system of AAV was achieved when the AAV Rep protein was used to direct the integration of integrase-de‐ fective retroviral vectors into human 19q13.42 locus [51]. The transfer of the locus-specific chromosomal integration apparatus of AAV2 to other vector types, e.g., plasmid gene vec‐ tors, can be accomplished as well [52].

**4.8. Top-up transgene administration to compensate for silenced transgenes**

**4.9. Selection of clones with stable non-silenced transgene expression**

transgene insertions or top-up transgene administrations).

**4.10. Small molecule enhancers of transgene expression**

format.

Normally, if the expression of therapeutic transgenes did die out, it is possible to perform a new round of gene transfer, thus achieving a new burst of transgene expression. Repeated vector administration can be particularly sought-after when the target cell population expe‐ riences programmed death, while the respective progenitor cell pool is poorly accessible for therapeutic gene transfer. This strategy can be used without hesitation in an *ex vivo* gene therapy setting where therapeutic genes are delivered *in vitro* to dividing cells derived from a patient's biopsy prior to autologous transplantation. In contrast, in an *in vivo* gene therapy setting, the drawbacks of vector re-administration include not only the increased complexity and cost of treatment, but also the realistic possibilities that immunity to elements of the vector might develop and that the effects of the toxic elements of the vector might build up to an unacceptable level. That is why low immunogenicity, low toxicity and the biodegrada‐ bility of auxiliary vector elements are important in the vector re-administration treatment

Silencing of Transgene Expression: A Gene Therapy Perspective

http://dx.doi.org/10.5772/53379

61

Reliable, robust and error-free site-specific integration into mammalian cells lacking pre-en‐ gineered integration sites is difficult to achieve. Simpler alternatives for attaining stable long-term transgene expression exist in the *ex vivo* gene therapy approach. In one of the treatment scenarios, transgenes are integrated randomly, e.g. using lentiviral vectors or nak‐ ed DNA vectors. It is then possible to select the best clone with minimal initial transgene silencing and minimal propensity for transgene expression shutdown among a heterogene‐ ous population of transfected or transduced cells. The preferred method for cell selection is antibody-based magnetic sorting, as this method allows processing of large numbers of cells without recourse to heterologous fluorescent proteins and mutagenic UV irradiation as in fluorescence activated cell sorting (FACS). Clearly, such a clone pre-selection strategy can be used in conjunction with some other counter-silencing strategies (e.g. multiple random

It is extremely attractive to use small molecule compounds to counteract transgene silenc‐ ing. Substances known to influence chromatin's state are prime candidates for this role. Thus, histon deacetylase inhibitors Trichostatin A, 4-phenylbutyric acid, butyric acid, valeric acid and caproic acid were successfully used to enhance transgene expression after transient trasfection [66]. Available data indicate that another histone deacetylase inhibitor, valproic acid, and also retinoic acid, which is known to act through a receptor-mediated mechanism, are epigenetically active substances and, therefore, in certain situations could be considered for use as transgene expression stimulants. Some small molecule enhancers could be specific for particular vectors used for gene transfer. Thus, hydroxyurea is known to boost transgene expression after delivery with AAV vectors [67]. In this case, transgene expression is likely to be spurred not through the inhibition of standard silencing mechanisms but rather

#### **4.6. Episomal localisation of a transgene**

Episomal maintenance of transgene expression cassettes is an attractive strategy to escape the control of some resident gene regulation systems, such as chromatin remodelling machi‐ nery, over transgene expression. The problem with this approach is that viral replicons, e.g., compact episomal replicons from SV40, polyoma, papilloma viruses, which are often com‐ pletely adequate for the research use of gene vectors, are rarely acceptable for therapeutic applications. Indeed, the expression of the large SV40 T-antigen and, hence, the malignant transformation of the recipient host cells is required for SV40-origin-based replication. Simi‐ larly, EBNA1-oriP DNA segment of Epstein-Barr Virus (EBV) can be used to support the maintenance of plasmid gene vectors in the nucleoplasm of dividing laboratory cells. Al‐ though EBNA1 expression does not result in a typical malignant transformation, it can still tilt the cells towards undesired immortalisation [53].

Alternative benign episomal replicons are being sought. Encouragingly, the scaffold/ matrix attachment region (S/MAR) from the human β-interferon gene was reported to support non-viral episomal replication when coupled to a promoter [54]. Thus, episomal maintenance mediated by S/MAR elements might be the reason behind the well-estab‐ lished beneficial effects of these elements on transgene expression [41,55,56]. Non-viral episomal vectors also include mammalian artificial chromosomes (MACs), which can be generated through both top-down and bottom-up approaches [57,58]. However, current progress with MACs is limited because of prohibiting costs associated with the genera‐ tion of these vectors.

#### **4.7. Employment of the locus control regions within the vectors**

Protection of integrated transgenes from encroaching heterochromatin can be achieved with chromatin insulators or other *cis*-acting locus control regions (LCRs) [59]. The mechanistic details of LCRs action are currently not clear and so the terminology in this area is some‐ what diffuse with, for example, 'chromatin boundary elements' and 'chromatin insulators' often being used synonymously [60,61]. Some enhancers have an important bearing on the state of chromatin and, therefore, can also be viewed as LCRs. Experiments with some known chromatin insulators show that their effects on transgene expression are not always positive and to a large extent depend on the cell context [62,63]. Nuclear 'matrix attachment region' elements (MARs) and the effectively synonymous nuclear 'scaffold attachment re‐ gion' elements (SARs) are known to possess some LCR activity. Some authors are trying to avoid the confusion between MARs and SARs using the joined names 'SAR/MARs', 'MAR/ SARs' or 'S/MARs'. Promising results in terms of sustained transgene expression were ach‐ ieved with MARs both within the scenario where two MAR elements are used 'to protect the transgene from the flanks' [64,65] and the scenario where a single promoter-MAR couple is driving the transgene's episomal replication [41].

#### **4.8. Top-up transgene administration to compensate for silenced transgenes**

chromosomal integration apparatus of AAV2 to other vector types, e.g., plasmid gene vec‐

Episomal maintenance of transgene expression cassettes is an attractive strategy to escape the control of some resident gene regulation systems, such as chromatin remodelling machi‐ nery, over transgene expression. The problem with this approach is that viral replicons, e.g., compact episomal replicons from SV40, polyoma, papilloma viruses, which are often com‐ pletely adequate for the research use of gene vectors, are rarely acceptable for therapeutic applications. Indeed, the expression of the large SV40 T-antigen and, hence, the malignant transformation of the recipient host cells is required for SV40-origin-based replication. Simi‐ larly, EBNA1-oriP DNA segment of Epstein-Barr Virus (EBV) can be used to support the maintenance of plasmid gene vectors in the nucleoplasm of dividing laboratory cells. Al‐ though EBNA1 expression does not result in a typical malignant transformation, it can still

Alternative benign episomal replicons are being sought. Encouragingly, the scaffold/ matrix attachment region (S/MAR) from the human β-interferon gene was reported to support non-viral episomal replication when coupled to a promoter [54]. Thus, episomal maintenance mediated by S/MAR elements might be the reason behind the well-estab‐ lished beneficial effects of these elements on transgene expression [41,55,56]. Non-viral episomal vectors also include mammalian artificial chromosomes (MACs), which can be generated through both top-down and bottom-up approaches [57,58]. However, current progress with MACs is limited because of prohibiting costs associated with the genera‐

Protection of integrated transgenes from encroaching heterochromatin can be achieved with chromatin insulators or other *cis*-acting locus control regions (LCRs) [59]. The mechanistic details of LCRs action are currently not clear and so the terminology in this area is some‐ what diffuse with, for example, 'chromatin boundary elements' and 'chromatin insulators' often being used synonymously [60,61]. Some enhancers have an important bearing on the state of chromatin and, therefore, can also be viewed as LCRs. Experiments with some known chromatin insulators show that their effects on transgene expression are not always positive and to a large extent depend on the cell context [62,63]. Nuclear 'matrix attachment region' elements (MARs) and the effectively synonymous nuclear 'scaffold attachment re‐ gion' elements (SARs) are known to possess some LCR activity. Some authors are trying to avoid the confusion between MARs and SARs using the joined names 'SAR/MARs', 'MAR/ SARs' or 'S/MARs'. Promising results in terms of sustained transgene expression were ach‐ ieved with MARs both within the scenario where two MAR elements are used 'to protect the transgene from the flanks' [64,65] and the scenario where a single promoter-MAR couple is

tors, can be accomplished as well [52].

60 Gene Therapy - Tools and Potential Applications

**4.6. Episomal localisation of a transgene**

tilt the cells towards undesired immortalisation [53].

**4.7. Employment of the locus control regions within the vectors**

driving the transgene's episomal replication [41].

tion of these vectors.

Normally, if the expression of therapeutic transgenes did die out, it is possible to perform a new round of gene transfer, thus achieving a new burst of transgene expression. Repeated vector administration can be particularly sought-after when the target cell population expe‐ riences programmed death, while the respective progenitor cell pool is poorly accessible for therapeutic gene transfer. This strategy can be used without hesitation in an *ex vivo* gene therapy setting where therapeutic genes are delivered *in vitro* to dividing cells derived from a patient's biopsy prior to autologous transplantation. In contrast, in an *in vivo* gene therapy setting, the drawbacks of vector re-administration include not only the increased complexity and cost of treatment, but also the realistic possibilities that immunity to elements of the vector might develop and that the effects of the toxic elements of the vector might build up to an unacceptable level. That is why low immunogenicity, low toxicity and the biodegrada‐ bility of auxiliary vector elements are important in the vector re-administration treatment format.

#### **4.9. Selection of clones with stable non-silenced transgene expression**

Reliable, robust and error-free site-specific integration into mammalian cells lacking pre-en‐ gineered integration sites is difficult to achieve. Simpler alternatives for attaining stable long-term transgene expression exist in the *ex vivo* gene therapy approach. In one of the treatment scenarios, transgenes are integrated randomly, e.g. using lentiviral vectors or nak‐ ed DNA vectors. It is then possible to select the best clone with minimal initial transgene silencing and minimal propensity for transgene expression shutdown among a heterogene‐ ous population of transfected or transduced cells. The preferred method for cell selection is antibody-based magnetic sorting, as this method allows processing of large numbers of cells without recourse to heterologous fluorescent proteins and mutagenic UV irradiation as in fluorescence activated cell sorting (FACS). Clearly, such a clone pre-selection strategy can be used in conjunction with some other counter-silencing strategies (e.g. multiple random transgene insertions or top-up transgene administrations).

#### **4.10. Small molecule enhancers of transgene expression**

It is extremely attractive to use small molecule compounds to counteract transgene silenc‐ ing. Substances known to influence chromatin's state are prime candidates for this role. Thus, histon deacetylase inhibitors Trichostatin A, 4-phenylbutyric acid, butyric acid, valeric acid and caproic acid were successfully used to enhance transgene expression after transient trasfection [66]. Available data indicate that another histone deacetylase inhibitor, valproic acid, and also retinoic acid, which is known to act through a receptor-mediated mechanism, are epigenetically active substances and, therefore, in certain situations could be considered for use as transgene expression stimulants. Some small molecule enhancers could be specific for particular vectors used for gene transfer. Thus, hydroxyurea is known to boost transgene expression after delivery with AAV vectors [67]. In this case, transgene expression is likely to be spurred not through the inhibition of standard silencing mechanisms but rather through the more active synthesis of the second DNA strand in the delivered single-strand‐ ed AAV vector DNA [67].

Clearly, the future solutions to transgene silencing enabling stable long-term expression of therapeutic transgenes will depend on the determined implementation of the above strat‐

[1] Eissenberg JC: Position effect variegation in drosophila: Towards a genetics of chro‐

[2] Dion V, Lin Y, Hubert L, Jr., Waterland RA, Wilson JH: Dnmt1 deficiency promotes cag repeat expansion in the mouse germline. Hum Mol Genet (2008) 17(9):1306-1317.

[3] Zaegel V, Guermann B, Le Ret M, Andres C, Meyer D, Erhardt M, Canaday J, Gual‐ berto JM, Imbault P: The plant-specific ssdna binding protein osb1 is involved in the stoichiometric transmission of mitochondrial DNA in arabidopsis. Plant Cell (2006)

[4] Kiwaki K, Kanegae Y, Saito I, Komaki S, Nakamura K, Miyazaki JI, Endo F, Matsuda I: Correction of ornithine transcarbamylase deficiency in adult spf(ash) mice and in otc-deficient human hepatocytes with recombinant adenoviruses bearing the cag

[5] Thum T, Catalucci D, Bauersachs J: Micrornas: Novel regulators in cardiac develop‐

[6] Yao S, Sukonnik T, Kean T, Bharadwaj RR, Pasceri P, Ellis J: Retrovirus silencing, var‐ iegation, extinction, and memory are controlled by a dynamic interplay of multiple

[7] Heard E, Colot V: Chromosome structural proteins and rna-mediated epigenetic si‐

[8] Okamoto I, Heard E: The dynamics of imprinted x inactivation during preimplanta‐ tion development in mice. Cytogenet Genome Res (2006) 113(1-4):318-324.

and Tanya Tolmachova2

Silencing of Transgene Expression: A Gene Therapy Perspective

http://dx.doi.org/10.5772/53379

63

egies and their effective combinations.

Oleg E. Tolmachov1\*, Tatiana Subkhankulova2

1 St. Mary's University College, Twickenham, UK

\*Address all correspondence to: 125317@live.smuc.ac.uk

matin assembly. Bioessays (1989) 11(1):14-17.

promoter. Hum Gene Ther (1996) 7(7):821-830.

ment and disease. Cardiovasc Res (2008) 79(4):562-570.

epigenetic modifications. Mol Ther (2004) 10(1):27-36.

lencing. Dev Cell (2008) 14(6):813-814.

2 National Heart and Lung Institute, Imperial College London, London, UK

**Author details**

**References**

18(12):3548-3563.

#### **4.11. Selection of low immunogenic vectors and transgene products**

The elimination of therapeutic gene vectors and transgenic cells by the immune system can imitate the silencing of transgene expression. Thus, the employment of low-immunogenic vectors is a preferred option. Vectors' epitopes should mimic the native epitopes of individ‐ ual patients and do not match their pre-existing immune profile. Coating vector particles with immunologically inert polymers like polyethyleneglycol is one of the strategies to es‐ cape immune surveillance. Alternatively, vector particles can be developed, which are able to mimic the immune-evasion strategy of some viruses that are capable of 'hiding' at the cell surface [68]. Non-immunogenic transgene products, e.g. exclusively human versions of pro‐ teins, should be chosen to prevent cell elimination via immune reactions *in vivo*. If required, transgene products should be re-engineered to achieve the 'stealth effect' and to tailor them to the immunological profiles of individual patients.

#### **5. Conclusion**

Epigenetic control by the target cells can result in permanent transgene silencing or in the instability of transgene expression. Thus, one needs to pursue therapeutic strategies, which can achieve long-term transgene expression by taking advantage of, circumventing or over‐ riding silencing favouritism of the resident gene expression control mechanisms.

There are many levels at which the longevity of transgene expression can be addressed through the gene vector choice, design and administration regimen, including: 1) em‐ ployment of non-nuclear vectors, e.g. mRNA or Sendai virus based vectors; 2) control of transgene modules' amenity to methylation (e.g. purposeful exclusion of methylationprone CpG islands); 3) employment of minimised DNA vectors such as minicircle DNA to avoid transgene silencing by the bacterial portion of the plasmid vectors; 4) choice of a suitable promoter-enhancer combination with the judicious use of tissue specific, indu‐ cible and ubiquitous promoters; 5) achieving a high number of randomly integrated transgenes; 6) control of the chromosomal integration sites via artificial site-preferences of retroviral integrases, transposases or via harnessing of site-specific integration sys‐ tems; 7) localisation of transgenes on nuclear episomes; 8) chromatin re-modelling con‐ trol via *cis*-acting elements such as insulator elements and other LCRs; 9) repeated vector administration; 10) selection of individual cell clones with transgenes integrated into favourable loci; 11) use of chemical reagents influencing the epigenetic state to ach‐ ieve higher and more long-term transgene expression; and 12) choice of non-immuno‐ genic transgene products to prevent the elimination of transgenic cells via immune reactions *in vivo*.

Clearly, the future solutions to transgene silencing enabling stable long-term expression of therapeutic transgenes will depend on the determined implementation of the above strat‐ egies and their effective combinations.

#### **Author details**

through the more active synthesis of the second DNA strand in the delivered single-strand‐

The elimination of therapeutic gene vectors and transgenic cells by the immune system can imitate the silencing of transgene expression. Thus, the employment of low-immunogenic vectors is a preferred option. Vectors' epitopes should mimic the native epitopes of individ‐ ual patients and do not match their pre-existing immune profile. Coating vector particles with immunologically inert polymers like polyethyleneglycol is one of the strategies to es‐ cape immune surveillance. Alternatively, vector particles can be developed, which are able to mimic the immune-evasion strategy of some viruses that are capable of 'hiding' at the cell surface [68]. Non-immunogenic transgene products, e.g. exclusively human versions of pro‐ teins, should be chosen to prevent cell elimination via immune reactions *in vivo*. If required, transgene products should be re-engineered to achieve the 'stealth effect' and to tailor them

Epigenetic control by the target cells can result in permanent transgene silencing or in the instability of transgene expression. Thus, one needs to pursue therapeutic strategies, which can achieve long-term transgene expression by taking advantage of, circumventing or over‐

There are many levels at which the longevity of transgene expression can be addressed through the gene vector choice, design and administration regimen, including: 1) em‐ ployment of non-nuclear vectors, e.g. mRNA or Sendai virus based vectors; 2) control of transgene modules' amenity to methylation (e.g. purposeful exclusion of methylationprone CpG islands); 3) employment of minimised DNA vectors such as minicircle DNA to avoid transgene silencing by the bacterial portion of the plasmid vectors; 4) choice of a suitable promoter-enhancer combination with the judicious use of tissue specific, indu‐ cible and ubiquitous promoters; 5) achieving a high number of randomly integrated transgenes; 6) control of the chromosomal integration sites via artificial site-preferences of retroviral integrases, transposases or via harnessing of site-specific integration sys‐ tems; 7) localisation of transgenes on nuclear episomes; 8) chromatin re-modelling con‐ trol via *cis*-acting elements such as insulator elements and other LCRs; 9) repeated vector administration; 10) selection of individual cell clones with transgenes integrated into favourable loci; 11) use of chemical reagents influencing the epigenetic state to ach‐ ieve higher and more long-term transgene expression; and 12) choice of non-immuno‐ genic transgene products to prevent the elimination of transgenic cells via immune

riding silencing favouritism of the resident gene expression control mechanisms.

**4.11. Selection of low immunogenic vectors and transgene products**

to the immunological profiles of individual patients.

ed AAV vector DNA [67].

62 Gene Therapy - Tools and Potential Applications

**5. Conclusion**

reactions *in vivo*.

Oleg E. Tolmachov1\*, Tatiana Subkhankulova2 and Tanya Tolmachova2

\*Address all correspondence to: 125317@live.smuc.ac.uk

1 St. Mary's University College, Twickenham, UK

2 National Heart and Lung Institute, Imperial College London, London, UK

#### **References**


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[49] Keravala A, Lee S, Thyagarajan B, Olivares EC, Gabrovsky VE, Woodard LE, Calos MP: Mutational derivatives of phic31 integrase with increased efficiency and specif‐

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**Section 2**

**Gene Therapy Tools: Synthetic**


**Gene Therapy Tools: Synthetic**

[63] Grandchamp N, Henriot D, Philippe S, Amar L, Ursulet S, Serguera C, Mallet J, Sar‐ kis C: Influence of insulators on transgene expression from integrating and non-inte‐

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68 Gene Therapy - Tools and Potential Applications

Biophys Res Commun (2004) 324(1):348-354.

Cancer Res (2011) 17(9):2767-2776.

**Chapter 4**

**Cellular Uptake Mechanism of Non-Viral Gene Delivery**

Non-viral delivery systems usually include mechanical, electrical, and chemical methods. Cationic liposomes and cationic polymers are two typical classes of non-viral vectors. Com‐ pared with viral vectors, non-viral ones are considered promising vehicles for gene therapy because of their low toxicity, biocompatibility, and controllability [1, 2], although their low efficacy limits their application as a mature gene delivery system. Improving the efficacy of non-viral vectors necessitates thorough understanding of their *in vivo* key steps. Non-viral vectors can complex with gene materials and help them access the target compartments within cells. Many barriers prevent gene materials from reaching their intended target and performing their functions [3], safe and effective delivery remains an important challenge

The delivery of pDNA or siRNA *in vivo* for therapeutic aims has been widely studied in re‐ cent years. However, non-viral delivery systems, which exhibit relatively low levels of effi‐ ciency, are not clinically applicable. Improving their efficiency is the main task of pDNA- or siRNA-based gene therapy. There are many barriers that hinder pDNA and siRNA from reaching their intended target in the plasma and performing their functions: First, gene ma‐ terials can be loaded into vectors. After *in vivo* administration, the vectors must be delivered to the blood vessels and should be stable in the blood; otherwise, they will be cleared by al‐ bumin because of their high surface charge and may also be uptaken by macrophages. The vectors must then pass through the epithelial tissue of the blood vessels and enter the target tissue. As it is very difficult for nanoparticles larger than 5 nm in diameter to pass through the epithelial tissue of blood vessels [5], it is crucial to study the cellular transport mecha‐ nism of epithelial cells through the caveolin-mediated endocytosis (CvME) pathway which is active in epithelial cells [6]. The distance between the extracellular matrix and target cells

> © 2013 Xiang and Zhang; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**and Means for Improving Transfection Efficiency**

Shengnan Xiang and Xiaoling Zhang

http://dx.doi.org/10.5772/53353

**1. Introduction**

Additional information is available at the end of the chapter

for the clinical development of non-viral vectors [4].

### **Cellular Uptake Mechanism of Non-Viral Gene Delivery and Means for Improving Transfection Efficiency**

Shengnan Xiang and Xiaoling Zhang

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53353

#### **1. Introduction**

Non-viral delivery systems usually include mechanical, electrical, and chemical methods. Cationic liposomes and cationic polymers are two typical classes of non-viral vectors. Com‐ pared with viral vectors, non-viral ones are considered promising vehicles for gene therapy because of their low toxicity, biocompatibility, and controllability [1, 2], although their low efficacy limits their application as a mature gene delivery system. Improving the efficacy of non-viral vectors necessitates thorough understanding of their *in vivo* key steps. Non-viral vectors can complex with gene materials and help them access the target compartments within cells. Many barriers prevent gene materials from reaching their intended target and performing their functions [3], safe and effective delivery remains an important challenge for the clinical development of non-viral vectors [4].

The delivery of pDNA or siRNA *in vivo* for therapeutic aims has been widely studied in re‐ cent years. However, non-viral delivery systems, which exhibit relatively low levels of effi‐ ciency, are not clinically applicable. Improving their efficiency is the main task of pDNA- or siRNA-based gene therapy. There are many barriers that hinder pDNA and siRNA from reaching their intended target in the plasma and performing their functions: First, gene ma‐ terials can be loaded into vectors. After *in vivo* administration, the vectors must be delivered to the blood vessels and should be stable in the blood; otherwise, they will be cleared by al‐ bumin because of their high surface charge and may also be uptaken by macrophages. The vectors must then pass through the epithelial tissue of the blood vessels and enter the target tissue. As it is very difficult for nanoparticles larger than 5 nm in diameter to pass through the epithelial tissue of blood vessels [5], it is crucial to study the cellular transport mecha‐ nism of epithelial cells through the caveolin-mediated endocytosis (CvME) pathway which is active in epithelial cells [6]. The distance between the extracellular matrix and target cells

© 2013 Xiang and Zhang; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. is great, and many vectors will be uptaken and cleared by macrophages after they do man‐ age to pass through the epithelial tissue of blood vessels.

gene therapy, highlight the importance of studying the intracellular fate of macromolecules, such as DNA and siRNA. In particular, in the case of gene therapy, intracellular events would be expected to be the major factors controlling the fate of the introduced gene and the efficiency of its expression. These authors attempted to establish an intracellular pharmaco‐ kinetic model of genes to study the intracellular events involved in gene therapy [7]. Under‐ standing the intracellular fate of a gene or vector is important for us to overcome the cellular

Cellular Uptake Mechanism of Non-Viral Gene Delivery and Means for Improving Transfection Efficiency

http://dx.doi.org/10.5772/53353

73

Of all intracellular events, the cellular uptake mechanism of non-viral vectors is the most es‐ sential to their efficiency and intracellular fate. Different cellular uptake pathways have dif‐ ferent intracellular fates. As the gene materials will be degraded in the endo-lysosomes (endosomes and lysosomes). One good example is that some endocytic pathways involve endo-lysosomes, but others that can bypass the endo-lysosomes have higher levels of deliv‐ ery efficiency. Polyethylenimine (PEI) is one of the most promising non-viral vectors [8]. Some researchers have shown that cellular uptake of PEI polyplexes affects other cellular processes and, consequently, transfection efficiency [9, 10]. These differences may depend on such factors as the size, surface properties, and shape of the particles [11], as well as dif‐

Research has shown that polyplexes and lipoplexes have different uptake mechanisms in A549 pneumocytes and HeLa cells. Lipoplex uptake proceeds only by clathrin-mediated en‐ docytosis (CME), whereas polyplexes are taken up by two mechanisms — one involving caveolae and another using clathrin-coated pits [10]. As the caveolae-mediated uptake mechanism has slower kinetics, the transfection process of polyplexes is slower than that of lipoplexes in A549 pneumocytes and HeLa cells. However, as the polyplexes uptaken via the caveolae escape the lysosomal compartment, polyplexes have a high level of transfection efficiency [10]. Taken together, these findings highlight the importance of studying the cellu‐ lar uptake of non-viral vectors, their intracellular fate, and their effects on transfection effi‐ ciency. Understanding cellular uptake mechanisms is crucial to engineering successful

The uptake pathways are divided into two groups: endocytic pathways and non-endocytic pathways. Inside endocytic group, there are two types of pathways: phagocytosis and non-

Phagocytosis is a special type of endocytic pathway which primarily exists in professional phagocytes such as macrophages, monocytes neutrophils, and dendritic cells (DCs) [13]. In comparison, other nonphagocytic pathways such as clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), and macropinocytosis occur in almost all kinds of cell types [14]. Phagocytic pathway is mediated by cup-like membrane extensions that are

barriers of DNA or siRNA delivery and rationally design efficient systems thereof.

ferent cell lines [9].

phagocytosis pathways [11].

**2.1. Phagocytosis**

reagents or vectors for non-viral gene transfection [12].

**2. Cellular uptake pathways of non-viral gene delivery**

Next, the vectors must attach to the cell membrane, which entails other issues altogether. First, the non-viral vectors should be able to identify specific cell types to ensure safety. They then enter cells mainly via endocytosis. Different endocytosis pathways yield different intracellular fates for vectors, which could potentially explain why the same vector differs in its transfection efficiency in various cell modes. After their entry into the cells, vectors must escape from the endosome or avoid the endo-lysosomal (endosomal and lysosomal) path‐ way through certain endocytosis pathways. After escaping the endosome and then entering the cytoplasm, vectors must release pDNA or siRNA and finally perform their function in the cytoplasm [5]. In addition, pDNA has to be transported into the nucleus. The key steps in non-viral delivery are shown in Figure 1.

**Figure 1.** Biological key steps of non-viral vectors

As discussed above, the cellular process (including uptake, transport, endosomal escape, and nuclear localization) is one of the most important steps for non-viral gene delivery. In 2001, Hideyoshi Harashima et al. stated that novel strategies of medical treatments, such as gene therapy, highlight the importance of studying the intracellular fate of macromolecules, such as DNA and siRNA. In particular, in the case of gene therapy, intracellular events would be expected to be the major factors controlling the fate of the introduced gene and the efficiency of its expression. These authors attempted to establish an intracellular pharmaco‐ kinetic model of genes to study the intracellular events involved in gene therapy [7]. Under‐ standing the intracellular fate of a gene or vector is important for us to overcome the cellular barriers of DNA or siRNA delivery and rationally design efficient systems thereof.

Of all intracellular events, the cellular uptake mechanism of non-viral vectors is the most es‐ sential to their efficiency and intracellular fate. Different cellular uptake pathways have dif‐ ferent intracellular fates. As the gene materials will be degraded in the endo-lysosomes (endosomes and lysosomes). One good example is that some endocytic pathways involve endo-lysosomes, but others that can bypass the endo-lysosomes have higher levels of deliv‐ ery efficiency. Polyethylenimine (PEI) is one of the most promising non-viral vectors [8]. Some researchers have shown that cellular uptake of PEI polyplexes affects other cellular processes and, consequently, transfection efficiency [9, 10]. These differences may depend on such factors as the size, surface properties, and shape of the particles [11], as well as dif‐ ferent cell lines [9].

Research has shown that polyplexes and lipoplexes have different uptake mechanisms in A549 pneumocytes and HeLa cells. Lipoplex uptake proceeds only by clathrin-mediated en‐ docytosis (CME), whereas polyplexes are taken up by two mechanisms — one involving caveolae and another using clathrin-coated pits [10]. As the caveolae-mediated uptake mechanism has slower kinetics, the transfection process of polyplexes is slower than that of lipoplexes in A549 pneumocytes and HeLa cells. However, as the polyplexes uptaken via the caveolae escape the lysosomal compartment, polyplexes have a high level of transfection efficiency [10]. Taken together, these findings highlight the importance of studying the cellu‐ lar uptake of non-viral vectors, their intracellular fate, and their effects on transfection effi‐ ciency. Understanding cellular uptake mechanisms is crucial to engineering successful reagents or vectors for non-viral gene transfection [12].

#### **2. Cellular uptake pathways of non-viral gene delivery**

The uptake pathways are divided into two groups: endocytic pathways and non-endocytic pathways. Inside endocytic group, there are two types of pathways: phagocytosis and nonphagocytosis pathways [11].

#### **2.1. Phagocytosis**

is great, and many vectors will be uptaken and cleared by macrophages after they do man‐

Next, the vectors must attach to the cell membrane, which entails other issues altogether. First, the non-viral vectors should be able to identify specific cell types to ensure safety. They then enter cells mainly via endocytosis. Different endocytosis pathways yield different intracellular fates for vectors, which could potentially explain why the same vector differs in its transfection efficiency in various cell modes. After their entry into the cells, vectors must escape from the endosome or avoid the endo-lysosomal (endosomal and lysosomal) path‐ way through certain endocytosis pathways. After escaping the endosome and then entering the cytoplasm, vectors must release pDNA or siRNA and finally perform their function in the cytoplasm [5]. In addition, pDNA has to be transported into the nucleus. The key steps

As discussed above, the cellular process (including uptake, transport, endosomal escape, and nuclear localization) is one of the most important steps for non-viral gene delivery. In 2001, Hideyoshi Harashima et al. stated that novel strategies of medical treatments, such as

age to pass through the epithelial tissue of blood vessels.

72 Gene Therapy - Tools and Potential Applications

in non-viral delivery are shown in Figure 1.

**Figure 1.** Biological key steps of non-viral vectors

Phagocytosis is a special type of endocytic pathway which primarily exists in professional phagocytes such as macrophages, monocytes neutrophils, and dendritic cells (DCs) [13]. In comparison, other nonphagocytic pathways such as clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), and macropinocytosis occur in almost all kinds of cell types [14]. Phagocytic pathway is mediated by cup-like membrane extensions that are usually larger than 1 µm to internalize large particles such as bacteria or dead cells. Under‐ standing of the mechanism of phagocytosis is very helpful to the non-viral gene therapy of macrophage-dominated immune diseases such as rheumatoid arthritis. In addition, a phag‐ ocytosis-like mechanism was proposed for the uptake of large lipoplexes and polyplexes that are larger than can be taken up by the classic CME pathway [15, 16].

complexes [26, 27]. In this pathway, a series of downstream events are activated after the recognition of ligands by receptors on the cell surface. Clathrins assemble in the polyhedral lattice right on the cytosolic surface of the cell membrane, which helps to deform the mem‐ brane into a coated pit with a size about 100–150 nm [27]. This process is mediated by GTPase dynamin. As the clathrin lattice formation continues, the pit becomes deeply invagi‐ nated until the vesicle fission occurs. In the next step of the CME pathway, the endocytosed vesicles internalized from the plasma membrane are integrated into late endosomes and fi‐

Cellular Uptake Mechanism of Non-Viral Gene Delivery and Means for Improving Transfection Efficiency

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75

CvME begins in a special flask-shaped structure on the cell membrane called caveola, which is a kind of cholesterol- and sphingolipidrich smooth invagination [28]. CvME usually hap‐ pens in the vessel wall lining monolayer of endothelial cells [7]. Caveolae have a diameter range of 50–100 nm [11] and are typically between 50 and 80 nmwith a neck of 10–50 nm [6]. CvME is also a type of cholesterol, dynamin-dependent, and receptor-mediated pathway [29]. The fission of the caveolae from the membrane is mediated by the GTPase dynamin, which locates in the neck of caveolae and then generates the cytosolic caveolar vesicle [11]. Some receptors located in caveolae, such as insulin receptors [30] and epidermal growth fac‐ tor receptor (a type of receptor in ovarian cancer) [31], can mediate CvME [32]. The vesicle budding from the caveolae, a type of caveolin-1-containing endosome is called caveosome [29]. The intracellular fate of the caveosome differs from that of CME. Compared with CME, CvME is generally considered as a alternative pathway which can deliver the vectors into Golgi and/or endoplasmic reticulum, thus avoiding the normal lysosomal degradation

Macropinocytosis is a type of distinct pathway that nonspecifically takes up a large amount of fluid-phase contents through the mode called fluid-phase endocytosis (FPE) [33]. Macro‐ pinocytosis is a signal dependent process that normally occurs when macrophages or cancer cells are in response to colony-stimulating factor-1 (CSF-1), epidermal growth factor (EGF) and platelet-derived growth factor or tumor-promoting factor, such as phorbol myristate acetate respectively [34-36]. However, this process occurs constitutively in antigen-present‐ ing cells [37]. Macropinocytosis occurs via the formation of actin-driven membrane protru‐ sions, which is similar to phagocytosis. However, in this case, the protrusions do not zipper up the ligand-coated particle; instead, they collapse onto and fuse with the plasma mem‐ brane [11]. The macropinosomes have no apparent coat structures and are heterogenous in size, but are generally considered larger than 0.2 µm in diameter [38, 39]. During this proc‐ ess, the small GTPase, Ras-related in brain (Rab) proteins are essential for the vesicle fission from the cell membrane [40]. The relationship between the macropinocytosis and lysosome

There are three technologies that are designed to mediate the non-endocytic pathways and successful transfect the gene. One is microinjection, by which each cell is injected with the gene materials using glass capillary pipettes. The second one is permeabiliza‐ tion by using pore-forming reagents such as streptolysin O or anionic peptides such as HA2 subunit of the influenza virus hemagglutinin. The third one is electroporation,

nally transported to lysosomes.

is still unknown. This will be discussed later.

**2.3. Non-endocytic pathways**

Phagocytosis depending on opsonins can be called as opsonic phagocytosis. There is also another phagocytosis which is opsonins independent. This will be discussed later. First, for opsonic phagocytosis, the complexes will be recognized by opsonins in the bloodstream. Then, the opsonized complexes adhere to professional phagocytes and are ultimately ingest‐ ed by them [11]. Opsonization is the key step of the phagocytosis pathway. It involves com‐ plexes tagged by some major opsonins including immunoglobulins G and M (IgG and IgM), as well as complement components C3, C4, and C5 in the bloodstream [11, 17]. These opson‐ ized complexes become visible to macrophages and bind to their surface through the inter‐ action between receptors (such as fragment crystallizable receptors (FcR) and complement receptors (CR)) and the constant fragment of particle-adsorbed immunoglobulins.

Other receptors that mediate phagocytosis pathway have also been reported. Mannose re‐ ceptor (MR) has been used in gene vaccine by targeting human DCs and macrophages through the phagocytic pathway [18]. Scavenger receptor (SR)-mediated delivery of anti‐ sense miniexon phosphorothioate oligonucleotide to leishmania-infected macrophages is proved to be selective and efficient in eliminating the parasite [19]. SR-A, macrophage recep‐ tor, and CD36 are the three SR subtypes. CD36 can mediate non-opsonic phagocytosis of pathogenic microbes [20]. Unlike opsonic phagocytosis, non-opsonic phagocytosis is directly mediated by the receptors on the cell surface without the help of opsonins. This kind of mechanism can also be used for gene delivery.

Then the activated Rho-family GTPases trigger actin assembly and cell surface extension for‐ mation. This surface extension finally zippers up around the complexes and engulfs them [11]. The phagosomes carrying the complexes fuse with lysosomes to form mature phagoly‐ sosomes [11]. In phagolysosomes, the complexes undergo a process of acidification and en‐ zymatic reaction. As the intracellular fate of phagocytosis is the transportation of complexes into the lysosome, the gene materials will be degraded by the nucleases inside it [21]. Endo‐ somes and lysosomes (endo-lysosomes) are very important biological barrier for gene deliv‐ ery. The vectors should have capability to escape form them, if gene materials loaded vectors entry into the cell via phagocytosis. The mechanisms of endo-lysosome escape will be discussed later.

#### **2.2. Non- phagocytosis pathways**

Non- phagocytosis pathways mainly include clathrin-mediated endocytosis (CME), caveo‐ lae-mediated endocytosis (CvME), and macropinocytosis. CME is the best-characterized type of endocytosis, which is receptor-dependent, clathrin-mediated, and GTPase dynaminrequired [22, 23]. The uptake of low-density lipoprotein and transferrin is typically via this endocytic pathway, and they are often used as the CME probes in many studies [24, 25]. Transferrin has also been used as a ligand of non-viral vectors to improve the endocytosis of complexes [26, 27]. In this pathway, a series of downstream events are activated after the recognition of ligands by receptors on the cell surface. Clathrins assemble in the polyhedral lattice right on the cytosolic surface of the cell membrane, which helps to deform the mem‐ brane into a coated pit with a size about 100–150 nm [27]. This process is mediated by GTPase dynamin. As the clathrin lattice formation continues, the pit becomes deeply invagi‐ nated until the vesicle fission occurs. In the next step of the CME pathway, the endocytosed vesicles internalized from the plasma membrane are integrated into late endosomes and fi‐ nally transported to lysosomes.

CvME begins in a special flask-shaped structure on the cell membrane called caveola, which is a kind of cholesterol- and sphingolipidrich smooth invagination [28]. CvME usually hap‐ pens in the vessel wall lining monolayer of endothelial cells [7]. Caveolae have a diameter range of 50–100 nm [11] and are typically between 50 and 80 nmwith a neck of 10–50 nm [6]. CvME is also a type of cholesterol, dynamin-dependent, and receptor-mediated pathway [29]. The fission of the caveolae from the membrane is mediated by the GTPase dynamin, which locates in the neck of caveolae and then generates the cytosolic caveolar vesicle [11]. Some receptors located in caveolae, such as insulin receptors [30] and epidermal growth fac‐ tor receptor (a type of receptor in ovarian cancer) [31], can mediate CvME [32]. The vesicle budding from the caveolae, a type of caveolin-1-containing endosome is called caveosome [29]. The intracellular fate of the caveosome differs from that of CME. Compared with CME, CvME is generally considered as a alternative pathway which can deliver the vectors into Golgi and/or endoplasmic reticulum, thus avoiding the normal lysosomal degradation

Macropinocytosis is a type of distinct pathway that nonspecifically takes up a large amount of fluid-phase contents through the mode called fluid-phase endocytosis (FPE) [33]. Macro‐ pinocytosis is a signal dependent process that normally occurs when macrophages or cancer cells are in response to colony-stimulating factor-1 (CSF-1), epidermal growth factor (EGF) and platelet-derived growth factor or tumor-promoting factor, such as phorbol myristate acetate respectively [34-36]. However, this process occurs constitutively in antigen-present‐ ing cells [37]. Macropinocytosis occurs via the formation of actin-driven membrane protru‐ sions, which is similar to phagocytosis. However, in this case, the protrusions do not zipper up the ligand-coated particle; instead, they collapse onto and fuse with the plasma mem‐ brane [11]. The macropinosomes have no apparent coat structures and are heterogenous in size, but are generally considered larger than 0.2 µm in diameter [38, 39]. During this proc‐ ess, the small GTPase, Ras-related in brain (Rab) proteins are essential for the vesicle fission from the cell membrane [40]. The relationship between the macropinocytosis and lysosome is still unknown. This will be discussed later.

#### **2.3. Non-endocytic pathways**

usually larger than 1 µm to internalize large particles such as bacteria or dead cells. Under‐ standing of the mechanism of phagocytosis is very helpful to the non-viral gene therapy of macrophage-dominated immune diseases such as rheumatoid arthritis. In addition, a phag‐ ocytosis-like mechanism was proposed for the uptake of large lipoplexes and polyplexes

Phagocytosis depending on opsonins can be called as opsonic phagocytosis. There is also another phagocytosis which is opsonins independent. This will be discussed later. First, for opsonic phagocytosis, the complexes will be recognized by opsonins in the bloodstream. Then, the opsonized complexes adhere to professional phagocytes and are ultimately ingest‐ ed by them [11]. Opsonization is the key step of the phagocytosis pathway. It involves com‐ plexes tagged by some major opsonins including immunoglobulins G and M (IgG and IgM), as well as complement components C3, C4, and C5 in the bloodstream [11, 17]. These opson‐ ized complexes become visible to macrophages and bind to their surface through the inter‐ action between receptors (such as fragment crystallizable receptors (FcR) and complement

receptors (CR)) and the constant fragment of particle-adsorbed immunoglobulins.

mechanism can also be used for gene delivery.

74 Gene Therapy - Tools and Potential Applications

be discussed later.

**2.2. Non- phagocytosis pathways**

Other receptors that mediate phagocytosis pathway have also been reported. Mannose re‐ ceptor (MR) has been used in gene vaccine by targeting human DCs and macrophages through the phagocytic pathway [18]. Scavenger receptor (SR)-mediated delivery of anti‐ sense miniexon phosphorothioate oligonucleotide to leishmania-infected macrophages is proved to be selective and efficient in eliminating the parasite [19]. SR-A, macrophage recep‐ tor, and CD36 are the three SR subtypes. CD36 can mediate non-opsonic phagocytosis of pathogenic microbes [20]. Unlike opsonic phagocytosis, non-opsonic phagocytosis is directly mediated by the receptors on the cell surface without the help of opsonins. This kind of

Then the activated Rho-family GTPases trigger actin assembly and cell surface extension for‐ mation. This surface extension finally zippers up around the complexes and engulfs them [11]. The phagosomes carrying the complexes fuse with lysosomes to form mature phagoly‐ sosomes [11]. In phagolysosomes, the complexes undergo a process of acidification and en‐ zymatic reaction. As the intracellular fate of phagocytosis is the transportation of complexes into the lysosome, the gene materials will be degraded by the nucleases inside it [21]. Endo‐ somes and lysosomes (endo-lysosomes) are very important biological barrier for gene deliv‐ ery. The vectors should have capability to escape form them, if gene materials loaded vectors entry into the cell via phagocytosis. The mechanisms of endo-lysosome escape will

Non- phagocytosis pathways mainly include clathrin-mediated endocytosis (CME), caveo‐ lae-mediated endocytosis (CvME), and macropinocytosis. CME is the best-characterized type of endocytosis, which is receptor-dependent, clathrin-mediated, and GTPase dynaminrequired [22, 23]. The uptake of low-density lipoprotein and transferrin is typically via this endocytic pathway, and they are often used as the CME probes in many studies [24, 25]. Transferrin has also been used as a ligand of non-viral vectors to improve the endocytosis of

that are larger than can be taken up by the classic CME pathway [15, 16].

There are three technologies that are designed to mediate the non-endocytic pathways and successful transfect the gene. One is microinjection, by which each cell is injected with the gene materials using glass capillary pipettes. The second one is permeabiliza‐ tion by using pore-forming reagents such as streptolysin O or anionic peptides such as HA2 subunit of the influenza virus hemagglutinin. The third one is electroporation, which uses an electric field to open pores in the cell. All of them are highly invasive and not ideal for in vivo gene delivery.

complexes into a single cell type. For example, transfection by branched PEI25kDa/DNA polyplexes was mediated by both CME and CvME pathways in HUH-7 and Hela cells [9]. Later, Hansjörg Hufnagel reported that the macropinocytosis is also very important for the uptake of branched PEI25kDa/DNA polyplexes into Hela and CHO-1 cells due to the exis‐ tence of large particles of polyplexes (>500 nm) [12]. Therefore, the heterogeneity of com‐ plexes has to be taken into consideration when the results are analyzed. Particle size is a very important factor for the pathway selection of complexes. As mentioned above, the la‐ beled cationic PAMAM can induce hole formation in the cell membrane. The holes induced by PAMAM are 15–40 nm in diameter [42]. The particle including the gene complex, which is smaller than these holes, can diffuse through the holes and be taken up by nonspecific non-endocytic rather than specific receptor mediated endocytic pathways. PEI/DNA com‐ plexes with sizes smaller than 500 nm are mainly taken up by CME and CvME according to a previous study [10]. While PEI/DNA complexes with sizes >500 nm are mainly internal‐

Cellular Uptake Mechanism of Non-Viral Gene Delivery and Means for Improving Transfection Efficiency

http://dx.doi.org/10.5772/53353

77

The charge density of a complex is also an important factor for its uptake. The cell mem‐ brane consists of a bilayer of lipid and anionic membrane proteins. These anionic proteins are very helpful to the uptake of cationic complexes. However, once the net positive charge falls to neutral, the uptake efficiency will be inhibited a lot. This is because the neutral charge density will weaken the interaction between complexes and membrane proteins, and it will also increase the aggregation of complexes, which will make them large and hard to be internalized. This change can be caused by the anionic proteins in the in vivo circulation of blood, and the serum used in the in vitro transfection medium. The modification of poly‐ ethylene glycol (PEG) can solve this problem with its high hydrophilicity, electrical neutral‐

As to the relationship between the shape of particles and the pathway selection, few studies have been made about this issue. A group once reported that the uptake of protein-coated spherical gold nanoparticles is more efficient than rod-shaped ones in Hela cells, SNB19 cells, and STO cells [46, 47]. However, as to the relationship of nonviral gene complexes and their uptake efficiency, it is not easy to draw such a conclusion, because the non-viral gene complexes are usually a group of nanoparticles with heterogeneous shapes, and their shapes are dependent on the experimental conditions. Taking chitosan as an example, the fraction of complexes that have nonaggregated, globular structures increases with increasing chain length of the chitosan oligomer, increasing charge ratio and reduction of pH (from 6.5 to 3.5) [48]. Because of this, this complicated issue leaves much room for researchers to discuss.

Cell type is another important factor that influences the pathway selection of non-viral gene complexes. Different types of cells can take up a kind of complex in different pathways. Most of the studies focused on COS-7 cells, which were used as a well-established model cell for gene delivery researches [28]. Some researchers also used other cell lines such as A549, Hela, and HUH-7 cells. Caveolae, which are a very important structure for CvME pathway, are present in many cell types, but they are particularly abundant in the vessel wall lining monolayer of endothelial cells. As a result, endothelial cells have been especially used in studies on CvME pathway. A study tested the endocytosis pathways involved in the

ized by macropinocytosis pathway [10].

ity and steric-repulsive propensity [45].

However, There are evidences which can prove the existing of other non-endocytic path‐ ways. One pathway is related to the formation of holes in the cell membrane, called "pene‐ tration". A class of cationic peptides with the protein transduction domains (PTDs), such as TAT, has the ability to be taken up without endocytic events [41]. These peptides can direct‐ ly penetrate cell membranes in a receptor-, and energy-independent way. In 2004, Hong et al. studied the hole formation on the cell membrane induced by poly(amidoamine) (PA‐ MAM). The results indicated that the hole formation can be induced by positively charged PAMAM, and labeled PAMAM can diffuse into the cells through small holes in the mem‐ brane. This mechanism is considered a nonspecific pathway, which is not receptor-mediated and lacks selective cellular uptake [42]. In 2010, Lee et al. used a PTD called Hph-1 to conju‐ gate vector PEI to deliver siRNA. The result showed that the complexes entered the cells through the non-endocytic pathway, which has a quicker dynamic behavior compared with the endocytosis pathways and is energy-independent because it has high transfection effi‐ ciency even in low temperature [43]. Another non-endocytic pathway is called "fusion", which is special for lipoplexes, as it can cause a direct release of DNA to the cytoplasm be‐ fore entering the endocytic pathways. However, more and more evidences suggest that fu‐ sion with the cell membrane contributes minimally to the overall uptake of lipoplexes, while the CME plays an important role in the uptake of lipoplexes [44]. there have been few stud‐ ies on non-endocytic pathways, and more efforts are needed to have a comprehensive un‐ derstanding of these pathways for the improvement of non-viral gene delivery.


**Table 1.** Cellular uptake mechanisms.

#### **3. Factors that influence the uptake pathways of non-viral gene delivery**

There are many factors that are involved in the selection of uptake pathways of non-viral gene complexes. These factors include particle size, particle surface charge, particle shape, cell type, and even culture condition. Because the complexes of non-viral gene vector/DNA or siRNA are usually a group of heterogeneous particles with diverse sizes, surface charges, and shapes, several uptake pathways may be involved in the internalization of one kind of complexes into a single cell type. For example, transfection by branched PEI25kDa/DNA polyplexes was mediated by both CME and CvME pathways in HUH-7 and Hela cells [9]. Later, Hansjörg Hufnagel reported that the macropinocytosis is also very important for the uptake of branched PEI25kDa/DNA polyplexes into Hela and CHO-1 cells due to the exis‐ tence of large particles of polyplexes (>500 nm) [12]. Therefore, the heterogeneity of com‐ plexes has to be taken into consideration when the results are analyzed. Particle size is a very important factor for the pathway selection of complexes. As mentioned above, the la‐ beled cationic PAMAM can induce hole formation in the cell membrane. The holes induced by PAMAM are 15–40 nm in diameter [42]. The particle including the gene complex, which is smaller than these holes, can diffuse through the holes and be taken up by nonspecific non-endocytic rather than specific receptor mediated endocytic pathways. PEI/DNA com‐ plexes with sizes smaller than 500 nm are mainly taken up by CME and CvME according to a previous study [10]. While PEI/DNA complexes with sizes >500 nm are mainly internal‐ ized by macropinocytosis pathway [10].

which uses an electric field to open pores in the cell. All of them are highly invasive and

However, There are evidences which can prove the existing of other non-endocytic path‐ ways. One pathway is related to the formation of holes in the cell membrane, called "pene‐ tration". A class of cationic peptides with the protein transduction domains (PTDs), such as TAT, has the ability to be taken up without endocytic events [41]. These peptides can direct‐ ly penetrate cell membranes in a receptor-, and energy-independent way. In 2004, Hong et al. studied the hole formation on the cell membrane induced by poly(amidoamine) (PA‐ MAM). The results indicated that the hole formation can be induced by positively charged PAMAM, and labeled PAMAM can diffuse into the cells through small holes in the mem‐ brane. This mechanism is considered a nonspecific pathway, which is not receptor-mediated and lacks selective cellular uptake [42]. In 2010, Lee et al. used a PTD called Hph-1 to conju‐ gate vector PEI to deliver siRNA. The result showed that the complexes entered the cells through the non-endocytic pathway, which has a quicker dynamic behavior compared with the endocytosis pathways and is energy-independent because it has high transfection effi‐ ciency even in low temperature [43]. Another non-endocytic pathway is called "fusion", which is special for lipoplexes, as it can cause a direct release of DNA to the cytoplasm be‐ fore entering the endocytic pathways. However, more and more evidences suggest that fu‐ sion with the cell membrane contributes minimally to the overall uptake of lipoplexes, while the CME plays an important role in the uptake of lipoplexes [44]. there have been few stud‐ ies on non-endocytic pathways, and more efforts are needed to have a comprehensive un‐

derstanding of these pathways for the improvement of non-viral gene delivery.

Phagocytosis Rho Dependent Dependent CME Dynamin Dependent Dependent CvME Dynamin Independent Dependent Macropinocytosis Rab In dispute Non-specific Non-endocytic Independent Independent Non-specific

**3. Factors that influence the uptake pathways of non-viral gene delivery**

There are many factors that are involved in the selection of uptake pathways of non-viral gene complexes. These factors include particle size, particle surface charge, particle shape, cell type, and even culture condition. Because the complexes of non-viral gene vector/DNA or siRNA are usually a group of heterogeneous particles with diverse sizes, surface charges, and shapes, several uptake pathways may be involved in the internalization of one kind of

**lysosome Receptors**

**Pathways GTPases Relationship with**

not ideal for in vivo gene delivery.

76 Gene Therapy - Tools and Potential Applications

**Table 1.** Cellular uptake mechanisms.

The charge density of a complex is also an important factor for its uptake. The cell mem‐ brane consists of a bilayer of lipid and anionic membrane proteins. These anionic proteins are very helpful to the uptake of cationic complexes. However, once the net positive charge falls to neutral, the uptake efficiency will be inhibited a lot. This is because the neutral charge density will weaken the interaction between complexes and membrane proteins, and it will also increase the aggregation of complexes, which will make them large and hard to be internalized. This change can be caused by the anionic proteins in the in vivo circulation of blood, and the serum used in the in vitro transfection medium. The modification of poly‐ ethylene glycol (PEG) can solve this problem with its high hydrophilicity, electrical neutral‐ ity and steric-repulsive propensity [45].

As to the relationship between the shape of particles and the pathway selection, few studies have been made about this issue. A group once reported that the uptake of protein-coated spherical gold nanoparticles is more efficient than rod-shaped ones in Hela cells, SNB19 cells, and STO cells [46, 47]. However, as to the relationship of nonviral gene complexes and their uptake efficiency, it is not easy to draw such a conclusion, because the non-viral gene complexes are usually a group of nanoparticles with heterogeneous shapes, and their shapes are dependent on the experimental conditions. Taking chitosan as an example, the fraction of complexes that have nonaggregated, globular structures increases with increasing chain length of the chitosan oligomer, increasing charge ratio and reduction of pH (from 6.5 to 3.5) [48]. Because of this, this complicated issue leaves much room for researchers to discuss.

Cell type is another important factor that influences the pathway selection of non-viral gene complexes. Different types of cells can take up a kind of complex in different pathways. Most of the studies focused on COS-7 cells, which were used as a well-established model cell for gene delivery researches [28]. Some researchers also used other cell lines such as A549, Hela, and HUH-7 cells. Caveolae, which are a very important structure for CvME pathway, are present in many cell types, but they are particularly abundant in the vessel wall lining monolayer of endothelial cells. As a result, endothelial cells have been especially used in studies on CvME pathway. A study tested the endocytosis pathways involved in the transfection of PEI/DNA complexes with different cell lines. The result showed that in COS-7 cells, the clathrin-dependent pathway was the main contributor to the transfection process for both linear and branch PEIs [9]. Another study suggests that macropinosomes have a higher propensity to deliver PEI/DNA cargo than do endosomes in CHO and Hela cells [12]. Therefore, different cell lines involve different endocytic pathways, and cell type is the important factor that must be considered in such studies.

100 µM), monodansylcadaverine (MDC), phenylarsine oxide. However, all of them have been shown to be able to inhibit macropinocytosis, thus cannot be used to distinguish the clathrin-mediated endocytic pathway and the macropinocytic pathway. Besides this, all these inhibitors can influence the cortical actin cytoskeleton more or less, which can cause non-specific cytotoxicity. However, potassium depletion, chlorpromazine, and MDC are the relatively better choices than the other ones for the initial discrimination of clathrin-mediat‐

Cellular Uptake Mechanism of Non-Viral Gene Delivery and Means for Improving Transfection Efficiency

http://dx.doi.org/10.5772/53353

79

As to caveolae-mediated endocytic pathway, the commonly used inhibitors and methods are: statins, methyl-β-cyclodextrin (MβCD), filipin, nystatin, genestein, and cholesterol oxi‐ dase. Among them, the incubation with filipin, nystatin, and cholesterol oxidase produce the fewest side effects. The chronic inhibition of cholesterol synthesis by statins or acute cho‐ lesterol depletion by MβCD nonspecifically disrupts intracellular vesicle trafficking and the actin cytoskeleton. Also, the specificity of genestein is still in doubt for its nonspecific dis‐ ruption of the actin network. That being so, appropriate controls should be included when

The inhibitors for the study of intracellular fates of complexes are also very important. Mon‐ ensin, bafilomycin A can inhibit the acidification of endosomes, thus preventing their matu‐ ration and fusion into lysosomes [52, 53]. Chloroquine is another inhibitor that accumulates in endosomes/lysosomes and causes the swelling and disruption of endocytic vesicles by os‐ motic effects [21]. Last but not least, the cell-dependence of inhibitors should be noted when experiments are carried out. For example, chlorpromazine treatment inhibited the uptake of transferrin, a marker for CME by ~50% in D407 and HUH-7 cells. However, it showed no or little significant inhibitory capacity in ARPE-19 and Vero cells or even an enhanced effect in COS-7 cells [54, 55]. Therefore, a range of concentration with lowest cytotoxicity and suffi‐ cient inhibitory efficiency should be determined first when the inhibitor is used on the cell for the first time. Then, the lack of absolute specificity can be compensated by the combined application of biological methods such as siRNA silencing, transient or stable expression of dominant-negative proteins, and reconstruction of proteins by knockout mutants, all of which aremore specific than classical chemical inhibitors. For example, mutant dynamin has been successfully used to prove the necessity of dynamin in the endocytic pathways of transferrin receptors and EGF receptors [55]. A constitutive knockdown technique through RNAi has been used to prove the role of an essential accessory protein "epsin" in the CME pathway [56]. Another efficient way of making up the pitfalls of nonspecific inhibitors is the combined usage of fluorescently labeled gene complexes and fluorescent probes that are

Except for inhibitors, molecular probes and markers are also important tools for the study of uptake pathways for non-viral gene complexes. They can be used together with the classical chemical inhibitors or biological inhibitors to make the results more convinc‐ ing. There are several classical molecular probes that are known to be specifically inter‐ nalized through each uptake pathway. Transferrin is often used as a probe of CME pathway in many studies [12, 57, 58]. Transferrin receptor (TFR) mediates transferrin up‐ take by CME, so that it can be used as a CME marker and detected by anti-TFR [59].

ed endocytic pathway [51].

filipin, nystatin, and cholesterol oxidase are used [51].

specifically internalized through certain uptake pathways.

#### **4. Tools for the study of uptake pathways**

The study on the mechanisms of uptake pathways is important to the rational design of nonviral gene vectors because this step can determine the intracellular fate of complexes. How‐ ever, because there are many factors that influence the pathway selection, how to conduct these studies is also a very complicated problem that needs to be discussed in detail.

Inhibitors are the effective tools to block specific pathway in order to determine whether it plays an important role in the uptake of complexes. However, none of the commonly used inhibitors of different uptake pathways is absolutely specific. All of them either affect the actin cytoskeleton with their side effects, or interfere with alternative uptake pathways si‐ multaneously. In addition, they usually show cell type variations. The scope of the usage of commonly used inhibitors will be introduced according to the classification of uptake path‐ ways in the following paragraphs of this section. The most direct way to distinguish endo‐ cytic pathways and non-endocytic pathways is to use the inhibitor or method of energy depletion, because most endocytic pathways are energy dependent. The commonly used in‐ hibitors and methods are: low temperature (4 °C) and sodium azide (an ATPase inhibitor). Low temperature and ATP inhibitor should be used together in some conditions because some of the non-endocytic pathways are also sensitive to low temperature [42, 49].

To distinguish the phagocytic and macropinocytic pathways with CME and CvME path‐ ways, the commonly used inhibitors and methods for phagocytic and macropinocytic path‐ ways are: inhibitors of sodium-proton exchange "amiloride and its derivatives", Factindepolymerizing drugs "cytochalasin D and latrunculins", inhibitors of phosphoinositide metabolism "wortmannin and LY290042", and protein kinase C activator "phorbol esters". Except phorbol esters, the specificity of all the inhibitors is still in doubt as depolymerizing F-actin and inhibition of phosphoinositide metabolism may also disrupt the other two endocytic pathways. For example, cytochalasin D is also used as the inhibitor for CvME [50]. Within these inhibitors, amiloride and its derivatives may be considered as the first choice for their fewest side effects. Rottlerin, a novel macropinocytosis inhibitor which is rapid acting, irreversible, and selective, was discovered in 2005. In 2009, Hufnagel et al. found that rottlerin can specifically inhibit the transfection efficiency of PEI (25 kDa)/DNA complexes on Hela and CHO-K1 cells up to 50%, which verified the important role of FPE in the non-viral gene delivery by PEI (25 kDa) [12].

The commonly used inhibitors and methods for clathrin-mediated endocytosis are: Hyper‐ tonic sucrose (0.4–0.5 M), potassium depletion, cytosolic acidification, chlorpromazine (50– 100 µM), monodansylcadaverine (MDC), phenylarsine oxide. However, all of them have been shown to be able to inhibit macropinocytosis, thus cannot be used to distinguish the clathrin-mediated endocytic pathway and the macropinocytic pathway. Besides this, all these inhibitors can influence the cortical actin cytoskeleton more or less, which can cause non-specific cytotoxicity. However, potassium depletion, chlorpromazine, and MDC are the relatively better choices than the other ones for the initial discrimination of clathrin-mediat‐ ed endocytic pathway [51].

transfection of PEI/DNA complexes with different cell lines. The result showed that in COS-7 cells, the clathrin-dependent pathway was the main contributor to the transfection process for both linear and branch PEIs [9]. Another study suggests that macropinosomes have a higher propensity to deliver PEI/DNA cargo than do endosomes in CHO and Hela cells [12]. Therefore, different cell lines involve different endocytic pathways, and cell type is

The study on the mechanisms of uptake pathways is important to the rational design of nonviral gene vectors because this step can determine the intracellular fate of complexes. How‐ ever, because there are many factors that influence the pathway selection, how to conduct

Inhibitors are the effective tools to block specific pathway in order to determine whether it plays an important role in the uptake of complexes. However, none of the commonly used inhibitors of different uptake pathways is absolutely specific. All of them either affect the actin cytoskeleton with their side effects, or interfere with alternative uptake pathways si‐ multaneously. In addition, they usually show cell type variations. The scope of the usage of commonly used inhibitors will be introduced according to the classification of uptake path‐ ways in the following paragraphs of this section. The most direct way to distinguish endo‐ cytic pathways and non-endocytic pathways is to use the inhibitor or method of energy depletion, because most endocytic pathways are energy dependent. The commonly used in‐ hibitors and methods are: low temperature (4 °C) and sodium azide (an ATPase inhibitor). Low temperature and ATP inhibitor should be used together in some conditions because

these studies is also a very complicated problem that needs to be discussed in detail.

some of the non-endocytic pathways are also sensitive to low temperature [42, 49].

To distinguish the phagocytic and macropinocytic pathways with CME and CvME path‐ ways, the commonly used inhibitors and methods for phagocytic and macropinocytic path‐ ways are: inhibitors of sodium-proton exchange "amiloride and its derivatives", Factindepolymerizing drugs "cytochalasin D and latrunculins", inhibitors of phosphoinositide metabolism "wortmannin and LY290042", and protein kinase C activator "phorbol esters". Except phorbol esters, the specificity of all the inhibitors is still in doubt as depolymerizing F-actin and inhibition of phosphoinositide metabolism may also disrupt the other two endocytic pathways. For example, cytochalasin D is also used as the inhibitor for CvME [50]. Within these inhibitors, amiloride and its derivatives may be considered as the first choice for their fewest side effects. Rottlerin, a novel macropinocytosis inhibitor which is rapid acting, irreversible, and selective, was discovered in 2005. In 2009, Hufnagel et al. found that rottlerin can specifically inhibit the transfection efficiency of PEI (25 kDa)/DNA complexes on Hela and CHO-K1 cells up to 50%, which verified the important role of FPE in

The commonly used inhibitors and methods for clathrin-mediated endocytosis are: Hyper‐ tonic sucrose (0.4–0.5 M), potassium depletion, cytosolic acidification, chlorpromazine (50–

the important factor that must be considered in such studies.

**4. Tools for the study of uptake pathways**

78 Gene Therapy - Tools and Potential Applications

the non-viral gene delivery by PEI (25 kDa) [12].

As to caveolae-mediated endocytic pathway, the commonly used inhibitors and methods are: statins, methyl-β-cyclodextrin (MβCD), filipin, nystatin, genestein, and cholesterol oxi‐ dase. Among them, the incubation with filipin, nystatin, and cholesterol oxidase produce the fewest side effects. The chronic inhibition of cholesterol synthesis by statins or acute cho‐ lesterol depletion by MβCD nonspecifically disrupts intracellular vesicle trafficking and the actin cytoskeleton. Also, the specificity of genestein is still in doubt for its nonspecific dis‐ ruption of the actin network. That being so, appropriate controls should be included when filipin, nystatin, and cholesterol oxidase are used [51].

The inhibitors for the study of intracellular fates of complexes are also very important. Mon‐ ensin, bafilomycin A can inhibit the acidification of endosomes, thus preventing their matu‐ ration and fusion into lysosomes [52, 53]. Chloroquine is another inhibitor that accumulates in endosomes/lysosomes and causes the swelling and disruption of endocytic vesicles by os‐ motic effects [21]. Last but not least, the cell-dependence of inhibitors should be noted when experiments are carried out. For example, chlorpromazine treatment inhibited the uptake of transferrin, a marker for CME by ~50% in D407 and HUH-7 cells. However, it showed no or little significant inhibitory capacity in ARPE-19 and Vero cells or even an enhanced effect in COS-7 cells [54, 55]. Therefore, a range of concentration with lowest cytotoxicity and suffi‐ cient inhibitory efficiency should be determined first when the inhibitor is used on the cell for the first time. Then, the lack of absolute specificity can be compensated by the combined application of biological methods such as siRNA silencing, transient or stable expression of dominant-negative proteins, and reconstruction of proteins by knockout mutants, all of which aremore specific than classical chemical inhibitors. For example, mutant dynamin has been successfully used to prove the necessity of dynamin in the endocytic pathways of transferrin receptors and EGF receptors [55]. A constitutive knockdown technique through RNAi has been used to prove the role of an essential accessory protein "epsin" in the CME pathway [56]. Another efficient way of making up the pitfalls of nonspecific inhibitors is the combined usage of fluorescently labeled gene complexes and fluorescent probes that are specifically internalized through certain uptake pathways.

Except for inhibitors, molecular probes and markers are also important tools for the study of uptake pathways for non-viral gene complexes. They can be used together with the classical chemical inhibitors or biological inhibitors to make the results more convinc‐ ing. There are several classical molecular probes that are known to be specifically inter‐ nalized through each uptake pathway. Transferrin is often used as a probe of CME pathway in many studies [12, 57, 58]. Transferrin receptor (TFR) mediates transferrin up‐ take by CME, so that it can be used as a CME marker and detected by anti-TFR [59]. Cholera toxin beta subunit (CTBs) is commonly used as a probe for CvME [12, 57]. How‐ ever, Lisa et al. argued that CTBs binds receptors that are contained in lipid-rich areas and are internalized via a mechanism similar to CvME, because CTBs uptake is unaffect‐ ed by a clathrin inhibitor and 33% uptake remains after treatment with a specific caveola inhibitor. Therefore, CTBs may enter into the cells via another unknown clathrin-inde‐ pendent mechanism [60]. In addition, caveolin-1 is also an important marker for CvME, as it is specifically involved in the formation of caveosome [29].

dyes for lysosomes. Cell light (red or green) are the widely used dyes for early endosomes. Combined with the confocal imaging technology, the colocalization of labeled non-viral gene complexes and intracellular compartments can be viewed intuitively. However, the classical confocal imaging technology can only provide the monolayer images, the informa‐ tion from which is not convincing enough. A novel three dimensionally integrated confocal technology is so strong that it can provide the intact information of a whole cell by scanning

Cellular Uptake Mechanism of Non-Viral Gene Delivery and Means for Improving Transfection Efficiency

Based on the current understanding of cellular uptake mechanisms, one can rationally de‐ sign vectors and improve their efficiency. Each pathway has advantages that need to be op‐

**Pathways Advantages Disadvantages**

CME Specific receptors Lysosome involved

Lysosome involved In vivo clearance

http://dx.doi.org/10.5772/53353

81

Membrane structure dependent Slower cellular uptaking

Specific receptors

Specific receptors

Macropinocytosis Larger particles uptaking. Non-specific Non-endocytic Bypass the lysosome Non-specific

Endo-lysosomal escape is one of the most crucial issues in non-viral vector design. Non-viral delivery systems, such as polyplexes and lipoplexes, will be trapped and degraded in the lysosomes if their cellular uptake pathways involve endo-lysosomes. As discussed above, some of the uptake pathways involve endo-lysosomes, such as CME and phagocytosis. CvME is known to bypass the endo-lysosomes. Similarly, macropinocytosis is known to not have any associations with endo-lysosomes [66, 67], but some studies have suggested that it involves lysosomes [67, 68]. These contradictory data may be dependent on cell type. Stimu‐ lating special pathway to bypass endo-lysosomes is a novel direction to improve efficiency.

A non-viral delivery system uptaken by endo-lysosomes dependent pathways must be ca‐ pable of escaping endo-lysosomes. From early endosome to late endosome transport, a ma‐ turation process involving compartment acidification by proton pumps located on the endosomal membrane exists. Some non-viral vectors exhibit the ability to escape the endo-

layer by layer.

**Table 3.** Characteristics of pathways.

**5.1. Endo-lysosomal escape**

This will be discussed later.

**5. Application of cellular uptake mechanism.**

timized and disadvantages that should be avoided (Table 3).

Phagocytosis Specific cell-type targeting

CvME Bypass the lysosome

Dextran is the popular probe for macropinocytosis in some studies because it can accumu‐ late in the endo-lysosome compartment [57]. As to phagocytosis, large (2 µm) microspheres are usually used as the probes. To solve the issue about the intracellular fate of complexes, a group of the specific markers or biological dyes are necessary to colocalize the non-viral gene complexes and intracellular organelles. TFR is used as a classical early endosome marker because it is transported into an early endosome when transferrin is internalized. EEA-1 is a hydrophilic peripheral membrane protein present in cytosol and membrane frac‐ tions. It colocalizes with TFR, and immunoelectron microscopy shows that it is associated with tubulovesicular early endosomes [61]. The lysosome-associated type 1 membrane gly‐ coproteins LAMP-1 and LAMP-2 are localized primarily on the periphery of the lysosome, and can be used as markers for lysosome [62, 63]. The different roles of EEA-1 and LAMP in the endolysosome pathway allow us to know the stage in which the uptake carries on. Other endosome or lysosome markers are the Rab family proteins. They are small GTPases that control multiple membrane trafficking events in the cell, and there are at least 60 Rab genes in the human genome [64]. Inside the Rab family, Rab5 and Rab7 are the most studied Rab variants, in which Rab5 is found to be the marker for early endosomes as it in part controls the invagination at the plasma membrane, endosomal fusion, motility, and signaling [63], and Rab7 is found to be the marker for late endosomes and lysosomes as it controls the ag‐ gregation, fusion, and maintenance of perinuclear lysosome compartment [65].


**Table 2.** Inhibitors and markers

The organelle specific dyes are other ideal tools for the detection of colocalization, and they are relatively convenient. LysoTracker (red) and Lyso Sensor (green) are the widely used dyes for lysosomes. Cell light (red or green) are the widely used dyes for early endosomes. Combined with the confocal imaging technology, the colocalization of labeled non-viral gene complexes and intracellular compartments can be viewed intuitively. However, the classical confocal imaging technology can only provide the monolayer images, the informa‐ tion from which is not convincing enough. A novel three dimensionally integrated confocal technology is so strong that it can provide the intact information of a whole cell by scanning layer by layer.

#### **5. Application of cellular uptake mechanism.**


Based on the current understanding of cellular uptake mechanisms, one can rationally de‐ sign vectors and improve their efficiency. Each pathway has advantages that need to be op‐ timized and disadvantages that should be avoided (Table 3).

**Table 3.** Characteristics of pathways.

Cholera toxin beta subunit (CTBs) is commonly used as a probe for CvME [12, 57]. How‐ ever, Lisa et al. argued that CTBs binds receptors that are contained in lipid-rich areas and are internalized via a mechanism similar to CvME, because CTBs uptake is unaffect‐ ed by a clathrin inhibitor and 33% uptake remains after treatment with a specific caveola inhibitor. Therefore, CTBs may enter into the cells via another unknown clathrin-inde‐ pendent mechanism [60]. In addition, caveolin-1 is also an important marker for CvME,

Dextran is the popular probe for macropinocytosis in some studies because it can accumu‐ late in the endo-lysosome compartment [57]. As to phagocytosis, large (2 µm) microspheres are usually used as the probes. To solve the issue about the intracellular fate of complexes, a group of the specific markers or biological dyes are necessary to colocalize the non-viral gene complexes and intracellular organelles. TFR is used as a classical early endosome marker because it is transported into an early endosome when transferrin is internalized. EEA-1 is a hydrophilic peripheral membrane protein present in cytosol and membrane frac‐ tions. It colocalizes with TFR, and immunoelectron microscopy shows that it is associated with tubulovesicular early endosomes [61]. The lysosome-associated type 1 membrane gly‐ coproteins LAMP-1 and LAMP-2 are localized primarily on the periphery of the lysosome, and can be used as markers for lysosome [62, 63]. The different roles of EEA-1 and LAMP in the endolysosome pathway allow us to know the stage in which the uptake carries on. Other endosome or lysosome markers are the Rab family proteins. They are small GTPases that control multiple membrane trafficking events in the cell, and there are at least 60 Rab genes in the human genome [64]. Inside the Rab family, Rab5 and Rab7 are the most studied Rab variants, in which Rab5 is found to be the marker for early endosomes as it in part controls the invagination at the plasma membrane, endosomal fusion, motility, and signaling [63], and Rab7 is found to be the marker for late endosomes and lysosomes as it controls the ag‐

gregation, fusion, and maintenance of perinuclear lysosome compartment [65].

Phagocytosis Amiloride, cytochalasin D, latrunculins,

CME Chlorpromazine, monodansylcadaverine,

CvME Filipin, nystatin, cholesterol oxidase, statins,

Macropinocytosis Rottlerin, amiloride, cytochalasin D, latrunculins,

**Table 2.** Inhibitors and markers

**Pathways inhibitors markers**

phenylarsine oxide, sodium azide

wortmannin, LY290042, sodium azide

The organelle specific dyes are other ideal tools for the detection of colocalization, and they are relatively convenient. LysoTracker (red) and Lyso Sensor (green) are the widely used

wortmannin, LY290042, sodium azide Large microspheres (2 μm)

genestein, MβCD, sodium azide CTBs, caveolin-1

Transferrin, lactosylceramide, TFR

Dextran

as it is specifically involved in the formation of caveosome [29].

80 Gene Therapy - Tools and Potential Applications

#### **5.1. Endo-lysosomal escape**

Endo-lysosomal escape is one of the most crucial issues in non-viral vector design. Non-viral delivery systems, such as polyplexes and lipoplexes, will be trapped and degraded in the lysosomes if their cellular uptake pathways involve endo-lysosomes. As discussed above, some of the uptake pathways involve endo-lysosomes, such as CME and phagocytosis. CvME is known to bypass the endo-lysosomes. Similarly, macropinocytosis is known to not have any associations with endo-lysosomes [66, 67], but some studies have suggested that it involves lysosomes [67, 68]. These contradictory data may be dependent on cell type. Stimu‐ lating special pathway to bypass endo-lysosomes is a novel direction to improve efficiency. This will be discussed later.

A non-viral delivery system uptaken by endo-lysosomes dependent pathways must be ca‐ pable of escaping endo-lysosomes. From early endosome to late endosome transport, a ma‐ turation process involving compartment acidification by proton pumps located on the endosomal membrane exists. Some non-viral vectors exhibit the ability to escape the endolysosome, called *proton sponge*, such as PEI [10, 69, 70]. PEI contains a nitrogen atom that can be protonated, and this serves to consume endosomal protons because endosomes acidify their microenvironment. As a result, an increase in endosomal chloride anion, which diffus‐ es into the endosomes with the protons, leads to an increase in osmotic pressure, thus induc‐ ing osmotic swelling [69]. Therefore, the endosome might break down and release PEI. This mode of action has been widely incorporated in recent non-viral vector designs. However, a pDAMA-based vector with endosomal buffering capacity has been reported to show no en‐ dosomal escape activity in cell-based assay, indicating that the proton sponge hypothesis may not be applicable in some cases. These findings warrant further elucidation and investi‐ gation of the mechanism of non-viral gene delivery [71].

These data demonstrate that the uptake mechanism and subsequent endocytic processing are important design parameters for gene delivery materials [76]. However, the key is con‐

Cellular Uptake Mechanism of Non-Viral Gene Delivery and Means for Improving Transfection Efficiency

http://dx.doi.org/10.5772/53353

83

Particle size is a very important factor for uptake mechanisms. In a previous study, three particles (20, 40, and 100 nm) were investigated for their uptake efficiency via CvME in en‐ dothelial cells. The results showed that the uptake efficiency levels of the 20- and 40-nm nanoparticles were 5–10 times greater than that of the 100-nm particles [6], indicating that small particles can be uptaken by CvME more efficiently compared with large ones. Howev‐ er, another study found that the uptake of microspheres with a diameter <200 nm in nonphagocytic B16 cells involved CME. With increasing size, a shift to a mechanism that relied on a caveolae-mediated pathway became apparent, which became the predominant path‐ way of entry for particles measuring 500 nm in size [77]. This can be attributed to the fact that the mechanism of CvME is cell type dependent in some cases. According to the target

CvME is a kind of receptor-mediated endocytosis pathway. As a result, some specific li‐ gands can mediate CvME via ligand–receptor binding. The insulin receptor [30], epidermal growth factor (EGF) receptor [31], transforming growth factor beta (TGFbeta) receptor [78] have been found to mediate this pathway. Another study used the cyclic Asn–Gly–Arg pep‐ tide to enhance gene transfection efficiency in CD13-positive vascular endothelial cells via CvME [79]. However, cyclic RGD ligands have been reported to facilitate CvME of thiolated c(RGDfK)-polyethylene glycol (PEG)-b-PLL micelles without high endosomal-disrupting properties and thus improve transfection efficiency [80]. The cyclic RGD peptide ligands c(RGDfK) can selectively recognize αvβ3 and αvβ5 integrin receptors on the cell surface. The receptors can mediate CvME and bypass endo-lysosomes. The αvβ3 and αvβ5 integrin receptors overexpressed on endothelial cells of tumor capillaries and neointimal tissues. As a result, the vectors with cyclic RGD peptide ligands can be used for cancer gene therapy. Cellular stress can also be used to control the cellular uptake mechanism. Heat shock and hyperosmotic shock can stimulate caveolin internalization [81]. Recent research has shown that hypertonic exposure of alveolar cells caused down-regulation of CME and fluid-phase endocytosis while stimulating CvME. An osmotic polymannitol-based gene transporter that can increase caveolae-mediated endocytosis was designed taking advantage of this mecha‐ nism [82]. The possible mechanisms have been discussed. Non-penetrating osmolytes tend to draw water from the intracellular space through an osmotic gradient, cause cell hyperton‐ ic stress accompanied by cell shrinkage. Responding the cellular hypertonic stress, phos‐ phorylation of caveolin-1 is mediated by Src-kinase. Src-kinase-mediated phosphorylation

cell type, the mechanism must be fully studied before designing a vector.

of caveolin-1 is required for caveolae budding. Finally the CvME is stimulated.

After *in vivo* administration, the non-viral delivery system can be uptaken by macrophag‐ es and then cleared. This macrophage clearance effect mainly via phagocytosis is one of the main barriers for non-viral gene delivery. Numerous methods are used to avoid phagocytosis of macrophages in vector design. Antibodies are being widely used for tar‐

trolling the uptake mechanism.

**5.3. Inhibition of phagocytosis**

For lipoplexes, the cationic liposome can interact with the anionic cytoplasmic facing mono‐ layer lipid of endosome and release the DNA from the endosome through the flip-flop mechanism [72]. 1,2-Dioleoylsn-glycero-3-phosphatidylethanolamine (DOPE), the pH-sensi‐ tive fusogenic lipid additive, is very helpful to the displacement of the anionic lipds from the cytoplasm-facingmonolayer of the endosomal membrane to the opposite direction via a flip-flop mechanism. However, the serum components are known to inactivate and destabi‐ lize the lipoplex structures that contain DOPE [73].

Viruses have the ability to destabilize the endosomal membrane, which explains why many proteins from different viruses are being used [69]. Some viruses are well known to use fusogenic peptides to cross the endosomal membrane and reach the cytosol [21]. The process by which viruses destabilize endosomal membranes in an acidification-de‐ pendent manner has been mimicked with synthetic peptides containing the amino-termi‐ nal 20-amino-acid sequence of the influenza virus HA [70]. Generally, short sequences of only 20 amino acids are needed for membrane destabilization, and they usually contain a high content of basic residues [74].

Cell-penetrating peptides (CPPs) are used to enhance endosomal escape. The HIV-1 Tat pro‐ tein is the first CPP to be discovered. It transactivates the transcription of the HIV-1 genome, has been observed to cross the plasma membrane by itself, leading to the identification of a peptide fragment (49–59 amino acids) that confers cell permeability to the protein (Tat pep‐ tide), and is one of the better characterized CPPs [75]. Most of the CPPs contain a high densi‐ ty of basic amino acids (arginines and/orlysines), which are proposed to interact with the anionic surface of the plasma membrane and enhance internalization of the peptides [75]. These peptides adopt an a -helical structure at endosomal pH leading to hydrophobic and hydrophilic faces that can interact with the endosomal membrane to cause disruption and pore formation [74].

#### **5.2. Optimization of CvME**

CvME is considered an alternative pathway that can bypass the endo-lysosomes. As gene materials will not be degraded in the lysosomal compartments, we can take advantage of CvME to improve the efficiency of transfection. For example, Nathan P. et al. targeted com‐ plexes (PEI–DNA) in CvME and CME with folic acid and transferrin, respectively; however, only vectors via CvME successfully delivered genes, as CvME is avoidant of lysosomes. These data demonstrate that the uptake mechanism and subsequent endocytic processing are important design parameters for gene delivery materials [76]. However, the key is con‐ trolling the uptake mechanism.

Particle size is a very important factor for uptake mechanisms. In a previous study, three particles (20, 40, and 100 nm) were investigated for their uptake efficiency via CvME in en‐ dothelial cells. The results showed that the uptake efficiency levels of the 20- and 40-nm nanoparticles were 5–10 times greater than that of the 100-nm particles [6], indicating that small particles can be uptaken by CvME more efficiently compared with large ones. Howev‐ er, another study found that the uptake of microspheres with a diameter <200 nm in nonphagocytic B16 cells involved CME. With increasing size, a shift to a mechanism that relied on a caveolae-mediated pathway became apparent, which became the predominant path‐ way of entry for particles measuring 500 nm in size [77]. This can be attributed to the fact that the mechanism of CvME is cell type dependent in some cases. According to the target cell type, the mechanism must be fully studied before designing a vector.

CvME is a kind of receptor-mediated endocytosis pathway. As a result, some specific li‐ gands can mediate CvME via ligand–receptor binding. The insulin receptor [30], epidermal growth factor (EGF) receptor [31], transforming growth factor beta (TGFbeta) receptor [78] have been found to mediate this pathway. Another study used the cyclic Asn–Gly–Arg pep‐ tide to enhance gene transfection efficiency in CD13-positive vascular endothelial cells via CvME [79]. However, cyclic RGD ligands have been reported to facilitate CvME of thiolated c(RGDfK)-polyethylene glycol (PEG)-b-PLL micelles without high endosomal-disrupting properties and thus improve transfection efficiency [80]. The cyclic RGD peptide ligands c(RGDfK) can selectively recognize αvβ3 and αvβ5 integrin receptors on the cell surface. The receptors can mediate CvME and bypass endo-lysosomes. The αvβ3 and αvβ5 integrin receptors overexpressed on endothelial cells of tumor capillaries and neointimal tissues. As a result, the vectors with cyclic RGD peptide ligands can be used for cancer gene therapy.

Cellular stress can also be used to control the cellular uptake mechanism. Heat shock and hyperosmotic shock can stimulate caveolin internalization [81]. Recent research has shown that hypertonic exposure of alveolar cells caused down-regulation of CME and fluid-phase endocytosis while stimulating CvME. An osmotic polymannitol-based gene transporter that can increase caveolae-mediated endocytosis was designed taking advantage of this mecha‐ nism [82]. The possible mechanisms have been discussed. Non-penetrating osmolytes tend to draw water from the intracellular space through an osmotic gradient, cause cell hyperton‐ ic stress accompanied by cell shrinkage. Responding the cellular hypertonic stress, phos‐ phorylation of caveolin-1 is mediated by Src-kinase. Src-kinase-mediated phosphorylation of caveolin-1 is required for caveolae budding. Finally the CvME is stimulated.

#### **5.3. Inhibition of phagocytosis**

lysosome, called *proton sponge*, such as PEI [10, 69, 70]. PEI contains a nitrogen atom that can be protonated, and this serves to consume endosomal protons because endosomes acidify their microenvironment. As a result, an increase in endosomal chloride anion, which diffus‐ es into the endosomes with the protons, leads to an increase in osmotic pressure, thus induc‐ ing osmotic swelling [69]. Therefore, the endosome might break down and release PEI. This mode of action has been widely incorporated in recent non-viral vector designs. However, a pDAMA-based vector with endosomal buffering capacity has been reported to show no en‐ dosomal escape activity in cell-based assay, indicating that the proton sponge hypothesis may not be applicable in some cases. These findings warrant further elucidation and investi‐

For lipoplexes, the cationic liposome can interact with the anionic cytoplasmic facing mono‐ layer lipid of endosome and release the DNA from the endosome through the flip-flop mechanism [72]. 1,2-Dioleoylsn-glycero-3-phosphatidylethanolamine (DOPE), the pH-sensi‐ tive fusogenic lipid additive, is very helpful to the displacement of the anionic lipds from the cytoplasm-facingmonolayer of the endosomal membrane to the opposite direction via a flip-flop mechanism. However, the serum components are known to inactivate and destabi‐

Viruses have the ability to destabilize the endosomal membrane, which explains why many proteins from different viruses are being used [69]. Some viruses are well known to use fusogenic peptides to cross the endosomal membrane and reach the cytosol [21]. The process by which viruses destabilize endosomal membranes in an acidification-de‐ pendent manner has been mimicked with synthetic peptides containing the amino-termi‐ nal 20-amino-acid sequence of the influenza virus HA [70]. Generally, short sequences of only 20 amino acids are needed for membrane destabilization, and they usually contain

Cell-penetrating peptides (CPPs) are used to enhance endosomal escape. The HIV-1 Tat pro‐ tein is the first CPP to be discovered. It transactivates the transcription of the HIV-1 genome, has been observed to cross the plasma membrane by itself, leading to the identification of a peptide fragment (49–59 amino acids) that confers cell permeability to the protein (Tat pep‐ tide), and is one of the better characterized CPPs [75]. Most of the CPPs contain a high densi‐ ty of basic amino acids (arginines and/orlysines), which are proposed to interact with the anionic surface of the plasma membrane and enhance internalization of the peptides [75]. These peptides adopt an a -helical structure at endosomal pH leading to hydrophobic and hydrophilic faces that can interact with the endosomal membrane to cause disruption and

CvME is considered an alternative pathway that can bypass the endo-lysosomes. As gene materials will not be degraded in the lysosomal compartments, we can take advantage of CvME to improve the efficiency of transfection. For example, Nathan P. et al. targeted com‐ plexes (PEI–DNA) in CvME and CME with folic acid and transferrin, respectively; however, only vectors via CvME successfully delivered genes, as CvME is avoidant of lysosomes.

gation of the mechanism of non-viral gene delivery [71].

82 Gene Therapy - Tools and Potential Applications

lize the lipoplex structures that contain DOPE [73].

a high content of basic residues [74].

pore formation [74].

**5.2. Optimization of CvME**

After *in vivo* administration, the non-viral delivery system can be uptaken by macrophag‐ es and then cleared. This macrophage clearance effect mainly via phagocytosis is one of the main barriers for non-viral gene delivery. Numerous methods are used to avoid phagocytosis of macrophages in vector design. Antibodies are being widely used for tar‐ geting non-viral gene delivery. However, the constant fragments can be recognized by phagocytosis and then uptaken by macrophages. Therefore, antibodies that lack constant fragments are sometimes used to help non-viral vectors avoid recognition and clearance by macrophages *in vivo* [83].

**Acknowledgements**

mission (No.10SG22).

**Author details**

**References**

651-8.

745-53.

18(1): p. 33-7.

2003. 92(2): p. 203-17.

Shengnan Xiang and Xiaoling Zhang

\*Address all correspondence to: xlzhang@sibs.ac.cn

(SIBS), Chinese Academy of Sciences (CAS), Shanghai, China

*peutics.* Nat Biotechnol, 2008. 26(5): p. 561-9.

*thelial cells.* ACS Nano, 2009. 3(12): p. 4110-6.

*siRNA delivery.* Nat Rev Drug Discov, 2009. 8(2): p. 129-38.

*ing liposomes as drug carriers.* Eur J Pharm Sci, 2001. 13(1): p. 85-9.

This work was supported by grants from The Ministry of Science and Technology of China (No.2011DFA30790), National Natural Science Foundation of China (No. 81190133), Chinese Academy of Sciences (No.XDA01030404, KSCX2-EW-Q-1-07), Science and Technology Com‐ mission of Shanghai Municipality (No.11QH1401600), Shanghai Municipal Education Com‐

Cellular Uptake Mechanism of Non-Viral Gene Delivery and Means for Improving Transfection Efficiency

http://dx.doi.org/10.5772/53353

85

The Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Jiao Tong University School of Medicine (SJTUSM) & Shanghai Institutes for Biological Sciences

[1] Gao, K. and L. Huang, *Nonviral methods for siRNA delivery.* Mol Pharm, 2009. 6(3): p.

[2] Luo, D. and W.M. Saltzman, *Synthetic DNA delivery systems.* Nat Biotechnol, 2000.

[3] Wiethoff, C.M. and C.R. Middaugh, *Barriers to nonviral gene delivery.* J Pharm Sci,

[4] Akinc, A., et al., *A combinatorial library of lipid-like materials for delivery of RNAi thera‐*

[5] Whitehead, K.A., R. Langer, and D.G. Anderson, *Knocking down barriers: advances in*

[6] Wang, Z., et al., *Size and dynamics of caveolae studied using nanoparticles in living endo‐*

[7] Harashima, H., Y. Shinohara, and H. Kiwada, *Intracellular control of gene trafficking us‐*

[8] Boussif, O., et al., *A versatile vector for gene and oligonucleotide transfer into cells in cul‐ ture and in vivo: polyethylenimine.* Proc Natl Acad Sci U S A, 1995. 92(16): p. 7297-301. [9] von Gersdorff, K., et al., *The internalization route resulting in successful gene expression depends on both cell line and polyethylenimine polyplex type.* Mol Ther, 2006. 14(5): p.

Other vectors can also be recognized by macrophages. As discussed above, some cationic polyplexes or lipoplexes will be tagged by some opsonins and then recognized *in vivo*. PE‐ Gylation is widely used to avoid the *in vivo* clearance effect by phagocytosis. The highly hy‐ drophilic nature of PEG produces a hydration shell around its conjugated partner, hence reducing intermolecular interactions and, consequently, toxicity [84]. As an effect of reduc‐ ing intermolecular interactions, PEGylation can effectively avoid phagocytosis; moreover, *in vivo* studies have reported on long circulating half-life of PEGylated vectors [85].

However, some studies have shown that PEGylation can reduce the efficiency of vectors [84] possibly because PEGylation may inhibit cellular uptake and endosomal escape of the vec‐ tors. One study compared non-PEGylated and PEGylated liposomes, with the data showing that PEGylated liposomes have poor endosomal escape capability as non-PEGylated lipo‐ somes can escape from endosome efficiently [86]. The inhibitory effects of PEGylation de‐ pend on some factors. A study about PEGylated cationic liposomes demonstrated that acidlabile PEGylation liposomes have higher transfection efficiency than acid-stable PEGylation ones, which can be ascribed to the more efficient endosomal escape activity of acid-labile PEGylation liposomes [87]. The possible mechanism involved here is that the PEG of acidlabile PEGylation liposomes can be cleaved under low pH (endosomal compartments), al‐ lowing the vector to fully interact with the endosomal membrane. So other biodegradable shielding methods should be better than classical PEGylation. According to this hypothesis, recently, an alternative to PEGylation was designed. This work reports, for the first time, the use of hydroxyethyl starch (HES) for the controlled shielding/deshielding of polyplexes. Non-viral delivery systems can be protected by HES shielding, and the HES can then be de‐ graded *in vivo*, indicating that HES shielding has less influence on the efficiency of vectors compared with PEGylation [88].

#### **6. Conclusion**

In summary, cellular uptake is the most important intracellular process. Understanding cel‐ lular uptake mechanisms is essential to determining the limits of gene delivery. Different pathways have different intracellular fates. Some vectors can enter cells via endo-lysosomal pathways. Thus, some methods have to be used to protect genes against degradation in ly‐ sosomes. Optimizing CvME can successfully deliver genes by avoiding endo-lysosomes. Each pathway has its own disadvantages, and learning how to inhibit certain pathways is significant in some cases. In conclusion, taking advantage of cellular uptake mechanisms and knowing how to control them hold considerable potential for improving the efficiency of gene delivery.

#### **Acknowledgements**

geting non-viral gene delivery. However, the constant fragments can be recognized by phagocytosis and then uptaken by macrophages. Therefore, antibodies that lack constant fragments are sometimes used to help non-viral vectors avoid recognition and clearance

Other vectors can also be recognized by macrophages. As discussed above, some cationic polyplexes or lipoplexes will be tagged by some opsonins and then recognized *in vivo*. PE‐ Gylation is widely used to avoid the *in vivo* clearance effect by phagocytosis. The highly hy‐ drophilic nature of PEG produces a hydration shell around its conjugated partner, hence reducing intermolecular interactions and, consequently, toxicity [84]. As an effect of reduc‐ ing intermolecular interactions, PEGylation can effectively avoid phagocytosis; moreover, *in*

However, some studies have shown that PEGylation can reduce the efficiency of vectors [84] possibly because PEGylation may inhibit cellular uptake and endosomal escape of the vec‐ tors. One study compared non-PEGylated and PEGylated liposomes, with the data showing that PEGylated liposomes have poor endosomal escape capability as non-PEGylated lipo‐ somes can escape from endosome efficiently [86]. The inhibitory effects of PEGylation de‐ pend on some factors. A study about PEGylated cationic liposomes demonstrated that acidlabile PEGylation liposomes have higher transfection efficiency than acid-stable PEGylation ones, which can be ascribed to the more efficient endosomal escape activity of acid-labile PEGylation liposomes [87]. The possible mechanism involved here is that the PEG of acidlabile PEGylation liposomes can be cleaved under low pH (endosomal compartments), al‐ lowing the vector to fully interact with the endosomal membrane. So other biodegradable shielding methods should be better than classical PEGylation. According to this hypothesis, recently, an alternative to PEGylation was designed. This work reports, for the first time, the use of hydroxyethyl starch (HES) for the controlled shielding/deshielding of polyplexes. Non-viral delivery systems can be protected by HES shielding, and the HES can then be de‐ graded *in vivo*, indicating that HES shielding has less influence on the efficiency of vectors

In summary, cellular uptake is the most important intracellular process. Understanding cel‐ lular uptake mechanisms is essential to determining the limits of gene delivery. Different pathways have different intracellular fates. Some vectors can enter cells via endo-lysosomal pathways. Thus, some methods have to be used to protect genes against degradation in ly‐ sosomes. Optimizing CvME can successfully deliver genes by avoiding endo-lysosomes. Each pathway has its own disadvantages, and learning how to inhibit certain pathways is significant in some cases. In conclusion, taking advantage of cellular uptake mechanisms and knowing how to control them hold considerable potential for improving the efficiency

*vivo* studies have reported on long circulating half-life of PEGylated vectors [85].

by macrophages *in vivo* [83].

84 Gene Therapy - Tools and Potential Applications

compared with PEGylation [88].

**6. Conclusion**

of gene delivery.

This work was supported by grants from The Ministry of Science and Technology of China (No.2011DFA30790), National Natural Science Foundation of China (No. 81190133), Chinese Academy of Sciences (No.XDA01030404, KSCX2-EW-Q-1-07), Science and Technology Com‐ mission of Shanghai Municipality (No.11QH1401600), Shanghai Municipal Education Com‐ mission (No.10SG22).

#### **Author details**

Shengnan Xiang and Xiaoling Zhang

\*Address all correspondence to: xlzhang@sibs.ac.cn

The Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Jiao Tong University School of Medicine (SJTUSM) & Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS), Shanghai, China

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**Chapter 5**

**Polylipid Nanoparticle,**

Additional information is available at the end of the chapter

Yahan Fan and Jian Wu

http://dx.doi.org/10.5772/54270

**1. Introduction**

**a Novel Lipid-Based Vector for Liver Gene Transfer**

Lipid nanoparticles (LNP) are invaluable carriers for drug and gene delivery, and they are classified as cationic, neutral and anionic depending on the electronic charges exist‐ ing on the surface of the vesicles [1]. These charges are originated from the charged lip‐ ids from which lipid nanoparticles are formulated. Cationic LNP are commonly used for DNA or RNA carriers due to their interaction with negatively-charged nucleotide. Both neutral and negatively-charged LNPs are used for drug delivery [2] and may be formu‐ lated as sterically stable LNPs (SSLNPs), which are amendable for cell type-specific or tissue-specific targeting delivery [3]. For liver drug delivery, tremendous efforts have been made to develop cell type-selective lipid-based drug carriers. Effective approaches in targeting hepatocytes, Kupffer cells and hepatic stellate cells have been evaluated in small animals [3, 4], and some of them may be translational to clinical application [5]. These approaches are referable when cationic LNPs are considered for cell type-selective gene delivery. A prerequisite for the success of gene therapy for liver disorders is the de‐ velopment of powerful gene carriers. Non-viral vectors have been very successful for gene transfer in an *in vitro* setting, in terms of efficiency of lipofection, applicability in variety of cell types, and amending ability of cell type-specific delivery (Fig. 1). The clini‐ cal application of LNP-mediated gene transfer has been hampered by low efficiency, in‐ stability in the bloodstream, short-term transgene expression and toxicity. These shortcomings are the bottle neck hindering the gene transfer employing LNPs as carriers for delivery of function gene(s) to solid organs, and are the challenges in moving from small to large animals of potential gene carriers and approaches, and in the translation to clinical application. However, the polylipid nanoparticles (PLNP) we have developed over the past decade represent one of the few formulations that are applicable for *in vivo*

> © 2013 Fan and Wu; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.


### **Polylipid Nanoparticle, a Novel Lipid-Based Vector for Liver Gene Transfer**

Yahan Fan and Jian Wu

[78] Razani, B., et al., *Caveolin-1 regulates transforming growth factor (TGF)-beta/SMAD sig‐ naling through an interaction with the TGF-beta type I receptor.* J Biol Chem, 2001. 276(9):

[79] Liu, C., et al., *Enhanced gene transfection efficiency in CD13-positive vascular endothelial cells with targeted poly(lactic acid)-poly(ethylene glycol) nanoparticles through caveolae-*

[80] Oba, M., et al., *Polyplex micelles with cyclic RGD peptide ligands and disulfide cross-links directing to the enhanced transfection via controlled intracellular trafficking.* Mol Pharm,

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[82] Park, T.E., et al., *Selective stimulation of caveolae-mediated endocytosis by an osmotic poly‐*

[83] Ikeda, Y. and K. Taira, *Ligand-targeted delivery of therapeutic siRNA.* Pharm Res, 2006.

[84] Fitzsimmons, R.E. and H. Uludag, *Specific effects of PEGylation on gene delivery efficacy of polyethylenimine: Interplay between PEG substitution and N/P ratio.* Acta Biomater,

[85] Chaudhari, K.R., et al., *Opsonization, biodistribution, cellular uptake and apoptosis study of PEGylated PBCA nanoparticle as potential drug delivery carrier.* Pharm Res, 2012. 29(1):

[86] Remaut, K., et al., *Pegylation of liposomes favours the endosomal degradation of the deliv‐ ered phosphodiester oligonucleotides.* J Control Release, 2007. 117(2): p. 256-66.

[87] Chan, C.L., et al., *Endosomal escape and transfection efficiency of PEGylated cationic lipo‐ some-DNA complexes prepared with an acid-labile PEG-lipid.* Biomaterials, 2012. 33(19):

[88] Noga, M., et al., *Controlled shielding and deshielding of gene delivery polyplexes using hy‐ droxyethyl starch (HES) and alpha-amylase.* J Control Release, 2012. 159(1): p. 92-103.

*mannitol-based gene transporter.* Biomaterials, 2012. 33(29): p. 7272-81.

*mediated endocytosis.* J Control Release, 2011. 151(2): p. 162-75.

p. 6727-38.

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23(8): p. 1631-40.

2012.

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p. 4928-35.

*shock.* Exp Cell Res, 2000. 255(2): p. 221-8.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54270

#### **1. Introduction**

Lipid nanoparticles (LNP) are invaluable carriers for drug and gene delivery, and they are classified as cationic, neutral and anionic depending on the electronic charges exist‐ ing on the surface of the vesicles [1]. These charges are originated from the charged lip‐ ids from which lipid nanoparticles are formulated. Cationic LNP are commonly used for DNA or RNA carriers due to their interaction with negatively-charged nucleotide. Both neutral and negatively-charged LNPs are used for drug delivery [2] and may be formu‐ lated as sterically stable LNPs (SSLNPs), which are amendable for cell type-specific or tissue-specific targeting delivery [3]. For liver drug delivery, tremendous efforts have been made to develop cell type-selective lipid-based drug carriers. Effective approaches in targeting hepatocytes, Kupffer cells and hepatic stellate cells have been evaluated in small animals [3, 4], and some of them may be translational to clinical application [5]. These approaches are referable when cationic LNPs are considered for cell type-selective gene delivery. A prerequisite for the success of gene therapy for liver disorders is the de‐ velopment of powerful gene carriers. Non-viral vectors have been very successful for gene transfer in an *in vitro* setting, in terms of efficiency of lipofection, applicability in variety of cell types, and amending ability of cell type-specific delivery (Fig. 1). The clini‐ cal application of LNP-mediated gene transfer has been hampered by low efficiency, in‐ stability in the bloodstream, short-term transgene expression and toxicity. These shortcomings are the bottle neck hindering the gene transfer employing LNPs as carriers for delivery of function gene(s) to solid organs, and are the challenges in moving from small to large animals of potential gene carriers and approaches, and in the translation to clinical application. However, the polylipid nanoparticles (PLNP) we have developed over the past decade represent one of the few formulations that are applicable for *in vivo*

© 2013 Fan and Wu; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

gene transfer [6], due to a high DNA-packaging capacity, an extremely low binding rate to serum proteins, low toxicity, and amendable synthetic approaches [7, 8]. This chapter intends to introduce the characteristics of this formulation, and to discuss our efforts in moving the non-viral gene transfer platform from small to large animals towards clinical applications.

ing cationic liposome-mediated gene transfer (NCT00004471) has been completed. A phase I trial of intratumoral epidermal growth factor receptor (EGFR) antisense DNA delivered by DC-Chol liposomes in advanced head and neck cancer, including oral squamous cell carci‐ noma (NCT00009841) and DOTAP-Chol-Fus1 liposome-mediated gene therapy for nonsmall cell lung cancer (NCT00059605] [9] were conducted respectively by University of Pittsburg and MD Anderson Cancer Center in collaboration with the National Cancer Insti‐ tute (NCI). Fus1 is a tumor suppressive gene that has been shown to be effective in sup‐ pressing the growth of original or metastatic lesions of non-small lung cancer when it is delivered locally or systemically [10]. Thus, it appears that genetic therapy using LNPs as gene carriers has the potential to be specially tailored for genetic disorders or cancers.

Polylipid Nanoparticle, a Novel Lipid-Based Vector for Liver Gene Transfer

http://dx.doi.org/10.5772/54270

93

Lipid-based gene carriers include liposomes (cationic or anionic), polymer and dendrimer nanoparticles. Cationic liposomes are capable of delivering genes to cells or tissues, and achieving maximal therapeutic efficiency with minimal adverse effects [1]. However, the use of cationic LNPs for *in vivo* DNA transfection is hindered by substantial problems; i.e. after intravenous administration, cationic LNPs bind to plasma protein and blood cells due to charge reaction. The resulting aggregates of carriers with proteins or cells block microcirculation or may be cleared rapidly [11, 12]. The common formulations for *in vivo* gene delivery are DOTMA or DOTAP-DOPE or DOTAP-cholesterol (Chol). These formulations are highly serum-reactive [6, 13]. Lungs are the major organ shown to be highly transfected probably due to the accumulation of aggregates of lipoplexes with se‐ rum proteins or blood cells when the lipoplexes are administrated intravenously [14]. For this reason, cationic LNPs were once used widely for gene delivery to the lungs; and lat‐ er for treating lung cancers and metastasis with further optimization [10, 15, 16]. LNPmediated gene delivery to the liver is more difficult than to lungs. For the development of the gene carriers, cationic LNP formulations, such as DC-Chol, DOTAP-Chol, are available for delivering genes to various tissues [17]. A few LNP formulations targeting hepatocellular carcinoma (HCC) have been developed for improving efficacies of drug therapy [18, 19]. In order to avoid the rapid clearance by the reticuloendothelial system (RES) and to increase the drug delivery through the enhanced permeability and retention (EPR) effect to a tumor site by passive targeting, novel strategies, such as reducing parti‐ cle size, minimizing rigidity of lipids, generating amphiphilic vesicles and shielding from the recognition by RES system, have been attempted in formulating lipid-based drug/ gene carriers [1, 2]. To reduce lysosomal degradation, pH-sensitive LNPs are prepared for drug or gene delivery [20]. These approaches may be instructive in the development

**2. Nanoparticle carriers for drug or gene delivery**

of LNPs for gene transfer at different stages of preclinical translation.

Polymeric non-viral vectors have exhibited additional advantages of lower toxicity and im‐ munogenicity [21, 22]. These vectors may offer the possibility of industrial production fol‐ lowing good manufacturing practice (GMP). Amphiphilic polyethylene glycol (PEG) has been engineered as a linker, most for coupling peptides to cationic lipids. Other polymers,

**Figure 1.** *In vivo* **gene transfer mediated by viral or non-viral vectors.** Viral vectors, such as lentiviral or retroviral vectors, which lead to integration of transgene in the host genome, give rise to long-term transgene expression. How‐ ever, they may cause insertion-induced mutation that is oncogenic. Adenoviral or adeno-associated viral (AAV) vectors often yield a high level of transgene expression in host organs. However, generation of antibodies against viral com‐ ponents is still a concern. Non-viral gene transfer may be achieved by direct plasmid administration with local electro‐ poration or a hydrodynamic approach. The latter is only applicable in mice. Lipid nanoparticle (LNP)-mediated gene transfer becomes a useful approach which is often very successful for *in vitro* gene transfer. Few formulations of cati‐ onic LNPs are valuable for *in vivo* gene transfer. There has been a still demand in improving their *in vivo* stability and gene transfer efficacy.

There are a number of critical components for a potential gene therapy product to move from one step to the next in this pipeline. Promisingly, LNP-mediated gene transfection for the treatment of genetic and metabolic disorders or tumors has been moved to clinical trial phases (http://clinicaltrials.gov). A phase I pilot study of gene therapy for cystic fibrosis us‐ ing cationic liposome-mediated gene transfer (NCT00004471) has been completed. A phase I trial of intratumoral epidermal growth factor receptor (EGFR) antisense DNA delivered by DC-Chol liposomes in advanced head and neck cancer, including oral squamous cell carci‐ noma (NCT00009841) and DOTAP-Chol-Fus1 liposome-mediated gene therapy for nonsmall cell lung cancer (NCT00059605] [9] were conducted respectively by University of Pittsburg and MD Anderson Cancer Center in collaboration with the National Cancer Insti‐ tute (NCI). Fus1 is a tumor suppressive gene that has been shown to be effective in sup‐ pressing the growth of original or metastatic lesions of non-small lung cancer when it is delivered locally or systemically [10]. Thus, it appears that genetic therapy using LNPs as gene carriers has the potential to be specially tailored for genetic disorders or cancers.

#### **2. Nanoparticle carriers for drug or gene delivery**

gene transfer [6], due to a high DNA-packaging capacity, an extremely low binding rate to serum proteins, low toxicity, and amendable synthetic approaches [7, 8]. This chapter intends to introduce the characteristics of this formulation, and to discuss our efforts in moving the non-viral gene transfer platform from small to large animals towards clinical

**Figure 1.** *In vivo* **gene transfer mediated by viral or non-viral vectors.** Viral vectors, such as lentiviral or retroviral vectors, which lead to integration of transgene in the host genome, give rise to long-term transgene expression. How‐ ever, they may cause insertion-induced mutation that is oncogenic. Adenoviral or adeno-associated viral (AAV) vectors often yield a high level of transgene expression in host organs. However, generation of antibodies against viral com‐ ponents is still a concern. Non-viral gene transfer may be achieved by direct plasmid administration with local electro‐ poration or a hydrodynamic approach. The latter is only applicable in mice. Lipid nanoparticle (LNP)-mediated gene transfer becomes a useful approach which is often very successful for *in vitro* gene transfer. Few formulations of cati‐ onic LNPs are valuable for *in vivo* gene transfer. There has been a still demand in improving their *in vivo* stability and

There are a number of critical components for a potential gene therapy product to move from one step to the next in this pipeline. Promisingly, LNP-mediated gene transfection for the treatment of genetic and metabolic disorders or tumors has been moved to clinical trial phases (http://clinicaltrials.gov). A phase I pilot study of gene therapy for cystic fibrosis us‐

applications.

92 Gene Therapy - Tools and Potential Applications

gene transfer efficacy.

Lipid-based gene carriers include liposomes (cationic or anionic), polymer and dendrimer nanoparticles. Cationic liposomes are capable of delivering genes to cells or tissues, and achieving maximal therapeutic efficiency with minimal adverse effects [1]. However, the use of cationic LNPs for *in vivo* DNA transfection is hindered by substantial problems; i.e. after intravenous administration, cationic LNPs bind to plasma protein and blood cells due to charge reaction. The resulting aggregates of carriers with proteins or cells block microcirculation or may be cleared rapidly [11, 12]. The common formulations for *in vivo* gene delivery are DOTMA or DOTAP-DOPE or DOTAP-cholesterol (Chol). These formulations are highly serum-reactive [6, 13]. Lungs are the major organ shown to be highly transfected probably due to the accumulation of aggregates of lipoplexes with se‐ rum proteins or blood cells when the lipoplexes are administrated intravenously [14]. For this reason, cationic LNPs were once used widely for gene delivery to the lungs; and lat‐ er for treating lung cancers and metastasis with further optimization [10, 15, 16]. LNPmediated gene delivery to the liver is more difficult than to lungs. For the development of the gene carriers, cationic LNP formulations, such as DC-Chol, DOTAP-Chol, are available for delivering genes to various tissues [17]. A few LNP formulations targeting hepatocellular carcinoma (HCC) have been developed for improving efficacies of drug therapy [18, 19]. In order to avoid the rapid clearance by the reticuloendothelial system (RES) and to increase the drug delivery through the enhanced permeability and retention (EPR) effect to a tumor site by passive targeting, novel strategies, such as reducing parti‐ cle size, minimizing rigidity of lipids, generating amphiphilic vesicles and shielding from the recognition by RES system, have been attempted in formulating lipid-based drug/ gene carriers [1, 2]. To reduce lysosomal degradation, pH-sensitive LNPs are prepared for drug or gene delivery [20]. These approaches may be instructive in the development of LNPs for gene transfer at different stages of preclinical translation.

Polymeric non-viral vectors have exhibited additional advantages of lower toxicity and im‐ munogenicity [21, 22]. These vectors may offer the possibility of industrial production fol‐ lowing good manufacturing practice (GMP). Amphiphilic polyethylene glycol (PEG) has been engineered as a linker, most for coupling peptides to cationic lipids. Other polymers, such as dendritic poly(L-lysine)-b-poly(L-lactide)-b-dendritic vector [23], poly (ethylenei‐ mine) (PEI) [24], poly (methacrylate) [25] and polyamidoamine dendrimers [26], have been demonstrated to be effective for *in vitro* gene delivery. However, striking issues still exist for cationic polymers regarding whether they are applicable for *in vivo* gene transfer to solid or‐ gans such as the liver, without significant adverse effects.

High density lipoprotein (HDL) has a high drug carry capacity, and can be recognized by HDL receptors on hepatocytes. Recombinant HDL was utilized to deliver an anti-HBV pep‐ tide (nosiheptide) to the liver, and it was shown to achieve a selective distribution in hepato‐ ma cells *in vitro* and a preferential liver distribution in rats [36]. Apolipoprotein E is cleared by hepatocytes, and it has been employed to be carriers for small interfering RNA (siRNA)

Polylipid Nanoparticle, a Novel Lipid-Based Vector for Liver Gene Transfer

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95

Given the fact that hepatic stellate cells (HSCs) are the major cell type responsible for hepatic fibrosis, a repairing process that causes excess production of extracellular matrix compo‐ nents and deposition of fibrotic scaring in chronically injured liver [38], much attention has been focused on targeting this cell type in the last decade. A couple of cell surface molecules that are overexpressed on activated HSCs during hepatic fibrogenesis, such as insulin growth factor receptor II [39], collagen type VI and platelet-derived growth factor (PDGF) receptor β-subunit [40] are selected as the cell surface targets. Drug carriers labeled with specific peptides recognizing these cell surface molecules, such as cyclic peptide containing arginine-glycine-aspartate (RGD)-labeled sterical lipid nanoparticles [3] or Mannose-6-phos‐ phate human serum albumin (M6P/HAS) [41] exhibited HSC-selective distribution. The RGD cyclic peptide was recently used as a targeting molecule for the recognition of activat‐ ed HSCs in two animal models for early diagnosis of hepatic fibrosis with a SPECT imaging modality [42]. Using the retinol binding protein (RBP) in activated HSCs seems to be very effective in delivering siRNA against gp46 (rat homolog of human heat shock protein 47),

Targeting approaches for drug or gene delivery to other non-parenchymal cell types, such as Kupffer cells or sinusoidal endothelial cells, are summarized recently [27]. These approaches are crucial in delivering agents which are anti-inflammatory or anti-oxidants to these cell types due to the fact that Kupffer cells are pivotal in the mediation of inflammatory respons‐

Compared to drug delivery, LNP-mediated *in vivo* gene delivery is still in its development stage; and many issues that affect delivery approaches and efficacy remain to be solved. The main issues include: 1) the formation of aggregates between cationic lipids and serum pro‐ teins bearing negative charges; 2) the administration routes of LNP-DNA complexes (lipo‐ plexes); 3) intracellular trafficking from the cytoplasm to the nucleus; 4) the proliferative state of cells to be transfected; and 5) transient transgene expression for a short duration [6, 45]. Substantial efforts have been made to address these issues in our previous studies and by others [8, 46, 47]. Particularly, we polymerized an acrylamide lipid to generate a polyca‐ tionic lipid (PCL), which was able to interact with plasmid DNA effectively and form com‐ pacted complexes as demonstrated by Raman microspectral analysis [8]. PCL has a unique molecular configuration and molecular weight distribution as indicated by mass spectro‐ photometrical analysis [8]. Moreover, this lipid can be synthesized in a multiple gram quan‐

delivery to hepatocytes [37].

and inhibiting fibrosis in two animal models [43].

**4. Polylipid nanoparticle-mediated liver gene delivery**

es and subsequent fibrogenesis [44].

#### **3. Liver-specific gene delivery**

Because of our interest in gene therapy of liver disorders, we have focused our efforts on improving liver-based gene delivery. The pathogenesis of liver injury and fibrosis in‐ volves complicated interactions among different cell populations in the liver, soluble fac‐ tors, such as cytokines and reactive oxygen species (ROS), and the extracellular matrix components. In order to improve the efficacy in preventing hepatocellular injury, the use of LNPs that are capable of delivering hepatoprotective agents to the liver, selectively to hepatocytes, will increase local concentration of therapeutic agents, reduce adverse effects, and achieve maximal therapeutic efficiency. The parenchymal cell type in the liver is hep‐ atocytes, which are responsible for an array of metabolic function in the body and are of‐ ten damaged in a variety of pathological processes. The asialoglycoprotein receptor (ASGP-R) on mammalian hepatocytes provides a unique means for the development of liver-specific drug or gene carriers. The abundant receptors on hepatocytes specifically recognize the natural ligands, lectin and asialofetuin (AF), as well as those with terminal galactose or N-acetylgalactosamine residues, and hepatocytes endocytose these ligands for an intracellular degradation process [27, 28]. The use of its natural or synthetic ligands, such as galactosylated cholesterol, glycolipids or galactosylated polymers to label LNPs has achieved significant targeting efficacy to the liver [4, 28]. AF-labeled LNPs have been used for improving liver-targeting gene transfer in small animals [29], yet there have not been successful reports available in the translation to large animals, such as pigs [30]. In‐ stead, plasmid DNA was directly administrated into the hepatic vein through a catheter with a balloon closure of hepatic vein blood flow [30]. One particular attention has been drawn in terms of the use of AF-labeled drug carriers for HCC targeting. The expression of ASGP-R in HCC cells varies depending on the differentiation status of HCC cells [31]. In general, well-differentiated HCC usually expresses relatively high levels of hepatocytespecific genes, including ASGP-R; whereas poorly-differentiated HCC expresses minimal or no hepatocyte-specific genes, including ASGP-R [32]. In most cases, there exists the dra‐ matic heterogeneity of liver-specific gene expression in human HCC tissues [33], and de‐ creased expression of ASGP-R was observed in liver cancer tissue [34]. Therefore, using AF or other galactosylated or lactosylated residues to label LNPs for drug or gene deliv‐ ery may not always be effective for patients with HCC, because HCC develops on a varie‐ ty of disease backgrounds and there is a striking variation in ASGP-R expression levels in HCC from different patients. Using well-differentiated hepatoma cells, such as HepG2, Hep3B and Huh-7 cells, as an *in vitro* screening tool may not necessarily reflect targeting efficacy to tumor-specific distribution *in vivo* [35].

High density lipoprotein (HDL) has a high drug carry capacity, and can be recognized by HDL receptors on hepatocytes. Recombinant HDL was utilized to deliver an anti-HBV pep‐ tide (nosiheptide) to the liver, and it was shown to achieve a selective distribution in hepato‐ ma cells *in vitro* and a preferential liver distribution in rats [36]. Apolipoprotein E is cleared by hepatocytes, and it has been employed to be carriers for small interfering RNA (siRNA) delivery to hepatocytes [37].

such as dendritic poly(L-lysine)-b-poly(L-lactide)-b-dendritic vector [23], poly (ethylenei‐ mine) (PEI) [24], poly (methacrylate) [25] and polyamidoamine dendrimers [26], have been demonstrated to be effective for *in vitro* gene delivery. However, striking issues still exist for cationic polymers regarding whether they are applicable for *in vivo* gene transfer to solid or‐

Because of our interest in gene therapy of liver disorders, we have focused our efforts on improving liver-based gene delivery. The pathogenesis of liver injury and fibrosis in‐ volves complicated interactions among different cell populations in the liver, soluble fac‐ tors, such as cytokines and reactive oxygen species (ROS), and the extracellular matrix components. In order to improve the efficacy in preventing hepatocellular injury, the use of LNPs that are capable of delivering hepatoprotective agents to the liver, selectively to hepatocytes, will increase local concentration of therapeutic agents, reduce adverse effects, and achieve maximal therapeutic efficiency. The parenchymal cell type in the liver is hep‐ atocytes, which are responsible for an array of metabolic function in the body and are of‐ ten damaged in a variety of pathological processes. The asialoglycoprotein receptor (ASGP-R) on mammalian hepatocytes provides a unique means for the development of liver-specific drug or gene carriers. The abundant receptors on hepatocytes specifically recognize the natural ligands, lectin and asialofetuin (AF), as well as those with terminal galactose or N-acetylgalactosamine residues, and hepatocytes endocytose these ligands for an intracellular degradation process [27, 28]. The use of its natural or synthetic ligands, such as galactosylated cholesterol, glycolipids or galactosylated polymers to label LNPs has achieved significant targeting efficacy to the liver [4, 28]. AF-labeled LNPs have been used for improving liver-targeting gene transfer in small animals [29], yet there have not been successful reports available in the translation to large animals, such as pigs [30]. In‐ stead, plasmid DNA was directly administrated into the hepatic vein through a catheter with a balloon closure of hepatic vein blood flow [30]. One particular attention has been drawn in terms of the use of AF-labeled drug carriers for HCC targeting. The expression of ASGP-R in HCC cells varies depending on the differentiation status of HCC cells [31]. In general, well-differentiated HCC usually expresses relatively high levels of hepatocytespecific genes, including ASGP-R; whereas poorly-differentiated HCC expresses minimal or no hepatocyte-specific genes, including ASGP-R [32]. In most cases, there exists the dra‐ matic heterogeneity of liver-specific gene expression in human HCC tissues [33], and de‐ creased expression of ASGP-R was observed in liver cancer tissue [34]. Therefore, using AF or other galactosylated or lactosylated residues to label LNPs for drug or gene deliv‐ ery may not always be effective for patients with HCC, because HCC develops on a varie‐ ty of disease backgrounds and there is a striking variation in ASGP-R expression levels in HCC from different patients. Using well-differentiated hepatoma cells, such as HepG2, Hep3B and Huh-7 cells, as an *in vitro* screening tool may not necessarily reflect targeting

gans such as the liver, without significant adverse effects.

efficacy to tumor-specific distribution *in vivo* [35].

**3. Liver-specific gene delivery**

94 Gene Therapy - Tools and Potential Applications

Given the fact that hepatic stellate cells (HSCs) are the major cell type responsible for hepatic fibrosis, a repairing process that causes excess production of extracellular matrix compo‐ nents and deposition of fibrotic scaring in chronically injured liver [38], much attention has been focused on targeting this cell type in the last decade. A couple of cell surface molecules that are overexpressed on activated HSCs during hepatic fibrogenesis, such as insulin growth factor receptor II [39], collagen type VI and platelet-derived growth factor (PDGF) receptor β-subunit [40] are selected as the cell surface targets. Drug carriers labeled with specific peptides recognizing these cell surface molecules, such as cyclic peptide containing arginine-glycine-aspartate (RGD)-labeled sterical lipid nanoparticles [3] or Mannose-6-phos‐ phate human serum albumin (M6P/HAS) [41] exhibited HSC-selective distribution. The RGD cyclic peptide was recently used as a targeting molecule for the recognition of activat‐ ed HSCs in two animal models for early diagnosis of hepatic fibrosis with a SPECT imaging modality [42]. Using the retinol binding protein (RBP) in activated HSCs seems to be very effective in delivering siRNA against gp46 (rat homolog of human heat shock protein 47), and inhibiting fibrosis in two animal models [43].

Targeting approaches for drug or gene delivery to other non-parenchymal cell types, such as Kupffer cells or sinusoidal endothelial cells, are summarized recently [27]. These approaches are crucial in delivering agents which are anti-inflammatory or anti-oxidants to these cell types due to the fact that Kupffer cells are pivotal in the mediation of inflammatory respons‐ es and subsequent fibrogenesis [44].

#### **4. Polylipid nanoparticle-mediated liver gene delivery**

Compared to drug delivery, LNP-mediated *in vivo* gene delivery is still in its development stage; and many issues that affect delivery approaches and efficacy remain to be solved. The main issues include: 1) the formation of aggregates between cationic lipids and serum pro‐ teins bearing negative charges; 2) the administration routes of LNP-DNA complexes (lipo‐ plexes); 3) intracellular trafficking from the cytoplasm to the nucleus; 4) the proliferative state of cells to be transfected; and 5) transient transgene expression for a short duration [6, 45]. Substantial efforts have been made to address these issues in our previous studies and by others [8, 46, 47]. Particularly, we polymerized an acrylamide lipid to generate a polyca‐ tionic lipid (PCL), which was able to interact with plasmid DNA effectively and form com‐ pacted complexes as demonstrated by Raman microspectral analysis [8]. PCL has a unique molecular configuration and molecular weight distribution as indicated by mass spectro‐ photometrical analysis [8]. Moreover, this lipid can be synthesized in a multiple gram quan‐ tity in a laboratory, and the synthetic approach is amendable for industrial production at a quantity sufficient enough for large animal use [8]. PLNP was formulated with a neutral lip‐ id, cholesterol. The PLNP size was reduced to approximately 100 nm in diameter [7], and the Zeta potential of PLNP was decreased to neutral by neutralizing extra-positive charges with excess plasmid DNA [8]. Not only was this formulation of PLNP non-toxic, but it also displayed transfection efficiency equivalent to other commercially available transfection agents, such as Lipofectamine in hepatoma cell lines [7]. Moreover, high-resolution fluores‐ cent deconvolution microscopy documented that PLNP-mediated gene transfection led to earlier GFP expression in hepatoma cells than Lipofectamine [8]. The unique feature of this formulation is that it is extremely serum-resistant, and exposure to cell culture medium con‐ taining 50% fetal bovine serum for 24 hours did not affect its size significantly. PLNP react‐ ed up to 30-fold less with serum proteins or blood cells after intravenous administration in comparison with DOTAP-DOPE or DOTAP-Chol formulations [6]. This feature makes PLNP formulation particularly useful for *in vivo* gene transfer. In the subsequent studies, we have proved that it is very effective in the transfer of reporter genes or function genes to normal mouse livers as demonstrated in Fig. 2 by bioluminescent imaging of firefly lucifer‐ ase gene expression 24 hours after portal vein injection of PLNP-plasmid DNA complexes (polyplexes) or preclinical models [48, 49].

We also developed an approach to promote normal hepatocytes to proliferate *in situ* without partial hepatectomy, which favors the transgene expression by lipofection but is not accepta‐ ble for clinical application [6]. Furthermore, placing an indwelling catheter in the portal vein allows repeated administration of polyplexes for sustained transgene expression [6]. All these efforts render our formulation of PLNP distinct from other lipid-based nanoparticles. Our animal experiments have clearly demonstrated that PLNP is characterized as extremely stable in the bloodstream, and highly effective in liver-based gene transfer when polyplexes are administrated through the portal vein [6, 17]. In comparison with other commonly used lipid formulations of nanoparticles, our formulation possesses the notable advantages essen‐

**Characteristics PCL PLNP Lipofectamine DOTAP-Chol**

Irrelevant 125±54 358 ±85 110±20

Irrelevant >10E7 >10E7 >10E7

10±3% (>5%)

Polylipid Nanoparticle, a Novel Lipid-Based Vector for Liver Gene Transfer

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97

Low Low Obvious 20-30-fold higher than

11±3.5% (>5%)

PLNP

*In vitro* transfection *In vivo* transfection

Cationic lipid Yes LNP LNP LNP

Size changes(50%FBS) Irrelevant 100±20nm 2206 ± 311 nm 1050±100 nm

*In vivo* stability Irrelevant Stable Not determined Instability

*In vitro* or *in vivo* transfection

The content in this table was summarized according to our previous publications [6-8]. FBS = fetal bovine serum. RLU =

In order to demonstrate that our PLNP formulation is effective in delivering functional genes to the liver, we established a liver injury model in mice caused by the treatment with D-galactosamine (D-Gal) and lipopolysaccharide (LPS). This combination of D-Gal/LPS treatment resulted in a profound acute liver injury characterized by massive liver cell death through apoptosis, elevation of serum alanine aminotransferase (ALT), significant oxidant stress, depletion of the reduced form of glutathione and enhanced lipid peroxidation [50]. In

tial for *in vivo* gene delivery as illustrated in Table 1.

Cytotoxicity (LDH release) Low or none Normal

PLNP

**5. Preclinical trials for proof of the concept**

**Table 1.** Comparison of common transfection agents for *in vitro* and *in vivo* application

Usage Raw material for

relative light unit. LDH = lactate dehydrogenase.

Particle size (nm)

(in RLU)

protein

*In vitro* transfection efficiency Luciferase activity

Binding rate to serum

**Figure 2. PLNP-mediated gene transfer into mice through portal vein injection.** One day after the intravenous injec‐ tion of polyplexes with pNDLux.2 plasmid encoding the firefly luciferase gene, the animal was imaged by CCD camera. The expression of luciferase was clearly shown in the liver area, demonstrating the effectiveness of this delivery approach and the applicability of a non-invasive imaging modality in the determination of transgene expression in animals.

We also developed an approach to promote normal hepatocytes to proliferate *in situ* without partial hepatectomy, which favors the transgene expression by lipofection but is not accepta‐ ble for clinical application [6]. Furthermore, placing an indwelling catheter in the portal vein allows repeated administration of polyplexes for sustained transgene expression [6]. All these efforts render our formulation of PLNP distinct from other lipid-based nanoparticles. Our animal experiments have clearly demonstrated that PLNP is characterized as extremely stable in the bloodstream, and highly effective in liver-based gene transfer when polyplexes are administrated through the portal vein [6, 17]. In comparison with other commonly used lipid formulations of nanoparticles, our formulation possesses the notable advantages essen‐ tial for *in vivo* gene delivery as illustrated in Table 1.


The content in this table was summarized according to our previous publications [6-8]. FBS = fetal bovine serum. RLU = relative light unit. LDH = lactate dehydrogenase.

**Table 1.** Comparison of common transfection agents for *in vitro* and *in vivo* application

#### **5. Preclinical trials for proof of the concept**

tity in a laboratory, and the synthetic approach is amendable for industrial production at a quantity sufficient enough for large animal use [8]. PLNP was formulated with a neutral lip‐ id, cholesterol. The PLNP size was reduced to approximately 100 nm in diameter [7], and the Zeta potential of PLNP was decreased to neutral by neutralizing extra-positive charges with excess plasmid DNA [8]. Not only was this formulation of PLNP non-toxic, but it also displayed transfection efficiency equivalent to other commercially available transfection agents, such as Lipofectamine in hepatoma cell lines [7]. Moreover, high-resolution fluores‐ cent deconvolution microscopy documented that PLNP-mediated gene transfection led to earlier GFP expression in hepatoma cells than Lipofectamine [8]. The unique feature of this formulation is that it is extremely serum-resistant, and exposure to cell culture medium con‐ taining 50% fetal bovine serum for 24 hours did not affect its size significantly. PLNP react‐ ed up to 30-fold less with serum proteins or blood cells after intravenous administration in comparison with DOTAP-DOPE or DOTAP-Chol formulations [6]. This feature makes PLNP formulation particularly useful for *in vivo* gene transfer. In the subsequent studies, we have proved that it is very effective in the transfer of reporter genes or function genes to normal mouse livers as demonstrated in Fig. 2 by bioluminescent imaging of firefly lucifer‐ ase gene expression 24 hours after portal vein injection of PLNP-plasmid DNA complexes

**Figure 2. PLNP-mediated gene transfer into mice through portal vein injection.** One day after the intravenous injec‐ tion of polyplexes with pNDLux.2 plasmid encoding the firefly luciferase gene, the animal was imaged by CCD camera. The expression of luciferase was clearly shown in the liver area, demonstrating the effectiveness of this delivery approach and

the applicability of a non-invasive imaging modality in the determination of transgene expression in animals.

(polyplexes) or preclinical models [48, 49].

96 Gene Therapy - Tools and Potential Applications

In order to demonstrate that our PLNP formulation is effective in delivering functional genes to the liver, we established a liver injury model in mice caused by the treatment with D-galactosamine (D-Gal) and lipopolysaccharide (LPS). This combination of D-Gal/LPS treatment resulted in a profound acute liver injury characterized by massive liver cell death through apoptosis, elevation of serum alanine aminotransferase (ALT), significant oxidant stress, depletion of the reduced form of glutathione and enhanced lipid peroxidation [50]. In separate studies we have demonstrated that anti-oxidant enzyme such as extracellular su‐ peroxide dismutase (EC-SOD), SOD mimetics (MnTBAP) and catalase are effective in the prevention of hepatic toxicity caused by xenobiotics in primary hepatocytes or hepatoma cells [51-53], and they improved recipient survival and graft function and growth after small-for-size liver transplantation in rats [54]. Therefore, we chose the human EC-SOD gene as a functional gene to prove the feasibility. The EC-SOD gene product was exclusively secreted into the extracellular space and functions as an ROS scavenger. ROS are generated in both intracellular and extracellular spaces, and superoxide anions and hydrogen peroxide (H2O2) are able to cross the plasmatic membrane to enter the extracellular space [17]. It was found that two days after portal vein injection of EC-SOD polyplexes, liver EC-SOD gene expression was increased approximately 50-fold compared to the group receiving injection of control plasmid polyplexes, and serum SOD activity was increased accordingly. On the other hand, serum ALT was reduced to nearly one third in mice receiving EC-SOD polyplex injection compared to those with D-Gal/LPS challenge, along with improved liver histology, restored glutathione levels and decreased lipid peroxidation [48]. The findings of this pre‐ clinical trial confirmed the effectiveness of PLNP-mediated EC-SOD gene delivery to the liv‐ er, and that the delivery protected the mice from oxidant stress-associated liver injury. The results also indicate that this anti-oxidant gene delivery approach could be useful in attenu‐ ating xenobiotics or drug metabolite-induced toxicity to the liver.

**6. Challenges in scaling-up and moving towards clinical applications**

Our preclinical studies were performed in mice, and there are certainly a number of issues to face when this anti-oxidant gene therapy approach is considered to be evaluated in mid‐ dle or large size animals such as rabbits, dogs, monkeys or pigs. The first issue is to scale-up, which includes the plasmid DNA generation, synthesis of PCL in a quantity, and formula‐ tion of PLNP at a volume sufficient enough for the use in large animals. More challenges exist regarding how to stimulate liver cells to proliferate in large animals and deliver poly‐ plexes locally to the liver. Using a catheter through the femoral vein or jugular vein for ret‐ rograde administration into hepatic vein or passing into the portal vein for administration similar to the transjugular intrahepatic portosystemic shunt (TIPS) procedure, which is used to lower portal hypertension in cirrhotic patients, should be feasible in large animals when angiography and the administration are performed by an experienced specialist with the availability of angiographic devices. The latter method was used to administer adenoviral vector in baboons [59]. One trial of plasmid DNA injection into the hepatic vein by blocking the hepatic vein out-flow with an inflated balloon achieved high gene expression levels in selected pig liver lobes [30]. Safety concerns include amount of polyplexes to be administrat‐ ed locally and the effects of the plasmid DNA, PLNP and polyplexes on the liver as well as systematically. LNP-mediated gene transfer is usually transient; therefore, there will be less concern for long-term effects of the transgene products on the host. However, immune reac‐ tion to human gene products in animals may occur if the transgene products are produced at sustained levels for a long period of time. It is preventable by administration of immuno‐ suppressive agents, such as FK506. Moreover, innate immunity to plasmid DNA with bacte‐ rial unmethylated CG dinucleotide (CpG) can be eliminated by using CpG-free plasmid [60].

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99

An additional concern is to establish a liver injury model to evaluate the effect of anti-oxi‐ dant gene transfer by PLNP in large animals. For pigs, exposure to a loading dose of 0.25 g/kg, maintaining the blood concentration of acetaminophen at 350-450 mg/dl, and adapting enteric maintenance dose of 1,000-3,000 mg/hour resulted in the onset of acute liver failure (prothrombin time value <30%) within 32±4.4 hours, and further mortality in 15.8±2.4 hours [61]. A large dose of acetaminophen intake causes significant oxidant stress and acute liver injury due to its metabolism and generation of an interactive metabolite, n-acetyl-p-benzo‐ quinone imine (NAPQI), which binds to the cytoplasmic membrane, leads to lipid peroxida‐ tion, depletion of antioxidants, such as glutathione, and results in hepatic injury. Not only will the delivery of antioxidant genes with PLNP in a pig model of liver injury assess the therapeutic efficacy, but also take advantage of a regenerative response to the injury for high transgene expression. Alternatively, small size graft liver transplantation (SSGLT) at ≤50% graft volume could be performed in rabbits or pigs to mimic living donor liver transplanta‐ tion in humans. Significant oxidant stress-associated injury and regenerative response in the small size grafts will be the best fit for the high transgene expression and ROS scavenging property of the gene product. Therefore, SSGLT may be considered to be a valuable model for evaluating the feasibility and efficacy of anti-oxidant gene transfer for small-for-size-as‐

sociated graft failure in a transplant setting.

Ischemia/reperfusion (I/R)-associated donor organ damage is inevitable in all solid organ transplantation, and is caused by enhanced oxidant stress with release of inflammatory cytokines, such as tumor growth factor-α (TNF-α) and interleukin 2 (IL-2). Although the precise molecular mechanism of the I/R-associated liver injury remains to be investigated, enhanced oxidant stress with release of superoxide anions or H2O2, depletion of the re‐ duced form of glutathione and increased lipid peroxidation has been the key element in the pathogenesis in orthotopic liver transplantation (OLT) or small size liver graft trans‐ plantation (SSLGT) [54-56]. Thus, it is rational to use of antioxidant gene transfer to mini‐ mize oxidant stress and improve the donor organ quality and function after the implantation. We delivered either EC-SOD, catalase gene or in combination, using the same approach as described above. Two days after the delivery, the transgene expression was increased for 10-50-fold, with increased SOD or catalase activity in the mouse liver. This delivery led to a marked decrease in superoxide anion levels and H2O2 release along with a decrease in serum ALT levels, liver lipid peroxidation and dramatic improvement of liver histology [49]. This study was positively commented by two well-known hepatol‐ ogists from Europe as an editorial, quoting "beyond a proof of the principle, the study could be the basis for studies with larger animals and may help bridge the gap between the basic understanding of pathophysiologic processes in animal models towards a practi‐ cal clinical application in liver transplantation" [57]. The findings are especially applicable in living donor liver transplantation, for which small or margin donor livers were used for transplantation. Much more pronounced oxidant stress, a higher rate of graft failure, and retarded graft growth are found in small size liver transplantation than OLT [54, 58]. The margin grafts with small size or steatosis and fibrotic deposition are often used for transplantation in clinics due to severe shortage of donor organs.

#### **6. Challenges in scaling-up and moving towards clinical applications**

separate studies we have demonstrated that anti-oxidant enzyme such as extracellular su‐ peroxide dismutase (EC-SOD), SOD mimetics (MnTBAP) and catalase are effective in the prevention of hepatic toxicity caused by xenobiotics in primary hepatocytes or hepatoma cells [51-53], and they improved recipient survival and graft function and growth after small-for-size liver transplantation in rats [54]. Therefore, we chose the human EC-SOD gene as a functional gene to prove the feasibility. The EC-SOD gene product was exclusively secreted into the extracellular space and functions as an ROS scavenger. ROS are generated in both intracellular and extracellular spaces, and superoxide anions and hydrogen peroxide (H2O2) are able to cross the plasmatic membrane to enter the extracellular space [17]. It was found that two days after portal vein injection of EC-SOD polyplexes, liver EC-SOD gene expression was increased approximately 50-fold compared to the group receiving injection of control plasmid polyplexes, and serum SOD activity was increased accordingly. On the other hand, serum ALT was reduced to nearly one third in mice receiving EC-SOD polyplex injection compared to those with D-Gal/LPS challenge, along with improved liver histology, restored glutathione levels and decreased lipid peroxidation [48]. The findings of this pre‐ clinical trial confirmed the effectiveness of PLNP-mediated EC-SOD gene delivery to the liv‐ er, and that the delivery protected the mice from oxidant stress-associated liver injury. The results also indicate that this anti-oxidant gene delivery approach could be useful in attenu‐

Ischemia/reperfusion (I/R)-associated donor organ damage is inevitable in all solid organ transplantation, and is caused by enhanced oxidant stress with release of inflammatory cytokines, such as tumor growth factor-α (TNF-α) and interleukin 2 (IL-2). Although the precise molecular mechanism of the I/R-associated liver injury remains to be investigated, enhanced oxidant stress with release of superoxide anions or H2O2, depletion of the re‐ duced form of glutathione and increased lipid peroxidation has been the key element in the pathogenesis in orthotopic liver transplantation (OLT) or small size liver graft trans‐ plantation (SSLGT) [54-56]. Thus, it is rational to use of antioxidant gene transfer to mini‐ mize oxidant stress and improve the donor organ quality and function after the implantation. We delivered either EC-SOD, catalase gene or in combination, using the same approach as described above. Two days after the delivery, the transgene expression was increased for 10-50-fold, with increased SOD or catalase activity in the mouse liver. This delivery led to a marked decrease in superoxide anion levels and H2O2 release along with a decrease in serum ALT levels, liver lipid peroxidation and dramatic improvement of liver histology [49]. This study was positively commented by two well-known hepatol‐ ogists from Europe as an editorial, quoting "beyond a proof of the principle, the study could be the basis for studies with larger animals and may help bridge the gap between the basic understanding of pathophysiologic processes in animal models towards a practi‐ cal clinical application in liver transplantation" [57]. The findings are especially applicable in living donor liver transplantation, for which small or margin donor livers were used for transplantation. Much more pronounced oxidant stress, a higher rate of graft failure, and retarded graft growth are found in small size liver transplantation than OLT [54, 58]. The margin grafts with small size or steatosis and fibrotic deposition are often used for

ating xenobiotics or drug metabolite-induced toxicity to the liver.

98 Gene Therapy - Tools and Potential Applications

transplantation in clinics due to severe shortage of donor organs.

Our preclinical studies were performed in mice, and there are certainly a number of issues to face when this anti-oxidant gene therapy approach is considered to be evaluated in mid‐ dle or large size animals such as rabbits, dogs, monkeys or pigs. The first issue is to scale-up, which includes the plasmid DNA generation, synthesis of PCL in a quantity, and formula‐ tion of PLNP at a volume sufficient enough for the use in large animals. More challenges exist regarding how to stimulate liver cells to proliferate in large animals and deliver poly‐ plexes locally to the liver. Using a catheter through the femoral vein or jugular vein for ret‐ rograde administration into hepatic vein or passing into the portal vein for administration similar to the transjugular intrahepatic portosystemic shunt (TIPS) procedure, which is used to lower portal hypertension in cirrhotic patients, should be feasible in large animals when angiography and the administration are performed by an experienced specialist with the availability of angiographic devices. The latter method was used to administer adenoviral vector in baboons [59]. One trial of plasmid DNA injection into the hepatic vein by blocking the hepatic vein out-flow with an inflated balloon achieved high gene expression levels in selected pig liver lobes [30]. Safety concerns include amount of polyplexes to be administrat‐ ed locally and the effects of the plasmid DNA, PLNP and polyplexes on the liver as well as systematically. LNP-mediated gene transfer is usually transient; therefore, there will be less concern for long-term effects of the transgene products on the host. However, immune reac‐ tion to human gene products in animals may occur if the transgene products are produced at sustained levels for a long period of time. It is preventable by administration of immuno‐ suppressive agents, such as FK506. Moreover, innate immunity to plasmid DNA with bacte‐ rial unmethylated CG dinucleotide (CpG) can be eliminated by using CpG-free plasmid [60].

An additional concern is to establish a liver injury model to evaluate the effect of anti-oxi‐ dant gene transfer by PLNP in large animals. For pigs, exposure to a loading dose of 0.25 g/kg, maintaining the blood concentration of acetaminophen at 350-450 mg/dl, and adapting enteric maintenance dose of 1,000-3,000 mg/hour resulted in the onset of acute liver failure (prothrombin time value <30%) within 32±4.4 hours, and further mortality in 15.8±2.4 hours [61]. A large dose of acetaminophen intake causes significant oxidant stress and acute liver injury due to its metabolism and generation of an interactive metabolite, n-acetyl-p-benzo‐ quinone imine (NAPQI), which binds to the cytoplasmic membrane, leads to lipid peroxida‐ tion, depletion of antioxidants, such as glutathione, and results in hepatic injury. Not only will the delivery of antioxidant genes with PLNP in a pig model of liver injury assess the therapeutic efficacy, but also take advantage of a regenerative response to the injury for high transgene expression. Alternatively, small size graft liver transplantation (SSGLT) at ≤50% graft volume could be performed in rabbits or pigs to mimic living donor liver transplanta‐ tion in humans. Significant oxidant stress-associated injury and regenerative response in the small size grafts will be the best fit for the high transgene expression and ROS scavenging property of the gene product. Therefore, SSGLT may be considered to be a valuable model for evaluating the feasibility and efficacy of anti-oxidant gene transfer for small-for-size-as‐ sociated graft failure in a transplant setting.

In summary, moving promising PLNP-mediated antioxidant gene transfer from small ani‐ mals to large animals may face more challenges than discussed above, and it is even more challenging when further considering for clinical use, in terms of safety concern and admin‐ istrative approval. Fig. 3 provides a schematic illustration of the roadmap from bench to bedsides of a potential biological therapy. The reality is that with limited funding opportu‐ nities from governmental or private agencies, to cope with multi-facet challenges at a large scale, it is less likely to reach the final goal in a short term. Attracting financial investments and taking advantages of cutting-edging technologies and vast resources from biopharma‐ ceutical companies may advance this process in a fast pace. In this context, the net benefits would be the early clinical application of this promising antioxidant gene transfer in pa‐ tients with critical needs and the financial return from the investment. We would foresee such a movement occurring in the near future.

vectors, such as lipid nanoparticles (LNPs) possess their own drawbacks when they are con‐ sidered for *in vivo* use. One prominent issue is the interaction of cationic LNPs with serum protein and blood cells, and this causes a series of issues, such as instability of the lipoplexes or polyplexes and adverse effects to the host, including non-preferential distribution, embo‐ lism of the aggregates of lipoplex-protein or blood cells, and inflammatory responses. For these reasons, many gene transfer agents are very effective in cell culture; whereas they have less applicability *in vivo*. Up to date, only a few formulations of cationic LNPs have proved to be effective and safe in animals and have reached the stage of clinical trials, such as DO‐ TAP-Chol and DC-Chol. Our PLNP formulation has a superior stability profile, and dis‐ played much less reactivity to serum proteins and blood cells when compared to other commercially available formulations. At the same time, it has proved to be the most effec‐ tive liver-based gene transfer agent [6]. Two preclinical trials with different models of oxi‐ dant-stress-associated liver injury have demonstrated the effectiveness of the anti-oxidant gene delivery in the liver, and the efficacy of the gene delivery in minimizing oxidant-stress, attenuating liver cell death, and improving liver histology [48, 49]. Further efforts have been made to move this promising PLNP-mediated anti-oxidant gene transfer technology from bench to bedside. The strategies in pushing this movement towards clinical trials include: 1) Scaling-up of the polycationic lipid production and generation of PLNPs; 2) Generation of specific antioxidant gene plasmids in a GMP facility at the standard for clinical use; 3) Estab‐ lishing large animal models for safety and efficacy assessment; and 4) Preparation for ob‐ taining administrative approval of clinical application. Although the clinical translation of this potential technology will need tremendous efforts, we anticipate that this technology will eventually reach to patients with critical needs as a novel therapy. Potential indications which may benefit from this therapy range from alcohol or drug toxicity to living donor liv‐ er transplantation with a margin graft. This technology is also applicable in oxidant stressassociated disorders in other systems, such as ischemic cardiac, pulmonary, brain or renal damage, etc. [17]. With the combination of our extensive expertise in drug and gene deliv‐ ery, advanced knowledge and skills in liver injury, fibrosis, transplant and cancer research and practice, in addition to the engine of financial investment from various sources, such as venture capital and governmental support in entrepreneurship, we are optimistic to foresee the benefits of this technology in indicated patients in a near future. Nevertheless, the road to reach this goal will not be smooth, and various challenges demand powerful solutions.

Polylipid Nanoparticle, a Novel Lipid-Based Vector for Liver Gene Transfer

http://dx.doi.org/10.5772/54270

101

The studies presented in the chapter were supported by the UC Davis Health System Award, American Liver Foundation Liver Scholar Award, UC Davis Technology Transfer Award, and the National Institute of Diabetes, Digestive and Kidney Diseases (DK069939) to JW. The commercialization and translation to clinical application of this technology is sup‐ ported by the Nanjing Municipal Innovative Technology Award (321 Plan). Yahan Fan is the

recipient of China Scholarship Council Award (201207610003).

**Acknowledgements**

**Figure 3. Translation roadmap of a potential gene therapy platform from bench to bedside.** This illustration summarizes the major steps in moving a potential gene transfer approach from laboratory research to clinical trials. The actual actions could be more complicated than described. However, for the guarantee of patient safety, each new therapeutic agent must be well characterized, and evaluated in preclinical settings, and then move to large animals for feasibility assessment. The balance between therapeutic benefits and potential risks of an innovative therapy plat‐ form always leans on the patient safety as the first priority.

#### **7. Conclusion and prospectives**

Non-viral vector-mediated gene transfer has less concern in terms of integration-associated long-term transgene expression and insertion-induced mutation. In general, non-viral vector elicits minimal immune responses in contrast to adenoviral vectors [17]. However, non-viral vectors, such as lipid nanoparticles (LNPs) possess their own drawbacks when they are con‐ sidered for *in vivo* use. One prominent issue is the interaction of cationic LNPs with serum protein and blood cells, and this causes a series of issues, such as instability of the lipoplexes or polyplexes and adverse effects to the host, including non-preferential distribution, embo‐ lism of the aggregates of lipoplex-protein or blood cells, and inflammatory responses. For these reasons, many gene transfer agents are very effective in cell culture; whereas they have less applicability *in vivo*. Up to date, only a few formulations of cationic LNPs have proved to be effective and safe in animals and have reached the stage of clinical trials, such as DO‐ TAP-Chol and DC-Chol. Our PLNP formulation has a superior stability profile, and dis‐ played much less reactivity to serum proteins and blood cells when compared to other commercially available formulations. At the same time, it has proved to be the most effec‐ tive liver-based gene transfer agent [6]. Two preclinical trials with different models of oxi‐ dant-stress-associated liver injury have demonstrated the effectiveness of the anti-oxidant gene delivery in the liver, and the efficacy of the gene delivery in minimizing oxidant-stress, attenuating liver cell death, and improving liver histology [48, 49]. Further efforts have been made to move this promising PLNP-mediated anti-oxidant gene transfer technology from bench to bedside. The strategies in pushing this movement towards clinical trials include: 1) Scaling-up of the polycationic lipid production and generation of PLNPs; 2) Generation of specific antioxidant gene plasmids in a GMP facility at the standard for clinical use; 3) Estab‐ lishing large animal models for safety and efficacy assessment; and 4) Preparation for ob‐ taining administrative approval of clinical application. Although the clinical translation of this potential technology will need tremendous efforts, we anticipate that this technology will eventually reach to patients with critical needs as a novel therapy. Potential indications which may benefit from this therapy range from alcohol or drug toxicity to living donor liv‐ er transplantation with a margin graft. This technology is also applicable in oxidant stressassociated disorders in other systems, such as ischemic cardiac, pulmonary, brain or renal damage, etc. [17]. With the combination of our extensive expertise in drug and gene deliv‐ ery, advanced knowledge and skills in liver injury, fibrosis, transplant and cancer research and practice, in addition to the engine of financial investment from various sources, such as venture capital and governmental support in entrepreneurship, we are optimistic to foresee the benefits of this technology in indicated patients in a near future. Nevertheless, the road to reach this goal will not be smooth, and various challenges demand powerful solutions.

#### **Acknowledgements**

In summary, moving promising PLNP-mediated antioxidant gene transfer from small ani‐ mals to large animals may face more challenges than discussed above, and it is even more challenging when further considering for clinical use, in terms of safety concern and admin‐ istrative approval. Fig. 3 provides a schematic illustration of the roadmap from bench to bedsides of a potential biological therapy. The reality is that with limited funding opportu‐ nities from governmental or private agencies, to cope with multi-facet challenges at a large scale, it is less likely to reach the final goal in a short term. Attracting financial investments and taking advantages of cutting-edging technologies and vast resources from biopharma‐ ceutical companies may advance this process in a fast pace. In this context, the net benefits would be the early clinical application of this promising antioxidant gene transfer in pa‐ tients with critical needs and the financial return from the investment. We would foresee

**Figure 3. Translation roadmap of a potential gene therapy platform from bench to bedside.** This illustration summarizes the major steps in moving a potential gene transfer approach from laboratory research to clinical trials. The actual actions could be more complicated than described. However, for the guarantee of patient safety, each new therapeutic agent must be well characterized, and evaluated in preclinical settings, and then move to large animals for feasibility assessment. The balance between therapeutic benefits and potential risks of an innovative therapy plat‐

Non-viral vector-mediated gene transfer has less concern in terms of integration-associated long-term transgene expression and insertion-induced mutation. In general, non-viral vector elicits minimal immune responses in contrast to adenoviral vectors [17]. However, non-viral

such a movement occurring in the near future.

100 Gene Therapy - Tools and Potential Applications

form always leans on the patient safety as the first priority.

**7. Conclusion and prospectives**

The studies presented in the chapter were supported by the UC Davis Health System Award, American Liver Foundation Liver Scholar Award, UC Davis Technology Transfer Award, and the National Institute of Diabetes, Digestive and Kidney Diseases (DK069939) to JW. The commercialization and translation to clinical application of this technology is sup‐ ported by the Nanjing Municipal Innovative Technology Award (321 Plan). Yahan Fan is the recipient of China Scholarship Council Award (201207610003).

#### **Abbreviations used in the chapter**

ASGP-R = asialoglycoprotein receptor; DOTAP = (dioleoyloxy)-3-(trimethylamonio) pro‐ pane; DOPE = L-a dioleoyl phosphatidylethanolamine; EC-SOD = extracellular superoxide dismutase; HCC = hepatocellular carcinoma; LDLT = Living donor liver transplantation; LNP = lipid nanoparticles; OLT = orthotopic liver transplantation; PEG = polyethylene gly‐ col; PLNP = polylipid nanoparticles; polyplex = PLNP-plasmid DNA complex; RES = reticu‐ loendothelial system.

[7] Wu J, Lizarzaburu ME, Kurth MJ, Liu L, Wege H, Zern MA, Nantz MH. Cationic lip‐ id polymerization as a novel approach for constructing new DNA delivery agents.

Polylipid Nanoparticle, a Novel Lipid-Based Vector for Liver Gene Transfer

http://dx.doi.org/10.5772/54270

103

[8] Nyunt MT, Dicus CW, Cui YY, Yappert MC, Huser TR, Nantz MH, Wu J. Physicochemical characterization of polylipid nanoparticles for gene delivery to the liver. Bi‐

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[10] Ito I, Ji L, Tanaka F, Saito Y, Gopalan B, Branch CD, Xu K, et al. Liposomal vector mediated delivery of the 3p FUS1 gene demonstrates potent antitumor activity

[11] Sakurai F, Nishioka T, Saito H, Baba T, Okuda A, Matsumoto O, Taga T, et al. Inter‐ action between DNA-cationic liposome complexes and erythrocytes is an important factor in systemic gene transfer via the intravenous route in mice: the role of the neu‐

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[15] Schleh C, Rothen-Rutishauser B, Kreyling WG. The influence of pulmonary surfac‐ tant on nanoparticulate drug delivery systems. Eur J Pharm Biopharm

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#### **Author details**

Yahan Fan1,2\* and Jian Wu1

\*Address all correspondence to: jdwu@ucdavis.edu.

1 Dept. of Internal Medicine, Division of Gastroenterology & Hepatology, University of California, Davis Medical Center, Sacramento, CA, USA

2 Dept. of Internal Medicine, Division of Gastroenterology, Xinqiao Hospital, The Third Military Medical University, Chongqin, P. R. China

#### **References**


[7] Wu J, Lizarzaburu ME, Kurth MJ, Liu L, Wege H, Zern MA, Nantz MH. Cationic lip‐ id polymerization as a novel approach for constructing new DNA delivery agents. Bioconjug Chem 2001;12:251-257.

**Abbreviations used in the chapter**

102 Gene Therapy - Tools and Potential Applications

\*Address all correspondence to: jdwu@ucdavis.edu.

Military Medical University, Chongqin, P. R. China

California, Davis Medical Center, Sacramento, CA, USA

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**Author details**

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ASGP-R = asialoglycoprotein receptor; DOTAP = (dioleoyloxy)-3-(trimethylamonio) pro‐ pane; DOPE = L-a dioleoyl phosphatidylethanolamine; EC-SOD = extracellular superoxide dismutase; HCC = hepatocellular carcinoma; LDLT = Living donor liver transplantation; LNP = lipid nanoparticles; OLT = orthotopic liver transplantation; PEG = polyethylene gly‐ col; PLNP = polylipid nanoparticles; polyplex = PLNP-plasmid DNA complex; RES = reticu‐

1 Dept. of Internal Medicine, Division of Gastroenterology & Hepatology, University of

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**Chapter 6**

**DNA Electrotransfer: An Effective Tool for Gene**

The concept of gene therapy was first introduced in the mid-80s, and is based on the deliv‐ ery of genetic material (DNA or RNA) in the nucleus of patient cells, so that it is expressed

**•** Correcting defective function by supplying a functional gene to the cells, thereby directly

**•** Transferring a gene encoding a therapeutic protein, in order to treat, prevent or slow the

**•** Introducing antisense DNA inhibiting the formation of a protein or the replication of a vi‐

Originally developed for monogenic diseases, and therefore associated with the compensa‐ tion of genes whose alteration is responsible for diseases, the concept of gene therapy has rapidly expanded to the use of DNA as a new type of drug. Therefore, gene therapy leads to indications which are far beyond the case of genetic diseases, since a DNA drug can, in prin‐ ciple, replace any medication which will control protein synthesis. Gene therapy seems an alternative choice to fight against diseases currently treated imperfectly, or not treated with

In addition, gene therapy has many advantages compared to the administration of recombi‐ nant proteins. Indeed, recombinant proteins are costly and their elimination from the blood

> © 2013 Burgain-Chain and Scherman; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is

Aurore Burgain-Chain and Daniel Scherman

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52528

and produces a therapeutic effect.

Different approaches can be considered:

progression of certain diseases.

conventional pharmaceutical approaches.

properly cited.

addressing the cause of a genetic disease.

**•** Introducing a gene leading to the death of a diseased cell

**1. Introduction**

rus

**Therapy**

## **DNA Electrotransfer: An Effective Tool for Gene Therapy**

Aurore Burgain-Chain and Daniel Scherman

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52528

**1. Introduction**

The concept of gene therapy was first introduced in the mid-80s, and is based on the deliv‐ ery of genetic material (DNA or RNA) in the nucleus of patient cells, so that it is expressed and produces a therapeutic effect.

Different approaches can be considered:


Originally developed for monogenic diseases, and therefore associated with the compensa‐ tion of genes whose alteration is responsible for diseases, the concept of gene therapy has rapidly expanded to the use of DNA as a new type of drug. Therefore, gene therapy leads to indications which are far beyond the case of genetic diseases, since a DNA drug can, in prin‐ ciple, replace any medication which will control protein synthesis. Gene therapy seems an alternative choice to fight against diseases currently treated imperfectly, or not treated with conventional pharmaceutical approaches.

In addition, gene therapy has many advantages compared to the administration of recombi‐ nant proteins. Indeed, recombinant proteins are costly and their elimination from the blood

© 2013 Burgain-Chain and Scherman; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

flow is fast, while gene therapy leads to a long-term and potentially regulated production of a therapeutic protein. Gene therapy also allows the localized expression of the transgene, avoiding any risk associated with the presence of a systemic exogenous protein.

lization and permeabilization of the plasma membrane of suspended cells, thus promoting the entry of exogenous DNA into these cells. Two years later [3], the confirmation of this re‐ sult opened the way for the development of electroporation (or electropermeabilization) into bacterial [4], fungal [5] vegetal or animal cells. This method is routinely used now. The opti‐ mization of electrical parameters is critical to allow transient permeabilization, together with

DNA Electrotransfer: An Effective Tool for Gene Therapy

http://dx.doi.org/10.5772/52528

111

In initial studies, *in vivo* DNA electrotransfer has been tested in the skin in 1991, by the use of exponentially decaying electrical pulses, and in 1996 in the liver using trains of short 100 µs pulses [7]. In 1998, four independent teams showed the effectiveness of electrotransfer using pulses of long duration (5-50ms): in skeletal muscle, our team in collaboration with that of Luis Mir [8] and Aihara [9], in tumors, Rols *et al.* [10] and in liver Suzuki *et al.* [11]. *In vivo* DNA electrotransfer has now been successfully used in a broad range of target tissues and organs including for example : arteries [12], skin [13], tendon [14], bladder [15], cornea

Electropermeabilization can also be used to deliver chemical drugs into the cells: e.g. electro‐ chemotherapy in tumors, with the use of bleomycin, developed since 1991 [20]. Several clini‐ cal trials are underway [21], primarily for the treatment of subcutaneous or skin tumors [22, 23] and recently for the treatment of breast cancer with cisplatin [24] (For a review see [25]).

The technique of electroporation for the transfer of nucleic acids has been used since the 80s, however its exact mechanism is not yet completely elucidated [26, 27]. At the cell level, it seems that two phenomena occur: first the permeabilization of the cell to small molecules, probably due to a destabilization of the cell membrane, and secondly the transport of DNA

The lipid bi-layer of the plasma membrane separates two solutions with very high ionic con‐ ductivity: the cytoplasm and the extracellular medium. Typically, at rest, the membrane po‐ tential difference (ΔVm0) is around -70mV. When an electric field is applied to a cell, the resulting current induces an accumulation of electric charges at the cell membrane which leads to a variation of thistransmembrane potential. And if the transmembrane potential ex‐ ceeds a certain threshold value, the cell membrane is disorganized and structural changes

Shall the cell be considered a hollow sphere where the thickness of the membrane is negligi‐ ble vis-à-vis the cell radius, then the transmembrane potential difference ΔVm induced by

a satisfactory cell survival rate [6].

by electrophoresis.

**3.1. Permeabilization**

[16], the retinal cells [17], spinal cord [18]and brain [19].

**3. Mechanism of electrotransfer at the cell level**

occur. That is a necessary condition for an effective gene transfer [28].

an electric field is, as described by Schwann's equation:

The main limitation of current gene therapy is the development of effective gene transfer. Indeed, in order to reach the cell nucleus, the therapeutic gene has to cross several biological barriers. Therefore, the success of any gene therapy requires the development of efficient and appropriate methods and vectors for introducing the gene of interest into target cells. The ideal vehicle for gene transfer must have the following properties: (1) specificity to tar‐ get cells, (2) localized gene delivery, (3) resistance to metabolic degradation and/or attack by the immune system, (4) minimum side effects, and (5) eventually controlled temporal trans‐ gene expression [1].

Many methods of *in vivo* gene transfer exist and are generally classified into two main cate‐ gories: viral and non viral. Viruses are very efficient vehicles for gene transfer; however their use is limited by high production costs and safety concerns, such as immune response, possible pathogen reversion, mutagenesis and carcinogenesis. Considering these limitations, the delivery of therapeutic genes to target cells by non viral approaches may be of great val‐ ue for the development of gene therapy. Among these approaches, *in vivo* electroporation, also called *in vivo* electropermeabilization or *in vivo* electrotransfer, has proven to be one of the simplest and most efficient methods for gene therapy, while at the same time being safe, cheap, and easy to perform.

*In vivo* electrotransfer is a recent physical technique for gene delivery in various tissues and organs, which relies on the combination of plasmid injection and delivery of short and de‐ fined electric pulses. This process results in the association between cell permeabilization and DNA electrophoresis. Skeletal muscle have now been frequently electrotransfered, since it offers promising treatment for muscle disorders, but also a way for systemic secretion of therapeutic proteins, by converting skeletal muscles into an endocrine organ: the protein produced can diffuse into the vascular system and circulate throughout the body to exert a physiological and potentially therapeutic effect. Many published studies have demonstrated that plasmid electrotransfer can lead to long-lasting therapeutic effects in various patholo‐ gies such as cancer, rheumatoid arthritis, muscle and blood disorders, cardiac diseases, etc... Indeed, the physical method of electrotransfer allows for greater efficiency of gene transfer after a single injection and improves protein expression by several orders of magnitude, as compared to DNA injected in the absence of electrotransfer. Therefore, plasmid electrotrans‐ fer can be considered a powerful tool for gene therapy.

The scope of this chapter encompasses the methods of electrotransfer, its implementation, mechanism, optimization and therapeutic applications.

#### **2. Description of the electrotransfer technique**

In 1982, E. Neumann and his collaborators demonstrated *in vitro* the possibility of introduc‐ ing DNA into cells using electrical pulses [2]. These electric pulses would cause the destabi‐ lization and permeabilization of the plasma membrane of suspended cells, thus promoting the entry of exogenous DNA into these cells. Two years later [3], the confirmation of this re‐ sult opened the way for the development of electroporation (or electropermeabilization) into bacterial [4], fungal [5] vegetal or animal cells. This method is routinely used now. The opti‐ mization of electrical parameters is critical to allow transient permeabilization, together with a satisfactory cell survival rate [6].

In initial studies, *in vivo* DNA electrotransfer has been tested in the skin in 1991, by the use of exponentially decaying electrical pulses, and in 1996 in the liver using trains of short 100 µs pulses [7]. In 1998, four independent teams showed the effectiveness of electrotransfer using pulses of long duration (5-50ms): in skeletal muscle, our team in collaboration with that of Luis Mir [8] and Aihara [9], in tumors, Rols *et al.* [10] and in liver Suzuki *et al.* [11]. *In vivo* DNA electrotransfer has now been successfully used in a broad range of target tissues and organs including for example : arteries [12], skin [13], tendon [14], bladder [15], cornea [16], the retinal cells [17], spinal cord [18]and brain [19].

Electropermeabilization can also be used to deliver chemical drugs into the cells: e.g. electro‐ chemotherapy in tumors, with the use of bleomycin, developed since 1991 [20]. Several clini‐ cal trials are underway [21], primarily for the treatment of subcutaneous or skin tumors [22, 23] and recently for the treatment of breast cancer with cisplatin [24] (For a review see [25]).

#### **3. Mechanism of electrotransfer at the cell level**

The technique of electroporation for the transfer of nucleic acids has been used since the 80s, however its exact mechanism is not yet completely elucidated [26, 27]. At the cell level, it seems that two phenomena occur: first the permeabilization of the cell to small molecules, probably due to a destabilization of the cell membrane, and secondly the transport of DNA by electrophoresis.

#### **3.1. Permeabilization**

flow is fast, while gene therapy leads to a long-term and potentially regulated production of a therapeutic protein. Gene therapy also allows the localized expression of the transgene,

The main limitation of current gene therapy is the development of effective gene transfer. Indeed, in order to reach the cell nucleus, the therapeutic gene has to cross several biological barriers. Therefore, the success of any gene therapy requires the development of efficient and appropriate methods and vectors for introducing the gene of interest into target cells. The ideal vehicle for gene transfer must have the following properties: (1) specificity to tar‐ get cells, (2) localized gene delivery, (3) resistance to metabolic degradation and/or attack by the immune system, (4) minimum side effects, and (5) eventually controlled temporal trans‐

Many methods of *in vivo* gene transfer exist and are generally classified into two main cate‐ gories: viral and non viral. Viruses are very efficient vehicles for gene transfer; however their use is limited by high production costs and safety concerns, such as immune response, possible pathogen reversion, mutagenesis and carcinogenesis. Considering these limitations, the delivery of therapeutic genes to target cells by non viral approaches may be of great val‐ ue for the development of gene therapy. Among these approaches, *in vivo* electroporation, also called *in vivo* electropermeabilization or *in vivo* electrotransfer, has proven to be one of the simplest and most efficient methods for gene therapy, while at the same time being safe,

*In vivo* electrotransfer is a recent physical technique for gene delivery in various tissues and organs, which relies on the combination of plasmid injection and delivery of short and de‐ fined electric pulses. This process results in the association between cell permeabilization and DNA electrophoresis. Skeletal muscle have now been frequently electrotransfered, since it offers promising treatment for muscle disorders, but also a way for systemic secretion of therapeutic proteins, by converting skeletal muscles into an endocrine organ: the protein produced can diffuse into the vascular system and circulate throughout the body to exert a physiological and potentially therapeutic effect. Many published studies have demonstrated that plasmid electrotransfer can lead to long-lasting therapeutic effects in various patholo‐ gies such as cancer, rheumatoid arthritis, muscle and blood disorders, cardiac diseases, etc... Indeed, the physical method of electrotransfer allows for greater efficiency of gene transfer after a single injection and improves protein expression by several orders of magnitude, as compared to DNA injected in the absence of electrotransfer. Therefore, plasmid electrotrans‐

The scope of this chapter encompasses the methods of electrotransfer, its implementation,

In 1982, E. Neumann and his collaborators demonstrated *in vitro* the possibility of introduc‐ ing DNA into cells using electrical pulses [2]. These electric pulses would cause the destabi‐

avoiding any risk associated with the presence of a systemic exogenous protein.

gene expression [1].

110 Gene Therapy - Tools and Potential Applications

cheap, and easy to perform.

fer can be considered a powerful tool for gene therapy.

mechanism, optimization and therapeutic applications.

**2. Description of the electrotransfer technique**

The lipid bi-layer of the plasma membrane separates two solutions with very high ionic con‐ ductivity: the cytoplasm and the extracellular medium. Typically, at rest, the membrane po‐ tential difference (ΔVm0) is around -70mV. When an electric field is applied to a cell, the resulting current induces an accumulation of electric charges at the cell membrane which leads to a variation of thistransmembrane potential. And if the transmembrane potential ex‐ ceeds a certain threshold value, the cell membrane is disorganized and structural changes occur. That is a necessary condition for an effective gene transfer [28].

Shall the cell be considered a hollow sphere where the thickness of the membrane is negligi‐ ble vis-à-vis the cell radius, then the transmembrane potential difference ΔVm induced by an electric field is, as described by Schwann's equation:

$$
\Delta \mathbf{V} \mathbf{m} = \text{f.g.r.E.cosq.(1-exp(-t/t))}\tag{1}
$$

The membrane is off-balance and becomes transiently permeable when the sum: ΔVm<sup>0</sup> (at rest) + ΔVm (induced) reaches a threshold value of about 200mV [29]. Thus, the greater the difference between the threshold value and the value applied, the greater the surface area is permeabilized. However for a given electric field, beyond a certain angle, the ΔVm falls be‐ low the threshold value of permeabilization. The relationship between the applied electric field and the permeabilized surface was demonstrated by *in vitro* fluorescent labeling of per‐ meabilized areas of the cell [30]. Moreover, these studies have shown that it is the face of the cell toward the anode side which is permeabilized first, the negative potential of the cell be‐

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113

One theory suggests that the DNA enters into the cell through pores which are generated by electrical stimulation [32]. The electropermeabilization creates relatively stable "electro‐ pores" [2, 33]. But these pores have never been visualized. The plasmid DNA may optionally

The second phenomenon necessary for gene transfer by electroporation is the electrophore‐

The occurrence of an electrophoretic process has been demonstrated *in vitro* [31]. Various stud‐ ies have shown this electrophoretic effect: Klenchin *et al.* demonstrated that DNA has to be present at the time of the pulses [31]. Furthermore, they showed that the transfection efficien‐ cy depends on the polarity of the electric field. Sukharev *et al.* also showed *in vitro* that short pulses of high voltage (HV) induce membrane permeabilization but not transfection, whereas long pulses at low voltage (LV) do not induce permeabilization or transfection. However, the sequence "high voltage pulses followed by low voltage pulses" provides a transfection. An hy‐ pothesis is proposed that transfection of cells permeabilized by high voltage is only possible if

The role of permeabilization and electrophoresis was demonstrated directly at the cell level by fluorescence microscopy [35]. This work shows that interaction between the membrane and electropermeabilized DNA is induced in response to electrical pulses of a few millisec‐ onds. DNA electrophoretically accumulates on the cathode side of the cell without immedi‐ ately moving into the cytosol (Figure 1). Thus DNA must be present during the pulse and electrophoresis induced by the electric field promotes its transfer through the membrane, but it is only during the following minute that DNA crosses the electropermeabilized mem‐ brane [36]. There is a direct relationship between the DNA/membrane interaction and trans‐ fection efficiency: the larger the contact surface between DNA and the membrane, the higher

In the early 90's, the first studies about *in vivo* electroporation appeared. They primarily con‐ cerned the transfer of chemical molecules. The first real demonstration of *in vivo* cellular

pass the membrane after a step of binding to the surface of the cell and by diffusion.

low voltage pulses can subsequently mediate DNA electrophoresis [34].

ing in addition to that induced by the external electric field.

sis of negatively charged DNA.

**3.2. DNA electrophoresis**

is the expression [27].

**4. Mechanism of** *in vivo* **electrotransfer**

Thus, the transmembrane potential difference ΔVm is proportional to


If the membrane is seen as a pure dielectric object, g is equal to 1. Under the conditions used for cellular electroporation, the pulse duration is significantly longer (of a few hundred mi‐ croseconds to a few milliseconds) than the charging time constant of the cell, which is of the order of a few microseconds. The equation can be simplified to:

$$
\Delta \text{Vm} = \text{f. r.E.cosq} \tag{2}
$$

This transmembrane potential difference ΔVm is not uniform on the surface of the cell: the induced transmembrane potential is maximal at the points of the cell facing the electrodes (θ = 0 and π).

**Figure 1.** Theoretical model of the cell for electroporation: E, the electrical potential induces ΔVm, a transmembrane potential difference which dependent on r, the radius of the cell and θ, the angle between the direction of electric field and the normal to the tangent of the membrane of the cell at this point

The membrane is off-balance and becomes transiently permeable when the sum: ΔVm<sup>0</sup> (at rest) + ΔVm (induced) reaches a threshold value of about 200mV [29]. Thus, the greater the difference between the threshold value and the value applied, the greater the surface area is permeabilized. However for a given electric field, beyond a certain angle, the ΔVm falls be‐ low the threshold value of permeabilization. The relationship between the applied electric field and the permeabilized surface was demonstrated by *in vitro* fluorescent labeling of per‐ meabilized areas of the cell [30]. Moreover, these studies have shown that it is the face of the cell toward the anode side which is permeabilized first, the negative potential of the cell be‐ ing in addition to that induced by the external electric field.

One theory suggests that the DNA enters into the cell through pores which are generated by electrical stimulation [32]. The electropermeabilization creates relatively stable "electro‐ pores" [2, 33]. But these pores have never been visualized. The plasmid DNA may optionally pass the membrane after a step of binding to the surface of the cell and by diffusion.

The second phenomenon necessary for gene transfer by electroporation is the electrophore‐ sis of negatively charged DNA.

#### **3.2. DNA electrophoresis**

D = Vm f.g.r.E.cosq. 1-exp -t( ( ))/t (1)

DVm = f. r.E.cosq (2)

Thus, the transmembrane potential difference ΔVm is proportional to

**•** the magnitude of the electric field (E) (expressed in volts/cm)

**•** the pulse duration for which the electric field is applied (t)

order of a few microseconds. The equation can be simplified to:

If the membrane is seen as a pure dielectric object, g is equal to 1. Under the conditions used for cellular electroporation, the pulse duration is significantly longer (of a few hundred mi‐ croseconds to a few milliseconds) than the charging time constant of the cell, which is of the

This transmembrane potential difference ΔVm is not uniform on the surface of the cell: the induced transmembrane potential is maximal at the points of the cell facing the electrodes (θ

**Figure 1.** Theoretical model of the cell for electroporation: E, the electrical potential induces ΔVm, a transmembrane potential difference which dependent on r, the radius of the cell and θ, the angle between the direction of electric

field and the normal to the tangent of the membrane of the cell at this point

**•** the cell radius (r)

**•** a cell shape factor (f)

= 0 and π).

**•** the cosine of (θ), its incidence angle,

112 Gene Therapy - Tools and Potential Applications

**•** the conductivity of the medium (g)

**•** the charging time constant of the cell (τ).

The occurrence of an electrophoretic process has been demonstrated *in vitro* [31]. Various stud‐ ies have shown this electrophoretic effect: Klenchin *et al.* demonstrated that DNA has to be present at the time of the pulses [31]. Furthermore, they showed that the transfection efficien‐ cy depends on the polarity of the electric field. Sukharev *et al.* also showed *in vitro* that short pulses of high voltage (HV) induce membrane permeabilization but not transfection, whereas long pulses at low voltage (LV) do not induce permeabilization or transfection. However, the sequence "high voltage pulses followed by low voltage pulses" provides a transfection. An hy‐ pothesis is proposed that transfection of cells permeabilized by high voltage is only possible if low voltage pulses can subsequently mediate DNA electrophoresis [34].

The role of permeabilization and electrophoresis was demonstrated directly at the cell level by fluorescence microscopy [35]. This work shows that interaction between the membrane and electropermeabilized DNA is induced in response to electrical pulses of a few millisec‐ onds. DNA electrophoretically accumulates on the cathode side of the cell without immedi‐ ately moving into the cytosol (Figure 1). Thus DNA must be present during the pulse and electrophoresis induced by the electric field promotes its transfer through the membrane, but it is only during the following minute that DNA crosses the electropermeabilized mem‐ brane [36]. There is a direct relationship between the DNA/membrane interaction and trans‐ fection efficiency: the larger the contact surface between DNA and the membrane, the higher is the expression [27].

#### **4. Mechanism of** *in vivo* **electrotransfer**

In the early 90's, the first studies about *in vivo* electroporation appeared. They primarily con‐ cerned the transfer of chemical molecules. The first real demonstration of *in vivo* cellular electropermeabilization was performed on tumors after injection of bleomycin, a cytotoxic anticancer agent, [22, 37]. The effectiveness of bleomycin depends on its intracellular con‐ centration, but this drug penetrates poorly into cells. Therefore, a better penetration of bleo‐ mycin was measured after application of electric pulses to tumors, leading to an enhanced desired cytotoxicity.

spective contribution could help to develop more effective electrotransfer strategies and

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115

The *in vivo* electrotransfer technique is particularly easy to implement: a solution of plasmid DNA (i. e. a circular nucleic acid) in isotonic saline (NaCl, 150mM) is injected into the target tissue with a syringe, and electric pulses are then delivered by means of electrodes placed on either side of the injection site and connected to a generator (Figure 2). Electrodes can be

**Generator**

This technique allows a site specific gene transfer. It is relatively efficient in skeletal muscle and is applicable to many other tissues such as brain, liver, skin, bladder, kidney, lung, cor‐ nea, retina, testis, tumor tissue etc... for more details see [46]. Electrotransfer can also be used in a wealth of animal models, ranging from rats and mice to sheeps [47] and cows [48]

The efficiency of gene transfer depends on the target tissue, the delivered DNA and electric pulses parameters. The aim is to deliver, into each tissue, electrical pulses that can cause the permeabilization of cell membranes and DNA transfer, while remaining below the toxic threshold. Otherwise, local cell death by necrosis of the treated cells would occur, followed

**Plates electrodes**

**Conductor gel**

**Figure 2.** Experimental set up for intramuscular plasmid electrotransfer in mice

protocols.

**DNA solution**

and even fish [49].

**5.1. Operating parameters**

**Tissue**

**5. Electrotransfer into practice**

either needles or plates.

Most studies are pointing to a mechanism of *in vivo* electrotransfer comparable to the mech‐ anism of *in vitro* electrotransfer described above,which can be extended to the whole tissue: several steps have to take place, including cell permeabilization beyond a threshold value of local electric field. In 1999, we evaluated on one hand cell permeabilization following the ap‐ plication of electrical pulses by measuring the ability of muscle cells to capture a small radi‐ oactive hydrophilic molecule complexe of EDTA Chelating 51 chromium (51Cr-EDTA), and on the other hand, transgene expression for evidence of DNA entry [38, 39]. The uptake of 51Cr-EDTA was similar whether injected thirty seconds before or after applying electrical pulses. In contrast, DNA injected after the electrical impulses does not penetrate into cells. This suggests that DNA must be present *in situ* at the time of electrical pulses to obtain an efficient cell transfection, and that there is a direct, active effect of the electric field on the DNA molecules to promote their entry into cells. Hence the current mechanistic hypothesis of gene electrotransfer necessitates not only a permeabilization of cell membranes but also a DNA electrophoresis.

This hypothesis is supported by the study of Bureau *et al.* [40] of gene electrotransfer in skel‐ etal muscle of mice with different combinations of long pulses of low voltage (LV, i.e. elec‐ trophoretic pulses) and short pulses of high voltage (HV, i.e. permeabilizing pulses). Only the combination of a HV-pulse followed by a LV-pulse provided efficient gene transfer. Fur‐ ther studies confirmed that HV-pulses are related to permeabilization, while LV-pulses are related to the efficiency of DNA electrophoresis [41]. The importance of cell permeabiliza‐ tion was also studied by magnetic resonance imaging using a gadolinium complex as con‐ trast agent (dimeglumine gadopentate): the zone of di meglumine gadopentate complex permeabilization is identical to the area expression of electrotransfered DNA [42].

The destabilization of cell membranes and the electrophoretic effect are probably not the on‐ ly mechanisms involved in gene transfer by electroporation. Scientists have discussed the importance of energy metabolism (ATP and ADP) for the passage of DNA through the per‐ meabilized membrane and its migration to the nucleus [28].

Other studies suggest a mechanism of DNA transport by endocytosis [43]. These same stud‐ ies show that transfection efficiency does not decrease if the electrical pulses are delivered up to four hours after injection of DNA, while other studies show that most of the injected DNA is degraded in first hours after injection [44]. We also confirmed that after an intra‐ muscular injection, most of the DNA is degraded and eliminated quickly. However, a small proportion of DNA is preserved and provides a source of stable DNA which can been elec‐ trotransfered [45].

In summary, the molecular mechanism of *in vivo* DNA electrotransfer is still under investi‐ gation. It likely corresponds to multiple steps whose elucidation and understanding of re‐ spective contribution could help to develop more effective electrotransfer strategies and protocols.

#### **5. Electrotransfer into practice**

electropermeabilization was performed on tumors after injection of bleomycin, a cytotoxic anticancer agent, [22, 37]. The effectiveness of bleomycin depends on its intracellular con‐ centration, but this drug penetrates poorly into cells. Therefore, a better penetration of bleo‐ mycin was measured after application of electric pulses to tumors, leading to an enhanced

Most studies are pointing to a mechanism of *in vivo* electrotransfer comparable to the mech‐ anism of *in vitro* electrotransfer described above,which can be extended to the whole tissue: several steps have to take place, including cell permeabilization beyond a threshold value of local electric field. In 1999, we evaluated on one hand cell permeabilization following the ap‐ plication of electrical pulses by measuring the ability of muscle cells to capture a small radi‐ oactive hydrophilic molecule complexe of EDTA Chelating 51 chromium (51Cr-EDTA), and on the other hand, transgene expression for evidence of DNA entry [38, 39]. The uptake of 51Cr-EDTA was similar whether injected thirty seconds before or after applying electrical pulses. In contrast, DNA injected after the electrical impulses does not penetrate into cells. This suggests that DNA must be present *in situ* at the time of electrical pulses to obtain an efficient cell transfection, and that there is a direct, active effect of the electric field on the DNA molecules to promote their entry into cells. Hence the current mechanistic hypothesis of gene electrotransfer necessitates not only a permeabilization of cell membranes but also a

This hypothesis is supported by the study of Bureau *et al.* [40] of gene electrotransfer in skel‐ etal muscle of mice with different combinations of long pulses of low voltage (LV, i.e. elec‐ trophoretic pulses) and short pulses of high voltage (HV, i.e. permeabilizing pulses). Only the combination of a HV-pulse followed by a LV-pulse provided efficient gene transfer. Fur‐ ther studies confirmed that HV-pulses are related to permeabilization, while LV-pulses are related to the efficiency of DNA electrophoresis [41]. The importance of cell permeabiliza‐ tion was also studied by magnetic resonance imaging using a gadolinium complex as con‐ trast agent (dimeglumine gadopentate): the zone of di meglumine gadopentate complex

The destabilization of cell membranes and the electrophoretic effect are probably not the on‐ ly mechanisms involved in gene transfer by electroporation. Scientists have discussed the importance of energy metabolism (ATP and ADP) for the passage of DNA through the per‐

Other studies suggest a mechanism of DNA transport by endocytosis [43]. These same stud‐ ies show that transfection efficiency does not decrease if the electrical pulses are delivered up to four hours after injection of DNA, while other studies show that most of the injected DNA is degraded in first hours after injection [44]. We also confirmed that after an intra‐ muscular injection, most of the DNA is degraded and eliminated quickly. However, a small proportion of DNA is preserved and provides a source of stable DNA which can been elec‐

In summary, the molecular mechanism of *in vivo* DNA electrotransfer is still under investi‐ gation. It likely corresponds to multiple steps whose elucidation and understanding of re‐

permeabilization is identical to the area expression of electrotransfered DNA [42].

meabilized membrane and its migration to the nucleus [28].

desired cytotoxicity.

114 Gene Therapy - Tools and Potential Applications

DNA electrophoresis.

trotransfered [45].

The *in vivo* electrotransfer technique is particularly easy to implement: a solution of plasmid DNA (i. e. a circular nucleic acid) in isotonic saline (NaCl, 150mM) is injected into the target tissue with a syringe, and electric pulses are then delivered by means of electrodes placed on either side of the injection site and connected to a generator (Figure 2). Electrodes can be either needles or plates.

**Figure 2.** Experimental set up for intramuscular plasmid electrotransfer in mice

This technique allows a site specific gene transfer. It is relatively efficient in skeletal muscle and is applicable to many other tissues such as brain, liver, skin, bladder, kidney, lung, cor‐ nea, retina, testis, tumor tissue etc... for more details see [46]. Electrotransfer can also be used in a wealth of animal models, ranging from rats and mice to sheeps [47] and cows [48] and even fish [49].

#### **5.1. Operating parameters**

The efficiency of gene transfer depends on the target tissue, the delivered DNA and electric pulses parameters. The aim is to deliver, into each tissue, electrical pulses that can cause the permeabilization of cell membranes and DNA transfer, while remaining below the toxic threshold. Otherwise, local cell death by necrosis of the treated cells would occur, followed by tissue regeneration, which would induce the loss of the benefit of the treatment (but with no toxicity at the level of the whole organism). Therefore, optimal conditions for the DNA electrotransfer in a targeted tissue result from a compromise between the efficiency of DNA transfer and minimal cellular toxicity.

damage threshold (irreversible), in order to define optimal electrical conditions for gene transfer with minimal toxic effects. Micklavic *et al.* have developed a system combining nu‐ merical predictions and experimental observations in order to determine these thresholds in

DNA Electrotransfer: An Effective Tool for Gene Therapy

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117

Different types of electrical pulses can be applied: unipolar square pulses, bipolar square pulses, or pulses with exponential decay [51]. The exponentially decaying pulses, colloquial‐ ly referred to as "exponential pulses" are mainly used *in vitro* with a time constant depend‐ ent on the resistance of the incubation media. The square pulses are preferred *in vivo*, since the voltage and pulse duration can be set independently of the electrical resistance of the targeted tissue. Unipolar square pulses are the most widely used for electrotransfer experi‐

Tissue damage can be caused by electrotransfer and thus limits the efficiency of transfection [53]. The cell permeabilization is the main toxicity factor: it leads to an inward diffusion of the external medium as well as leakage of intracellular content, thus changing the composi‐ tion of the latter. This toxicity can be reduced by minimizing the duration and the extent of

Other factors of toxicity have been described such as oxidative stress due to the formation of free radicals near the electropermeabilized membrane [6, 54]. It was also shown that electro‐ transfer induced muscle damage dependent on the amount of DNA injected [55]; these le‐

In our laboratory, histological analyzes of muscle slices have shown that the application of electric fields optimized for gene transfer does not induce gene expression markers of stress and cellular toxicity [56]. Other experiments have allowed to conclude that, even in opti‐ mized conditions, very little muscle damage is generated: few inflammatory lesions are ob‐ served with a maximum in the first seven days after the electrotransfer, but these disappear

It is also possible to reduce the extent of damage by increasing the accessibility of DNA to target cells. Indeed, studies have shown that improving the plasmid distribution leads to an increase in transgene expression. Thus, the value of the electric field used can be reduced. Better distribution can be obtained for example by pre-injection of hyaluronidase [59], an en‐ zyme that degrades hyaluronic acid, which is a major component of the extracellular matrix [60]. This pretreatment allows for the same expression level, using lower voltages while re‐ ducing muscle damage [61]. A pre-injection of sucrose may also improve the distribution of DNA, by creating spaces between the muscle fibers [62]. Similarly, a pre-injection of poly-Lglutamate, an anionic polymer, seems to increase the internalization of the plasmid inside the cell and/or to reduce its degradation [63], and therefore increases the expression of exog‐

the case of needle electrodes used in rat liver for drug delivery [50].

**5.2. Toxicity**

permeabilization.

enous gene.

sions disappear within two months after injection.

rapidly in less than three weeks [57-58].

ments, while bipolar squares pulses are rather used for electrophysiology [52].

#### *5.1.1. The electrodes*

The choice of electrodes depends on the target tissue and the size of the treated animal. It is critically important and should be carefully considered. For an electrotransfer on a small an‐ imal in a tissue such as skeletal muscle, or liver tumor, most experimenters use electrodes made of two plates attached to a clamp (Figure 3, left). Indeed, this type of electrodes can be easily applied externally on each side of the interested tissue. Because one key parameter is the electric field, which is related to the ratio between the voltage applied and the distance between electrodes, this latter distance should not be too large in order to avoid prohibitive high voltage *in vivo* delivery. Thus, for animals of larger size, needle electrodes (Figure 3, right) are more often used than external plates.

#### *5.1.2. Electrical parameters*

Knowing the magnitude and distribution of electric field is very important for both efficient gene transfer and reduced toxicity. The distribution of the electric field is dependent on both the tissue and the type of electrodes, which causes variations in the effective magnitude of the field in the tissue area of interest. The electric field distribution is more homogeneous when using plate electrodes than with needle electrodes, and for a given setting, the result‐ ing electric field is lower with needle electrodes that with electrode plates [38].

Moreover, it is necessary to determine, for each tissue and each species, the threshold values of the electric field magnitude, i.e. the permeabilization threshold (reversible) and the cell damage threshold (irreversible), in order to define optimal electrical conditions for gene transfer with minimal toxic effects. Micklavic *et al.* have developed a system combining nu‐ merical predictions and experimental observations in order to determine these thresholds in the case of needle electrodes used in rat liver for drug delivery [50].

Different types of electrical pulses can be applied: unipolar square pulses, bipolar square pulses, or pulses with exponential decay [51]. The exponentially decaying pulses, colloquial‐ ly referred to as "exponential pulses" are mainly used *in vitro* with a time constant depend‐ ent on the resistance of the incubation media. The square pulses are preferred *in vivo*, since the voltage and pulse duration can be set independently of the electrical resistance of the targeted tissue. Unipolar square pulses are the most widely used for electrotransfer experi‐ ments, while bipolar squares pulses are rather used for electrophysiology [52].

#### **5.2. Toxicity**

by tissue regeneration, which would induce the loss of the benefit of the treatment (but with no toxicity at the level of the whole organism). Therefore, optimal conditions for the DNA electrotransfer in a targeted tissue result from a compromise between the efficiency of DNA

The choice of electrodes depends on the target tissue and the size of the treated animal. It is critically important and should be carefully considered. For an electrotransfer on a small an‐ imal in a tissue such as skeletal muscle, or liver tumor, most experimenters use electrodes made of two plates attached to a clamp (Figure 3, left). Indeed, this type of electrodes can be easily applied externally on each side of the interested tissue. Because one key parameter is the electric field, which is related to the ratio between the voltage applied and the distance between electrodes, this latter distance should not be too large in order to avoid prohibitive high voltage *in vivo* delivery. Thus, for animals of larger size, needle electrodes (Figure 3,

**Figure 3.** Examples of electrode plates for external use (left) and needle electrodes for internal use, designed by the

Knowing the magnitude and distribution of electric field is very important for both efficient gene transfer and reduced toxicity. The distribution of the electric field is dependent on both the tissue and the type of electrodes, which causes variations in the effective magnitude of the field in the tissue area of interest. The electric field distribution is more homogeneous when using plate electrodes than with needle electrodes, and for a given setting, the result‐

Moreover, it is necessary to determine, for each tissue and each species, the threshold values of the electric field magnitude, i.e. the permeabilization threshold (reversible) and the cell

ing electric field is lower with needle electrodes that with electrode plates [38].

transfer and minimal cellular toxicity.

116 Gene Therapy - Tools and Potential Applications

right) are more often used than external plates.

*5.1.1. The electrodes*

company Sphergen (right).

*5.1.2. Electrical parameters*

Tissue damage can be caused by electrotransfer and thus limits the efficiency of transfection [53]. The cell permeabilization is the main toxicity factor: it leads to an inward diffusion of the external medium as well as leakage of intracellular content, thus changing the composi‐ tion of the latter. This toxicity can be reduced by minimizing the duration and the extent of permeabilization.

Other factors of toxicity have been described such as oxidative stress due to the formation of free radicals near the electropermeabilized membrane [6, 54]. It was also shown that electro‐ transfer induced muscle damage dependent on the amount of DNA injected [55]; these le‐ sions disappear within two months after injection.

In our laboratory, histological analyzes of muscle slices have shown that the application of electric fields optimized for gene transfer does not induce gene expression markers of stress and cellular toxicity [56]. Other experiments have allowed to conclude that, even in opti‐ mized conditions, very little muscle damage is generated: few inflammatory lesions are ob‐ served with a maximum in the first seven days after the electrotransfer, but these disappear rapidly in less than three weeks [57-58].

It is also possible to reduce the extent of damage by increasing the accessibility of DNA to target cells. Indeed, studies have shown that improving the plasmid distribution leads to an increase in transgene expression. Thus, the value of the electric field used can be reduced. Better distribution can be obtained for example by pre-injection of hyaluronidase [59], an en‐ zyme that degrades hyaluronic acid, which is a major component of the extracellular matrix [60]. This pretreatment allows for the same expression level, using lower voltages while re‐ ducing muscle damage [61]. A pre-injection of sucrose may also improve the distribution of DNA, by creating spaces between the muscle fibers [62]. Similarly, a pre-injection of poly-Lglutamate, an anionic polymer, seems to increase the internalization of the plasmid inside the cell and/or to reduce its degradation [63], and therefore increases the expression of exog‐ enous gene.

#### **5.3. Target tissues**

During recent years, electrotransfer has been applied in various animal species to many tis‐ sues, including skeletal muscle, skin, liver, lungs, kidneys, joints, brain, retina, cornea, etc... [64]. The optimal parameters of a given electrotransfer should be determined based on the cell type and species, since these parameters strongly depend on tissue organization and the size of the transfected cells.

the skin from a single injection could still be observed [70, 71]. Moreover Dujardin *et al*. have shown that square or exponential pulses induce moderate and reversible effects on the skin without inflammation or necrosis, while transiently permeabilizing the skin and thus allow‐

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119

An important goal for gene transfer applications is the level and duration of gene expres‐ sion. To determine optimal conditions which maximize efficiency while reducing tissue damage, different protocols have been used to improve the access of plasmids to targeted cells. As already described, improved plasmid distribution in the skeletal muscle leads to an increase in DNA expression. Accordingly, Cemazar *et al.* showed recently enhanced trans‐ fection efficiency of gene transfer by pretreatment of tumors with hyaluronidase and/or col‐

A secretion signal can be also added to the transgene sequence : we have recently shown that by modifying the cellular localization of the produced protein by adding a secretory signal, the production and secretion of this protein is enhanced, thus enhancing biological

We have also shown that codon optimization of the transgene (i.e. retaining the natural ami‐ no acid sequence but using the preferred host animal codons) leads to increase in the expres‐

Another method to increase the stability of the protein produced in the blood circulation is to increase its size in order to avoid kidney excretion. Thus, the construction of fusion pro‐ teins, for instance by fusing a therapeutic protein with an IgG constant [75], appears a sim‐ ple way to deliver enhanced levels of secreted proteins without altering their biological

The enhanced protein expression, and so their biological effects, also depends of the injec‐

DNA electrotransfer is a recent technique of has not yet successfully completed all stages of clinical development, but this is progressing. The first Phase I human clinical trial has been initiated in U.S. by the company Inovio Biomedicals, for the treatment of skin cancer [76]. Since then, the delivery of plasmid DNA encoding therapeutic genes has been tested exten‐

Applications designated as "therapeutic" which are mainly reported in the literature have been demonstrated on animal models of human diseases. The main potential therapeutic areas cover cancer [78], cardiovascular diseases [75], autoimmune diseases [79], monogenic diseases [80], organ-specific disorders [81] and vaccination [82, 83]. Different examples show

lagenase, two enzymes which modulate components of the extracellular matrix [73].

ing the passage of molecules [72].

sion of the protein of interest [74].

tion regimen and the administered plasmid dose [74].

**6. Applications of plasmid electrotransfer**

sively in preclinical melanoma models [77].

effect [74].

activities.

**5.4. Optimization of** *in vivo* **electrotransfer conditions**

#### *5.3.1. Skeletal muscle*

One of the most widely used tissues for electrotransfer is skeletal muscle. The DNA electro‐ transfer into skeletal muscle was discovered independently by three teams [8, 9, 52]. Indeed, skeletal muscle offers many advantages:


Combined together, these features can turn muscle into systemic drug delivery system for distant targets [65]. Interestingly, the cotransfection of multiple unlinked genes can be easily performed by electroporation [66]. For examples of electrotransfer in skeletal muscle in vari‐ ous mammalian species see [46].

#### *5.3.2. The skin*

The skin is, as muscle, also a widely used tissue for DNA electrotransfer, mostly because:


However, skin structure [67] does not facilitate gene transfer. In particular, the top layer (stratum corneum or horny layer) is a major barrier [68, 69]. But a high level of expression in the skin from a single injection could still be observed [70, 71]. Moreover Dujardin *et al*. have shown that square or exponential pulses induce moderate and reversible effects on the skin without inflammation or necrosis, while transiently permeabilizing the skin and thus allow‐ ing the passage of molecules [72].

#### **5.4. Optimization of** *in vivo* **electrotransfer conditions**

**5.3. Target tissues**

size of the transfected cells.

118 Gene Therapy - Tools and Potential Applications

skeletal muscle offers many advantages:

ous mammalian species see [46].

that reach the bloodstream;

brief period of expression.

which facilitates DNA trafficking to the nucleus;

sence of regeneration due to injury or cytotoxic immune response;

**•** this tissue is easily accessible and a large area of tissue can be treated;

and is therefore an organ of choice for applications in DNA vaccination;

*5.3.1. Skeletal muscle*

**•** a large, easy access;

the fibers;

*5.3.2. The skin*

During recent years, electrotransfer has been applied in various animal species to many tis‐ sues, including skeletal muscle, skin, liver, lungs, kidneys, joints, brain, retina, cornea, etc... [64]. The optimal parameters of a given electrotransfer should be determined based on the cell type and species, since these parameters strongly depend on tissue organization and the

One of the most widely used tissues for electrotransfer is skeletal muscle. The DNA electro‐ transfer into skeletal muscle was discovered independently by three teams [8, 9, 52]. Indeed,

**•** sets of muscle fibers are parallel to each other: many fibers might have an optimal orienta‐ tion relative to the electric field, which promotes even transfer across the entire length of

**•** unlike other cells, muscle cells have multiple nuclei flattened against the cell membrane,

**•** muscle fibers do not divide, ensuring long-term gene expression, notwithstanding the ab‐

**•** finally, a major advantage of skeletal muscle lies in its ability to produce and release bio‐

Combined together, these features can turn muscle into systemic drug delivery system for distant targets [65]. Interestingly, the cotransfection of multiple unlinked genes can be easily performed by electroporation [66]. For examples of electrotransfer in skeletal muscle in vari‐

The skin is, as muscle, also a widely used tissue for DNA electrotransfer, mostly because:

**•** keratinocytes, which are epidermal cells, can synthesize and secrete therapeutic proteins

**•** by its natural function of a biological barrier, the skin contains cells that present antigens

**•** the epidermal cells have a short lifespan, which can be useful for treatments requiring a

However, skin structure [67] does not facilitate gene transfer. In particular, the top layer (stratum corneum or horny layer) is a major barrier [68, 69]. But a high level of expression in

logically active proteins into the bloodstream, due to the strong vascularisation.

An important goal for gene transfer applications is the level and duration of gene expres‐ sion. To determine optimal conditions which maximize efficiency while reducing tissue damage, different protocols have been used to improve the access of plasmids to targeted cells. As already described, improved plasmid distribution in the skeletal muscle leads to an increase in DNA expression. Accordingly, Cemazar *et al.* showed recently enhanced trans‐ fection efficiency of gene transfer by pretreatment of tumors with hyaluronidase and/or col‐ lagenase, two enzymes which modulate components of the extracellular matrix [73].

A secretion signal can be also added to the transgene sequence : we have recently shown that by modifying the cellular localization of the produced protein by adding a secretory signal, the production and secretion of this protein is enhanced, thus enhancing biological effect [74].

We have also shown that codon optimization of the transgene (i.e. retaining the natural ami‐ no acid sequence but using the preferred host animal codons) leads to increase in the expres‐ sion of the protein of interest [74].

Another method to increase the stability of the protein produced in the blood circulation is to increase its size in order to avoid kidney excretion. Thus, the construction of fusion pro‐ teins, for instance by fusing a therapeutic protein with an IgG constant [75], appears a sim‐ ple way to deliver enhanced levels of secreted proteins without altering their biological activities.

The enhanced protein expression, and so their biological effects, also depends of the injec‐ tion regimen and the administered plasmid dose [74].

#### **6. Applications of plasmid electrotransfer**

DNA electrotransfer is a recent technique of has not yet successfully completed all stages of clinical development, but this is progressing. The first Phase I human clinical trial has been initiated in U.S. by the company Inovio Biomedicals, for the treatment of skin cancer [76]. Since then, the delivery of plasmid DNA encoding therapeutic genes has been tested exten‐ sively in preclinical melanoma models [77].

Applications designated as "therapeutic" which are mainly reported in the literature have been demonstrated on animal models of human diseases. The main potential therapeutic areas cover cancer [78], cardiovascular diseases [75], autoimmune diseases [79], monogenic diseases [80], organ-specific disorders [81] and vaccination [82, 83]. Different examples show

the efficiency of plasmid electrotransfer to produce therapeutic proteins in various patholo‐ gies [46]: all these experiments showed an improvement in symptoms of the relative disor‐ der.

**6.2. Monogenic diseases**

electrotransfer [98].

tivity of monomer [103].

with small fluctuation [105].

**6.3. Hematopoietic factor deficiency**

Monogenic diseases with an identified defective gene have been the first diseases targeted by gene therapy approaches. Among these diseases, Duchenne muscular dystrophy (DMD), which is characterized by the absence of dystrophin, is a good model, since even a small amount of dystrophin would be sufficient to reverse the clinical phenotype of the disease. An approach to eventually restore this protein in patients with DMD is to introduce into their muscles a plasmid encoding dystrophinc DNA. Pichavant *et al.* were the first to dem‐ onstrate local restoration of full-length dog dystrophin in dystrophic dog muscle by DNA

DNA Electrotransfer: An Effective Tool for Gene Therapy

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121

Erythropoietin (EPO) is another good candidate for gene therapy applications because a small amount will produce the desired physiological effect of raising the hematocrit. Nu‐ merous studies, in particular by our own group, report efficient EPO secretion after plasmid electrotransfer, with a therapeutic effect in anemia and beta thalassemia. The use of intra‐ muscular plasmid electrotransfer for EPO gene delivery in mice increased approximately 10 to 100-fold the expression of this gene, as compared to naked DNA alone [99, 100]. More‐ over with this method, the protein in circulation and hematocrit levels were stable for 2 to 6 months after a single injection of minimal amounts (as little as 1 µg) of a plasmid carrying the mouse EPO cDNA. Several studies also showed that EPO expression could be regulated, for instance by co-administering an EPO encoding plasmid under the control of a tetracy‐ cline-inducible promotor and a second plasmid carrying the reverse tetracycline-dependent transactivator protein [100, 101]. All these studies exemplified that plasmid DNA electro‐

transfer can efficiently produce enough amounts of transgenic EPO in normal mice.

In collaboration with the group of Y. Beuzard, we have demonstrated the relevance of intra‐ muscular electroporation of an EPO-expressing plasmid in a mouse model of human β-tha‐ lassemia, a severe genetic disease, leading to a durable and dose-dependent phenotypic correction of this severe genetic disease [102]. In addition, we have also shown that it is pos‐ sible to produce fusion protein by plasmid DNA electrotransfer [103]: indeed since the bridging of two adjacent EPO receptors triggers a conformational change that initiates signal transduction [104], we have hypothesized that the fusion of two EPO molecules might lead an increase in intrinsic activity of EPO. Thus, we demonstrated that the injection of EPO dimer encoding plasmid by electrotransfer in a skeletal muscle of β-thalassemic mice indu‐ ces an increase in the biologic specific activity of this EPO dimer in comparison with the ac‐

Furthermore the secretion peak of therapeutic protein following DNA administration is po‐ tentially deleterious. We reported that muscular electrotransfer of low doses of plasmid can be repeated several times to weeks or even months after the initial injection, and that this strategy leads to efficient, long-lasting and non-toxic treatment of β-thalassemic mouse ane‐ mia avoiding the deleterious initial hematocrit peak and maintaining a normal hematocrit

#### **6.1. Cancer**

Cancer accounts for major field of application trials of gene therapy. Different strategies can be broadly grouped into four main categories:


These strategies can be combined to obtain synergistic results. For example, a combination of HSV-TK-suicide gene therapy and IL-21 immune gene therapy byelectrotransfer im‐ proves antitumor responses in mice [90]. Moreover, *in vivo* electrotransfer could be used in combination with other strategies such as chemotherapy, because these two approaches use different mechanisms to kill cancer cells, and thus a synergistic effect may be obtained.

Actually, electroporation of DNA encoding cytokines into tumors is extensively studied: IL-12 [91], IL-18 [92], IFN-α [93] have been shown to reduce tumor growth and increase sur‐ vival times in different tumor models. Other interesting results are represented by the inhib‐ ition of tumor growth in various models with plasmids encoding metaloproteinase-3 inhibitor for the treatment of prostate cancers [94], or encoding endostatin for his therapeu‐ tic efficacy in mouse-transplanted tumors [95].

All these experiments show the potential of in vivo electrotranfer for cancer treatment. And the strategy used, i.e. the direct intra-tumoral plasmid electrotransfer, is well suited for the local production of therapeutic proteins. However, since the efficacy of gene transfer into tumor cells *in vivo* is generally low, intramuscular electrotransfer can also be efficiently used for distal tumor treatment. Indeed, an important application of the technique of plasmid electrotransfer is the protein secretion by skeletal muscle: the pro‐ duced protein, such as, for instant, an immunostimulating cytokine, can diffuse into the vascular system and circulate throughout the body to exert a physiological effect, partic‐ ularly therapeutic. This distal approach may be very powerful for surgically inaccessible tumors, such as head and neck tumors.

Finally, the intramuscular electrotransfer of a plasmid encoding the prostate membrane spe‐ cific antigen (PMSA) has been tested in a human clinical trial of prostate cancer active im‐ munotherapy [96]. DNA fusion-gene vaccination in patients with prostate cancer induces high-frequency CD8 (+) T-cell responses and increases PSA doubling time [97].

#### **6.2. Monogenic diseases**

the efficiency of plasmid electrotransfer to produce therapeutic proteins in various patholo‐ gies [46]: all these experiments showed an improvement in symptoms of the relative disor‐

Cancer accounts for major field of application trials of gene therapy. Different strategies can

**c.** Repair cell cycle defects caused by the loss of tumor suppressor genes or oncogene acti‐

These strategies can be combined to obtain synergistic results. For example, a combination of HSV-TK-suicide gene therapy and IL-21 immune gene therapy byelectrotransfer im‐ proves antitumor responses in mice [90]. Moreover, *in vivo* electrotransfer could be used in combination with other strategies such as chemotherapy, because these two approaches use different mechanisms to kill cancer cells, and thus a synergistic effect may be obtained.

Actually, electroporation of DNA encoding cytokines into tumors is extensively studied: IL-12 [91], IL-18 [92], IFN-α [93] have been shown to reduce tumor growth and increase sur‐ vival times in different tumor models. Other interesting results are represented by the inhib‐ ition of tumor growth in various models with plasmids encoding metaloproteinase-3 inhibitor for the treatment of prostate cancers [94], or encoding endostatin for his therapeu‐

All these experiments show the potential of in vivo electrotranfer for cancer treatment. And the strategy used, i.e. the direct intra-tumoral plasmid electrotransfer, is well suited for the local production of therapeutic proteins. However, since the efficacy of gene transfer into tumor cells *in vivo* is generally low, intramuscular electrotransfer can also be efficiently used for distal tumor treatment. Indeed, an important application of the technique of plasmid electrotransfer is the protein secretion by skeletal muscle: the pro‐ duced protein, such as, for instant, an immunostimulating cytokine, can diffuse into the vascular system and circulate throughout the body to exert a physiological effect, partic‐ ularly therapeutic. This distal approach may be very powerful for surgically inaccessible

Finally, the intramuscular electrotransfer of a plasmid encoding the prostate membrane spe‐ cific antigen (PMSA) has been tested in a human clinical trial of prostate cancer active im‐ munotherapy [96]. DNA fusion-gene vaccination in patients with prostate cancer induces

high-frequency CD8 (+) T-cell responses and increases PSA doubling time [97].

der.

**6.1. Cancer**

be broadly grouped into four main categories:

**d.** Inhibition of tumor angiogenesis [89].

tic efficacy in mouse-transplanted tumors [95].

tumors, such as head and neck tumors.

**b.** Use of suicide genes [85-87];

120 Gene Therapy - Tools and Potential Applications

vation [88],

**a.** Stimulation of the immune response against a tumor [84],

Monogenic diseases with an identified defective gene have been the first diseases targeted by gene therapy approaches. Among these diseases, Duchenne muscular dystrophy (DMD), which is characterized by the absence of dystrophin, is a good model, since even a small amount of dystrophin would be sufficient to reverse the clinical phenotype of the disease. An approach to eventually restore this protein in patients with DMD is to introduce into their muscles a plasmid encoding dystrophinc DNA. Pichavant *et al.* were the first to dem‐ onstrate local restoration of full-length dog dystrophin in dystrophic dog muscle by DNA electrotransfer [98].

#### **6.3. Hematopoietic factor deficiency**

Erythropoietin (EPO) is another good candidate for gene therapy applications because a small amount will produce the desired physiological effect of raising the hematocrit. Nu‐ merous studies, in particular by our own group, report efficient EPO secretion after plasmid electrotransfer, with a therapeutic effect in anemia and beta thalassemia. The use of intra‐ muscular plasmid electrotransfer for EPO gene delivery in mice increased approximately 10 to 100-fold the expression of this gene, as compared to naked DNA alone [99, 100]. More‐ over with this method, the protein in circulation and hematocrit levels were stable for 2 to 6 months after a single injection of minimal amounts (as little as 1 µg) of a plasmid carrying the mouse EPO cDNA. Several studies also showed that EPO expression could be regulated, for instance by co-administering an EPO encoding plasmid under the control of a tetracy‐ cline-inducible promotor and a second plasmid carrying the reverse tetracycline-dependent transactivator protein [100, 101]. All these studies exemplified that plasmid DNA electro‐ transfer can efficiently produce enough amounts of transgenic EPO in normal mice.

In collaboration with the group of Y. Beuzard, we have demonstrated the relevance of intra‐ muscular electroporation of an EPO-expressing plasmid in a mouse model of human β-tha‐ lassemia, a severe genetic disease, leading to a durable and dose-dependent phenotypic correction of this severe genetic disease [102]. In addition, we have also shown that it is pos‐ sible to produce fusion protein by plasmid DNA electrotransfer [103]: indeed since the bridging of two adjacent EPO receptors triggers a conformational change that initiates signal transduction [104], we have hypothesized that the fusion of two EPO molecules might lead an increase in intrinsic activity of EPO. Thus, we demonstrated that the injection of EPO dimer encoding plasmid by electrotransfer in a skeletal muscle of β-thalassemic mice indu‐ ces an increase in the biologic specific activity of this EPO dimer in comparison with the ac‐ tivity of monomer [103].

Furthermore the secretion peak of therapeutic protein following DNA administration is po‐ tentially deleterious. We reported that muscular electrotransfer of low doses of plasmid can be repeated several times to weeks or even months after the initial injection, and that this strategy leads to efficient, long-lasting and non-toxic treatment of β-thalassemic mouse ane‐ mia avoiding the deleterious initial hematocrit peak and maintaining a normal hematocrit with small fluctuation [105].

In addition, Gothelf *et al.* demonstrate that gene electrotransfer to skin of even small amounts of EPO DNA can lead to systemically therapeutic levels of EPO protein [106].

tine palmitoyltransferase 1 (CPT1), the enzyme that controls the entry of long-chain fatty ac‐ yl CoA into mitochondria: an overexpression of CPT1 led to enhance rates of fatty acid betaoxidation and improved insulin action in muscle in high-fat diet insulin-resistant rats [114]. In the same model, electrotransfer of the orphan nuclear receptor (Nur77) significantly amel‐

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123

The prospect of inducing an immune response to a protein expressed *in vivo* directly from administered DNA vaccine represents an attractive alternative to other modes of vaccina‐ tion. Plasmid electrotransfer has been used in genetic immunization to produce antigenic proteins. It is now well established that genetic immunization induces both durable cellular and humoral responses [116]. This type of immunization is often developed for vaccination (virus or antibacterial), for anticancer active immunotherapy, and also to induce in animals

Since electrotransfer efficiently transfers genes compared to a single injection of plasmid, improving antigenic protein expression by several orders of magnitude, the antibody tit‐ er and the quality of the immune response are also improved [117], with an increasing factor of 100 in mice after electrotransfer of a plasmid encoding a surface antigen of hep‐ atitis B [118]. High titers of antibodies were also obtained in mice and rabbits after i.m. electrotransfer of a plasmid encoding an envelope glycoprotein of hepatitis C [119], and in mice after electrotransfer of a plasmid encoding a protein of Mycobacterium tubercu‐ losis [120]. In the laboratory, it was shown that i.m. electrotransfer of a plasmid encod‐ ing the influenza hemagglutinin induces a better immune response in mice that a single i.m. injection [121]. And recently, we have assessed the potential of i.m. electrotransferin mouse to produce neutralizing antibodies, with high titer, against botulinum toxins, the most powerful poison in the world in present time [74]. We have optimized DNA elec‐ trotransfer for genetic immunization against botulinum antigen. This DNA immunization has been used in rabbits to induce antibodies production which is compatible with in‐ dustrial development of antiserum production for a human therapeutic use (Burgain *et al.*, unpublished results).These examples show that it is possible to obtain high titers neu‐

Monoclonal antibodies are increasingly being used in a wide range of clinical applications in the field of autoimmune disease, cancer and infectious disease. The production and secre‐ tion by electrotransfered muscle of monoclonal antibodies has been demonstrated by our group and the one of I. Mathiesen, independently [83, 122]. These studies demonstrated that the co-transfection of two naked plasmids encoding the heavy and light G immunoglobulin chains led to the secretion of fully assembled and functional immunoglobulin molecules. The successful neutralization of various pathogens resulted from monoclonal antibody se‐ cretion by electrotransfered muscle, raising the possibility of clinical passive immunization

iorates the effect of this protein on glucose metabolism [115].

**6.7. Vaccination and passive immunization by antibody production**

the production with high yield of antibodies against a given antigen.

tralizing antibodies in animals by DNA electrotransfer.

applications.

#### **6.4. Cardiovascular diseases**

Gene therapy is an attractive strategy for the treatment of cardiovascular disease. How‐ ever, using current methods, the induction of gene expression at therapeutic levels is of‐ ten inefficient. Therefore DNA electrotransfer directly into heart may enhance the delivery of therapeutic protein as shown the team of R. Heller : the electroporation meth‐ od ameliorates the delivery of a plasmid encoding an angiogenic growth factor (vascular endothelial growth factor, VEGF), which is a molecule previously documented to stimu‐ late revascularization in coronary artery disease [107]. Ayuni *et al.* demonstrated that, un‐ like the usual methods to treat coronary artery diseases, electrotransfer applied directly into the beating heart enhances the delivery of a plasmid injected via the coronary veins after transient occlusion of the coronary sinus [108]. These results show that *in vivo* elec‐ troporation mediated gene transfer is feasible and safe,in particular to the heart. Finally, in skin, D. Dean reported that using electroporation in skin enhances delivery of plas‐ mid DNA encoding fibroblast growth factor-2 (FGF-2) to induce neovascularization as atherapy for ischemia in a rat model [109].

#### **6.5. Eye diseases**

The eye is an isolated organ difficult to reach via systemic administration. Eye diseases are treated with intra- or periocular injections and these repeated injections bear the risk of ad‐ verse effects, mainly infections, and are poorly tolerated by the patients. The use of DNA electrotransfer technique is therefore possible to deliver a local treatment. Our team associat‐ ed with an ophtalmology group has developed electrotransfer to the ciliary muscle, which is a particular smooth muscle with some characteristics of striated skeletal muscle, for the local treatment of inflammatory eye disease. This approach led to production and secretion of therapeutic levels of TNFα soluble receptor in the ocular media, and not in the serum, thus preventing clinical and histological signs in a rat uveitis model [110, 111]. Recently, supra‐ choroidal electrotransfer with a reporter plasmid to transfect the choroid and the retina without detaching the retina has been reported [112]. Not only choroidal cells but also RPE, and potentially photoreceptors, were efficiently transduced for at least a month, without oc‐ ular complications. This minimally invasive non-viral gene therapy method may open new prospects for human retinal therapies.

#### **6.6. Obesity and diabetes**

As mentioned above, skeletal muscle can be an efficient platform for the secretion of eryth‐ ropoietin (EPO), which displays a variety of metabolic effects when it is expressed in supraphysiological levels. Hojman *et al.* have proposed to overexpress EPO in muscle by electrotransfer of plasmid in the aim to protect mice against diet-induced obesity and nor‐ malize glucose sensitivity, associated with a shift to increased fat metabolism in the muscles [113]. Similar results were obtained after DNA electrotransfer of plasmid encoding the carni‐

tine palmitoyltransferase 1 (CPT1), the enzyme that controls the entry of long-chain fatty ac‐ yl CoA into mitochondria: an overexpression of CPT1 led to enhance rates of fatty acid betaoxidation and improved insulin action in muscle in high-fat diet insulin-resistant rats [114]. In the same model, electrotransfer of the orphan nuclear receptor (Nur77) significantly amel‐ iorates the effect of this protein on glucose metabolism [115].

#### **6.7. Vaccination and passive immunization by antibody production**

In addition, Gothelf *et al.* demonstrate that gene electrotransfer to skin of even small amounts of EPO DNA can lead to systemically therapeutic levels of EPO protein [106].

Gene therapy is an attractive strategy for the treatment of cardiovascular disease. How‐ ever, using current methods, the induction of gene expression at therapeutic levels is of‐ ten inefficient. Therefore DNA electrotransfer directly into heart may enhance the delivery of therapeutic protein as shown the team of R. Heller : the electroporation meth‐ od ameliorates the delivery of a plasmid encoding an angiogenic growth factor (vascular endothelial growth factor, VEGF), which is a molecule previously documented to stimu‐ late revascularization in coronary artery disease [107]. Ayuni *et al.* demonstrated that, un‐ like the usual methods to treat coronary artery diseases, electrotransfer applied directly into the beating heart enhances the delivery of a plasmid injected via the coronary veins after transient occlusion of the coronary sinus [108]. These results show that *in vivo* elec‐ troporation mediated gene transfer is feasible and safe,in particular to the heart. Finally, in skin, D. Dean reported that using electroporation in skin enhances delivery of plas‐ mid DNA encoding fibroblast growth factor-2 (FGF-2) to induce neovascularization as

The eye is an isolated organ difficult to reach via systemic administration. Eye diseases are treated with intra- or periocular injections and these repeated injections bear the risk of ad‐ verse effects, mainly infections, and are poorly tolerated by the patients. The use of DNA electrotransfer technique is therefore possible to deliver a local treatment. Our team associat‐ ed with an ophtalmology group has developed electrotransfer to the ciliary muscle, which is a particular smooth muscle with some characteristics of striated skeletal muscle, for the local treatment of inflammatory eye disease. This approach led to production and secretion of therapeutic levels of TNFα soluble receptor in the ocular media, and not in the serum, thus preventing clinical and histological signs in a rat uveitis model [110, 111]. Recently, supra‐ choroidal electrotransfer with a reporter plasmid to transfect the choroid and the retina without detaching the retina has been reported [112]. Not only choroidal cells but also RPE, and potentially photoreceptors, were efficiently transduced for at least a month, without oc‐ ular complications. This minimally invasive non-viral gene therapy method may open new

As mentioned above, skeletal muscle can be an efficient platform for the secretion of eryth‐ ropoietin (EPO), which displays a variety of metabolic effects when it is expressed in supraphysiological levels. Hojman *et al.* have proposed to overexpress EPO in muscle by electrotransfer of plasmid in the aim to protect mice against diet-induced obesity and nor‐ malize glucose sensitivity, associated with a shift to increased fat metabolism in the muscles [113]. Similar results were obtained after DNA electrotransfer of plasmid encoding the carni‐

**6.4. Cardiovascular diseases**

122 Gene Therapy - Tools and Potential Applications

atherapy for ischemia in a rat model [109].

prospects for human retinal therapies.

**6.6. Obesity and diabetes**

**6.5. Eye diseases**

The prospect of inducing an immune response to a protein expressed *in vivo* directly from administered DNA vaccine represents an attractive alternative to other modes of vaccina‐ tion. Plasmid electrotransfer has been used in genetic immunization to produce antigenic proteins. It is now well established that genetic immunization induces both durable cellular and humoral responses [116]. This type of immunization is often developed for vaccination (virus or antibacterial), for anticancer active immunotherapy, and also to induce in animals the production with high yield of antibodies against a given antigen.

Since electrotransfer efficiently transfers genes compared to a single injection of plasmid, improving antigenic protein expression by several orders of magnitude, the antibody tit‐ er and the quality of the immune response are also improved [117], with an increasing factor of 100 in mice after electrotransfer of a plasmid encoding a surface antigen of hep‐ atitis B [118]. High titers of antibodies were also obtained in mice and rabbits after i.m. electrotransfer of a plasmid encoding an envelope glycoprotein of hepatitis C [119], and in mice after electrotransfer of a plasmid encoding a protein of Mycobacterium tubercu‐ losis [120]. In the laboratory, it was shown that i.m. electrotransfer of a plasmid encod‐ ing the influenza hemagglutinin induces a better immune response in mice that a single i.m. injection [121]. And recently, we have assessed the potential of i.m. electrotransferin mouse to produce neutralizing antibodies, with high titer, against botulinum toxins, the most powerful poison in the world in present time [74]. We have optimized DNA elec‐ trotransfer for genetic immunization against botulinum antigen. This DNA immunization has been used in rabbits to induce antibodies production which is compatible with in‐ dustrial development of antiserum production for a human therapeutic use (Burgain *et al.*, unpublished results).These examples show that it is possible to obtain high titers neu‐ tralizing antibodies in animals by DNA electrotransfer.

Monoclonal antibodies are increasingly being used in a wide range of clinical applications in the field of autoimmune disease, cancer and infectious disease. The production and secre‐ tion by electrotransfered muscle of monoclonal antibodies has been demonstrated by our group and the one of I. Mathiesen, independently [83, 122]. These studies demonstrated that the co-transfection of two naked plasmids encoding the heavy and light G immunoglobulin chains led to the secretion of fully assembled and functional immunoglobulin molecules. The successful neutralization of various pathogens resulted from monoclonal antibody se‐ cretion by electrotransfered muscle, raising the possibility of clinical passive immunization applications.

#### **7. Conclusion**

*In vivo* electrotransfer is a non-viral technique which has emerged as an efficient, userfriendly and cheap gene transfer method which issuited for a wide range of tissues and spe‐ cies. Moreover, *in vivo* electrotransfer can be used for either local or distal effect by secretion of the transgenic protein into the bloodstream. The skeletal muscle is able to produce func‐ tional proteins with adequate post-translational modifications, which means that the muscle can be used as an endocrine organ for the production of therapeutic secreted proteins target‐ ing systemic diseases. It is now established that therapeutic levels of circulating proteins can be reached in animal models. And since DNA does not induce any immune response, plas‐ mid electrotransfer can be repeated as often as desired (Scherman *et al.*, unpublished re‐ sults).

[4] Calvin NM, Hanawalt PC. High-efficiency transformation of bacterial cells by elec‐

DNA Electrotransfer: An Effective Tool for Gene Therapy

http://dx.doi.org/10.5772/52528

125

[5] Ganeva V, Galutzov B, Teissie J. Fast kinetic studies of plasmid DNA transfer in in‐ tact yeast cells mediated by electropulsation. BiochemBiophys Res Commun. 1995

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[9] Aihara H, Miyazaki J. Gene transfer into muscle by electroporation in vivo. Nat Bio‐

[10] Rols MP, Delteil C, Golzio M, Dumond P, Cros S, Teissie J. In vivo electrically medi‐ ated protein and gene transfer in murine melanoma. Nat Biotechnol. 1998 Feb;16(2):

[11] Suzuki T, Shin BC, Fujikura K, Matsuzaki T, Takata K. Direct gene transfer into rat

[12] Matsumoto T, Komori K, Shoji T, Kuma S, Kume M, Yamaoka T, et al. Successful and optimized in vivo gene transfer to rabbit carotid artery mediated by electronic pulse.

[13] Maruyama H, Ataka K, Higuchi N, Sakamoto F, Gejyo F, Miyazaki J. Skin-targeted gene transfer using in vivo electroporation. Gene Ther. 2001 Dec;8(23):1808-12.

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[16] Blair-Parks K, Weston BC, Dean DA. High-level gene transfer to the cornea using

[17] Dezawa M, Takano M, Negishi H, Mo X, Oshitari T, Sawada H. Gene transfer into retinal ganglion cells by in vivo electroporation: a new approach. Micron. 2002;33(1):

electroporation. J Gene Med. 2002 Jan-Feb;4(1):92-100.

liver cells by in vivo electroporation. FEBS Lett. 1998 Apr 3;425(3):436-40.

troporation. J Bacteriol. 1988 Jun;170(6):2796-801.

BiochimBiophysActa. 1999 Nov 9;1461(1):123-34.

C R AcadSci III. 1998 Nov;321(11):893-9.

technol. 1998 Sep;16(9):867-70.

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Med. 2003 Jul;5(7):618-24.

168-71.

1-6.

Sep 25;214(3):825-32.

The understanding of the precise mechanism of electrotransfer, the optimization of its reali‐ zation, the improvement of plasmids and of the structure of the encoded protein will bring more efficiency and above all more safety to the method, should it be applied to humans. Several clinical trials have been conducted and/or are still in progress. For more details see http://www.clinicaltrials.gov/ct2/results?term=electroporation. These clinical trials are main‐ ly conducted against infectious diseases such AIDS, hepatitis B, malaria, dengue, influenza... and various cancer types such as ovarian cancer or renal cancer, melanoma, cancers caused by human papillomavirus... Thus, DNA electrotransfer appears as a powerful and promis‐ ing tool not only for gene therapy, but also for in vivo gene delivery at the laboratory level within the frame of physiological studies.

#### **Author details**

Aurore Burgain-Chain and Daniel Scherman

Unit of Chemical and Genetic Pharmacology and of Bioimaging, CNRS Paris, Université Paris Descartes; Chimie Paris Tech; Paris-Sorbonne PRES, France

#### **References**


[4] Calvin NM, Hanawalt PC. High-efficiency transformation of bacterial cells by elec‐ troporation. J Bacteriol. 1988 Jun;170(6):2796-801.

**7. Conclusion**

124 Gene Therapy - Tools and Potential Applications

sults).

within the frame of physiological studies.

Aurore Burgain-Chain and Daniel Scherman

Paris Descartes; Chimie Paris Tech; Paris-Sorbonne PRES, France

NatlAcadSci U S A. 1984 Nov;81(22):7161-5.

**Author details**

**References**

*In vivo* electrotransfer is a non-viral technique which has emerged as an efficient, userfriendly and cheap gene transfer method which issuited for a wide range of tissues and spe‐ cies. Moreover, *in vivo* electrotransfer can be used for either local or distal effect by secretion of the transgenic protein into the bloodstream. The skeletal muscle is able to produce func‐ tional proteins with adequate post-translational modifications, which means that the muscle can be used as an endocrine organ for the production of therapeutic secreted proteins target‐ ing systemic diseases. It is now established that therapeutic levels of circulating proteins can be reached in animal models. And since DNA does not induce any immune response, plas‐ mid electrotransfer can be repeated as often as desired (Scherman *et al.*, unpublished re‐

The understanding of the precise mechanism of electrotransfer, the optimization of its reali‐ zation, the improvement of plasmids and of the structure of the encoded protein will bring more efficiency and above all more safety to the method, should it be applied to humans. Several clinical trials have been conducted and/or are still in progress. For more details see http://www.clinicaltrials.gov/ct2/results?term=electroporation. These clinical trials are main‐ ly conducted against infectious diseases such AIDS, hepatitis B, malaria, dengue, influenza... and various cancer types such as ovarian cancer or renal cancer, melanoma, cancers caused by human papillomavirus... Thus, DNA electrotransfer appears as a powerful and promis‐ ing tool not only for gene therapy, but also for in vivo gene delivery at the laboratory level

Unit of Chemical and Genetic Pharmacology and of Bioimaging, CNRS Paris, Université

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[74] Trollet C, Pereira Y, Burgain A, Litzler E, Mezrahi M, Seguin J, et al. Generation of high-titer neutralizing antibodies against botulinum toxins A, B, and E by DNA elec‐

[75] Adachi O, Nakano A, Sato O, Kawamoto S, Tahara H, Toyoda N, et al. Gene transfer of Fc-fusion cytokine by in vivo electroporation: application to gene therapy for viral

[76] Daud AI, DeConti RC, Andrews S, Urbas P, Riker AI, Sondak VK, et al. Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma. J Cli‐

[77] Heller LC, Heller R. Electroporation gene therapy preclinical and clinical trials for

[78] Bettan M, Ivanov MA, Mir LM, Boissiere F, Delaere P, Scherman D. Efficient DNA

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[80] Gollins H, McMahon J, Wells KE, Wells DJ. High-efficiency plasmid gene transfer in‐

[81] Tanaka T, Ichimaru N, Takahara S, Yazawa K, Hatori M, Suzuki K, et al. In vivo gene transfer of hepatocyte growth factor to skeletal muscle prevents changes in rat kid‐

[82] Bakker JM, Bleeker WK, Parren PW. Therapeutic antibody gene transfer: an active

[83] Perez N, Bigey P, Scherman D, Danos O, Piechaczyk M, Pelegrin M. Regulatable sys‐ temic production of monoclonal antibodies by in vivo muscle electroporation. Genet

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[98] Pichavant C, Chapdelaine P, Cerri DG, Bizario JC, Tremblay JP. Electrotransfer of the full-length dog dystrophin into mouse and dystrophic dog muscles. Hum Gene Ther. Nov;21(11):1591-601.

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[114] Bruce CR, Hoy AJ, Turner N, Watt MJ, Allen TL, Carpenter K, et al. Overexpression of carnitine palmitoyltransferase-1 in skeletal muscle is sufficient to enhance fatty acid oxidation and improve high-fat diet-induced insulin resistance. Diabetes. 2009

[115] Kanzleiter T, Preston E, Wilks D, Ho B, Benrick A, Reznick J, et al. Overexpression of the orphan receptor Nur77 alters glucose metabolism in rat muscle cells and rat mus‐

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[118] Widera G, Austin M, Rabussay D, Goldbeck C, Barnett SW, Chen M, et al. Increased DNA vaccine delivery and immunogenicity by electroporation in vivo. J Immunol.

[119] Zucchelli S, Capone S, Fattori E, Folgori A, Di Marco A, Casimiro D, et al. Enhancing B- and T-cell immune response to a hepatitis C virus E2 DNA vaccine by intramuscu‐

[120] Tollefsen S, Tjelle T, Schneider J, Harboe M, Wiker H, Hewinson G, et al. Improved cellular and humoral immune responses against Mycobacterium tuberculosis anti‐ gens after intramuscular DNA immunisation combined with muscle electroporation.

[121] Bachy M, Boudet F, Bureau M, Girerd-Chambaz Y, Wils P, Scherman D, et al. Electric pulses increase the immunogenicity of an influenza DNA vaccine injected intramusc‐

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lar electrical gene transfer. J Virol. 2000 Dec;74(24):11598-607.

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roid and the Retina Without Detaching the Retina. MolTher. 2012 Jan 17.

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[98] Pichavant C, Chapdelaine P, Cerri DG, Bizario JC, Tremblay JP. Electrotransfer of the full-length dog dystrophin into mouse and dystrophic dog muscles. Hum Gene Ther.

[99] Kreiss P, Bettan M, Crouzet J, Scherman D. Erythropoietin secretion and physiologi‐ cal effect in mouse after intramuscular plasmid DNA electrotransfer. J Gene Med.

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[102] Payen E, Bettan M, Rouyer-Fessard P, Beuzard Y, Scherman D. Improvement of mouse beta-thalassemia by electrotransfer of erythropoietin cDNA. ExpHematol.

[103] Dalle B, Henri A, Rouyer-Fessard P, Bettan M, Scherman D, Beuzard Y, et al. Dimeric erythropoietin fusion protein with enhanced erythropoietic activity in vitro and in

[104] Livnah O, Stura EA, Middleton SA, Johnson DL, Jolliffe LK, Wilson IA. Crystallo‐ graphic evidence for preformed dimers of erythropoietin receptor before ligand acti‐

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[106] Gothelf A, Hojman P, Gehl J. Therapeutic levels of erythropoietin (EPO) achieved af‐ ter gene electrotransfer to skin in mice. Gene Ther. 2010 Sep;17(9):1077-84.

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**Chapter 7**

**siRNA and Gene Formulation for Efficient Gene Therapy**

Whilst small interfering RNA (siRNA, also known as short interfering RNA) has a somewhat chequered history with regard to its discovery and initial usage, the "mammalian" research community singularly neither reading nor citing the output from the "plant" research community, it is now recognised in terms of \$bn being invested and spent that RNA interfer‐ ence (RNAi), sequence specific post-transcriptional gene silencing (PTGS) by siRNA, has many potential therapeutic applications [1] as well as being an important tool in the study of functional genomics. The site and mechanism of action of siRNA requires that these short double-stranded nucleic acids are delivered to the cytosol of target cells. Therefore, formula‐ tion is required in a strategy similar to that for gene therapy, although not requiring access to the nucleus. Efficient medicines design should come with an understanding of the problem at the molecular level. Our contributions are aimed at the use of non-viral gene therapy and this

siRNA is a double-stranded RNA (dsRNA) typically of 21-25 nucleotides per strand. siRNA operates as a part of the cellular mechanism called RNAi, which was first noticed in petunia flowers (*Petunia hybrida*) which showed reduced pigmentation on the introduction of exoge‐ nous genes that were meant to increase pigmentation [2, 3]. These experiments aimed at increasing the pigmentation of the petunia flowers by means of introducing additional gene constructs expressing either chalcone synthase [2, 3] or dihydroflavonol-4-reductase [2]. However, the resultant plants produced completely white flowers and/or flowers with white

> © 2013 Blagbrough and Metwally; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Ian S. Blagbrough and Abdelkader A. Metwally

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55518

Chapter therefore has such a focus.

**2.1. History and mechanism of RNA interference**

**2. RNA interference**

**1. Introduction**

### **siRNA and Gene Formulation for Efficient Gene Therapy**

Ian S. Blagbrough and Abdelkader A. Metwally

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55518

#### **1. Introduction**

Whilst small interfering RNA (siRNA, also known as short interfering RNA) has a somewhat chequered history with regard to its discovery and initial usage, the "mammalian" research community singularly neither reading nor citing the output from the "plant" research community, it is now recognised in terms of \$bn being invested and spent that RNA interfer‐ ence (RNAi), sequence specific post-transcriptional gene silencing (PTGS) by siRNA, has many potential therapeutic applications [1] as well as being an important tool in the study of functional genomics. The site and mechanism of action of siRNA requires that these short double-stranded nucleic acids are delivered to the cytosol of target cells. Therefore, formula‐ tion is required in a strategy similar to that for gene therapy, although not requiring access to the nucleus. Efficient medicines design should come with an understanding of the problem at the molecular level. Our contributions are aimed at the use of non-viral gene therapy and this Chapter therefore has such a focus.

#### **2. RNA interference**

#### **2.1. History and mechanism of RNA interference**

siRNA is a double-stranded RNA (dsRNA) typically of 21-25 nucleotides per strand. siRNA operates as a part of the cellular mechanism called RNAi, which was first noticed in petunia flowers (*Petunia hybrida*) which showed reduced pigmentation on the introduction of exoge‐ nous genes that were meant to increase pigmentation [2, 3]. These experiments aimed at increasing the pigmentation of the petunia flowers by means of introducing additional gene constructs expressing either chalcone synthase [2, 3] or dihydroflavonol-4-reductase [2]. However, the resultant plants produced completely white flowers and/or flowers with white

© 2013 Blagbrough and Metwally; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

or pale sectors on a pigmented background. The exact mechanism was not identified at the time and was simply termed co-suppression. The transcription level of the suppressed chalcone synthase genes in petunia flowers was found to be similar to that of the nonsuppressed genes, and thus the co-suppression must have been at the post-transcriptional level [4]. Later in 1997, the suppression of chalcone synthase endogene in petunia flowers was suggested to be related to formation of RNA duplexes by intermolecular pairing of comple‐ mentary sequences between the coding sequence and the 3′-UTR sequence of the transgene mRNA [5]. Indeed, the seminal contributions the plant RNAi community have made to this RNAi field are also reflected in the research of Hamilton and Sir David C. Baulcombe in the Sainsbury Laboratory, Norwich, UK, on PTGS as a nucleotide sequence-specific defence mechanism that can target both cellular and viral mRNAs with RNA molecules of a uniform length, ~25 nucleotides [6]. That RNA silencing involves the processing of dsRNA into 21-26 long siRNA to mediate gene suppression (correspondingly complementary to the dsRNA) was demonstrated in Arabidopsis, "RNA silencing pathways in plants that may also apply in animals" [7]. That Arabidopsis ARGONAUTE1 RNA-binding protein is an RNA slicer that selectively recruits microRNAs and siRNAs was shown to be by a key mechanism similar to but different from that found in animals [8]. In 1998, Fire, Mello and co-workers reported the reduction or inhibition (hence genetic "interference") of the expression of the *unc-22* gene *in Caenorhabditis elegans* by means of dsRNA that is homologous to 742 nucleotides in the targeted gene [9], a discovery that was awarded the Nobel Prize in medicine or physiology in 2006. The target gene expresses an abundant although nonessential myofilament protein. Decreasing *unc-22* activity resulted in an increasingly severe twitching phenotype, while complete inhibition resulted in impaired motility and muscle structural defects. The target gene inhibition was best achieved with dsRNA, while using the individual sense or anti-sense RNA strands resulted only in modest silencing. The authors also noticed that only few copies of the dsRNA are required per cell to initiate a potent and specific response, rejecting the hypothesis that the mechanism of interaction with target gene mRNA is stoichiometric in nature, and thus the role of the dsRNA in the interference machinery must be catalytic or amplifying.

plex. This core complex which carries-out mRNA degradation is the RNA induced silencing complex (RISC) [18-20]. The degradation process requires the key argonaute family of proteins, which contain a domain with RNase H (endonuclease) type of activity that catalyse cleavage of the phosphodiester bonds of the targeted mRNA. RISC assembly and subsequently its function to mediate sequence specific mRNA degradation occur in the cytoplasm of the cell [16]. The source of the dsRNA segments incorporated in RISC can be endogenously processed microRNA (miRNA), short hairpin RNA (shRNA), or synthetic siRNA. miRNA is produced from endogenous DNA through the action of RNA polymer‐ ase II resulting in the formation of non-coding RNA called primary miRNA (pri-miR‐ NA), which is processed in the nucleus by a protein complex containing an enzyme known as Drosha and a dsRNA binding protein cofactor called Pasha (DGCR8). Drosha cleaves pri-miRNA to produce (pre-miRNA), a dsRNA of 70-90 nucleotides and having a hairpin loop, which binds to Exportin 5 protein and is transferred from the nucleus into the cytoplasm. Pre-miRNA is processed by Dicer (RNase III enzyme) in the cytoplasm to give miRNA, typically of 22 nucleotides in length and having two nucleotide overhangs at the 3'-position [16, 21], shRNA is produced by transcription from an exogenous DNA that is delivered to the nucleus, and codes for a hairpin shaped RNA with segments of length 19-29 nucleotides and loop of 9 nucleotides [22, 23] which can then be processed by Dicer

siRNA and Gene Formulation for Efficient Gene Therapy

http://dx.doi.org/10.5772/55518

137

Once in the cytoplasm, the processed dsRNA (miRNA, processed shRNA, or siRNA) is then incorporated into a protein complex (RISC-loading complex, RLC). In *Drosophila* the RLC is composed of the dsRNA, heterodimer protein DCR2 (Dicer variant)/R2D2, possi‐ bly including the catalytic argonaute proteins as well in this complex. The active RISC is formed when one of the RNA strands in the complex is cleaved (the passenger strand) and the strand with the less thermodynamic stable 5'-end (guide/anti-sense strand) remains in the complex. The mRNA with complementary sequence to the guide strand binds to the active RISC and is cleaved by the endoribonuclease activity of the argonaute component

RNA is a polymer of ribonucleotides. Each RNA nucleotide is composed of one nucleo‐ base, the monosaccharide pentose ribose, and one phosphate group. The nucleobases in RNA are adenine (purine base), guanine (purine base), uracil (pyrimidine base), and cytosine (pyrimidine base) (Figure 2). A nucleoside is formed when each base is connect‐ ed via a glycosidic bond to the anomeric carbon 1' of ribose, thus when glycosylated, adenine, guanine, uracil, and cytosine nucleobases give adenosine, guanosine, uridine, and cytidine nucleosides. Each two nucleosides are connected via a phosphate diester bond between the 3' of one nucleoside and 5' of the next nucleoside to form the RNA polynucleo‐ tide strand. The main differences in the primary structure of RNA and DNA are that RNA pentose is ribose while DNA pentose is 2'-deoxyribose, and the RNA incorporates the

and incorporated in the RNAi machinery.

of the complex (Figure 1).

**2.2. RNA duplex structure**

nucleobase uracil instead of thymine.

Elbashir et al. reported in 2001 that sequence-specific gene silencing of endogenous and heterologous genes with 21 nucleotide siRNA occurs in mammalian cell cultures [10]. The reporter genes coding for sea pansy (*Renilla reniformis*) and firefly (*Photinus pyralis*) luciferases were silenced successfully in different cell lines including human embryonic kidney cells (293) and the cervix cancer cells (HeLa cell line, the first human cell line grown in vitro with success [11]), as well as the endogenous gene coding for the nuclear enve‐ lope proteins lamin A and lamin C in HeLa cells. The authors used dsRNA of length 21 or 22 nucleotides with 3'-symmetrical 2-nucleotide overhangs on each strand, as dsRNA with length >30 nucleotides initiates an immune response e.g. inducing interferon synthe‐ sis) that leads to non-specific mRNA degradation, which was evident from non-specific silencing of luciferase with 50 and 500 nucleotides dsRNA in HeLa S3 cells, COS-7 cells (kidney cells of the African green monkey), and NIH/3T3 cells (mouse fibroblasts) [10]. The RNAi mechanism of action continues to be investigated in detail and reviewed thorough‐ ly [12-17]. The RNAi mechanism involves the incorporation of dsRNA segments (e.g. siRNA) that have a sequence complementary to the targeted mRNA in a protein com‐

plex. This core complex which carries-out mRNA degradation is the RNA induced silencing complex (RISC) [18-20]. The degradation process requires the key argonaute family of proteins, which contain a domain with RNase H (endonuclease) type of activity that catalyse cleavage of the phosphodiester bonds of the targeted mRNA. RISC assembly and subsequently its function to mediate sequence specific mRNA degradation occur in the cytoplasm of the cell [16]. The source of the dsRNA segments incorporated in RISC can be endogenously processed microRNA (miRNA), short hairpin RNA (shRNA), or synthetic siRNA. miRNA is produced from endogenous DNA through the action of RNA polymer‐ ase II resulting in the formation of non-coding RNA called primary miRNA (pri-miR‐ NA), which is processed in the nucleus by a protein complex containing an enzyme known as Drosha and a dsRNA binding protein cofactor called Pasha (DGCR8). Drosha cleaves pri-miRNA to produce (pre-miRNA), a dsRNA of 70-90 nucleotides and having a hairpin loop, which binds to Exportin 5 protein and is transferred from the nucleus into the cytoplasm. Pre-miRNA is processed by Dicer (RNase III enzyme) in the cytoplasm to give miRNA, typically of 22 nucleotides in length and having two nucleotide overhangs at the 3'-position [16, 21], shRNA is produced by transcription from an exogenous DNA that is delivered to the nucleus, and codes for a hairpin shaped RNA with segments of length 19-29 nucleotides and loop of 9 nucleotides [22, 23] which can then be processed by Dicer and incorporated in the RNAi machinery.

Once in the cytoplasm, the processed dsRNA (miRNA, processed shRNA, or siRNA) is then incorporated into a protein complex (RISC-loading complex, RLC). In *Drosophila* the RLC is composed of the dsRNA, heterodimer protein DCR2 (Dicer variant)/R2D2, possi‐ bly including the catalytic argonaute proteins as well in this complex. The active RISC is formed when one of the RNA strands in the complex is cleaved (the passenger strand) and the strand with the less thermodynamic stable 5'-end (guide/anti-sense strand) remains in the complex. The mRNA with complementary sequence to the guide strand binds to the active RISC and is cleaved by the endoribonuclease activity of the argonaute component of the complex (Figure 1).

#### **2.2. RNA duplex structure**

or pale sectors on a pigmented background. The exact mechanism was not identified at the time and was simply termed co-suppression. The transcription level of the suppressed chalcone synthase genes in petunia flowers was found to be similar to that of the nonsuppressed genes, and thus the co-suppression must have been at the post-transcriptional level [4]. Later in 1997, the suppression of chalcone synthase endogene in petunia flowers was suggested to be related to formation of RNA duplexes by intermolecular pairing of comple‐ mentary sequences between the coding sequence and the 3′-UTR sequence of the transgene mRNA [5]. Indeed, the seminal contributions the plant RNAi community have made to this RNAi field are also reflected in the research of Hamilton and Sir David C. Baulcombe in the Sainsbury Laboratory, Norwich, UK, on PTGS as a nucleotide sequence-specific defence mechanism that can target both cellular and viral mRNAs with RNA molecules of a uniform length, ~25 nucleotides [6]. That RNA silencing involves the processing of dsRNA into 21-26 long siRNA to mediate gene suppression (correspondingly complementary to the dsRNA) was demonstrated in Arabidopsis, "RNA silencing pathways in plants that may also apply in animals" [7]. That Arabidopsis ARGONAUTE1 RNA-binding protein is an RNA slicer that selectively recruits microRNAs and siRNAs was shown to be by a key mechanism similar to but different from that found in animals [8]. In 1998, Fire, Mello and co-workers reported the reduction or inhibition (hence genetic "interference") of the expression of the *unc-22* gene *in Caenorhabditis elegans* by means of dsRNA that is homologous to 742 nucleotides in the targeted gene [9], a discovery that was awarded the Nobel Prize in medicine or physiology in 2006. The target gene expresses an abundant although nonessential myofilament protein. Decreasing *unc-22* activity resulted in an increasingly severe twitching phenotype, while complete inhibition resulted in impaired motility and muscle structural defects. The target gene inhibition was best achieved with dsRNA, while using the individual sense or anti-sense RNA strands resulted only in modest silencing. The authors also noticed that only few copies of the dsRNA are required per cell to initiate a potent and specific response, rejecting the hypothesis that the mechanism of interaction with target gene mRNA is stoichiometric in nature, and thus

136 Gene Therapy - Tools and Potential Applications

the role of the dsRNA in the interference machinery must be catalytic or amplifying.

Elbashir et al. reported in 2001 that sequence-specific gene silencing of endogenous and heterologous genes with 21 nucleotide siRNA occurs in mammalian cell cultures [10]. The reporter genes coding for sea pansy (*Renilla reniformis*) and firefly (*Photinus pyralis*) luciferases were silenced successfully in different cell lines including human embryonic kidney cells (293) and the cervix cancer cells (HeLa cell line, the first human cell line grown in vitro with success [11]), as well as the endogenous gene coding for the nuclear enve‐ lope proteins lamin A and lamin C in HeLa cells. The authors used dsRNA of length 21 or 22 nucleotides with 3'-symmetrical 2-nucleotide overhangs on each strand, as dsRNA with length >30 nucleotides initiates an immune response e.g. inducing interferon synthe‐ sis) that leads to non-specific mRNA degradation, which was evident from non-specific silencing of luciferase with 50 and 500 nucleotides dsRNA in HeLa S3 cells, COS-7 cells (kidney cells of the African green monkey), and NIH/3T3 cells (mouse fibroblasts) [10]. The RNAi mechanism of action continues to be investigated in detail and reviewed thorough‐ ly [12-17]. The RNAi mechanism involves the incorporation of dsRNA segments (e.g. siRNA) that have a sequence complementary to the targeted mRNA in a protein com‐

RNA is a polymer of ribonucleotides. Each RNA nucleotide is composed of one nucleo‐ base, the monosaccharide pentose ribose, and one phosphate group. The nucleobases in RNA are adenine (purine base), guanine (purine base), uracil (pyrimidine base), and cytosine (pyrimidine base) (Figure 2). A nucleoside is formed when each base is connect‐ ed via a glycosidic bond to the anomeric carbon 1' of ribose, thus when glycosylated, adenine, guanine, uracil, and cytosine nucleobases give adenosine, guanosine, uridine, and cytidine nucleosides. Each two nucleosides are connected via a phosphate diester bond between the 3' of one nucleoside and 5' of the next nucleoside to form the RNA polynucleo‐ tide strand. The main differences in the primary structure of RNA and DNA are that RNA pentose is ribose while DNA pentose is 2'-deoxyribose, and the RNA incorporates the nucleobase uracil instead of thymine.

**Figure 1.** RNAi mechanism in a eukaryotic cell. The source of the antisense strand incorporated in RISC can be miRNA, processed exogenous long dsRNA, or synthetic siRNA delivered to the cell.

**Figure 3.** siRNA duplex 22-mer targeting the enhanced green fluorescent protein (EGFP) mRNA. The two deoxythymi‐ dine residues at the 3'-end of the sense strand are not shown. Sense strand: 5'-GCAAGCUGACCCUGAAGUUCAUTT-3' Anti-sense strand: 5'-AUGAACUUCAGGGUCAGCUUGCCG-3' Target DNA sequence: 5'-CGGCAAGCTGACCCTGAAGTT‐

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139

In order to form an RNA duplex (Figure 3), the strands with complementary nucleotide sequence bind together by hydrogen bonds. Adenine is bound to uracil with two hydrogen bonds while guanine is bound to cytosine with three hydrogen bonds, thus forming what is known as Watson-Crick base pairs. RNA duplexes under normal physiological conditions are

The presence of the 2'-hydroxyl group of the ribose and the lack of the methyl group on the nucleotide uridine (in contrast to the methylated thymidine) results in structural differences between RNA and DNA, with the 2'-hydroxyl group of RNA being the major cause of the differences. The sugar phosphate backbone of RNA duplexes is stabilized by the 2'-hydroxyl in the C3'-endo position, while DNA adopts the C2'-endo position (Figure 4). Thus, the RNA duplex takes the A-helix form while the DNA helix takes the B-form. The A-helix form is suggested to have a greater hydration shell, giving RNA duplexes more thermodynamic stability and more rigidity compared to DNA duplexes [24-26]. RNA A-helix completes one complete rotation in 11-12 base pair (bp) compared to 10 bp for DNA, with a rise of 2.7 Å per bp of RNA [27]. The A-helix geometry has been suggested to be the major factor explaining why dsRNA and not dsDNA is involved in the RNAi machinery [28], where the A-helix geometry between the guide strand and the complementary target mRNA is essential for the

As a result of the presence of a hydroxyl group in the 2'-position of the ribose in the RNA backbone, the RNA phosphodiester backbone is more susceptible to hydrolysis by nucleases compared to the DNA which lacks the 2'-hydroxyl in its 2'-deoxyribose [29]. Incubation of

in the form of A-helix. This type of duplex is a right-handed helix [24-26].

catalytic activity of the argonaute 2 protein in the RISC.

CAT-3'

**Figure 2.** Nucleobases and pentoses of RNA and DNA.

*Nucleus*

*Cytoplasm*

mRNA cleavage

2-Deoxyribose

H H

O

NH

H

O

N H

H

OH O H

O H H

**RISC Argonaute**

**miRNA siRNA**

**RISC**

Adenine, A Guanine, G

O

N H

Ribose

H H

O H

O H H

OH O H

O

NH2

N

N

O

Cytosine, C Uracil, U Thymidine, T

O

NH2 <sup>N</sup>

NH

N

O

NH

H

N

H O

**Figure 1.** RNAi mechanism in a eukaryotic cell. The source of the antisense strand incorporated in RISC can be miRNA,

**Pri-miRNA Pre-miRNA**

**Pre-miRNA dsRNA**

DNA transcription

**mRNA**

**Dicer Dicer**

Sense strand cleavage

138 Gene Therapy - Tools and Potential Applications

processed exogenous long dsRNA, or synthetic siRNA delivered to the cell.

NH2

N H

N

N

N H

**Figure 2.** Nucleobases and pentoses of RNA and DNA.

**Figure 3.** siRNA duplex 22-mer targeting the enhanced green fluorescent protein (EGFP) mRNA. The two deoxythymi‐ dine residues at the 3'-end of the sense strand are not shown. Sense strand: 5'-GCAAGCUGACCCUGAAGUUCAUTT-3' Anti-sense strand: 5'-AUGAACUUCAGGGUCAGCUUGCCG-3' Target DNA sequence: 5'-CGGCAAGCTGACCCTGAAGTT‐ CAT-3'

In order to form an RNA duplex (Figure 3), the strands with complementary nucleotide sequence bind together by hydrogen bonds. Adenine is bound to uracil with two hydrogen bonds while guanine is bound to cytosine with three hydrogen bonds, thus forming what is known as Watson-Crick base pairs. RNA duplexes under normal physiological conditions are in the form of A-helix. This type of duplex is a right-handed helix [24-26].

The presence of the 2'-hydroxyl group of the ribose and the lack of the methyl group on the nucleotide uridine (in contrast to the methylated thymidine) results in structural differences between RNA and DNA, with the 2'-hydroxyl group of RNA being the major cause of the differences. The sugar phosphate backbone of RNA duplexes is stabilized by the 2'-hydroxyl in the C3'-endo position, while DNA adopts the C2'-endo position (Figure 4). Thus, the RNA duplex takes the A-helix form while the DNA helix takes the B-form. The A-helix form is suggested to have a greater hydration shell, giving RNA duplexes more thermodynamic stability and more rigidity compared to DNA duplexes [24-26]. RNA A-helix completes one complete rotation in 11-12 base pair (bp) compared to 10 bp for DNA, with a rise of 2.7 Å per bp of RNA [27]. The A-helix geometry has been suggested to be the major factor explaining why dsRNA and not dsDNA is involved in the RNAi machinery [28], where the A-helix geometry between the guide strand and the complementary target mRNA is essential for the catalytic activity of the argonaute 2 protein in the RISC.

As a result of the presence of a hydroxyl group in the 2'-position of the ribose in the RNA backbone, the RNA phosphodiester backbone is more susceptible to hydrolysis by nucleases compared to the DNA which lacks the 2'-hydroxyl in its 2'-deoxyribose [29]. Incubation of

**siRNA Disease**

ALN-RSV01 Respiratory syncytial

CALAA-01 Solid tumour/melanoma

Diabetic macular oedema

Age-related macular degeneration

virus infection

Colorectal cancer metastasizing to the liver

Protection from acute kidney injury after cardiac bypass surgery

Against PLK1 gene product in patients with hepatic cancer

Wound healing Nanoparticles/

Cand5/ Bevasiranib

Cand5/ Bevasiranib

Atu027

Two siRNA against TGFBI and COX-2 STP705

I5NP

TKM-080301

**Vector**/ *Route*

None/ *Intravitreal*

None/ *Intravitreal*

None/ *Intranasal*

Cyclodextrin nanoparticles/ *Intravenous*

> AtuPlex-Liposome/ *Intravenous*

> *Intravenous*

None/ *Intravenous*

Lipid nanoparticles/ *Hepatic intraarterial administration*

**Table 1.** Representative clinical trials using siRNA (http://clinicaltrials.gov/ct2/home, accessed on 5/8/2012).

injection [45], making it useful only if the target organ is the kidney.

The therapeutic application of siRNA requires overcoming several barriers (Figure 5) for its intracellular delivery and the subsequent functional gene silencing activity [42-44]. Those barriers are mainly due to siRNA specific characteristics, most important are having a highly negative charge due to their phosphate backbone (on average 40-50 negative charges per siRNA), being susceptible to degradation by nucleases, and having relatively large molecular weight (13-15 kDa) compared to conventional small drug molecules. First, local delivery (such as intravitreal) is different from intravenous delivery, where the latter will subject the siRNA to the serum ribonucleases, which results in degrading non-modified siRNA within time periods that vary from minutes to hours [31]. siRNA injected intravenously in rats was reported to be cleared rapidly from circulation and accumulates in kidneys within minutes of

In order to gain access into the cytoplasm where siRNA can exert its biological activity, the polyribonucleotide must pass first through the interstitial space then through the target cell membrane. This will be a difficult task, since both the extracellular matrix in many tissue types

**Phase Sponsor**

siRNA and Gene Formulation for Efficient Gene Therapy

Phase II Opko Health

Phase II Alnylam Pharmaceuticals

Phase I Silence Therapeutics

Phase I Quark Pharmaceuticals

Phase II (Phase III halted)

Phase I

Phase I

Phase I

(Miami, USA)

http://dx.doi.org/10.5772/55518

141

Opko Health (Miami, USA)

(Cambridge, USA)

Calando (Pasadena, CA, USA)

(London, UK)

Sirnaomics (Gaithersburg, MD, USA)

(Fremont, USA)

NCI (Maryland, USA)

**Figure 4.** 3'-endo ribose configuration of RNA (left) vs 2'-endo (right) of 2'-deoxyribose in DNA. Shown is cytidine (RNA) and deoxycytidine (DNA) with the 3'-hydroxyl phosphorylated. The hydrogen atoms at C2' and C3' are not dis‐ played for clarity.

siRNA in fetal bovine or human serum at 37 °C resulted in the degradation and partial or complete loss of activity [30]. When incubated in human plasma at 37 °C, more than 50% of the unmodified siRNA was degraded within one minute, and practically all siRNA was completely degraded within 4 hours [31]. Although Ribonuclease A (RNase A, an endoribo‐ nuclease) cleaves single stranded RNA, siRNA degradation in serum was reported to be mainly due to RNase-like activity[32], which is suggested to occur during transient breaking of the hydrogen bonds joining the two siRNA strands. In addition to the RNase A family of enzymes, blood serum contains phosphatases and exoribonucleases which can also affect degradation of siRNA at nuclease-sensitive sites on both strands [33].

#### **2.3. Therapeutic potential of RNAi based therapies**

RNAi based therapies emerged in the period following its discovery in 1998 in plants, and are promising therapeutic candidates to treat various types of diseases, ranging from age related macular oedema to respiratory tract infections to various types of cancer [34-36]. In addition to siRNA based therapies, shRNA [37, 38] and miRNA [39] are potential therapeutic tools. siRNA based therapeutics are already in phase I and phase II clinical trials; representative examples of clinical trials involving siRNA are shown in Table 1. The basic concept is the reduction or inhibition of the expression of a protein that is involved in the pathophysiological pathway of the target disease (silencing/knocking-down the target gene). This concept is evident from using Cand5 siRNA targeting the mRNA translating the vascular endothelial growth factor (VEGF), thus reducing/inhibiting angiogenesis and preventing progression of wet age related macular oedema (Table 1) [40]. Atu027 siRNA targets the biosynthesis of protein kinase N3 which plays a role in cancer metastasis [41].


**Table 1.** Representative clinical trials using siRNA (http://clinicaltrials.gov/ct2/home, accessed on 5/8/2012).

siRNA in fetal bovine or human serum at 37 °C resulted in the degradation and partial or complete loss of activity [30]. When incubated in human plasma at 37 °C, more than 50% of the unmodified siRNA was degraded within one minute, and practically all siRNA was completely degraded within 4 hours [31]. Although Ribonuclease A (RNase A, an endoribo‐ nuclease) cleaves single stranded RNA, siRNA degradation in serum was reported to be mainly due to RNase-like activity[32], which is suggested to occur during transient breaking of the hydrogen bonds joining the two siRNA strands. In addition to the RNase A family of enzymes, blood serum contains phosphatases and exoribonucleases which can also affect

**Figure 4.** 3'-endo ribose configuration of RNA (left) vs 2'-endo (right) of 2'-deoxyribose in DNA. Shown is cytidine (RNA) and deoxycytidine (DNA) with the 3'-hydroxyl phosphorylated. The hydrogen atoms at C2' and C3' are not dis‐

Deoxycytidine in DNA

O– O P O

O–

O

NH2

N

H

N

O

2'-endo

3'-exo

OH

H

RNAi based therapies emerged in the period following its discovery in 1998 in plants, and are promising therapeutic candidates to treat various types of diseases, ranging from age related macular oedema to respiratory tract infections to various types of cancer [34-36]. In addition to siRNA based therapies, shRNA [37, 38] and miRNA [39] are potential therapeutic tools. siRNA based therapeutics are already in phase I and phase II clinical trials; representative examples of clinical trials involving siRNA are shown in Table 1. The basic concept is the reduction or inhibition of the expression of a protein that is involved in the pathophysiological pathway of the target disease (silencing/knocking-down the target gene). This concept is evident from using Cand5 siRNA targeting the mRNA translating the vascular endothelial growth factor (VEGF), thus reducing/inhibiting angiogenesis and preventing progression of wet age related macular oedema (Table 1) [40]. Atu027 siRNA targets the biosynthesis of

degradation of siRNA at nuclease-sensitive sites on both strands [33].

Cytidine in RNA

O–

<sup>O</sup> O H

O

2'-exo

O

NH2

N

H

N

3'-endo

O– O P

H

OH

140 Gene Therapy - Tools and Potential Applications

played for clarity.

**2.3. Therapeutic potential of RNAi based therapies**

protein kinase N3 which plays a role in cancer metastasis [41].

The therapeutic application of siRNA requires overcoming several barriers (Figure 5) for its intracellular delivery and the subsequent functional gene silencing activity [42-44]. Those barriers are mainly due to siRNA specific characteristics, most important are having a highly negative charge due to their phosphate backbone (on average 40-50 negative charges per siRNA), being susceptible to degradation by nucleases, and having relatively large molecular weight (13-15 kDa) compared to conventional small drug molecules. First, local delivery (such as intravitreal) is different from intravenous delivery, where the latter will subject the siRNA to the serum ribonucleases, which results in degrading non-modified siRNA within time periods that vary from minutes to hours [31]. siRNA injected intravenously in rats was reported to be cleared rapidly from circulation and accumulates in kidneys within minutes of injection [45], making it useful only if the target organ is the kidney.

In order to gain access into the cytoplasm where siRNA can exert its biological activity, the polyribonucleotide must pass first through the interstitial space then through the target cell membrane. This will be a difficult task, since both the extracellular matrix in many tissue types and the cell membrane incorporate negatively charged glycosaminoglycans (e.g. heparan sulfate) [46]. In addition, cell membranes contain negatively charged phospholipids (e.g. phosphatidyl serine) therefore the membrane is negatively charged [46, 47]. The net result is an unfavourable repulsive interaction with naked siRNA. As a result, different strategies are being developed to overcome the barriers to reproducible and functional siRNA delivery, and these approaches fall into two general categories. One category is modifying the siRNA, the other is deploying a vector to protect the siRNA and increase its efficiency of delivery.

another common modification. Locked nucleic acids (LNAs) have a methylene bridge connecting the 2'-*O* to the 4'-C of the ribose unit, locking the sugar in the 3'-endo conformation. These modifications led to increased ribonuclease resistance [48, 49]. Modifications at the phosphate backbone include phosphorthioate, boranophosphate, and methylphosphonate linkages [48, 49] and is reported to increase siRNA stability against various ribonucleases and phophodiesterases [50]. siRNA nucleotides can be substituted with DNA nucleotides to increase stability and/or decrease unwanted siRNA off-target effects [51]. Modifications of the 3'-overhangs (usually two nucleotides in length) include incorporating deoxyribonucleotides to reduce costs and increase stability towards 3-exoribonucleases. The 5'-terminus chemical phosphorylation of the antisense strand results in higher gene silencing efficiency, while blunt ended duplexes were reported to be more resistant to exonucleases. The advantages of each of the aforementioned techniques, other modification strategies, as well as the considerations related to the degree of modification and its effect on gene silencing efficiency and associated

siRNA and Gene Formulation for Efficient Gene Therapy

http://dx.doi.org/10.5772/55518

143

The conjugation of drug molecules, aptamers, lipids, polymers, and peptides/proteins to siRNA could enhance in vivo delivery [55]. The main aims of such conjugations are: to enhance siRNA stability, increase in vivo half-life, control biodistribution, increase efficiency of

One strategy is to increase the hydrophobicity of the siRNA. Cholesterol was conjugated to the 5'-terminus of siRNA, the cholesterol-siRNA conjugate (chol-siRNA) resulted in better intracellular delivery compared to unmodified siRNA and retained gene silencing activity in vitro in *β*-galactosidase expressing liver cells [56]. When cholesterol was conjugated to the 3' terminus of the sense (passenger) strand of siRNA, the conjugate had improved in vivo pharmacokinetics as the intravenous administration of chol-siRNA in mice resulted in its distribution and detection in the fat tissues, heart, kidneys, liver, and lungs, even 24 h after intravenous injection [57]. No significant amounts of unmodified siRNA were detected in the tissues 24 h after the intravenous injection. Conjugation of siRNA to bile acids and long-chain fatty acids, in addition to cholesterol, mediates siRNA uptake into cells and gene silencing in vivo [58]. The medium chain fatty-acid conjugates, namely lauroyl (C12), myristoyl (C14) and palmitoyl (C16), did not silence the target apolipoprotein B mRNA levels in mouse livers after intravenous injection. However, siRNA fatty-acid conjugates having long saturated chains, stearoyl (C18) and docosanoyl (C22), significantly reduced apolipoprotein B mRNA levels. Cell penetrating peptides (CPPs) are used to facilitate cellular membrane crossing of many molecules displaying various properties such as antisense oligonucleotides, peptides, and proteins and are already being tested in vivo [59]. siRNA was conjugated to penetratin and transportin, to silence luciferase and green fluorescent protein (GFP) in different types of mammalian cells [60]. However, in vivo lung delivery in mouse of siRNA conjugated to penetratin and TAT(48-60), targeting p38 MAP kinase mRNA showed that the reduction in gene expression was peptide induced and the penetratin conjugated siRNA resulted in innate

siRNA functioning against the VEGF mRNA was conjugated to poly(ethylene glycol) (PEG, 25 kDa) via a disulfide bond at the 3'-terminus of the sense strand [62]. The siRNA-PEG

cytotoxic effects have been reviewed thoroughly [48, 52-54].

immunity response [61].

intracellular delivery, while maintaining the gene silencing activity.

Figure 5. Summary of barriers to successful gene-silencing mediated by siRNA after intravenous injection, whether delivered naked or incorporated in nanoparticles. **Figure 5.** Summary of barriers to successful gene-silencing mediated by siRNA after intravenous injection, whether delivered naked or incorporated in nanoparticles.

#### **3. Strategies to achieve efficient siRNA delivery and gene silencing**

**3. Strategies to achieve efficient siRNA delivery and gene silencing** 

silencing efficiency and associated cytotoxic effects have been reviewed thoroughly [48, 52-54].

efficiency of intracellular delivery, while maintaining the gene silencing activity.

#### **3.1. siRNA modifications**  siRNA modifications include those carried out at the ribose residue, at the phosphate backbone, at the RNA nucleotides, the siRNA **3.1. siRNA modifications**

gene silencing activity in vitro in

termini, and/or by conjugation of other molecules to the siRNA molecule. Modifications to the ribose at the 2'-position are common [48], and include 2'-*O*-alkylation (e.g. 2'-*O*-methyl and 2'-*O*-methylethoxy) modifications. 2'-Fluoro RNA is another common modification. Locked nucleic acids (LNAs) have a methylene bridge connecting the 2'-*O* to the 4'-C of the ribose unit, locking the sugar in the 3'-endo conformation. These modifications led to increased ribonuclease resistance [48, 49]. Modifications at the phosphate backbone include phosphorthioate, boranophosphate, and methylphosphonate linkages [48, 49] and is reported to increase siRNA stability against various ribonucleases and phophodiesterases [50]. siRNA nucleotides can be substituted with siRNA modifications include those carried out at the ribose residue, at the phosphate back‐ bone, at the RNA nucleotides, the siRNA termini, and/or by conjugation of other molecules to the siRNA molecule. Modifications to the ribose at the 2'-position are common [48], and include 2'-*O*-alkylation (e.g. 2'-*O*-methyl and 2'-*O*-methylethoxy) modifications. 2'-Fluoro RNA is

DNA nucleotides to increase stability and/or decrease unwanted siRNA off-target effects [51]. Modifications of the 3'-overhangs (usually two nucleotides in length) include incorporating deoxyribonucleotides to reduce costs and increase stability towards 3 exoribonucleases. The 5'-terminus chemical phosphorylation of the antisense strand results in higher gene silencing efficiency, while blunt ended duplexes were reported to be more resistant to exonucleases. The advantages of each of the aforementioned techniques, other modification strategies, as well as the considerations related to the degree of modification and its effect on gene

The conjugation of drug molecules, aptamers, lipids, polymers, and peptides/proteins to siRNA could enhance in vivo delivery [55]. The main aims of such conjugations are: to enhance siRNA stability, increase in vivo half-life, control biodistribution, increase

One strategy is to increase the hydrophobicity of the siRNA. Cholesterol was conjugated to the 5'-terminus of siRNA, the cholesterol-siRNA conjugate (chol-siRNA) resulted in better intracellular delivery compared to unmodified siRNA and retained

the sense (passenger) strand of siRNA, the conjugate had improved in vivo pharmacokinetics as the intravenous administration of chol-siRNA in mice resulted in its distribution and detection in the fat tissues, heart, kidneys, liver, and lungs, even 24 h after intravenous injection [57]. No significant amounts of unmodified siRNA were detected in the tissues 24 h after the intravenous


another common modification. Locked nucleic acids (LNAs) have a methylene bridge connecting the 2'-*O* to the 4'-C of the ribose unit, locking the sugar in the 3'-endo conformation. These modifications led to increased ribonuclease resistance [48, 49]. Modifications at the phosphate backbone include phosphorthioate, boranophosphate, and methylphosphonate linkages [48, 49] and is reported to increase siRNA stability against various ribonucleases and phophodiesterases [50]. siRNA nucleotides can be substituted with DNA nucleotides to increase stability and/or decrease unwanted siRNA off-target effects [51]. Modifications of the 3'-overhangs (usually two nucleotides in length) include incorporating deoxyribonucleotides to reduce costs and increase stability towards 3-exoribonucleases. The 5'-terminus chemical phosphorylation of the antisense strand results in higher gene silencing efficiency, while blunt ended duplexes were reported to be more resistant to exonucleases. The advantages of each of the aforementioned techniques, other modification strategies, as well as the considerations related to the degree of modification and its effect on gene silencing efficiency and associated cytotoxic effects have been reviewed thoroughly [48, 52-54].

and the cell membrane incorporate negatively charged glycosaminoglycans (e.g. heparan sulfate) [46]. In addition, cell membranes contain negatively charged phospholipids (e.g. phosphatidyl serine) therefore the membrane is negatively charged [46, 47]. The net result is an unfavourable repulsive interaction with naked siRNA. As a result, different strategies are being developed to overcome the barriers to reproducible and functional siRNA delivery, and these approaches fall into two general categories. One category is modifying the siRNA, the other is deploying a vector to protect the siRNA and increase its efficiency of delivery.

**RISC**

Figure 5. Summary of barriers to successful gene-silencing mediated by siRNA after intravenous injection, whether delivered naked or

**Figure 5.** Summary of barriers to successful gene-silencing mediated by siRNA after intravenous injection, whether

**3. Strategies to achieve efficient siRNA delivery and gene silencing**

siRNA modifications include those carried out at the ribose residue, at the phosphate backbone, at the RNA nucleotides, the siRNA termini, and/or by conjugation of other molecules to the siRNA molecule. Modifications to the ribose at the 2'-position are common [48], and include 2'-*O*-alkylation (e.g. 2'-*O*-methyl and 2'-*O*-methylethoxy) modifications. 2'-Fluoro RNA is another common modification. Locked nucleic acids (LNAs) have a methylene bridge connecting the 2'-*O* to the 4'-C of the ribose unit, locking the sugar in the 3'-endo conformation. These modifications led to increased ribonuclease resistance [48, 49]. Modifications at the phosphate backbone include phosphorthioate, boranophosphate, and methylphosphonate linkages [48, 49] and is reported to increase siRNA stability against various ribonucleases and phophodiesterases [50]. siRNA nucleotides can be substituted with DNA nucleotides to increase stability and/or decrease unwanted siRNA off-target effects [51]. Modifications of the 3'-overhangs (usually two nucleotides in length) include incorporating deoxyribonucleotides to reduce costs and increase stability towards 3 exoribonucleases. The 5'-terminus chemical phosphorylation of the antisense strand results in higher gene silencing efficiency, while blunt ended duplexes were reported to be more resistant to exonucleases. The advantages of each of the aforementioned techniques, other modification strategies, as well as the considerations related to the degree of modification and its effect on gene

siRNA modifications include those carried out at the ribose residue, at the phosphate back‐ bone, at the RNA nucleotides, the siRNA termini, and/or by conjugation of other molecules to the siRNA molecule. Modifications to the ribose at the 2'-position are common [48], and include 2'-*O*-alkylation (e.g. 2'-*O*-methyl and 2'-*O*-methylethoxy) modifications. 2'-Fluoro RNA is

The conjugation of drug molecules, aptamers, lipids, polymers, and peptides/proteins to siRNA could enhance in vivo delivery [55]. The main aims of such conjugations are: to enhance siRNA stability, increase in vivo half-life, control biodistribution, increase

One strategy is to increase the hydrophobicity of the siRNA. Cholesterol was conjugated to the 5'-terminus of siRNA, the cholesterol-siRNA conjugate (chol-siRNA) resulted in better intracellular delivery compared to unmodified siRNA and retained

the sense (passenger) strand of siRNA, the conjugate had improved in vivo pharmacokinetics as the intravenous administration of chol-siRNA in mice resulted in its distribution and detection in the fat tissues, heart, kidneys, liver, and lungs, even 24 h after intravenous injection [57]. No significant amounts of unmodified siRNA were detected in the tissues 24 h after the intravenous


**Crossing cell membrane**

**Serum degradation**

**Renal clearance**

**Intravenous Injection of naked siRNA**

**3. Strategies to achieve efficient siRNA delivery and gene silencing** 

**Endosome/lysosome degradation**

**siRNA release**

**Serum destabilization**

**Non-specific distribution**

**Vascular endothelium**

**Intravenous Injection of siRNA nanoparticles**

silencing efficiency and associated cytotoxic effects have been reviewed thoroughly [48, 52-54].

efficiency of intracellular delivery, while maintaining the gene silencing activity.

incorporated in nanoparticles.

**Extracellular**

**Cell membrane**

**Intracellular**

142 Gene Therapy - Tools and Potential Applications

**3.1. siRNA modifications** 

**3.1. siRNA modifications**

delivered naked or incorporated in nanoparticles.

gene silencing activity in vitro in

The conjugation of drug molecules, aptamers, lipids, polymers, and peptides/proteins to siRNA could enhance in vivo delivery [55]. The main aims of such conjugations are: to enhance siRNA stability, increase in vivo half-life, control biodistribution, increase efficiency of intracellular delivery, while maintaining the gene silencing activity.

One strategy is to increase the hydrophobicity of the siRNA. Cholesterol was conjugated to the 5'-terminus of siRNA, the cholesterol-siRNA conjugate (chol-siRNA) resulted in better intracellular delivery compared to unmodified siRNA and retained gene silencing activity in vitro in *β*-galactosidase expressing liver cells [56]. When cholesterol was conjugated to the 3' terminus of the sense (passenger) strand of siRNA, the conjugate had improved in vivo pharmacokinetics as the intravenous administration of chol-siRNA in mice resulted in its distribution and detection in the fat tissues, heart, kidneys, liver, and lungs, even 24 h after intravenous injection [57]. No significant amounts of unmodified siRNA were detected in the tissues 24 h after the intravenous injection. Conjugation of siRNA to bile acids and long-chain fatty acids, in addition to cholesterol, mediates siRNA uptake into cells and gene silencing in vivo [58]. The medium chain fatty-acid conjugates, namely lauroyl (C12), myristoyl (C14) and palmitoyl (C16), did not silence the target apolipoprotein B mRNA levels in mouse livers after intravenous injection. However, siRNA fatty-acid conjugates having long saturated chains, stearoyl (C18) and docosanoyl (C22), significantly reduced apolipoprotein B mRNA levels.

Cell penetrating peptides (CPPs) are used to facilitate cellular membrane crossing of many molecules displaying various properties such as antisense oligonucleotides, peptides, and proteins and are already being tested in vivo [59]. siRNA was conjugated to penetratin and transportin, to silence luciferase and green fluorescent protein (GFP) in different types of mammalian cells [60]. However, in vivo lung delivery in mouse of siRNA conjugated to penetratin and TAT(48-60), targeting p38 MAP kinase mRNA showed that the reduction in gene expression was peptide induced and the penetratin conjugated siRNA resulted in innate immunity response [61].

siRNA functioning against the VEGF mRNA was conjugated to poly(ethylene glycol) (PEG, 25 kDa) via a disulfide bond at the 3'-terminus of the sense strand [62]. The siRNA-PEG conjugate formed polyelectrolyte complex (PEC) micelles by electrostatic interaction with the cationic polymer polyethylenimine (PEI). The formed VEGF siRNA-PEG/PEI PEC micelles showed enhanced stability against nuclease degradation compared to the unmodified siRNA. These micelles efficiently silenced VEGF gene expression in prostate carcinoma cells (PC-3) and showed superior VEGF gene silencing compared to VEGF siRNA/PEI complexes in the presence of serum. PEG conjugation on its own enhanced the stability of the siRNA in serum containing medium. The prolonged stability of the PEC micelles was suggested to be due to the presence of PEG chains in the outer micellar shell layer, thus sterically hindering nuclease access into the siRNA in the micelle core [62]. Targeting molecules such as antibodies [63] and aptamers (peptides or single stranded DNA or RNA that have selective affinities toward target proteins) [64] have also been conjugated to siRNA, with the aim of increasing the efficiency of siRNA delivery to the target tissues.

Conjugating molecules to siRNA requires specific considerations. First, the site of conjugation (3'- and/or 5'-terminus, on sense and/or antisense strand) should be chosen such that it does not affect the activity of the siRNA and its ability to be incorporated in the RISC, or its ability to bind the target mRNA in the correct helix conformation. Second, the conjugated siRNA might have new properties that were not present in the unmodified parent siRNA. An example is the in vivo immune response resulting from the penetratin-siRNA conjugate [61]. Third, the conjugation process is multi-step, and the chemical reaction intermediates and products require efficient purification in order to meet the specifications of in vivo applications. These steps need to be repeated for each siRNA under investigation, which can be costly and time consuming. Thus, although there are clear advantages to synthesize siRNA conjugates, there are also disadvantages, and conjugation is therefore only one of two valuable approaches in the toolbox for preparing siRNA based therapies. The other valuable tool is complexation or incorporating the siRNA in a vector.

insertional mutagenesis [68, 71]. shRNA expression cassette delivered by a retroviral vector was used in rats to silence a RAS oncogene in order to suppress tumour growth [72]. Herpes virus was used successfully to deliver shRNA targeting exogenous *β*-galactosidase or endog‐ enous trpv1 gene mRNA in the peripheral neurons in mice by injecting once directly into the

**Table 2.** Summary of properties of viral vectors that are commonly used in gene therapy (adapted from http://

\* Lentiviral vectors can infect non-dividing cells as their pre-integration complex can traverse the nuclear membrane pores (NMP), in contrast to retrovirus pre-integration complex which does not traverse NMP, requiring the host-cell

**Retrovirus/ Lentivirus**

**Infection tropism** Dividing\* Dividing/

**Adenovirus**

**Genome** ssRNA dsDNA ssDNA dsDNA **Capsid** Icosahedral Icosahedral Icosahedral Icosahedral **Envelope** Enveloped None None Enveloped **Viral Polymerase** Positive Negative Negative Negative **Diameter (nm)** 80-130 70-90 18-26 150-200 **Genome size (kb)** 7-10 38 5 120-200

Non-dividing

**expression** Lasting Transient Lasting Transient

**integration** Integrating Non-integrating Integrating Non-integrating

7-8 8 4.5 >30

**Adenoassociated virus**

siRNA and Gene Formulation for Efficient Gene Therapy

Dividing/ Non-dividing **Herpes virus**

145

http://dx.doi.org/10.5772/55518

Dividing/ Non-dividing

Unlike other retroviruses, lentiviruses can infect dividing as well as differentiated and nondividing cells. The lentiviral genome can accommodate 7.5 kb [66], and their genome is integrated in the host cell genome, lentiviral vectors are generally preferred for long-term expression of transgenes, and efficient delivery in vivo to the brain, eye, and liver to induce long-term transgene expression as reported [74]. A lentiviral vector was used to deliver shRNA targeting *Smad3* gene mRNA, and enhanced myogenesis of old and injured muscles [75].

Adenoviruses are non-enveloped viruses, with linear double stranded DNA. They preferably infect the upper respiratory tract and the ocular tissue. Their genome can accommodate up to 8 kb which can be extended to ≥25 kb in modified viruses that have their viral genes deleted [68]. These viruses can infect post mitotic cells and thus are good candidates for neurological diseases. Unless delivering genes that can exist as episomes in host cells, adenoviruses result only in transient expression of their cargo. However, although the host cells with the episome can express the delivered genes for the cell life time, these cells will eventually be removed by the host immune system [68]. shRNA targeting VEGF that was delivered by an adenoviral

vector resulted in potent inhibition of angiogenesis and tumour growth in mice [76].

sciatic nerve of the animals [73].

**Virus genome**

**Transgene**

**Packaging capacity (kb)**

division to integrate the retroviral genome [67].

www.genetherapynet.com/viral-vectors.html, accessed on 5/8/2012).

**Viral vector properties**

**Gene therapy related**

#### **3.2. Viral vectors for shRNA delivery**

Vectors for RNAi based therapies are either viral or non-viral vectors. Viral vectors (Table 2) are used to deliver genes encoding hairpin RNA structures such as shRNA and miRNA, which are then processed by the cellular RNAi machinery to the functional silencing dsRNA [65, 66].

Viral vectors offer two main advantages, the first is the very high efficiency compared to nonviral vectors [68], which can reach few orders of magnitude more than that achieved with nonviral vectors, and the second is the potential of long term expression of the delivered RNAi therapeutic, which is very useful in the treatment of chronic diseases such as HIV infection and viral hepatitis [69, 70]. Retroviruses are enveloped, single stranded RNA viruses and have a genome capacity of 7-10 kilobases (kb). They preferentially target dividing cells which limits their use to mitotic tissues (thus for example excluding brain and neurons). Retroviruses integrate their DNA in the host genome using an integrase enzyme, which provides the advantage of stable long term expression of the delivered transgene in the host cell and its descendants. However, integrating new DNA sequences into host genome carries the risk of


conjugate formed polyelectrolyte complex (PEC) micelles by electrostatic interaction with the cationic polymer polyethylenimine (PEI). The formed VEGF siRNA-PEG/PEI PEC micelles showed enhanced stability against nuclease degradation compared to the unmodified siRNA. These micelles efficiently silenced VEGF gene expression in prostate carcinoma cells (PC-3) and showed superior VEGF gene silencing compared to VEGF siRNA/PEI complexes in the presence of serum. PEG conjugation on its own enhanced the stability of the siRNA in serum containing medium. The prolonged stability of the PEC micelles was suggested to be due to the presence of PEG chains in the outer micellar shell layer, thus sterically hindering nuclease access into the siRNA in the micelle core [62]. Targeting molecules such as antibodies [63] and aptamers (peptides or single stranded DNA or RNA that have selective affinities toward target proteins) [64] have also been conjugated to siRNA, with the aim of increasing the efficiency of

Conjugating molecules to siRNA requires specific considerations. First, the site of conjugation (3'- and/or 5'-terminus, on sense and/or antisense strand) should be chosen such that it does not affect the activity of the siRNA and its ability to be incorporated in the RISC, or its ability to bind the target mRNA in the correct helix conformation. Second, the conjugated siRNA might have new properties that were not present in the unmodified parent siRNA. An example is the in vivo immune response resulting from the penetratin-siRNA conjugate [61]. Third, the conjugation process is multi-step, and the chemical reaction intermediates and products require efficient purification in order to meet the specifications of in vivo applications. These steps need to be repeated for each siRNA under investigation, which can be costly and time consuming. Thus, although there are clear advantages to synthesize siRNA conjugates, there are also disadvantages, and conjugation is therefore only one of two valuable approaches in the toolbox for preparing siRNA based therapies. The other valuable tool is complexation or

Vectors for RNAi based therapies are either viral or non-viral vectors. Viral vectors (Table 2) are used to deliver genes encoding hairpin RNA structures such as shRNA and miRNA, which are then processed by the cellular RNAi machinery to the functional silencing

Viral vectors offer two main advantages, the first is the very high efficiency compared to nonviral vectors [68], which can reach few orders of magnitude more than that achieved with nonviral vectors, and the second is the potential of long term expression of the delivered RNAi therapeutic, which is very useful in the treatment of chronic diseases such as HIV infection and viral hepatitis [69, 70]. Retroviruses are enveloped, single stranded RNA viruses and have a genome capacity of 7-10 kilobases (kb). They preferentially target dividing cells which limits their use to mitotic tissues (thus for example excluding brain and neurons). Retroviruses integrate their DNA in the host genome using an integrase enzyme, which provides the advantage of stable long term expression of the delivered transgene in the host cell and its descendants. However, integrating new DNA sequences into host genome carries the risk of

siRNA delivery to the target tissues.

144 Gene Therapy - Tools and Potential Applications

incorporating the siRNA in a vector.

**3.2. Viral vectors for shRNA delivery**

dsRNA [65, 66].

\* Lentiviral vectors can infect non-dividing cells as their pre-integration complex can traverse the nuclear membrane pores (NMP), in contrast to retrovirus pre-integration complex which does not traverse NMP, requiring the host-cell division to integrate the retroviral genome [67].

**Table 2.** Summary of properties of viral vectors that are commonly used in gene therapy (adapted from http:// www.genetherapynet.com/viral-vectors.html, accessed on 5/8/2012).

insertional mutagenesis [68, 71]. shRNA expression cassette delivered by a retroviral vector was used in rats to silence a RAS oncogene in order to suppress tumour growth [72]. Herpes virus was used successfully to deliver shRNA targeting exogenous *β*-galactosidase or endog‐ enous trpv1 gene mRNA in the peripheral neurons in mice by injecting once directly into the sciatic nerve of the animals [73].

Unlike other retroviruses, lentiviruses can infect dividing as well as differentiated and nondividing cells. The lentiviral genome can accommodate 7.5 kb [66], and their genome is integrated in the host cell genome, lentiviral vectors are generally preferred for long-term expression of transgenes, and efficient delivery in vivo to the brain, eye, and liver to induce long-term transgene expression as reported [74]. A lentiviral vector was used to deliver shRNA targeting *Smad3* gene mRNA, and enhanced myogenesis of old and injured muscles [75].

Adenoviruses are non-enveloped viruses, with linear double stranded DNA. They preferably infect the upper respiratory tract and the ocular tissue. Their genome can accommodate up to 8 kb which can be extended to ≥25 kb in modified viruses that have their viral genes deleted [68]. These viruses can infect post mitotic cells and thus are good candidates for neurological diseases. Unless delivering genes that can exist as episomes in host cells, adenoviruses result only in transient expression of their cargo. However, although the host cells with the episome can express the delivered genes for the cell life time, these cells will eventually be removed by the host immune system [68]. shRNA targeting VEGF that was delivered by an adenoviral vector resulted in potent inhibition of angiogenesis and tumour growth in mice [76].

Adeno-associated virus (AAV) is a single stranded DNA non-pathogenic virus that can accommodate a 4.7 kb genome. They can infect dividing or non-dividing cells. The replication of AAV requires co-infection with adenovirus. The viral genome integrates into the host cell genome at a specific location on chromosome 19 [68]. Direct intracerebellar injection in a mouse model of spinocerebellar ataxia of an AAV vector delivering a cargo expressing shRNA targeting polyglutamine induced neurodegeneration significantly restored cerebellar mor‐ phology and improved motor coordination in mice [77].

Although highly efficient in delivering their cargo, viral vectors have their disadvantages. Adenoviral vectors have the disadvantage of triggering a strong immune (adaptive and innate) response by repeated administration, in addition to target organ immunotoxicity, specially hepatotoxicity [78-80], which resulted in 1999 in the death of one 18-year-old male who received high dose of adenovirus that was delivered directly in the hepatic artery in a clinical gene therapy safety study [81]. Clonal T-cell acute lymphoblastic leukemia caused by inser‐ tional mutagenesis in a gene therapy completed clinical trial involving patients suffering Xlinked severe combined immunodeficiency (SCID-X1) was reported in one out of the 10 patients using a retroviral vector [82]. Integration of the vector genome material in the antisense orientation 35 kb upstream of the protooncogene (LMO2) caused over expression of the gene in the leukemic cells. In a similar study, 4 out of 9 patients developed leukemia within 3-6 years post-treatment mainly due to vector-mediated upregulation of host cellular oncogenes [83, 84]. In addition, immune responses (whether adaptive or innate) of varying degrees depending on the type of vector, dose, and target organs were reported for lentiviral, adenoviral, adenoassociated viral vectors [80].

It was reported by Frankel and Pabo in 1988 that the HIV-1 derived TAT protein could be taken up by cells growing in tissue culture [102], and that a small basic region of TAT (48-60) was essential for uptake by the cells [103]. PTDs include antennapedia homeodomain protein (Antp, penetratin), mitogen-activated protein (MAP), poly-arginine, transportan, VP22 [59, 92]. Two major pathways are involved in the uptake of PTDs and PTD-cargos: direct translo‐ cation at 4 °C and 37 °C and endocytosis-translocation at 37 °C. These mechanisms depend on many factors: cargo size, cell line, PTD concentration, and the type of PTD [59, 104, 105]. siRNA can be conjugated covalently to the CPP or can be complexed with the cationic groups of basic amino acids that are present in the backbone of the CPP. As a representative example of noncovalent complexation, CADY [94], which is basic due to its five arginine residues can complex with the negatively charged siRNA. Another example of non-covalent complexation is the

CADY GLWRALWRLLRSLWRLLWRA Non-covalent GAPDH, p53 [94] EB1 LIRLWSHLIHIWFQNRRLKWKKK Non-covalent Luc [95]

Poly-arginine RRRRRRRRR Non-covalent VEGF [98]

Transportan LIKKALAALAKLNIKLLYGASNLTWG Covalent Luc, EGFP [100] TAT GRKKRRQRRRPPQ Covalent EGFP, CDK9 [101]

MPG GALFLGFLGAAGSTMGAWSQPKKKRKV Non-covalent Luc, GAPDH [96] Oct-3/4 [97]

**Type of association with siRNA**

> Covalent Covalent Covalent

**Target mRNA**

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147

siRNA and Gene Formulation for Efficient Gene Therapy

Luciferase (Luc), EGFP [60] SOD1, caspase-3 [99] Luc, p38 MAP kinase [61, 100]

PEI (Figure 6) is an efficient, but toxic, plasmid DNA delivery vector. However, as a siRNA delivery vector PEI is reported to be much less efficient [106, 107]. This decreased efficiency is due to the dissociation of the siRNA/PEI complex upon interaction with the negatively charged cell membrane, which is suggested to be because of the short length of siRNA and the associated weak electrostatic interaction with PEI [108, 109]. Another drawback of PEI is its relatively high toxicity [110]. Thus, in addition to linear PEI, PEI polymers with a wide range of molecular weights were developed to increase PEI efficiency and/or decrease toxicity, although not all PEI are suitable for siRNA delivery [111]. The main advantage of PEI is the ability of its variety of amino groups to be protonated at lower pH (inside endosomes) leading to what is known as the "proton-sponge effect" [112], and efficient escape of the nucleic acid

One approach to enhance siRNA delivery with PEI is increasing the hydrophobicity of PEI by covalently conjugating alkyl chains [113], where increasing the hydrophobic alkyl chain length generally improved the stability of the PEI/siRNA complex. In a similar strategy, cholesterol was conjugated to PEI with decreased toxicity of the conjugates [114]. Low molecular weight PEI (MW < 5 kDa) is less toxic than the higher molecular weight PEI (≈25 kDa), but less efficient

poly-arginine CPP [98].

**CPP Sequence of CPP**

Penetratin RQIKIWFQNRRMKWKK

**Table 3.** Selected CPPs used for siRNA delivery [59].

cargo from endosomes.

Current research on viral vectors for gene therapy is focussed on approaches such as vector engineering e.g. modifying the viral capsid or pseuodotyping the envelope, different delivery strategies, and administration to immune-privileged sites that can tolerate the delivered viral vectors without responding with an inflammatory response [80, 85]. Other research focusses on the essential scaling-up process of vector production and increasing the packaging effi‐ ciency of the vectors [85], the processes without which, the wide spread and successful therapeutic use of the viral vectors will be very difficult to achieve.

#### **3.3. Non-viral vectors**

Non-viral vectors for gene and siRNA delivery are an alternative to viral vectors, as they do not suffer many of the disadvantages of the viral vectors, especially immunogenicity and tumourigenicity. The non-viral vectors can be classified generally as peptides, polymeric based vectors, carbohydrate based, and lipid based [86]. CPPs, also known as peptide transduction domains (PTDs), have shown the ability to cross the cellular membrane despite their relatively high molecular weight and size (Table 3).

PTDs generally are short amphipathic and/or cationic peptides that can transport many hydrophilic molecules across the cell membrane. A wide range of molecules including liposomes [87, 88], peptides, proteins [89], peptide nucleic acids [90] and polynucleotides [91] are delivered intracellularly using PTDs and they have also been applied in vivo [59, 92, 93].


**Table 3.** Selected CPPs used for siRNA delivery [59].

Adeno-associated virus (AAV) is a single stranded DNA non-pathogenic virus that can accommodate a 4.7 kb genome. They can infect dividing or non-dividing cells. The replication of AAV requires co-infection with adenovirus. The viral genome integrates into the host cell genome at a specific location on chromosome 19 [68]. Direct intracerebellar injection in a mouse model of spinocerebellar ataxia of an AAV vector delivering a cargo expressing shRNA targeting polyglutamine induced neurodegeneration significantly restored cerebellar mor‐

Although highly efficient in delivering their cargo, viral vectors have their disadvantages. Adenoviral vectors have the disadvantage of triggering a strong immune (adaptive and innate) response by repeated administration, in addition to target organ immunotoxicity, specially hepatotoxicity [78-80], which resulted in 1999 in the death of one 18-year-old male who received high dose of adenovirus that was delivered directly in the hepatic artery in a clinical gene therapy safety study [81]. Clonal T-cell acute lymphoblastic leukemia caused by inser‐ tional mutagenesis in a gene therapy completed clinical trial involving patients suffering Xlinked severe combined immunodeficiency (SCID-X1) was reported in one out of the 10 patients using a retroviral vector [82]. Integration of the vector genome material in the antisense orientation 35 kb upstream of the protooncogene (LMO2) caused over expression of the gene in the leukemic cells. In a similar study, 4 out of 9 patients developed leukemia within 3-6 years post-treatment mainly due to vector-mediated upregulation of host cellular oncogenes [83, 84]. In addition, immune responses (whether adaptive or innate) of varying degrees depending on the type of vector, dose, and target organs were reported for lentiviral, adenoviral, adeno-

Current research on viral vectors for gene therapy is focussed on approaches such as vector engineering e.g. modifying the viral capsid or pseuodotyping the envelope, different delivery strategies, and administration to immune-privileged sites that can tolerate the delivered viral vectors without responding with an inflammatory response [80, 85]. Other research focusses on the essential scaling-up process of vector production and increasing the packaging effi‐ ciency of the vectors [85], the processes without which, the wide spread and successful

Non-viral vectors for gene and siRNA delivery are an alternative to viral vectors, as they do not suffer many of the disadvantages of the viral vectors, especially immunogenicity and tumourigenicity. The non-viral vectors can be classified generally as peptides, polymeric based vectors, carbohydrate based, and lipid based [86]. CPPs, also known as peptide transduction domains (PTDs), have shown the ability to cross the cellular membrane despite their relatively

PTDs generally are short amphipathic and/or cationic peptides that can transport many hydrophilic molecules across the cell membrane. A wide range of molecules including liposomes [87, 88], peptides, proteins [89], peptide nucleic acids [90] and polynucleotides [91] are delivered intracellularly using PTDs and they have also been applied in vivo [59, 92, 93].

therapeutic use of the viral vectors will be very difficult to achieve.

phology and improved motor coordination in mice [77].

associated viral vectors [80].

146 Gene Therapy - Tools and Potential Applications

**3.3. Non-viral vectors**

high molecular weight and size (Table 3).

It was reported by Frankel and Pabo in 1988 that the HIV-1 derived TAT protein could be taken up by cells growing in tissue culture [102], and that a small basic region of TAT (48-60) was essential for uptake by the cells [103]. PTDs include antennapedia homeodomain protein (Antp, penetratin), mitogen-activated protein (MAP), poly-arginine, transportan, VP22 [59, 92]. Two major pathways are involved in the uptake of PTDs and PTD-cargos: direct translo‐ cation at 4 °C and 37 °C and endocytosis-translocation at 37 °C. These mechanisms depend on many factors: cargo size, cell line, PTD concentration, and the type of PTD [59, 104, 105]. siRNA can be conjugated covalently to the CPP or can be complexed with the cationic groups of basic amino acids that are present in the backbone of the CPP. As a representative example of noncovalent complexation, CADY [94], which is basic due to its five arginine residues can complex with the negatively charged siRNA. Another example of non-covalent complexation is the poly-arginine CPP [98].

PEI (Figure 6) is an efficient, but toxic, plasmid DNA delivery vector. However, as a siRNA delivery vector PEI is reported to be much less efficient [106, 107]. This decreased efficiency is due to the dissociation of the siRNA/PEI complex upon interaction with the negatively charged cell membrane, which is suggested to be because of the short length of siRNA and the associated weak electrostatic interaction with PEI [108, 109]. Another drawback of PEI is its relatively high toxicity [110]. Thus, in addition to linear PEI, PEI polymers with a wide range of molecular weights were developed to increase PEI efficiency and/or decrease toxicity, although not all PEI are suitable for siRNA delivery [111]. The main advantage of PEI is the ability of its variety of amino groups to be protonated at lower pH (inside endosomes) leading to what is known as the "proton-sponge effect" [112], and efficient escape of the nucleic acid cargo from endosomes.

One approach to enhance siRNA delivery with PEI is increasing the hydrophobicity of PEI by covalently conjugating alkyl chains [113], where increasing the hydrophobic alkyl chain length generally improved the stability of the PEI/siRNA complex. In a similar strategy, cholesterol was conjugated to PEI with decreased toxicity of the conjugates [114]. Low molecular weight PEI (MW < 5 kDa) is less toxic than the higher molecular weight PEI (≈25 kDa), but less efficient

simple complexation and adsorption of siRNA onto chitosan [118]. Although chitosan has good potential as a non-viral gene delivery vector, widespread use is largely limited by its poor solubility (because of their p*K*a, chitosan amino groups are only partially protonated at the physiological pH 7.4), poor stability of its siRNA complexes at the physiological pH, and low transfection efficiency. Various strategies have been adopted to overcome these draw‐ backs, such as covalently conjugating PEG to chitosan and binding targeting ligands to enhance

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Cyclodextrins (CD) are cyclic oligosaccharides composed of 6, 7, or 8 D-(+)-glucose units, known as α-CD, β-CD, γ-CD respectively, bound through α-1,4-linkages. Polymers conjugated to β-CD lack immunogenicity and hence are attractive vectors for polynucleotide delivery. β-CD have a hydrophilic outer surface and a hydrophobic inner cavity which enable them to form inclusion complexes. Efficient cellular transfection of siRNA labelled with a fluorescent tag into human embryonic lung fibroblasts (MRC-5 cells) was observed by siRNA complexes with the β-CD guanidine derivatized bis-(guanidinium)-tetrakis-(β-cyclodextrin) tetrapod (having four β-CD units) [119]. The ability of β-CD to form inclusion complexes was used to develop a siRNA delivery vector. β-CD was covalently bound to a polycationic segment (to electrostatically bind siRNA), while adamantane-PEG-transferrin (adamantane can fit in the β-CD cavity) formed an inclusion complex which can enhance the stability of siRNA nano‐ particles in vivo [120]. This system was used to deliver siRNA silencing the *EWS-FLI1* gene thus inhibiting tumour growth in a murine model of metastatic Ewing's sarcoma. The first experimental siRNA therapeutic to provide targeted delivery in humans was reported by Davis and co-workers [121]. siRNA was formulated into a nanoparticle (CALAA-01), which consisted of a cyclodextrin-containing polymer that contains amidine and primary amine functional groups, a PEG for steric stabilization in the in vivo environment (via inclusion complexes of β-CD with adamantine-PEG conjugate), and human transferrin (Tf) as the targeting ligand to binds to the transferrin receptors that are over-expressed on cancer cells. The siRNA/nanoparticles components self-assembled in the pharmacy. CALAA-01 was administered intravenously to the first patient with a solid cancer in a phase I clinical trial (safety study) in May 2008 [121]. Tumour biopsies from patients' melanoma after treatment (phase I clinical trial) showed the presence of intracellular nanoparticles. Reductions in the levels of both the specific mRNA (M2 subunit of ribonucleotide reductase, RRM2) and the protein (RRM2) were found when compared to levels in pre-dosing tissues. These results demonstrated that siRNA nanoparticles administered systemically to a human patient can produce a specific gene knock-down via an RNAi mechanism of action [122]. A recent and novel approach to the synthesis of cationic or neutral PEGylated amphiphilic β-CD used copper-catalysed "click" chemistry to modify selectively the secondary 2-hydroxyl group of the β-CD. The 6-position of these β-CD conjugates was conjugated to a dodecane alkyl chain. Complexation of cationic β-CD alone with siRNA resulted in good silencing of the luciferase reporter gene in Caco2 cells in culture. Co-formulation of cationic β-CD with a PEGylated β-CD and siRNA resulted in lower surface charges and reduced aggregation. The transfection efficiency of the cationic β-CD vector was lowered by co-formulation with the PEGylated β-CD, although the siRNA binding was not affected and the surface charge of the complexes did

cell specificity [116].

not reach complete neutrality [123].

**Figure 6.** Representative examples of polyamines used in siRNA delivery either as in lipid conjugates of polyamine al‐ kaloids e.g. spermine and spermidine, or as in polyethylenimine (PEI), a cationic polymer.

in polynucleotide delivery, thus, cross-linking of the low molecular weight PEI with disulfide bonds which are cleaved in the reducing environment of the cytoplasm increased the efficiency of siRNA delivery through the enhanced release of siRNA in the cytoplasm [115].

Chitosan is a biocompatible and biodegradable polysaccharide that is a copolymer of *N*-acetyl-D-glucosamine and D-glucosamine. Chitosan has weakly basic properties due to the presence of the D-glucosamine residue with a p*K*a value 6.2-7.0. The molecular weight of chitosan affects the complex stability, size, zeta-potential and in vitro gene knock-down of siRNA/chitosan nanoparticles [116]. High molecular weight (64.8-170 kDa) chitosan formed stable complexes with siRNA and resulted in high gene knock-down efficiency in human lung carcinoma (H1299) cells, while low molecular weight (10 kDa) chitosan could not complex the siRNA into stable nanoparticles and showed almost no knock-down [117]. The method of association affects gene silencing efficiency, where chitosan-TPP/siRNA nanoparticles (siRNA entrapped inside the nanoparticles, and TPP is sodium tripolyphosphate and used as a polyanion to crosslink with the cationic chitosan groups by electrostatic interactions) showed high siRNA binding and better gene silencing in vitro compared to siRNA/chitosan particles prepared by simple complexation and adsorption of siRNA onto chitosan [118]. Although chitosan has good potential as a non-viral gene delivery vector, widespread use is largely limited by its poor solubility (because of their p*K*a, chitosan amino groups are only partially protonated at the physiological pH 7.4), poor stability of its siRNA complexes at the physiological pH, and low transfection efficiency. Various strategies have been adopted to overcome these draw‐ backs, such as covalently conjugating PEG to chitosan and binding targeting ligands to enhance cell specificity [116].

Cyclodextrins (CD) are cyclic oligosaccharides composed of 6, 7, or 8 D-(+)-glucose units, known as α-CD, β-CD, γ-CD respectively, bound through α-1,4-linkages. Polymers conjugated to β-CD lack immunogenicity and hence are attractive vectors for polynucleotide delivery. β-CD have a hydrophilic outer surface and a hydrophobic inner cavity which enable them to form inclusion complexes. Efficient cellular transfection of siRNA labelled with a fluorescent tag into human embryonic lung fibroblasts (MRC-5 cells) was observed by siRNA complexes with the β-CD guanidine derivatized bis-(guanidinium)-tetrakis-(β-cyclodextrin) tetrapod (having four β-CD units) [119]. The ability of β-CD to form inclusion complexes was used to develop a siRNA delivery vector. β-CD was covalently bound to a polycationic segment (to electrostatically bind siRNA), while adamantane-PEG-transferrin (adamantane can fit in the β-CD cavity) formed an inclusion complex which can enhance the stability of siRNA nano‐ particles in vivo [120]. This system was used to deliver siRNA silencing the *EWS-FLI1* gene thus inhibiting tumour growth in a murine model of metastatic Ewing's sarcoma. The first experimental siRNA therapeutic to provide targeted delivery in humans was reported by Davis and co-workers [121]. siRNA was formulated into a nanoparticle (CALAA-01), which consisted of a cyclodextrin-containing polymer that contains amidine and primary amine functional groups, a PEG for steric stabilization in the in vivo environment (via inclusion complexes of β-CD with adamantine-PEG conjugate), and human transferrin (Tf) as the targeting ligand to binds to the transferrin receptors that are over-expressed on cancer cells. The siRNA/nanoparticles components self-assembled in the pharmacy. CALAA-01 was administered intravenously to the first patient with a solid cancer in a phase I clinical trial (safety study) in May 2008 [121]. Tumour biopsies from patients' melanoma after treatment (phase I clinical trial) showed the presence of intracellular nanoparticles. Reductions in the levels of both the specific mRNA (M2 subunit of ribonucleotide reductase, RRM2) and the protein (RRM2) were found when compared to levels in pre-dosing tissues. These results demonstrated that siRNA nanoparticles administered systemically to a human patient can produce a specific gene knock-down via an RNAi mechanism of action [122]. A recent and novel approach to the synthesis of cationic or neutral PEGylated amphiphilic β-CD used copper-catalysed "click" chemistry to modify selectively the secondary 2-hydroxyl group of the β-CD. The 6-position of these β-CD conjugates was conjugated to a dodecane alkyl chain. Complexation of cationic β-CD alone with siRNA resulted in good silencing of the luciferase reporter gene in Caco2 cells in culture. Co-formulation of cationic β-CD with a PEGylated β-CD and siRNA resulted in lower surface charges and reduced aggregation. The transfection efficiency of the cationic β-CD vector was lowered by co-formulation with the PEGylated β-CD, although the siRNA binding was not affected and the surface charge of the complexes did not reach complete neutrality [123].

in polynucleotide delivery, thus, cross-linking of the low molecular weight PEI with disulfide bonds which are cleaved in the reducing environment of the cytoplasm increased the efficiency

**Figure 6.** Representative examples of polyamines used in siRNA delivery either as in lipid conjugates of polyamine al‐

NH2

N

N H

<sup>N</sup> NH2 <sup>H</sup>

NH2

NH2

n

N H NH2

2NH

N H

> N H

> > n

N NH2 N H

Chitosan is a biocompatible and biodegradable polysaccharide that is a copolymer of *N*-acetyl-D-glucosamine and D-glucosamine. Chitosan has weakly basic properties due to the presence of the D-glucosamine residue with a p*K*a value 6.2-7.0. The molecular weight of chitosan affects the complex stability, size, zeta-potential and in vitro gene knock-down of siRNA/chitosan nanoparticles [116]. High molecular weight (64.8-170 kDa) chitosan formed stable complexes with siRNA and resulted in high gene knock-down efficiency in human lung carcinoma (H1299) cells, while low molecular weight (10 kDa) chitosan could not complex the siRNA into stable nanoparticles and showed almost no knock-down [117]. The method of association affects gene silencing efficiency, where chitosan-TPP/siRNA nanoparticles (siRNA entrapped inside the nanoparticles, and TPP is sodium tripolyphosphate and used as a polyanion to crosslink with the cationic chitosan groups by electrostatic interactions) showed high siRNA binding and better gene silencing in vitro compared to siRNA/chitosan particles prepared by

of siRNA delivery through the enhanced release of siRNA in the cytoplasm [115].

N

N H

N H

kaloids e.g. spermine and spermidine, or as in polyethylenimine (PEI), a cationic polymer.

N H

Spermine

Spermidine

2NH

2NH

2NH

2NH

148 Gene Therapy - Tools and Potential Applications

Linear polyethylenimine

N H

N H

N H

Branched polyethylenimine

N H Dendrimers have a central core to which are connected several branched arms in a manner that can be symmetrical or asymmetrical. During the synthesis of dendrimers, arms (branches) are added to the core structure. Each addition is called a generation and increases the previous generation number by one. Due to their unique structure, dendrimers can have a planar, elliptical, or spherical shape depending on generation number. Among the most widely used dendrimers are polyamidoamine (PAMAM) and polypropylenimine (PPI) dendrimers [124]. Dendrimers which have positively charged cationic groups on their outer surface are com‐ monly used for polynucleotide delivery. The transfection efficiency of dendrimers increases with increasing the charge density or generation number [125]. However, dendrimers with high generation number are generally more cytotoxic compared to dendrimers with low generation number [126]. Usually the inner space near the core is larger compared to outer space near the surface due to the lower density of molecules (less number of arms) near the core, which allow small molecules to be incorporated in the inner space. Owing to the relatively large molecular weight of polynucleotides, they are usually bound to the surface of cationic dendrimers and not in the inner space of the dendrimer. Generally, the toxicity of dendrimers is lower than that of PEI or poly-L-lysine (PLL) [127]. One advantage of dendrimers is that they have pH buffering capacity (proton-sponge effect), an important feature for endosomal escape and enhancing the release of polynucleotides [125, 128].

liposomes containing the cationic lipid *N*-[1-(2,3-dioleyloxy)propyl]-*N,N,N*-trimethyl ammo‐ nium chloride (DOTMA) was reported to spontaneously complex DNA completely entrapping the DNA, and enhanced fusion with the cell membrane in vitro in cell cultures, resulting in efficient delivery and expression of the delivered DNA. The lipofection was 5-100-fold more effective than the commonly used transfection techniques at the time by either calcium phosphate or DEAE-dextran (diethylaminoethyl-dextran), depending on the cell line used [131]. Cationic lipids have polar and non-polar domains and thus are amphiphilic in nature, with three general structural domains: (a) a cationic hydrophilic head-group (positively charged). The head-group can carry a permanent positive charge as in quaternary ammonium groups, or can be protonated at the physiological pH 7.4, such as primary and secondary amine groups. There can be one cationic group per lipid molecule (monovalent cationic lipids) or more than one cationic group per lipid molecule (multivalent cationic lipids); (b) a hydropho‐ bic domain covalently attached by a linker to the cationic head-group. This domain can be in the form of either alkyl chains (commonly 2 chains) of various chain lengths (with various oxidation states) or can be a steroid such as cholesterol; (c) the linker between the head-group and the hydrophobic domain [132, 133]. This linker controls the biodegradation of the cationic lipid and its stability under different conditions according to the type of chemical bonds (e.g. ester, ether, or amide). Each domain can be controlled to change a specific character of the cationic lipid, e.g. using a disulfide functional group as a bioresponsive linker [134] which is reduced in the intracellular environment by glutathione/glutathione reductase and enhance

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The cationic head-group's main function is to bind electrostatically the negatively charged phosphates of the polynucleotides. The complexes of cationic lipids with polynucleotides such as DNA and siRNA are called lipoplexes. This requires the presence of a positive charge on the head-group at the physiological pH 7.4, i.e. the p*K*a of the head-group is ideally at least one unit higher than the physiological pH. The most commonly used head-groups contain nitrogen (e.g. amines or guanidines). However other head-groups, e.g. arsonium and phos‐ phonium have been reported [135]. Arsonium is less toxic than arsenic (III), and in vitro cytotoxicity evaluation showed that arsonium and phosphonium are surprisingly less toxic

One property that can be changed by controlling the type of the head-group is the head-group cross-sectional area. The greater the difference between the cross-sectional area of the polar head-group and that of the hydrophobic domain, when the former is designed to be smaller than the latter, the greater is the ability of the cationic lipid to fuse with the cell membrane and endosomal membrane and the greater is the release of polynucleotides from their complex with the cationic lipid due to the decreased structural stability of the lipid assembly [133, 137]. The hydration of the head-group affects its cross-sectional area, thus, the conjugation of groups which decrease the hydration state (such as hydroxyalkyl groups that form intermo‐

Thus, gene delivery by DOTMA and DOTAP (1,2-dioleoyloxy-3-(trimethylammonio) propane) was enhanced by incorporation of a hydroxyethyl group to yield the lipids DORIE

biodegradation characters of the lipid and decrease its cytotoxicity.

lecular H-bonds) decreases the head-group cross-sectional area.

**4.2. The cationic head-group**

than the ammonium group [135, 136].

PPI dendrimers with high generation numbers (4 and 5) were more efficient in forming discrete nanoparticles with siRNA and in gene silencing in human lung cancer (A549) cells than lower generation dendrimers (2 and 3). Generation 5 PPI dendrimers were more toxic, probably due to the increased positive charge density per dendrimer, than generation 4 dendrimers [129]. Complex formation between PAMAM dendrimers with an ethylenediamine core and siRNA as a function of three variables has been reported [130]. The ionic strength of the medium (without or with 150 mM NaCl), the generation number (4, 5, 6 and 7) and the *N/P* ratio (ratio of positively charged amine groups per negative phosphate) were varied. The size of the complexes depended on the ionic strength of the media, with the strong electrostatic interac‐ tions in medium without NaCl making siRNA/dendrimer complexes smaller than those obtained in 150 mM NaCl. Both the intracellular delivery and the silencing of EGFP expression in cell culture was dependent on complex size, with smaller complexes efficiently delivered, and resulting in the highest silencing of EGFP expression. siRNA complexed with generation 7 dendrimers resulted in the highest silencing of EGFP expression both in human brain tumour cell line T98G-EGFP (35%) and mouse macrophage cell line J-774-EGFP (45%) cells, in spite of having lower protection of siRNA against degradation with RNase A, showing the importance of formulation procedures on the efficiency of transfection [130].

#### **4. Cationic lipids as non-viral vectors for siRNA and DNA delivery**

#### **4.1. Gene delivery by cationic lipids**

Gene delivery (DNA transfection) with cationic lipids (Figure 7) dates back to 1987 when it was reported by Felgner et al. [131], and the term "lipofection" was coined. Small unilamellar liposomes containing the cationic lipid *N*-[1-(2,3-dioleyloxy)propyl]-*N,N,N*-trimethyl ammo‐ nium chloride (DOTMA) was reported to spontaneously complex DNA completely entrapping the DNA, and enhanced fusion with the cell membrane in vitro in cell cultures, resulting in efficient delivery and expression of the delivered DNA. The lipofection was 5-100-fold more effective than the commonly used transfection techniques at the time by either calcium phosphate or DEAE-dextran (diethylaminoethyl-dextran), depending on the cell line used [131]. Cationic lipids have polar and non-polar domains and thus are amphiphilic in nature, with three general structural domains: (a) a cationic hydrophilic head-group (positively charged). The head-group can carry a permanent positive charge as in quaternary ammonium groups, or can be protonated at the physiological pH 7.4, such as primary and secondary amine groups. There can be one cationic group per lipid molecule (monovalent cationic lipids) or more than one cationic group per lipid molecule (multivalent cationic lipids); (b) a hydropho‐ bic domain covalently attached by a linker to the cationic head-group. This domain can be in the form of either alkyl chains (commonly 2 chains) of various chain lengths (with various oxidation states) or can be a steroid such as cholesterol; (c) the linker between the head-group and the hydrophobic domain [132, 133]. This linker controls the biodegradation of the cationic lipid and its stability under different conditions according to the type of chemical bonds (e.g. ester, ether, or amide). Each domain can be controlled to change a specific character of the cationic lipid, e.g. using a disulfide functional group as a bioresponsive linker [134] which is reduced in the intracellular environment by glutathione/glutathione reductase and enhance biodegradation characters of the lipid and decrease its cytotoxicity.

#### **4.2. The cationic head-group**

Dendrimers have a central core to which are connected several branched arms in a manner that can be symmetrical or asymmetrical. During the synthesis of dendrimers, arms (branches) are added to the core structure. Each addition is called a generation and increases the previous generation number by one. Due to their unique structure, dendrimers can have a planar, elliptical, or spherical shape depending on generation number. Among the most widely used dendrimers are polyamidoamine (PAMAM) and polypropylenimine (PPI) dendrimers [124]. Dendrimers which have positively charged cationic groups on their outer surface are com‐ monly used for polynucleotide delivery. The transfection efficiency of dendrimers increases with increasing the charge density or generation number [125]. However, dendrimers with high generation number are generally more cytotoxic compared to dendrimers with low generation number [126]. Usually the inner space near the core is larger compared to outer space near the surface due to the lower density of molecules (less number of arms) near the core, which allow small molecules to be incorporated in the inner space. Owing to the relatively large molecular weight of polynucleotides, they are usually bound to the surface of cationic dendrimers and not in the inner space of the dendrimer. Generally, the toxicity of dendrimers is lower than that of PEI or poly-L-lysine (PLL) [127]. One advantage of dendrimers is that they have pH buffering capacity (proton-sponge effect), an important feature for endosomal

PPI dendrimers with high generation numbers (4 and 5) were more efficient in forming discrete nanoparticles with siRNA and in gene silencing in human lung cancer (A549) cells than lower generation dendrimers (2 and 3). Generation 5 PPI dendrimers were more toxic, probably due to the increased positive charge density per dendrimer, than generation 4 dendrimers [129]. Complex formation between PAMAM dendrimers with an ethylenediamine core and siRNA as a function of three variables has been reported [130]. The ionic strength of the medium (without or with 150 mM NaCl), the generation number (4, 5, 6 and 7) and the *N/P* ratio (ratio of positively charged amine groups per negative phosphate) were varied. The size of the complexes depended on the ionic strength of the media, with the strong electrostatic interac‐ tions in medium without NaCl making siRNA/dendrimer complexes smaller than those obtained in 150 mM NaCl. Both the intracellular delivery and the silencing of EGFP expression in cell culture was dependent on complex size, with smaller complexes efficiently delivered, and resulting in the highest silencing of EGFP expression. siRNA complexed with generation 7 dendrimers resulted in the highest silencing of EGFP expression both in human brain tumour cell line T98G-EGFP (35%) and mouse macrophage cell line J-774-EGFP (45%) cells, in spite of having lower protection of siRNA against degradation with RNase A, showing the importance

escape and enhancing the release of polynucleotides [125, 128].

150 Gene Therapy - Tools and Potential Applications

of formulation procedures on the efficiency of transfection [130].

**4.1. Gene delivery by cationic lipids**

**4. Cationic lipids as non-viral vectors for siRNA and DNA delivery**

Gene delivery (DNA transfection) with cationic lipids (Figure 7) dates back to 1987 when it was reported by Felgner et al. [131], and the term "lipofection" was coined. Small unilamellar The cationic head-group's main function is to bind electrostatically the negatively charged phosphates of the polynucleotides. The complexes of cationic lipids with polynucleotides such as DNA and siRNA are called lipoplexes. This requires the presence of a positive charge on the head-group at the physiological pH 7.4, i.e. the p*K*a of the head-group is ideally at least one unit higher than the physiological pH. The most commonly used head-groups contain nitrogen (e.g. amines or guanidines). However other head-groups, e.g. arsonium and phos‐ phonium have been reported [135]. Arsonium is less toxic than arsenic (III), and in vitro cytotoxicity evaluation showed that arsonium and phosphonium are surprisingly less toxic than the ammonium group [135, 136].

One property that can be changed by controlling the type of the head-group is the head-group cross-sectional area. The greater the difference between the cross-sectional area of the polar head-group and that of the hydrophobic domain, when the former is designed to be smaller than the latter, the greater is the ability of the cationic lipid to fuse with the cell membrane and endosomal membrane and the greater is the release of polynucleotides from their complex with the cationic lipid due to the decreased structural stability of the lipid assembly [133, 137]. The hydration of the head-group affects its cross-sectional area, thus, the conjugation of groups which decrease the hydration state (such as hydroxyalkyl groups that form intermo‐ lecular H-bonds) decreases the head-group cross-sectional area.

Thus, gene delivery by DOTMA and DOTAP (1,2-dioleoyloxy-3-(trimethylammonio) propane) was enhanced by incorporation of a hydroxyethyl group to yield the lipids DORIE (1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide) and DORI (*N*-[1-(2,3 dioleoyloxy)propyl-*N*-[1-(2-hydroxy)ethyl]-*N,N*-dimethyl ammonium iodide) respectively [138, 139]. The head-group cross-sectional area can be also controlled by subtle changes to the head-group structure. The DC-Chol (3β(*N*-(*N*',*N*'-dimethylaminoethane)carbamoyl)cholester‐ ol) with dimethylamino head-group resulted in more efficient transfection compared to DC-Chol with diethylamino or diisopropylamino head-groups, probably due to increased steric repulsion of the head-groups.

The in vitro gene transfer with six non-cholesterol-based cationic lipids (each having two alkyl chains) with a single guanidinium head-group in Chinese hamster ovary (CHO), COS-1, MCF-7, A549, and HepG2 cells has been reported [140]. These lipids were able to form lipoplexes with size-range 200-600 nm and ζ-potential +3.4 to -34 mV. The efficiencies of the lipids which had an extra quaternized cationic centre were 2-4-fold more than that of the commercially available reagent Lipofectamine in transfecting COS-1, CHO, A-549, and MCF-7 cells. MTT viability assay in CHO cells showed high (>75%) cell viabilities at the lipid/DNA charge ratios used. DNase I protection assays showed that the lipids having the extra quater‐ nized centre protected DNA better against enzyme catalysed hydrolysis. These results shed light on the importance of choosing the type of head-group and number of cationic centres in designing cationic lipids [140].

1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE)

1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE)

<sup>N</sup> <sup>N</sup> <sup>H</sup>

N NH

O N O N H

> H H H

HN H2N

> NH2 NH

N NH2 H N <sup>H</sup> <sup>N</sup>

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n

polyspermine imidazole-4,5-imine (PSI)

N <sup>H</sup> <sup>N</sup>

dioctadecylamidoglycylspermine (DOGS)

cholesteryl-spermidine

N NH <sup>O</sup>

O

cholesteryl-3-carboxyamidoethylene-*N*-hydroxyethylamine

O

 *N*-hydroxyethylaminopropanecarbamoyl cholesterol

H H H

 *N*-[1-(2,3-dioleoyloxy)propyl-*N*-[1-(2-hydroxy)ethyl]-*N,N*-dimethylammonium iodide (DORI)

Br–

O O

O O

H N+ N <sup>H</sup> <sup>O</sup>

O O

O O N+

O O

<sup>N</sup><sup>+</sup> Cl–

O H

Cl–

Cl –

N<sup>+</sup> I –

> O O

H2N N H N <sup>H</sup> <sup>N</sup>

O H N+

> O O

N NH

2NH

OH <sup>N</sup> H N H O O

**Figure 7.** Representative examples of cationic lipids used in DNA and siRNA delivery.

OH <sup>N</sup> H N H H H H

2NH

O H <sup>N</sup><sup>+</sup> Br–

O

 *N*-[1-(2,3-dioleyloxy)propyl]-*N,N,N*-trimethylammonium chloride (DOTMA)

1,2-dioleoyloxy-3-(trimethylammonio)-propane chloride (DOTAP)

3(*N*-(*N',N*'-dimethylaminoethane)carbamoyl)-cholesterol hydrochloride (DC-Chol)

H H H

A series of cationic cholesterol derivatives were synthesized by covalently attaching the heterocycles imidazole, piperazine, pyridine, and morpholine groups (the head-groups) to the parent cholesterol via a biodegradable carbamoyl linker [141]. These lipids were compared with the parent DC-Chol with the linear amine head-group, and they generally showed better or comparable transfection efficiency of pCMV-luciferase into human HepG2 cells (a human liver cancer cell line) in the presence or absence of FCS. The most efficient two of these lipids were with morpholine and piperazine head-groups, and they gave higher levels of gene expression in HepG2 and human melanoma cell line (KZ2) which are generally very hard to transfect with the commonly used reagents e.g. DC-Chol, Lipofectamine, and PEI. In vivo studies with lipids having morpholine and piperazine head-groups resulted in successful delivery of the reporter gene to the target cells through intrasplenic injection [141]. Cationic lipids which have more than one cationic head-group (multivalent cationic lipids) have more surface charge density than their monovalent (with one head-group) analogues, and they are generally expected to better bind and complex polynucleotides. Many multivalent cationic lipids contain a natural occurring polyamine such as spermidine and spermine, which are believed to interact with the minor groove of B-DNA [142].

The triamine spermidine and the tetramine spermine (Figure 6), and their diamine precursor putrescine, are organic polycations that are widely but unevenly distributed in both mamma‐ lian and non-mammalian cells and tissues. They have an essential role in controlling DNA, RNA and protein synthesis during normal and neoplastic growth, in cell differentiation, and tissue regeneration [143]. These polyamines exhibit many metabolic and neurophysiological effects in the nervous system, and are important for the developing and mature nervous system and affect modulation of ionic channels and calcium-dependent transmitter release [143-149].

#### siRNA and Gene Formulation for Efficient Gene Therapy http://dx.doi.org/10.5772/55518 153

(1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide) and DORI (*N*-[1-(2,3 dioleoyloxy)propyl-*N*-[1-(2-hydroxy)ethyl]-*N,N*-dimethyl ammonium iodide) respectively [138, 139]. The head-group cross-sectional area can be also controlled by subtle changes to the head-group structure. The DC-Chol (3β(*N*-(*N*',*N*'-dimethylaminoethane)carbamoyl)cholester‐ ol) with dimethylamino head-group resulted in more efficient transfection compared to DC-Chol with diethylamino or diisopropylamino head-groups, probably due to increased steric

The in vitro gene transfer with six non-cholesterol-based cationic lipids (each having two alkyl chains) with a single guanidinium head-group in Chinese hamster ovary (CHO), COS-1, MCF-7, A549, and HepG2 cells has been reported [140]. These lipids were able to form lipoplexes with size-range 200-600 nm and ζ-potential +3.4 to -34 mV. The efficiencies of the lipids which had an extra quaternized cationic centre were 2-4-fold more than that of the commercially available reagent Lipofectamine in transfecting COS-1, CHO, A-549, and MCF-7 cells. MTT viability assay in CHO cells showed high (>75%) cell viabilities at the lipid/DNA charge ratios used. DNase I protection assays showed that the lipids having the extra quater‐ nized centre protected DNA better against enzyme catalysed hydrolysis. These results shed light on the importance of choosing the type of head-group and number of cationic centres in

A series of cationic cholesterol derivatives were synthesized by covalently attaching the heterocycles imidazole, piperazine, pyridine, and morpholine groups (the head-groups) to the parent cholesterol via a biodegradable carbamoyl linker [141]. These lipids were compared with the parent DC-Chol with the linear amine head-group, and they generally showed better or comparable transfection efficiency of pCMV-luciferase into human HepG2 cells (a human liver cancer cell line) in the presence or absence of FCS. The most efficient two of these lipids were with morpholine and piperazine head-groups, and they gave higher levels of gene expression in HepG2 and human melanoma cell line (KZ2) which are generally very hard to transfect with the commonly used reagents e.g. DC-Chol, Lipofectamine, and PEI. In vivo studies with lipids having morpholine and piperazine head-groups resulted in successful delivery of the reporter gene to the target cells through intrasplenic injection [141]. Cationic lipids which have more than one cationic head-group (multivalent cationic lipids) have more surface charge density than their monovalent (with one head-group) analogues, and they are generally expected to better bind and complex polynucleotides. Many multivalent cationic lipids contain a natural occurring polyamine such as spermidine and spermine, which are

The triamine spermidine and the tetramine spermine (Figure 6), and their diamine precursor putrescine, are organic polycations that are widely but unevenly distributed in both mamma‐ lian and non-mammalian cells and tissues. They have an essential role in controlling DNA, RNA and protein synthesis during normal and neoplastic growth, in cell differentiation, and tissue regeneration [143]. These polyamines exhibit many metabolic and neurophysiological effects in the nervous system, and are important for the developing and mature nervous system and affect modulation of ionic channels and calcium-dependent transmitter release [143-149].

repulsion of the head-groups.

152 Gene Therapy - Tools and Potential Applications

designing cationic lipids [140].

believed to interact with the minor groove of B-DNA [142].

1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE)

<sup>N</sup> <sup>N</sup> <sup>H</sup>

N NH

H2N

N NH2 H N <sup>H</sup> <sup>N</sup>

n

polyspermine imidazole-4,5-imine (PSI)

N <sup>H</sup> <sup>N</sup>

N NH

**Figure 7.** Representative examples of cationic lipids used in DNA and siRNA delivery.

H2N N H N <sup>H</sup> <sup>N</sup> Spermine is incorporated in the cationic polymer polyspermine imidazole-4,5-imine (PSI) and in dioctadecylamidoglycyl-spermine (DOGS) [150] (Figure 7); spermidine is bound in cholesterylspermidine [151]. The free amine groups of spermine in cholesteryl-spermine conjugates have different p*K*a values and provide a buffering effect in the endosomes facilitating the escape of lipoplex from the endosomes [152]. The length of the linear polyamine attached to the hydropho‐ bic domain and the charge distribution on it affects the transfection efficiency of the cationic lipid [153]. Addition of amine groups separated by methylene groups to the linear polyamine attach‐ ed to a cholesterol residue did not automatically increase transfection efficiency regardless of the increased charge density. Molecular modelling simulations suggested that increasing chain length led to an increased number of folded conformations due to greater flexibility of the conjugates, which is unfavourable for interaction with DNA [132, 153].

the shorter chain conjugates [163]. A series of multivalent Gemini-surfactants with the hydrophobic chains systematically varied resulted in the conjugates with C18 oleoyl chains to be better in transfection than the C16 and C14 alkyl chains [164]. Chain saturation was also shown to affect the efficiency of transfection. The results of studies on a set of cationic triester phosphatidyl choline derivatives (each having two alkyl chains) show a strong dependence of their transfection efficiency on the lipid hydrocarbon chain characteristics, where transfection activity increases with increasing chain unsaturation from fully saturated to having two double bonds. Transfection efficiency decreased with increasing chain length (increasing the total number of carbons per lipid molecule ~30-50). Maximum transfection was with monounsatu‐ rated myristoleoyl 14:1 chains [156]. The data obtained from transfection experiments with 20 cationic phosphatidylcholine (PC) derivatives show that hydrocarbon chain variations results in transfection efficiencies that varies by more than 2 orders of magnitude. The most important variables were chain saturation state and total number of carbon atoms in the lipid chains. Transfection increased with decreasing chain length and increasing chain unsaturation. Best transfection efficiency was found for cationic lipids with monounsaturated (myristoleoyl) 14:1 chains [154]. Higher levels of transfection were also reported with lipids having oleoyl chains in comparison with stearoyl chains [157, 158]. Unsaturated chains promote lipid fusion between the lipoplexes and the various cellular membranes, which is essential for delivery and

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Cholesterol derivatives with various cationic head-groups were synthesized to investigate their efficiency as siRNA delivery vectors. The transfection efficiencies of siRNA lipoplexes prepared with the cationic cholesterol derivatives DC-Chol, cholesteryl-3β-carboxyamidoethylene-*N*-hydroxyethylamine (OH-Chol), and *N*-hydroxyethylaminopropane carbamoyl cholesterol (HAPC) was investigated in human prostate tumour cells that stably express the luciferase gene (PC-3-Luc). When lipoplexes were prepared in water, HAPC was more effective in knocking-down luciferase activity than OH-Chol and DC-Chol [166]. The presence of NaCl while preparing the lipoplexes increased the gene silencing efficiency of luciferase, while it did not affect efficiency of HAPC. The commercially available transfection reagent, Lipofectamine 2000 (a cationic lipid liposomal preparation) resulted in strong gene silencing by siRNA, but exhibited increased toxicity (~40% cell viability), in contrast to OH-Chol, DC-Chol, and HAPC lipoplexes (~80–100% cell viability). These results indicated that siRNA lipoplexes prepared with OH-Chol, and HAPC can efficiently suppress gene expression

The linker is dependent upon the type (hence properties) of the functional group and its length (number of carbon atoms). The linker has two main functions: (a) to conjugate covalently the polar head-group to the hydrophobic domain; (b) to control the biodegradability of the cationic lipid and/or introduce a new property to the cationic lipid, e.g. responding to the intracellular reducing environment [133, 167]. The most commonly used linker functional groups are:

endosomal escape [133, 154, 165].

without increased cytotoxicity [166].

amide, carbamate, ester, ether, ortho ester, and disulfide.

**4.4. The linker**

The central tetramethylene motif of spermine was reported to be essential in conferring high transfection efficiency in a series of cholesterol-polyamine conjugates [152]. It was suggested that the tetramethylene segment of spermine can bridge between the DNA complementary strands, while the polyamine with a central trimethylene segment would only bind with the adjacent phosphates on the same DNA strand. These results point to the importance of the structure of the polyamine head-group and the relation between its amine groups, and also point to the fact that increasing efficiency of transfection is not only a matter of increasing the number of positive charges per head-group.

#### **4.3. The hydrophobic domain**

The length, saturation state and type of the hydrophobic chains conjugated to cationic lipids affect their transfection efficiency [154-156]. Although these factors were studied extensively for the effect on transfection, and although the majority of studies accepted that the type of alkyl chains influence the outcome of transfection, it is difficult to set a definitive set of rules to describe the best type of alkyl chains to be conjugated to the polar head-groups. The contribution of the alkyl chains (and the hydrophobic domain) to the cationic lipid properties as a whole is what determines the transfection efficiency of the lipid.

Results obtained with DMRIE (1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl-ammonium bromide) [157], glycine betaine conjugates [138] with two alkyl chains, alkyl acyl carnitine esters having chains of length C12 to C18 [158], lactic acid conjugates of *N,N*-dialkyl amine group [159], lipids related to DOTAP with two alkyl chains (C12-C18) linked to the head-group through ether bonds [160], and cationic lipids with different hydroxyethyl or dihydroxypropyl ammonium backbones and esterified hydrocarbon chains and hydroxyl substituents [161] showed that a comparison of the cationic lipids based only on the lengths of the two saturated aliphatic chains led to the observation of the superior transfection efficiency of C14 chains over the longer C16 and C18 chains [132, 133]. It was proposed that a shorter chain length facilitates mixing with cellular membranes [138] which is important for endosomal escape [162].

In another set of experiments, we showed the longer chain C18 oleoyl (with one *cis*-double bond) to be more efficient than cationic lipids with shorter chain lengths. Varying the chain length in *N*<sup>4</sup> ,*N*<sup>9</sup> -diacyl spermines from C10 to C18, for plasmid DNA delivery, resulted in us establishing that the conjugate with C18 oleoyl chains is both more efficient and less toxic than the shorter chain conjugates [163]. A series of multivalent Gemini-surfactants with the hydrophobic chains systematically varied resulted in the conjugates with C18 oleoyl chains to be better in transfection than the C16 and C14 alkyl chains [164]. Chain saturation was also shown to affect the efficiency of transfection. The results of studies on a set of cationic triester phosphatidyl choline derivatives (each having two alkyl chains) show a strong dependence of their transfection efficiency on the lipid hydrocarbon chain characteristics, where transfection activity increases with increasing chain unsaturation from fully saturated to having two double bonds. Transfection efficiency decreased with increasing chain length (increasing the total number of carbons per lipid molecule ~30-50). Maximum transfection was with monounsatu‐ rated myristoleoyl 14:1 chains [156]. The data obtained from transfection experiments with 20 cationic phosphatidylcholine (PC) derivatives show that hydrocarbon chain variations results in transfection efficiencies that varies by more than 2 orders of magnitude. The most important variables were chain saturation state and total number of carbon atoms in the lipid chains. Transfection increased with decreasing chain length and increasing chain unsaturation. Best transfection efficiency was found for cationic lipids with monounsaturated (myristoleoyl) 14:1 chains [154]. Higher levels of transfection were also reported with lipids having oleoyl chains in comparison with stearoyl chains [157, 158]. Unsaturated chains promote lipid fusion between the lipoplexes and the various cellular membranes, which is essential for delivery and endosomal escape [133, 154, 165].

Cholesterol derivatives with various cationic head-groups were synthesized to investigate their efficiency as siRNA delivery vectors. The transfection efficiencies of siRNA lipoplexes prepared with the cationic cholesterol derivatives DC-Chol, cholesteryl-3β-carboxyamidoethylene-*N*-hydroxyethylamine (OH-Chol), and *N*-hydroxyethylaminopropane carbamoyl cholesterol (HAPC) was investigated in human prostate tumour cells that stably express the luciferase gene (PC-3-Luc). When lipoplexes were prepared in water, HAPC was more effective in knocking-down luciferase activity than OH-Chol and DC-Chol [166]. The presence of NaCl while preparing the lipoplexes increased the gene silencing efficiency of luciferase, while it did not affect efficiency of HAPC. The commercially available transfection reagent, Lipofectamine 2000 (a cationic lipid liposomal preparation) resulted in strong gene silencing by siRNA, but exhibited increased toxicity (~40% cell viability), in contrast to OH-Chol, DC-Chol, and HAPC lipoplexes (~80–100% cell viability). These results indicated that siRNA lipoplexes prepared with OH-Chol, and HAPC can efficiently suppress gene expression without increased cytotoxicity [166].

#### **4.4. The linker**

Spermine is incorporated in the cationic polymer polyspermine imidazole-4,5-imine (PSI) and in dioctadecylamidoglycyl-spermine (DOGS) [150] (Figure 7); spermidine is bound in cholesterylspermidine [151]. The free amine groups of spermine in cholesteryl-spermine conjugates have different p*K*a values and provide a buffering effect in the endosomes facilitating the escape of lipoplex from the endosomes [152]. The length of the linear polyamine attached to the hydropho‐ bic domain and the charge distribution on it affects the transfection efficiency of the cationic lipid [153]. Addition of amine groups separated by methylene groups to the linear polyamine attach‐ ed to a cholesterol residue did not automatically increase transfection efficiency regardless of the increased charge density. Molecular modelling simulations suggested that increasing chain length led to an increased number of folded conformations due to greater flexibility of the

The central tetramethylene motif of spermine was reported to be essential in conferring high transfection efficiency in a series of cholesterol-polyamine conjugates [152]. It was suggested that the tetramethylene segment of spermine can bridge between the DNA complementary strands, while the polyamine with a central trimethylene segment would only bind with the adjacent phosphates on the same DNA strand. These results point to the importance of the structure of the polyamine head-group and the relation between its amine groups, and also point to the fact that increasing efficiency of transfection is not only a matter of increasing the

The length, saturation state and type of the hydrophobic chains conjugated to cationic lipids affect their transfection efficiency [154-156]. Although these factors were studied extensively for the effect on transfection, and although the majority of studies accepted that the type of alkyl chains influence the outcome of transfection, it is difficult to set a definitive set of rules to describe the best type of alkyl chains to be conjugated to the polar head-groups. The contribution of the alkyl chains (and the hydrophobic domain) to the cationic lipid properties

Results obtained with DMRIE (1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl-ammonium bromide) [157], glycine betaine conjugates [138] with two alkyl chains, alkyl acyl carnitine esters having chains of length C12 to C18 [158], lactic acid conjugates of *N,N*-dialkyl amine group [159], lipids related to DOTAP with two alkyl chains (C12-C18) linked to the head-group through ether bonds [160], and cationic lipids with different hydroxyethyl or dihydroxypropyl ammonium backbones and esterified hydrocarbon chains and hydroxyl substituents [161] showed that a comparison of the cationic lipids based only on the lengths of the two saturated aliphatic chains led to the observation of the superior transfection efficiency of C14 chains over the longer C16 and C18 chains [132, 133]. It was proposed that a shorter chain length facilitates mixing with cellular membranes [138] which is important for endosomal escape [162].

In another set of experiments, we showed the longer chain C18 oleoyl (with one *cis*-double bond) to be more efficient than cationic lipids with shorter chain lengths. Varying the chain

establishing that the conjugate with C18 oleoyl chains is both more efficient and less toxic than


conjugates, which is unfavourable for interaction with DNA [132, 153].

as a whole is what determines the transfection efficiency of the lipid.

number of positive charges per head-group.

**4.3. The hydrophobic domain**

154 Gene Therapy - Tools and Potential Applications

length in *N*<sup>4</sup>

,*N*<sup>9</sup>

The linker is dependent upon the type (hence properties) of the functional group and its length (number of carbon atoms). The linker has two main functions: (a) to conjugate covalently the polar head-group to the hydrophobic domain; (b) to control the biodegradability of the cationic lipid and/or introduce a new property to the cationic lipid, e.g. responding to the intracellular reducing environment [133, 167]. The most commonly used linker functional groups are: amide, carbamate, ester, ether, ortho ester, and disulfide.

Both amides and ester bonds are biodegradable and hence are hypothesized to be less toxic than other non-biodegradable bonds (e.g. ethers) [168]. Lipids with a pyridinium head-group (with palmitoyl 16:0 hydrophobic domains and with ester and amide linkers) were used to prepare liposomes with either DOPE or cholesterol at the cationic lipid/helper-lipid molar ratio of 1:1. Following transfection of CHO cells with lipoplexes delivering plasmids expressing EGFP, the cationic lipids having amide linkers significantly increased transfection efficiency in all liposomal formulations compared to their counterparts having the ester linker [169]. The high transfection efficiency of lipids with amide linker was suggested to be due to their lower phase-transition temperature which makes the liposome's bilayer structure more stable in aqueous media during the transfection process as well as liposome storage. The phasetransition temperature of a lipid is the temperature at which there is a change in the lipid's physical state from the ordered gel phase (where the hydrocarbon chains are closely packed and fully extended) to the disordered liquid crystalline phase (where the hydrocarbon chains are fluid and randomly orientated) [169].

Transfection with DNA lipoplexes of three thiocholesterol-derived gemini cationic lipids possessing disulfide linkages incorporated between the cationic head-group and the thiocho‐ lesterol backbone in order to render the lipids biodegradable has been reported [173]. Com‐ paring transfection in a prostate cancer line (PC3AR) and a human keratinocyte cell line (HaCat) with two commercially available reagents showed comparable or better expression of GFP in the transfected cells. Cytotoxicity studies showed the nontoxic property of these lipid-DNA complexes at different *N/P* ratios used for transfection studies. The rationale behind this design was to ensure the destabilization of the lipid-polynucleotide lipoplexes in the cytoplasm after reduction of the disulfide linker by the intracellular glutathione (GSH), which is the most abundant low molecular weight thiol present in cells and is involved in controlling cellular redox environment. GSH is found at very high intracellular concentrations and at comparatively low extracellular concentrations e.g. blood plasma concentrations (2 µM) are 1000-fold less than concentration in erythrocytes (2 mM). This large difference between intraand extracellular environments provides a potential mechanism for release of polynucleotides from lipoplexes of lipids that have a disulfide functional group linker and is now a well-

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In our research, symmetrical and asymmetrical acyl polyamine derivatives (fatty acid amides of spermine) [152] have been synthesized, characterized, and evaluated as non-viral vectors for siRNA [163, 174-177]. The intracellular delivery of siRNA and the subsequent sequence specific gene silencing has been quantified by flow cytometry techniques (FACS analysis) [163]. The ability of the spermine conjugates to bind siRNA and form nanoparticles has been investigated and the effect of the complexes of siRNA lipoplexes on the cell viability 48 h posttransfection has been quantified. Our SAR studies allow the identification of the most efficient

fatty acids in terms of high gene-silencing efficiency and high cell viability [174-178].

Whilst we were completing this Chapter, four interesting papers, each one on a different aspect of this topic, were published. Langer, Anderson and co-workers at MIT reported on the delivery of immunostimulatory RNA (isRNA) to Toll-like receptor (TLR)-expressing cells to drive innate and adaptive immune responses. The specific activation of TLRs has potential for a variety of therapeutic indications including antiviral immunotherapy and as vaccine adjuvants. Effective lipidoid-isRNA nanoparticles, when tested in mice, stimulated strong IFN-α responses following subcutaneous injection, had robust antiviral activity that sup‐ pressed influenza virus replication, and enhanced antiovalbumin humoral and cell-mediated responses when used as a vaccine adjuvant. Their lipidoid formulations, designed specifically for the delivery of isRNA to TLRs, were superior to the commonly used *N*-[1-(2,3-dioleoy‐ loxy)propyl]-*N*,*N*,*N*-trimethylammonium methylsulfate-RNA delivery system and may provide new tools for the manipulation of TLR responses in vitro and in vivo [179]. This paper follows after their other recent major contribution on delivering naked siRNA as part of a selfassembled (due to DNA complementarity) tetrahedral nanoparticle construct considering the presentation of folate as a cancer targeting ligand [180]. These monodisperse nanoparticles of

trodden research path [115, 134, 173].

**5. Conclusions and future avenues**

Depending on the structure of the cationic lipid, the linker influence on transfection efficiency can be more than on cytotoxicity. Cholesterol-based cationic lipids that have different nitro‐ genous heterocyclic head-groups (*N*-methylimidazole, *N*-methylmorpholine, and pyridine) and acid-labile linkers (carbamate, ester, and *N,O*-acetal ether) were used to transfect human embryonic kidney 293 (HEK 293) cells with EGFP plasmid [170]. Choosing those linkers was based on the concept that incorporation of acid-labile bonds in the cationic lipid structure enhances the release of polynucleotides from the endosomes, therefore increasing the trans‐ fection efficiency [171]. *N,O*-Acetals are known to undergo hydrolysis in acidic environment [170, 171]. The results showed that the structure of these lipids only slightly affected their cytotoxicity but largely changes the efficiency of intracellular accumulation of the polynu‐ cleotides. The lipids having the cationic head-groups pyridine and/or methylimidazole headgroups with either an ester or a carbamate linker resulted in better transfection efficiency as compared with the cationic lipids with either the *N*-methylmorpholine head-groups and/or an ether linker. The lipid that has a pyridine head-group and a carbamate linker to deliver EGFP plasmid resulted in comparable transfection efficiency with that achieved with com‐ mercially available Lipofectamine 2000.

Two cleavable cationic lipids having a linear or a cyclic ortho-ester linker between the cationic head-group and the unsaturated hydrophobic domain (two oleoyl chains) were previously reported [172]. It is hypothesized that the acidic pH in the endosomes catalyzes the hydrolysis of the linker group to result in fragmentation products that destabilize the endosomal mem‐ branes. At pH 7.4, the lipids (with DOPE) formed stable lipoplexes with plasmid DNA. Decreas‐ ing the pH enhanced the hydrolysis of the ortho ester linkers which removed the cationic headgroups and caused lipoplex aggregation. At pH 5.5, the cationic lipid *N*-[2-methyl-2-(1',2' dioleylglyceroxy)dioxolan-4-yl]methyl-*N,N,N*-trimethylammonium iodide that have a cyclic ortho-ester linker showed increased pH-sensitivity and caused the permeation of its lipoplexes to model biomembranes within the time span of endosomal processing before the lysosomal degradation. This lipid markedly increased gene transfection (~3-50-fold) of the luciferase reporter protein in monkey kidney fibroblast (CV-1) and human breast cancer (HTB-129) cells in culture compared to the pH-insensitive control lipid DOTAP lipoplexes [172].

Transfection with DNA lipoplexes of three thiocholesterol-derived gemini cationic lipids possessing disulfide linkages incorporated between the cationic head-group and the thiocho‐ lesterol backbone in order to render the lipids biodegradable has been reported [173]. Com‐ paring transfection in a prostate cancer line (PC3AR) and a human keratinocyte cell line (HaCat) with two commercially available reagents showed comparable or better expression of GFP in the transfected cells. Cytotoxicity studies showed the nontoxic property of these lipid-DNA complexes at different *N/P* ratios used for transfection studies. The rationale behind this design was to ensure the destabilization of the lipid-polynucleotide lipoplexes in the cytoplasm after reduction of the disulfide linker by the intracellular glutathione (GSH), which is the most abundant low molecular weight thiol present in cells and is involved in controlling cellular redox environment. GSH is found at very high intracellular concentrations and at comparatively low extracellular concentrations e.g. blood plasma concentrations (2 µM) are 1000-fold less than concentration in erythrocytes (2 mM). This large difference between intraand extracellular environments provides a potential mechanism for release of polynucleotides from lipoplexes of lipids that have a disulfide functional group linker and is now a welltrodden research path [115, 134, 173].

#### **5. Conclusions and future avenues**

Both amides and ester bonds are biodegradable and hence are hypothesized to be less toxic than other non-biodegradable bonds (e.g. ethers) [168]. Lipids with a pyridinium head-group (with palmitoyl 16:0 hydrophobic domains and with ester and amide linkers) were used to prepare liposomes with either DOPE or cholesterol at the cationic lipid/helper-lipid molar ratio of 1:1. Following transfection of CHO cells with lipoplexes delivering plasmids expressing EGFP, the cationic lipids having amide linkers significantly increased transfection efficiency in all liposomal formulations compared to their counterparts having the ester linker [169]. The high transfection efficiency of lipids with amide linker was suggested to be due to their lower phase-transition temperature which makes the liposome's bilayer structure more stable in aqueous media during the transfection process as well as liposome storage. The phasetransition temperature of a lipid is the temperature at which there is a change in the lipid's physical state from the ordered gel phase (where the hydrocarbon chains are closely packed and fully extended) to the disordered liquid crystalline phase (where the hydrocarbon chains

Depending on the structure of the cationic lipid, the linker influence on transfection efficiency can be more than on cytotoxicity. Cholesterol-based cationic lipids that have different nitro‐ genous heterocyclic head-groups (*N*-methylimidazole, *N*-methylmorpholine, and pyridine) and acid-labile linkers (carbamate, ester, and *N,O*-acetal ether) were used to transfect human embryonic kidney 293 (HEK 293) cells with EGFP plasmid [170]. Choosing those linkers was based on the concept that incorporation of acid-labile bonds in the cationic lipid structure enhances the release of polynucleotides from the endosomes, therefore increasing the trans‐ fection efficiency [171]. *N,O*-Acetals are known to undergo hydrolysis in acidic environment [170, 171]. The results showed that the structure of these lipids only slightly affected their cytotoxicity but largely changes the efficiency of intracellular accumulation of the polynu‐ cleotides. The lipids having the cationic head-groups pyridine and/or methylimidazole headgroups with either an ester or a carbamate linker resulted in better transfection efficiency as compared with the cationic lipids with either the *N*-methylmorpholine head-groups and/or an ether linker. The lipid that has a pyridine head-group and a carbamate linker to deliver EGFP plasmid resulted in comparable transfection efficiency with that achieved with com‐

Two cleavable cationic lipids having a linear or a cyclic ortho-ester linker between the cationic head-group and the unsaturated hydrophobic domain (two oleoyl chains) were previously reported [172]. It is hypothesized that the acidic pH in the endosomes catalyzes the hydrolysis of the linker group to result in fragmentation products that destabilize the endosomal mem‐ branes. At pH 7.4, the lipids (with DOPE) formed stable lipoplexes with plasmid DNA. Decreas‐ ing the pH enhanced the hydrolysis of the ortho ester linkers which removed the cationic headgroups and caused lipoplex aggregation. At pH 5.5, the cationic lipid *N*-[2-methyl-2-(1',2' dioleylglyceroxy)dioxolan-4-yl]methyl-*N,N,N*-trimethylammonium iodide that have a cyclic ortho-ester linker showed increased pH-sensitivity and caused the permeation of its lipoplexes to model biomembranes within the time span of endosomal processing before the lysosomal degradation. This lipid markedly increased gene transfection (~3-50-fold) of the luciferase reporter protein in monkey kidney fibroblast (CV-1) and human breast cancer (HTB-129) cells in

culture compared to the pH-insensitive control lipid DOTAP lipoplexes [172].

are fluid and randomly orientated) [169].

156 Gene Therapy - Tools and Potential Applications

mercially available Lipofectamine 2000.

In our research, symmetrical and asymmetrical acyl polyamine derivatives (fatty acid amides of spermine) [152] have been synthesized, characterized, and evaluated as non-viral vectors for siRNA [163, 174-177]. The intracellular delivery of siRNA and the subsequent sequence specific gene silencing has been quantified by flow cytometry techniques (FACS analysis) [163]. The ability of the spermine conjugates to bind siRNA and form nanoparticles has been investigated and the effect of the complexes of siRNA lipoplexes on the cell viability 48 h posttransfection has been quantified. Our SAR studies allow the identification of the most efficient fatty acids in terms of high gene-silencing efficiency and high cell viability [174-178].

Whilst we were completing this Chapter, four interesting papers, each one on a different aspect of this topic, were published. Langer, Anderson and co-workers at MIT reported on the delivery of immunostimulatory RNA (isRNA) to Toll-like receptor (TLR)-expressing cells to drive innate and adaptive immune responses. The specific activation of TLRs has potential for a variety of therapeutic indications including antiviral immunotherapy and as vaccine adjuvants. Effective lipidoid-isRNA nanoparticles, when tested in mice, stimulated strong IFN-α responses following subcutaneous injection, had robust antiviral activity that sup‐ pressed influenza virus replication, and enhanced antiovalbumin humoral and cell-mediated responses when used as a vaccine adjuvant. Their lipidoid formulations, designed specifically for the delivery of isRNA to TLRs, were superior to the commonly used *N*-[1-(2,3-dioleoy‐ loxy)propyl]-*N*,*N*,*N*-trimethylammonium methylsulfate-RNA delivery system and may provide new tools for the manipulation of TLR responses in vitro and in vivo [179]. This paper follows after their other recent major contribution on delivering naked siRNA as part of a selfassembled (due to DNA complementarity) tetrahedral nanoparticle construct considering the presentation of folate as a cancer targeting ligand [180]. These monodisperse nanoparticles of essentially naked DNA, carrying siRNA as the cargo, have a defined size of only a few nm. They show that at least three folate molecules per nanoparticle are required for optimal delivery of the siRNA into cells and that gene silencing only occurs when the ligands are appropriately orientated. In vivo, these naked DNA nanoparticles showed a longer blood circulation time than the parent siRNA [180]. In another exciting development, Geall and coworkers at Novartis have also advanced the field of nucleic acid vaccines by taking advantage of the recent innovations in non-viral systemic delivery of siRNA using lipid nanoparticles (LNPs) to develop a self-amplifying RNA vaccine. This technology elicited broad, potent, and protective immune responses, comparable with those achieved by a viral delivery system, but without the inherent limitations of viral vectors [181]. Even today, a biologically responsive cationic polymer system based on spermine has been reported for the intracellular delivery of siRNA [182]. This polyspermine imidazole-4,5-imine (PSI) (Figure 7) carrier is designed to be hydrolysed at the mildly acidic pH found in the endosome.

[2] van der Krol AR, Mur LA, Beld M, Mol J, Stuitje AR. Flavonoid Genes in Petunia: Addition of a Limited Number of Gene Copies May Lead to a Suppression of Gene

siRNA and Gene Formulation for Efficient Gene Therapy

http://dx.doi.org/10.5772/55518

159

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It is clear that both ssRNA to activate the immune system and RNAi brought about by siRNA delivery have high therapeutic potential. The major remaining barrier, that of efficient and potentially selective delivery to target cells in now being addressed. The non-viral delivery of siRNAisamajortoolinmodernfunctionalgenomics.Medicinesdesign,theformulationofdrugs, in this case siRNA and plasmid DNA, is an essential requirement for efficient gene therapy.

#### **Acknowledgements**

We thank the Egyptian Government for a fully-funded studentship to AAM.

#### **Author details**

Ian S. Blagbrough1 and Abdelkader A. Metwally1,2

\*Address all correspondence to: prsisb@bath.ac.uk

1 Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK

2 Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Abbasya, Cairo, Egypt

#### **References**

[1] Blagbrough IS, Zara C. Animal Models for Target Diseases in Gene Therapy - Using DNA and siRNA Delivery Strategies. Pharmaceutical Research 2009;26(1) 1-18.

[2] van der Krol AR, Mur LA, Beld M, Mol J, Stuitje AR. Flavonoid Genes in Petunia: Addition of a Limited Number of Gene Copies May Lead to a Suppression of Gene Expression. The Plant Cell Online 1990;2(4) 291-299.

essentially naked DNA, carrying siRNA as the cargo, have a defined size of only a few nm. They show that at least three folate molecules per nanoparticle are required for optimal delivery of the siRNA into cells and that gene silencing only occurs when the ligands are appropriately orientated. In vivo, these naked DNA nanoparticles showed a longer blood circulation time than the parent siRNA [180]. In another exciting development, Geall and coworkers at Novartis have also advanced the field of nucleic acid vaccines by taking advantage of the recent innovations in non-viral systemic delivery of siRNA using lipid nanoparticles (LNPs) to develop a self-amplifying RNA vaccine. This technology elicited broad, potent, and protective immune responses, comparable with those achieved by a viral delivery system, but without the inherent limitations of viral vectors [181]. Even today, a biologically responsive cationic polymer system based on spermine has been reported for the intracellular delivery of siRNA [182]. This polyspermine imidazole-4,5-imine (PSI) (Figure 7) carrier is designed to be

It is clear that both ssRNA to activate the immune system and RNAi brought about by siRNA delivery have high therapeutic potential. The major remaining barrier, that of efficient and potentially selective delivery to target cells in now being addressed. The non-viral delivery of siRNAisamajortoolinmodernfunctionalgenomics.Medicinesdesign,theformulationofdrugs, in this case siRNA and plasmid DNA, is an essential requirement for efficient gene therapy.

We thank the Egyptian Government for a fully-funded studentship to AAM.

1 Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK

2 Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams

[1] Blagbrough IS, Zara C. Animal Models for Target Diseases in Gene Therapy - Using DNA and siRNA Delivery Strategies. Pharmaceutical Research 2009;26(1) 1-18.

and Abdelkader A. Metwally1,2

\*Address all correspondence to: prsisb@bath.ac.uk

University, Abbasya, Cairo, Egypt

hydrolysed at the mildly acidic pH found in the endosome.

**Acknowledgements**

158 Gene Therapy - Tools and Potential Applications

**Author details**

Ian S. Blagbrough1

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[180] Lee H, Lytton-Jean AKR, Chen Y, Love KT, Park AI, Karagiannis ED, Sehgal A, Querbes W, Zurenko CS, Jayaraman M, Peng CG, Charisse K, Borodovsky A, Mano‐ haran M, Donahoe JS, Truelove J, Nahrendorf M, Langer R, Anderson DG. Molecu‐ larly Self-assembled Nucleic Acid Nanoparticles for Targeted in Vivo siRNA Delivery. Nature Nanotechnology 2012;7(6) 389-393. DOI: 10.1038/NNANO.2012.73.

Lipids, Gemini Surfactants, and Lipophilic Oligomers for Gene Delivery. Journal of

[169] Zhu L, Lu Y, Miller DD, Mahato RI. Structural and Formulation Factors Influencing Pyridinium Lipid-Based Gene Transfer. Bioconjugate Chemistry 2008;19(12)

[170] Medvedeva DA, Maslov MA, Serikov RN, Morozova NG, Serebrenikova GA, She‐ glov DV, Latyshev AV, Vlassov VV, Zenkova MA. Novel Cholesterol-Based Cationic Lipids for Gene Delivery. Journal of Medicinal Chemistry 2009;52(21) 6558-6568.

[171] Guo X, Szoka FC. Chemical Approaches to Triggerable Lipid Vesicles for Drug and

[172] Chen HG, Zhang HZ, McCallum CM, Szoka FC, Guo X. Unsaturated Cationic Ortho Esters for Endosome Permeation in Gene Delivery. Journal of Medicinal Chemistry

[173] Bajaj A, Kondaiah P, Bhattacharya S. Effect of the Nature of the Spacer on Gene Transfer Efficacies of Novel Thiocholesterol Derived Gemini Lipids in Different Cell Lines: A Structure-Activity Investigation. Journal of Medicinal Chemistry 2008;51(8)

[174] Metwally AA, Pourzand C, Blagbrough IS. Efficient Gene Silencing by Self-assem‐ bled Complexes of siRNA and Symmetrical Fatty Acid Amides of Spermine, Phar‐

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[176] Blagbrough IS, Metwally AA, Ghonaim HM, Asymmetrical N4,N9-Diacyl Sper‐ mines: SAR studies of Nonviral Lipopolyamine Vectors for Efficient siRNA Delivery with Silencing of EGFP Reporter Gene, Molecular Pharmaceutics 2012;9 1853-1861.

[177] Metwally AA, Reelfs O, Pourzand C, Blagbrough IS, Efficient Silencing of EGFP Re‐ porter Gene with siRNA Delivered by Asymmetrical N4,N9-Diacyl Spermines, Mo‐

[178] Metwally AA, Blagbrough IS, Mantell JM, Quantitative Silencing of EGFP Reporter Gene by Self-assembled siRNA Lipoplexes of LinOS and Cholesterol, Molecular

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2499-2512.

172 Gene Therapy - Tools and Potential Applications

2007;50(18) 4269-4278.

2533-2540.


**Section 3**

**Gene Therapy Tools: Biological**

**Gene Therapy Tools: Biological**

**Chapter 8**

**Mesenchymal Stem Cells as Gene Delivery Vehicles**

Mesenchymal stem cells (MSCs) possess a battery of unique properties which make them ideally suited not only for cellular therapies/regenerative medicine, but also as vehicles for gene delivery in a wide array of clinical settings. These include: 1) widespread distribution throughout the body; 2) ease of isolation and ability to be extensively expanded in culture without loss of potential; 3) the ability to differentiate into a wide array of functional cell types in vitro and in vivo; 4) they exert pronounced anti-inflammatory and immunomodula‐ tory effects upon transplantation; and 5) the ability to home to damaged tissues, solid tu‐

In this Chapter, we will summarize the latest research in the use of MSC in regenerative medicine, focusing predominantly on their use as vehicles for transferring exogenous genes. To highlight the immense potential these cells possess for gene therapy applications, we will attempt to paint as broad a canvas as possible, starting with a discussion about the basic bi‐ ology of MSC, and their unique properties which combine to make MSC one of the most promising stem cell populations for use in gene therapy studies and trials. We will reveal the versatility of MSC as gene delivery vehicles by summarizing some of the most recent studies showing the ease with which MSC can be modified with a wide range of both viral and non-viral vector systems, and highlighting some of the advantages to delivering trans‐ genes within a cellular vehicle, as opposed to administering vectors directly into the body. We will then discuss the engineering of MSC to enhance their natural abilities to mediate repair within various tissues; one of the most popular uses of MSC to-date in the gene thera‐ py arena. We will discuss our recent work, and that of others, using MSC to deliver coagula‐ tion factors to treat the hemophilias, with hemophilia A serving as a paradigm for how MSC could be used to deliver a therapeutic transgene, and thereby correct essentially any inherit‐ ed disease. The Chapter will conclude with a discussion of MSC's ability to selectively mi‐ grate to forming solid tumors following intravenous administration, and to actively seek out

> © 2013 Porada and Almeida-Porada; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is

Christopher D. Porada and Graça Almeida-Porada

Additional information is available at the end of the chapter

mors, and metastases following in vivo administration.

properly cited.

http://dx.doi.org/10.5772/53240

**1. Introduction**

### **Mesenchymal Stem Cells as Gene Delivery Vehicles**

Christopher D. Porada and Graça Almeida-Porada

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53240

#### **1. Introduction**

Mesenchymal stem cells (MSCs) possess a battery of unique properties which make them ideally suited not only for cellular therapies/regenerative medicine, but also as vehicles for gene delivery in a wide array of clinical settings. These include: 1) widespread distribution throughout the body; 2) ease of isolation and ability to be extensively expanded in culture without loss of potential; 3) the ability to differentiate into a wide array of functional cell types in vitro and in vivo; 4) they exert pronounced anti-inflammatory and immunomodula‐ tory effects upon transplantation; and 5) the ability to home to damaged tissues, solid tu‐ mors, and metastases following in vivo administration.

In this Chapter, we will summarize the latest research in the use of MSC in regenerative medicine, focusing predominantly on their use as vehicles for transferring exogenous genes. To highlight the immense potential these cells possess for gene therapy applications, we will attempt to paint as broad a canvas as possible, starting with a discussion about the basic bi‐ ology of MSC, and their unique properties which combine to make MSC one of the most promising stem cell populations for use in gene therapy studies and trials. We will reveal the versatility of MSC as gene delivery vehicles by summarizing some of the most recent studies showing the ease with which MSC can be modified with a wide range of both viral and non-viral vector systems, and highlighting some of the advantages to delivering trans‐ genes within a cellular vehicle, as opposed to administering vectors directly into the body. We will then discuss the engineering of MSC to enhance their natural abilities to mediate repair within various tissues; one of the most popular uses of MSC to-date in the gene thera‐ py arena. We will discuss our recent work, and that of others, using MSC to deliver coagula‐ tion factors to treat the hemophilias, with hemophilia A serving as a paradigm for how MSC could be used to deliver a therapeutic transgene, and thereby correct essentially any inherit‐ ed disease. The Chapter will conclude with a discussion of MSC's ability to selectively mi‐ grate to forming solid tumors following intravenous administration, and to actively seek out

© 2013 Porada and Almeida-Porada; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

metastases at sites far removed from the primary site of the tumor. We will summarize ex‐ citing recent work showing that it is possible to exploit this property to achieve sustained, high-level expression of pro-apoptotic gene products within the tumor, obtaining greatly improved anti-tumor effects, while essentially eliminating the systemic toxicities that plague current radiation/chemotherapy-based treatments.

geneous, investigators will likely either have to await the development of novel antibodies that recognize as yet unidentified antigens that are unique to primitive MSC, or employ strategies in which multiple antibodies are combined to allow for positive selection of MSC and depletion of cells of other lineages that share expression of the antigens recognized by

Mesenchymal Stem Cells as Gene Delivery Vehicles

http://dx.doi.org/10.5772/53240

179

Although much of the work to date has focused on MSC isolated from adult bone marrow, we and others have isolated cells that appear phenotypically and functionally to be MSC, from numerous tissues including brain, liver, lung, fetal blood, umbilical cord blood, kid‐ ney, and even liposuction material [19-26]. The broad distribution of MSC throughout the body leads one to postulate that MSC may play a critical role in organ homeostasis by pro‐ viding supportive factors and/or mediating maintenance/repair within their respective tis‐ sue. Importantly, although MSC from each of these tissues appear similar with respect to phenotype and overall differentiative potential, studies at the RNA and protein level have now revealed that subtle differences exist between MSC from these various tissues, with MSC from each tissue possessing a molecular fingerprint indicative of their tissue of origin [21, 22, 27-31]. Using a non-injury fetal sheep transplantation model, we showed that these differences result in a bias for human MSC to home to and give rise to cells of their tissue of origin in vivo [32, 33]. This suggests that, to use MSC as therapeutics or as gene delivery ve‐ hicles, the ideal source of MSC may differ depending on the specific disease to be treated

Despite the apparent presence of MSC within many of the major organs of the body, the rel‐ atively non-invasive fashion with which adipose tissue or bone marrow can be obtained, and the fact that both these tissues could readily be obtained autologously, combine to sug‐ gest that these two tissues will likely be the predominant source of MSC employed in clini‐ cal applications in the foreseeable future. However, additional experiments will need to be performed to rigorously assess the inherent safety of adipose tissue-derived MSC before these cells will see widespread clinical use, since several recent studies have suggested that they may be inherently less genetically stable than MSC isolated from bone marrow [34], ex‐ hibiting aneuploidy [35, 36] and undergoing transformation [37, 38] upon prolonged propa‐ gation in vitro. However, another recent study provided evidence that, even if genomic instability is intentionally induced with genotoxic agents, adipose tissue-derived MSC re‐ spond to this insult by undergoing terminal adipogenic differentiation rather than transfor‐ mation [39]. The dramatically conflicting nature of the results from these different studies could, perhaps, be due to differing methods employed for isolating and culturing MSC, dif‐ fering levels of contaminating non-MSC cells in the cultures, as well as the duration of the culture (i.e., the number of times the cells have been passaged). Bearing this possible insta‐ bility in mind, the recommendation has been put forward to only make clinical use of cells that have been passaged fewer than 25 times in culture, regardless of the source of MSC [40].

the MSC antibody in question, as we have done with Stro-1, CD45, and GlyA.

**3. Sources of MSC**

and the desired target organ.

#### **2. Isolation and characterization of MSC**

More than 30 years ago, Friedenstein pioneered the concept that the marrow microenviron‐ ment resided within the so-called stromal cells of the marrow, by demonstrating that fibro‐ blastoid cells obtained from the bone marrow were capable of transferring the hematopoietic microenvironment to ectopic sites [1, 2]. Years later, scientists began to ex‐ plore the full potential of these microenvironmental cells, and results of these studies led to the realization that this population harbored cells with properties of true stem cells. These cells were officially termed mesenchymal stem cells (MSC) [3]. MSC are now recognized to be a key part of the microenvironment/niche that supports the hematopoietic stem cell and drives the process of hematopoiesis, yet despite serving this vital function, MSC only com‐ prise ~0.001-0.01% of cells within the marrow [4], making methods for isolating/enriching and for expanding these cells essential to their study and their ultimate clinical use. The most straightforward method for obtaining MSC is to exploit their propensity to adhere to plastic and their ability to be passaged with trypsin (contaminating hematopoietic cells do not passage with trypsin) to rapidly obtain a relatively morphologically homogeneous pop‐ ulation of fibroblastic cells from a bulk mononuclear cell preparation [5-7]. Unfortunately, true MSC (defined by function) account for only a small percentage of the highly heteroge‐ neous resultant population, making results obtained with cells prepared in this fashion diffi‐ cult to interpret, and inconsistent from experiment-to-experiment and from group-to-group. The identification of antigens that are unique to MSC would eliminate this problem. Human MSC do not express markers which have been associated with other stem cell populations (like hematopoietic stem cells) such as CD34, CD133, or c-kit, nor hematopoietic markers such as CD45, CD14, and CD19. Moreover, no marker has been identified to date that specif‐ ically identifies MSC. Nevertheless, several surface antigens have proven useful for obtain‐ ing highly enriched MSC populations. The first of these markers to be identified was Stro-1, an antibody that reacts with non-hematopoietic bone marrow stromal precursor cells [8]. Al‐ though the antigen recognized by this antibody has not yet been identified, we and others have found that by tri-labeling bone marrow cells with Stro-1, anti-CD45, and anti-GlyA, and selecting the Stro-1+CD45-GlyA- cells, it is possible to consistently obtain a homogene‐ ous population that is highly enriched for MSC [9-15]. In addition to Stro-1, antibodies such as SB-10, SH2, SH3, and SH4 have been developed over the years and numerous surface an‐ tigens such CD13, CD29, CD44, CD63, CD73, CD90, CD105, and CD166 have been used to attempt to identify and isolate MSC [16-18]. Unfortunately, all of these antigens appear to be expressed on a wide range of cell types within the body in addition to MSC. This lack of a unique marker suggests that to obtain a pure population of MSC that are functionally homo‐ geneous, investigators will likely either have to await the development of novel antibodies that recognize as yet unidentified antigens that are unique to primitive MSC, or employ strategies in which multiple antibodies are combined to allow for positive selection of MSC and depletion of cells of other lineages that share expression of the antigens recognized by the MSC antibody in question, as we have done with Stro-1, CD45, and GlyA.

#### **3. Sources of MSC**

metastases at sites far removed from the primary site of the tumor. We will summarize ex‐ citing recent work showing that it is possible to exploit this property to achieve sustained, high-level expression of pro-apoptotic gene products within the tumor, obtaining greatly improved anti-tumor effects, while essentially eliminating the systemic toxicities that plague

More than 30 years ago, Friedenstein pioneered the concept that the marrow microenviron‐ ment resided within the so-called stromal cells of the marrow, by demonstrating that fibro‐ blastoid cells obtained from the bone marrow were capable of transferring the hematopoietic microenvironment to ectopic sites [1, 2]. Years later, scientists began to ex‐ plore the full potential of these microenvironmental cells, and results of these studies led to the realization that this population harbored cells with properties of true stem cells. These cells were officially termed mesenchymal stem cells (MSC) [3]. MSC are now recognized to be a key part of the microenvironment/niche that supports the hematopoietic stem cell and drives the process of hematopoiesis, yet despite serving this vital function, MSC only com‐ prise ~0.001-0.01% of cells within the marrow [4], making methods for isolating/enriching and for expanding these cells essential to their study and their ultimate clinical use. The most straightforward method for obtaining MSC is to exploit their propensity to adhere to plastic and their ability to be passaged with trypsin (contaminating hematopoietic cells do not passage with trypsin) to rapidly obtain a relatively morphologically homogeneous pop‐ ulation of fibroblastic cells from a bulk mononuclear cell preparation [5-7]. Unfortunately, true MSC (defined by function) account for only a small percentage of the highly heteroge‐ neous resultant population, making results obtained with cells prepared in this fashion diffi‐ cult to interpret, and inconsistent from experiment-to-experiment and from group-to-group. The identification of antigens that are unique to MSC would eliminate this problem. Human MSC do not express markers which have been associated with other stem cell populations (like hematopoietic stem cells) such as CD34, CD133, or c-kit, nor hematopoietic markers such as CD45, CD14, and CD19. Moreover, no marker has been identified to date that specif‐ ically identifies MSC. Nevertheless, several surface antigens have proven useful for obtain‐ ing highly enriched MSC populations. The first of these markers to be identified was Stro-1, an antibody that reacts with non-hematopoietic bone marrow stromal precursor cells [8]. Al‐ though the antigen recognized by this antibody has not yet been identified, we and others have found that by tri-labeling bone marrow cells with Stro-1, anti-CD45, and anti-GlyA, and selecting the Stro-1+CD45-GlyA- cells, it is possible to consistently obtain a homogene‐ ous population that is highly enriched for MSC [9-15]. In addition to Stro-1, antibodies such as SB-10, SH2, SH3, and SH4 have been developed over the years and numerous surface an‐ tigens such CD13, CD29, CD44, CD63, CD73, CD90, CD105, and CD166 have been used to attempt to identify and isolate MSC [16-18]. Unfortunately, all of these antigens appear to be expressed on a wide range of cell types within the body in addition to MSC. This lack of a unique marker suggests that to obtain a pure population of MSC that are functionally homo‐

current radiation/chemotherapy-based treatments.

178 Gene Therapy - Tools and Potential Applications

**2. Isolation and characterization of MSC**

Although much of the work to date has focused on MSC isolated from adult bone marrow, we and others have isolated cells that appear phenotypically and functionally to be MSC, from numerous tissues including brain, liver, lung, fetal blood, umbilical cord blood, kid‐ ney, and even liposuction material [19-26]. The broad distribution of MSC throughout the body leads one to postulate that MSC may play a critical role in organ homeostasis by pro‐ viding supportive factors and/or mediating maintenance/repair within their respective tis‐ sue. Importantly, although MSC from each of these tissues appear similar with respect to phenotype and overall differentiative potential, studies at the RNA and protein level have now revealed that subtle differences exist between MSC from these various tissues, with MSC from each tissue possessing a molecular fingerprint indicative of their tissue of origin [21, 22, 27-31]. Using a non-injury fetal sheep transplantation model, we showed that these differences result in a bias for human MSC to home to and give rise to cells of their tissue of origin in vivo [32, 33]. This suggests that, to use MSC as therapeutics or as gene delivery ve‐ hicles, the ideal source of MSC may differ depending on the specific disease to be treated and the desired target organ.

Despite the apparent presence of MSC within many of the major organs of the body, the rel‐ atively non-invasive fashion with which adipose tissue or bone marrow can be obtained, and the fact that both these tissues could readily be obtained autologously, combine to sug‐ gest that these two tissues will likely be the predominant source of MSC employed in clini‐ cal applications in the foreseeable future. However, additional experiments will need to be performed to rigorously assess the inherent safety of adipose tissue-derived MSC before these cells will see widespread clinical use, since several recent studies have suggested that they may be inherently less genetically stable than MSC isolated from bone marrow [34], ex‐ hibiting aneuploidy [35, 36] and undergoing transformation [37, 38] upon prolonged propa‐ gation in vitro. However, another recent study provided evidence that, even if genomic instability is intentionally induced with genotoxic agents, adipose tissue-derived MSC re‐ spond to this insult by undergoing terminal adipogenic differentiation rather than transfor‐ mation [39]. The dramatically conflicting nature of the results from these different studies could, perhaps, be due to differing methods employed for isolating and culturing MSC, dif‐ fering levels of contaminating non-MSC cells in the cultures, as well as the duration of the culture (i.e., the number of times the cells have been passaged). Bearing this possible insta‐ bility in mind, the recommendation has been put forward to only make clinical use of cells that have been passaged fewer than 25 times in culture, regardless of the source of MSC [40].

#### **4. MSC as vehicles for delivering therapeutic genes**

While MSC possess tremendous therapeutic potential by virtue of their ability to lodge/ engraft within multiple tissues in the body and both give rise to tissue-specific cells and re‐ lease trophic factors that trigger the tissue's own endogenous repair pathways [41-59], gene therapists have realized that these properties are just the beginning of the therapeutic appli‐ cations for MSC [24, 60, 61]. By using gene therapy to engineer MSC to either augment their own natural production of specific desired proteins or to enable them to express proteins outside of their native repertoire, it is possible to greatly broaden the spectrum of diseases for which MSC could provide therapeutic benefit. Unlike hematopoietic stem cells which are notoriously difficult to modify with most viral vectors while preserving their in vivo poten‐ tial, MSC can be readily transduced with all of the major clinically prevalent viral vector sys‐ tems including those based upon adenovirus [62-64], the murine retroviruses [64-68], lentiviruses [69-74], and AAV [75, 76], and efficiently produce a wide range of cytoplasmic, membrane-bound, and secreted protein products. This ease of transduction coupled with the ability to subsequently select and expand only the gene-modified cells in vitro to gener‐ ate adequate numbers for transplantation combine to make MSC one of the most promising stem cell populations for use in gene therapy studies and trials.

with persons exhibiting less than 1% normal factor (<0.01 IU/mL) being considered to have severe hemophilia, persons with 1-5% normal factor moderately severe, and persons with 5%-40% of the normal FVIII levels mild [114-116]. Up to 70% of hemophilia A patients present with the severe form of the disease, and suffer from frequent hemorrhaging, leading to chronic debilitating arthropathy, hematomas of subcutaneous connective tissue/muscle, and internal bleeding. Over time, the collective complications of recurrent hemorrhaging re‐ sult in chronic pain, absences from school and work, and permanent disability [114]. Cur‐ rent state-of-the-art treatment consists of frequent prophylactic infusions of plasma-derived or recombinant FVIII protein to maintain hemostasis, and has greatly increased life expect‐

Mesenchymal Stem Cells as Gene Delivery Vehicles

http://dx.doi.org/10.5772/53240

181

This treatment approach is, however, far from ideal, due to the need for lifelong intravenous infusions and the high treatment cost. Moreover, this treatment is unavailable to a large per‐ centage of the world's hemophiliacs, placing these patients at great risk of severe, perma‐ nent disabilities and life-threatening bleeds. Furthermore, even among the patients who are fortunate enough to have access to, and the financial means to afford, prophylactic FVIII in‐ fusions, approximately 30% will form FVIII inhibitors [117]. The formation of these inhibi‐ tors greatly reduces the efficacy of subsequent FVIII infusions, and can ultimately lead to treatment failure, placing the patient at risk of life-threatening hemorrhage. There is thus a

In the past three decades, the remarkable progress in the understanding of the molecular ba‐ sis of the disease, the identification and characterization of FVIII gene, structure, and biolo‐ gy has heightened the interest and feasibility of treating hemophilia A with gene therapy. In contrast to current protein-based therapeutics, lifelong improvement or permanent cure of hemophilia A is theoretically possible after only a single gene therapy treatment; indeed, several aspects of hemophilia A make it ideally suited for correction by gene therapy [118-126]. First, in contrast to many other genetic diseases, the missing protein (coagulation FVIII) does not need to be expressed in either a cell- or tissue-specific fashion to mediate cor‐ rection. Although the liver is thought to be the primary natural site of synthesis of FVIII, ex‐ pression of this factor in other tissues exerts no deleterious effects. As long as the protein is expressed in cells which have ready access to the circulation, it can be secreted into the bloodstream and exert its appropriate clotting activity. Second, even modest levels (3-5%) of FVIII-expressing cells would be expected to convert severe hemophilia A to a moderate/ mild phenotype, reducing or eliminating episodes of spontaneous bleeding and greatly im‐ proving quality of life. Thus, even with the low levels of transduction that are routinely ob‐ tained with many of the current viral-based gene delivery systems, a marked clinical improvement would be anticipated in patients with hemophilia A. Conversely, even supra‐ physiologic levels of FVIII as high as 150% of normal are predicted to be well tolerated, making the therapeutic window extremely wide [116]. Based on this knowledge, the Ameri‐ can Society of Gene and Cell Therapy (www.ASGCT.org) recently provided NIH director, Dr. Francis Collins, with a roadmap of disease indications that it feels will be viable gene therapy products within the next 5-7 years. The hemophilias were identified as belonging to

ancy and quality of life for many hemophilia A patients.

the most promising, "Target 10", group of diseases.

significant need to develop novel, longer-lasting hemophilia A therapies.

To date, the majority of studies using gene-modified MSC have been undertaken with the purpose of enhancing the natural abilities of MSC to mediate repair within various tissues. Using the heart as an example, once investigators discovered the identity of some of the key trophic factors responsible for MSC's beneficial effect on the injured myocardium, they un‐ dertook studies using MSC engineered to overexpress a number of these factors [69, 77-86]. As anticipated, the "gene-enhanced" MSC were substantially more effective than their un‐ modified counterparts, producing greatly enhanced therapeutic effects. Similar studies have also been performed to repair the damaged/diseased CNS using MSC engineered to pro‐ duce neurotrophic factors [87-94], to repair the injured liver using MSC expressing proteins involved in hepatocyte differentiation and/or proliferation [95, 96], to repair ischemia/reper‐ fusion injury [97-102], and to repair the kidney [103-105]. In each case thus far, MSC engi‐ neered to express higher levels of proteins known to be beneficial for the tissue in question and/or to promote survival have produced markedly better results than unmodified MSC.

Despite the many advantages of using MSC as gene delivery vehicles, however, relatively few studies have thus far explored this potential for the treatment of genetic diseases. One disease for which we and others are actively investigating MSC for delivery of a therapeutic gene is hemophilia A [106-112].

#### **5. Hemophilia A as a paradigm for the use of gene-modified msc to correct genetic diseases**

Hemophilia A represents the most common inheritable deficiency of the coagulation pro‐ teins [113]. The severity of hemophilia A is traditionally based on plasma levels of FVIII, with persons exhibiting less than 1% normal factor (<0.01 IU/mL) being considered to have severe hemophilia, persons with 1-5% normal factor moderately severe, and persons with 5%-40% of the normal FVIII levels mild [114-116]. Up to 70% of hemophilia A patients present with the severe form of the disease, and suffer from frequent hemorrhaging, leading to chronic debilitating arthropathy, hematomas of subcutaneous connective tissue/muscle, and internal bleeding. Over time, the collective complications of recurrent hemorrhaging re‐ sult in chronic pain, absences from school and work, and permanent disability [114]. Cur‐ rent state-of-the-art treatment consists of frequent prophylactic infusions of plasma-derived or recombinant FVIII protein to maintain hemostasis, and has greatly increased life expect‐ ancy and quality of life for many hemophilia A patients.

**4. MSC as vehicles for delivering therapeutic genes**

180 Gene Therapy - Tools and Potential Applications

stem cell populations for use in gene therapy studies and trials.

gene is hemophilia A [106-112].

**correct genetic diseases**

While MSC possess tremendous therapeutic potential by virtue of their ability to lodge/ engraft within multiple tissues in the body and both give rise to tissue-specific cells and re‐ lease trophic factors that trigger the tissue's own endogenous repair pathways [41-59], gene therapists have realized that these properties are just the beginning of the therapeutic appli‐ cations for MSC [24, 60, 61]. By using gene therapy to engineer MSC to either augment their own natural production of specific desired proteins or to enable them to express proteins outside of their native repertoire, it is possible to greatly broaden the spectrum of diseases for which MSC could provide therapeutic benefit. Unlike hematopoietic stem cells which are notoriously difficult to modify with most viral vectors while preserving their in vivo poten‐ tial, MSC can be readily transduced with all of the major clinically prevalent viral vector sys‐ tems including those based upon adenovirus [62-64], the murine retroviruses [64-68], lentiviruses [69-74], and AAV [75, 76], and efficiently produce a wide range of cytoplasmic, membrane-bound, and secreted protein products. This ease of transduction coupled with the ability to subsequently select and expand only the gene-modified cells in vitro to gener‐ ate adequate numbers for transplantation combine to make MSC one of the most promising

To date, the majority of studies using gene-modified MSC have been undertaken with the purpose of enhancing the natural abilities of MSC to mediate repair within various tissues. Using the heart as an example, once investigators discovered the identity of some of the key trophic factors responsible for MSC's beneficial effect on the injured myocardium, they un‐ dertook studies using MSC engineered to overexpress a number of these factors [69, 77-86]. As anticipated, the "gene-enhanced" MSC were substantially more effective than their un‐ modified counterparts, producing greatly enhanced therapeutic effects. Similar studies have also been performed to repair the damaged/diseased CNS using MSC engineered to pro‐ duce neurotrophic factors [87-94], to repair the injured liver using MSC expressing proteins involved in hepatocyte differentiation and/or proliferation [95, 96], to repair ischemia/reper‐ fusion injury [97-102], and to repair the kidney [103-105]. In each case thus far, MSC engi‐ neered to express higher levels of proteins known to be beneficial for the tissue in question and/or to promote survival have produced markedly better results than unmodified MSC. Despite the many advantages of using MSC as gene delivery vehicles, however, relatively few studies have thus far explored this potential for the treatment of genetic diseases. One disease for which we and others are actively investigating MSC for delivery of a therapeutic

**5. Hemophilia A as a paradigm for the use of gene-modified msc to**

Hemophilia A represents the most common inheritable deficiency of the coagulation pro‐ teins [113]. The severity of hemophilia A is traditionally based on plasma levels of FVIII, This treatment approach is, however, far from ideal, due to the need for lifelong intravenous infusions and the high treatment cost. Moreover, this treatment is unavailable to a large per‐ centage of the world's hemophiliacs, placing these patients at great risk of severe, perma‐ nent disabilities and life-threatening bleeds. Furthermore, even among the patients who are fortunate enough to have access to, and the financial means to afford, prophylactic FVIII in‐ fusions, approximately 30% will form FVIII inhibitors [117]. The formation of these inhibi‐ tors greatly reduces the efficacy of subsequent FVIII infusions, and can ultimately lead to treatment failure, placing the patient at risk of life-threatening hemorrhage. There is thus a significant need to develop novel, longer-lasting hemophilia A therapies.

In the past three decades, the remarkable progress in the understanding of the molecular ba‐ sis of the disease, the identification and characterization of FVIII gene, structure, and biolo‐ gy has heightened the interest and feasibility of treating hemophilia A with gene therapy. In contrast to current protein-based therapeutics, lifelong improvement or permanent cure of hemophilia A is theoretically possible after only a single gene therapy treatment; indeed, several aspects of hemophilia A make it ideally suited for correction by gene therapy [118-126]. First, in contrast to many other genetic diseases, the missing protein (coagulation FVIII) does not need to be expressed in either a cell- or tissue-specific fashion to mediate cor‐ rection. Although the liver is thought to be the primary natural site of synthesis of FVIII, ex‐ pression of this factor in other tissues exerts no deleterious effects. As long as the protein is expressed in cells which have ready access to the circulation, it can be secreted into the bloodstream and exert its appropriate clotting activity. Second, even modest levels (3-5%) of FVIII-expressing cells would be expected to convert severe hemophilia A to a moderate/ mild phenotype, reducing or eliminating episodes of spontaneous bleeding and greatly im‐ proving quality of life. Thus, even with the low levels of transduction that are routinely ob‐ tained with many of the current viral-based gene delivery systems, a marked clinical improvement would be anticipated in patients with hemophilia A. Conversely, even supra‐ physiologic levels of FVIII as high as 150% of normal are predicted to be well tolerated, making the therapeutic window extremely wide [116]. Based on this knowledge, the Ameri‐ can Society of Gene and Cell Therapy (www.ASGCT.org) recently provided NIH director, Dr. Francis Collins, with a roadmap of disease indications that it feels will be viable gene therapy products within the next 5-7 years. The hemophilias were identified as belonging to the most promising, "Target 10", group of diseases.

#### **6. Mesenchymal Stem Cells (MSC) as hemophilia A therapeutics**

mobility, and proteolytic activation pattern that was virtually identical to that of FVIII pro‐ duced by other commercial cell lines [109]. Given the widespread distribution and engraft‐ ment of MSC following their systemic infusion, the ability of MSC to give rise, in vivo, to cells of numerous tissue types, and their ability to efficiently process and secrete high amounts of biologically active FVIII, they are, not surprisingly, being viewed as ideal vehi‐ cles for delivering a FVIII transgene throughout the body and thus providing long-term/

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183

In addition to their widespread engraftment and their ability to serve as delivery vehicles for the FVIII gene, the rather unique immunological properties of MSC may further increase their utility for treating hemophilia A. MSC do not normally express MHC class II or the costimulatory molecules CD80 and CD82, unless they are stimulated with IFN-γ, and are thus viewed as being relatively hypo-immunogenic. As such, they do not provoke the prolifera‐ tion of allogeneic lymphocytes or serve as very effective targets for lysis by cytotoxic T cells or NK cells. In fact, a large body of evidence is now accumulating that MSC can be readily transplanted across allogeneic barriers without eliciting an immune response [163, 164]. Thus, if one wished to use MSC to treat hemophilia A, off-the-shelf MSC from an unrelated donor could theoretically be used, greatly increasing the feasibility of obtaining and using

Perhaps even more important from the standpoint of their potential use as hemophilia A therapeutics, more recent studies have provided evidence that MSC also appear to have the ability to exert both immunosuppressive and anti-inflammatory properties both in vitro and in vivo. These properties appear to result from MSC's ability to intervene, at multiple levels, with the generation and propagation of an immune response. To name just a few examples, MSC have been demonstrated to interfere with the generation and maturation of cytotoxic and helper T cells [165-174], dendritic cells [175-178], and B cells [179]. In addition to actively shutting down the generation of immune effector cells, MSC also have the ability to induce the formation of potent Tregs, although the mechanism by which this comes about is still the subject of active research [40, 180-182]. MSC are also known to express a battery of factors [40, 168-170, 180, 183-187] that reduce local inflammation, blunt immune response, and counteract the chemotactic signals responsible for recruiting immune cells to sites of injury/ inflammation. One could thus envision these immune-dampening properties enabling the delivery of FVIII without eliciting an immune response and subsequent inhibitor formation, thus overcoming one of the major hurdles that plague current treatment/management of he‐ mophilia A. As will be discussed in the next section, however, our postnatal studies in the hemophilic sheep suggest that further work will be required to discover how to obtain these potential immune benefits in the context of the ongoing injury/inflammation present in ani‐

In addition to the aforementioned properties, preclinical animal studies examining the po‐ tential of MSC isolated from adult tissues have also highlighted another interesting and clin‐ ically valuable characteristic of MSC; their ability to selectively navigate to sites of injury and/or inflammation within the body. Once reaching these specific sites, the MSC then me‐ diate repair both by engrafting and generating tissue-specific cells within the injured tissue

permanent correction of hemophilia A [106-109, 162].

mals/patients with clinically advanced hemophilia A.

these stem cells for therapy.

As discussed in the preceding section, the liver is thought to be the primary site of FVIII syn‐ thesis within the body. We and others have devoted a great deal of energy to demonstrating the ability of MSC from various sources to serve as therapeutics for liver disease [11, 13, 14, 33, 96, 127-152]. It is now clear that, not only do MSC have the ability to generate, in vitro and in vivo, cells which are indistinguishable from native hepatocytes, but transplantation of MSC in a range of model systems can result in fairly robust formation of hepatocytes which repair a variety of inborn genetic defects, toxin-induced injuries, and even fibrosis. The fetal sheep model provides a unique system in which to explore the full differentiative potential of various stem cell populations, since the continuous need for new cells within all of the organs during fetal development provides a permissive milieu in which gene-modi‐ fied donor cells can engraft, proliferate, and differentiate. Furthermore, by performing the transplant at a stage in gestation when the fetus is considered to be largely immuno-naïve, it is possible to engraft human cells at significant levels, which persist for the lifespan of the animal due to induction of donor-specific tolerance [130-132]. Indeed, in ongoing studies, we have found that, after transplantation into fetal sheep, human MSC engraft at levels of up to 12% within the recipient liver [11, 131, 132, 153-158], and contribute to both the paren‐ chyma and the perivascular zones of the engrafted organs, placing them ideally for deliver‐ ing FVIII into the circulation. Since FVIII levels of 3-5% of normal would convert a patient with severe hemophilia A to a moderate or mild phenotype, these levels of engraftment should be highly therapeutic. These collective results thus suggest that MSC may represent an ideal cell type for treating hemophilia A.

However, although MSC engrafted (following transplantation in utero) at significant levels within organs that are natural sites of FVIII synthesis, only a small percent expressed endog‐ enous FVIII, suggesting that simply transplanting "healthy" MSC will not likely provide an effective means of treating/curing hemophilia A. By using gene therapy to engineer MSC to express FVIII, however, it is highly probable that the levels of engrafted MSC we have thus far achieved in utero would provide marked therapeutic benefit in hemophilia A. By trans‐ ducing the MSC in vitro, rather than performing gene therapy by injecting the vector direct‐ ly, as is the current practice in clinical gene therapy trials, there is no risk of off-target transduction, and the vector being employed simply needs a strong constitutively active promoter to ensure that all cells derived from the transplanted MSC continue to express FVIII and mediate a therapeutic effect. Importantly, the only documented cases of retroviralinduced insertional mutagenesis have been observed following genetic modification of hem‐ atopoietic stem cells [159-161]; there is no evidence that MSC transform or progress to clonal dominance following transduction, suggesting they represent safe cellular vehicles for deliv‐ ering FVIII (or other transgenes).

Importantly, critical proof-of-principle studies have already shown that MSC can be trans‐ duced with FVIII-expressing viral vectors and secrete high levels of FVIII protein in vitro and following transplantation in vivo [106-109]. FVIII purified from the conditioned medi‐ um of the transduced MSC was proven to have a specific activity, relative electrophoretic mobility, and proteolytic activation pattern that was virtually identical to that of FVIII pro‐ duced by other commercial cell lines [109]. Given the widespread distribution and engraft‐ ment of MSC following their systemic infusion, the ability of MSC to give rise, in vivo, to cells of numerous tissue types, and their ability to efficiently process and secrete high amounts of biologically active FVIII, they are, not surprisingly, being viewed as ideal vehi‐ cles for delivering a FVIII transgene throughout the body and thus providing long-term/ permanent correction of hemophilia A [106-109, 162].

**6. Mesenchymal Stem Cells (MSC) as hemophilia A therapeutics**

an ideal cell type for treating hemophilia A.

182 Gene Therapy - Tools and Potential Applications

ering FVIII (or other transgenes).

As discussed in the preceding section, the liver is thought to be the primary site of FVIII syn‐ thesis within the body. We and others have devoted a great deal of energy to demonstrating the ability of MSC from various sources to serve as therapeutics for liver disease [11, 13, 14, 33, 96, 127-152]. It is now clear that, not only do MSC have the ability to generate, in vitro and in vivo, cells which are indistinguishable from native hepatocytes, but transplantation of MSC in a range of model systems can result in fairly robust formation of hepatocytes which repair a variety of inborn genetic defects, toxin-induced injuries, and even fibrosis. The fetal sheep model provides a unique system in which to explore the full differentiative potential of various stem cell populations, since the continuous need for new cells within all of the organs during fetal development provides a permissive milieu in which gene-modi‐ fied donor cells can engraft, proliferate, and differentiate. Furthermore, by performing the transplant at a stage in gestation when the fetus is considered to be largely immuno-naïve, it is possible to engraft human cells at significant levels, which persist for the lifespan of the animal due to induction of donor-specific tolerance [130-132]. Indeed, in ongoing studies, we have found that, after transplantation into fetal sheep, human MSC engraft at levels of up to 12% within the recipient liver [11, 131, 132, 153-158], and contribute to both the paren‐ chyma and the perivascular zones of the engrafted organs, placing them ideally for deliver‐ ing FVIII into the circulation. Since FVIII levels of 3-5% of normal would convert a patient with severe hemophilia A to a moderate or mild phenotype, these levels of engraftment should be highly therapeutic. These collective results thus suggest that MSC may represent

However, although MSC engrafted (following transplantation in utero) at significant levels within organs that are natural sites of FVIII synthesis, only a small percent expressed endog‐ enous FVIII, suggesting that simply transplanting "healthy" MSC will not likely provide an effective means of treating/curing hemophilia A. By using gene therapy to engineer MSC to express FVIII, however, it is highly probable that the levels of engrafted MSC we have thus far achieved in utero would provide marked therapeutic benefit in hemophilia A. By trans‐ ducing the MSC in vitro, rather than performing gene therapy by injecting the vector direct‐ ly, as is the current practice in clinical gene therapy trials, there is no risk of off-target transduction, and the vector being employed simply needs a strong constitutively active promoter to ensure that all cells derived from the transplanted MSC continue to express FVIII and mediate a therapeutic effect. Importantly, the only documented cases of retroviralinduced insertional mutagenesis have been observed following genetic modification of hem‐ atopoietic stem cells [159-161]; there is no evidence that MSC transform or progress to clonal dominance following transduction, suggesting they represent safe cellular vehicles for deliv‐

Importantly, critical proof-of-principle studies have already shown that MSC can be trans‐ duced with FVIII-expressing viral vectors and secrete high levels of FVIII protein in vitro and following transplantation in vivo [106-109]. FVIII purified from the conditioned medi‐ um of the transduced MSC was proven to have a specific activity, relative electrophoretic In addition to their widespread engraftment and their ability to serve as delivery vehicles for the FVIII gene, the rather unique immunological properties of MSC may further increase their utility for treating hemophilia A. MSC do not normally express MHC class II or the costimulatory molecules CD80 and CD82, unless they are stimulated with IFN-γ, and are thus viewed as being relatively hypo-immunogenic. As such, they do not provoke the prolifera‐ tion of allogeneic lymphocytes or serve as very effective targets for lysis by cytotoxic T cells or NK cells. In fact, a large body of evidence is now accumulating that MSC can be readily transplanted across allogeneic barriers without eliciting an immune response [163, 164]. Thus, if one wished to use MSC to treat hemophilia A, off-the-shelf MSC from an unrelated donor could theoretically be used, greatly increasing the feasibility of obtaining and using these stem cells for therapy.

Perhaps even more important from the standpoint of their potential use as hemophilia A therapeutics, more recent studies have provided evidence that MSC also appear to have the ability to exert both immunosuppressive and anti-inflammatory properties both in vitro and in vivo. These properties appear to result from MSC's ability to intervene, at multiple levels, with the generation and propagation of an immune response. To name just a few examples, MSC have been demonstrated to interfere with the generation and maturation of cytotoxic and helper T cells [165-174], dendritic cells [175-178], and B cells [179]. In addition to actively shutting down the generation of immune effector cells, MSC also have the ability to induce the formation of potent Tregs, although the mechanism by which this comes about is still the subject of active research [40, 180-182]. MSC are also known to express a battery of factors [40, 168-170, 180, 183-187] that reduce local inflammation, blunt immune response, and counteract the chemotactic signals responsible for recruiting immune cells to sites of injury/ inflammation. One could thus envision these immune-dampening properties enabling the delivery of FVIII without eliciting an immune response and subsequent inhibitor formation, thus overcoming one of the major hurdles that plague current treatment/management of he‐ mophilia A. As will be discussed in the next section, however, our postnatal studies in the hemophilic sheep suggest that further work will be required to discover how to obtain these potential immune benefits in the context of the ongoing injury/inflammation present in ani‐ mals/patients with clinically advanced hemophilia A.

In addition to the aforementioned properties, preclinical animal studies examining the po‐ tential of MSC isolated from adult tissues have also highlighted another interesting and clin‐ ically valuable characteristic of MSC; their ability to selectively navigate to sites of injury and/or inflammation within the body. Once reaching these specific sites, the MSC then me‐ diate repair both by engrafting and generating tissue-specific cells within the injured tissue [188-190], and by releasing trophic factors that blunt the inflammatory response and often promote healing by activating the tissue's own endogenous repair mechanisms. While the mechanisms responsible for this trafficking to sites of injury are still being elucidated [191-193], this observation raises the exciting possibility that, following systemic infusion, FVIII-expressing MSC could efficiently migrate to sites of active bleeding/injury, thereby re‐ leasing FVIII locally and focusing the therapy where it is most needed. As will be discussed in the following section, over the past 2-3 years, we have begun exploring whether it is pos‐ sible to exploit these many advantages of MSC as a cellular vehicle for delivering a FVIII gene by testing the ability of FVIII-expressing MSC to correct hemophilia A in a new large animal model; sheep.

toms of pain in standing up, restricting nursing activity. Stronger injuries that arose when animals were not placed in carefully controlled isolation resulted in heavy bleeding and in‐ tensive pain. Laboratory tests showed increased PTT, and FVIII levels (as assessed by aPTT) of about 1% of control animals. Replacement therapy with human FVIII (hFVIII) concentrate or fresh sheep plasma resulted in remission of disease and rapid clinical improvement.

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Unfortunately, due to the expense and effort of maintaining these sheep, the Swiss investi‐ gators allowed the line to die out, saving only a few straws of semen prior to allowing this valuable resource to pass into extinction. We recently used a variety of reproductive tech‐ nologies to successfully re-establish this line of hemophilia A sheep, and fully characterized both the clinical parameters and the precise molecular basis for their disease [211-216]. In similarity to mutations seen in many human patients [217], these animals possess a prema‐ ture stop codon with a frameshift mutation. This is the only animal model of hemophilia A with this clinically relevant mutation-type, providing a unique opportunity to study thera‐ pies in this context. All ten animals to-date have exhibited bleeding from the umbilical cord, prolonged tail and "cuticle" bleeding time, and multiple episodes of severe spontaneous bleeding including hemarthroses, muscle hematomas, and hematuria, all of which have re‐ sponded to human FVIII. Just like human patients with severe hemophilia A, a hallmark symptom in these sheep is repeated spontaneous joint bleeds, which lead to chronic, debili‐ tating arthropathies and reduced mobility. Importantly, chromogenic assays performed in‐ dependently at the BloodCenter of Wisconsin and Emory University revealed undetectable FVIII activity in the circulation of these sheep, explaining their severe, life-threatening phe‐

In addition to the value of another large animal model of hemophilia A and the uniqueness of the mutation, sheep possess many characteristics that make them an ideal preclinical model for gene therapy. The first of these is the size. Sheep are fairly close in size to humans, weighing roughly 8lbs at birth and 150-200lbs as adults, likely obviating the need for scaleup of cell/vector dose to move from experiments in sheep to trials in humans. This is in marked contrast to mice which are ~2800 times smaller than a typical human patient [218]. Secondly, sheep share many important physiological and developmental characteristics with humans; for example, the pattern of fetal to adult hemoglobin switching, and the natu‐ rally occurring changes in the primary sites of hematopoiesis from yolk sac to fetal liver and finally to the bone marrow near the end of gestation. Thirdly, sheep are outbred, and thus represent a wide spectrum of genetic determinants of the immune response, as do humans. As the immune response to both the vector and the vector–encoded FVIII are likely to play a key role in FVIII inhibitor formation (or lack thereof), this represents an advantage not found in most other models, with the possible exception of the dog, which could conceiva‐ bly be outbred as well to achieve a broader genetic spectrum. In addition, the development of the sheep immune system has been investigated in detail [219-225], making sheep well suited for studying the immunological aspects of gene-based therapies for hemophilia A. Importantly, the large size of the sheep, their long life span (9-12 years), and their relative ease of maintenance and breeding make it possible to conduct long-term studies in relative‐ ly large numbers of animals to fully evaluate the efficacy and safety issues related to gene

notype.

#### **7. Establishment of a new preclinical model of hemophilia A and success with MSC-based treatment**

A number of animal models have been developed to evaluate new methods of not only treatment of coagulation disorders, but also the prevention and treatment of inhibitor for‐ mation. Transient hemophilic rabbit models induced by infusion of plasma containing in‐ hibitors have been used to evaluate the effect of different bypass products to factor VIII [194], but these models, while valuable for inhibitor studies, do not accurately recapitulate the human disease, precluding their use for gene therapy studies. Fortunately, dog models of hemophilia A with congenital deficiency [195, 196] and mouse models obtained by gene targeting and knockout technology [197] are available to study FVIII function and gene ther‐ apy approaches for treating hemophilia A. Therapeutic benefit has been obtained in numer‐ ous studies using a variety of vector systems in the murine model [121, 122, 198-204], and phenotypic correction of hemophilia A in the dog has been achieved, but has proven to be far more difficult than in mice [205, 206]. Despite promising results in both canine and mur‐ ine models, however, no clinical gene therapy trial has shown phenotypic/clinical improve‐ ment of hemophilia A in human patients. This is in marked contrast to the recent clinical successes with gene therapy for hemophilia B [207]. The reasons for the disparity in the effi‐ cacy of gene therapy for treating hemophilia A versus B is not presently clear. Nonetheless, based on the disappointing results to-date, there are currently no active hemophilia A clini‐ cal gene therapy trials, even though hemophilia A accounts for roughly 80% of all cases of hemophilia.

The difficulties seen thus far translating success in animal models into therapeutic benefit in human patients underscores the importance of preclinical animal models that both precisely mimic the disease process of hemophilia A, and closely parallel normal human immunology and physiology. To this end, between 1979 and 1982, a number of male offspring of a single white alpine ewe at the Swiss Federal Institute of Technology all died several hours postpartum due to severe bleeding from the umbilical cord [208-210]. Daughters and grand‐ daughters of this ewe also gave birth to lambs exhibiting the same pathology. Investigation of the affected animals showed extensive subcutaneous and intramuscular hematomas. Spontaneous hemarthroses were also frequent, leading to reduced locomotion and symp‐ toms of pain in standing up, restricting nursing activity. Stronger injuries that arose when animals were not placed in carefully controlled isolation resulted in heavy bleeding and in‐ tensive pain. Laboratory tests showed increased PTT, and FVIII levels (as assessed by aPTT) of about 1% of control animals. Replacement therapy with human FVIII (hFVIII) concentrate or fresh sheep plasma resulted in remission of disease and rapid clinical improvement.

[188-190], and by releasing trophic factors that blunt the inflammatory response and often promote healing by activating the tissue's own endogenous repair mechanisms. While the mechanisms responsible for this trafficking to sites of injury are still being elucidated [191-193], this observation raises the exciting possibility that, following systemic infusion, FVIII-expressing MSC could efficiently migrate to sites of active bleeding/injury, thereby re‐ leasing FVIII locally and focusing the therapy where it is most needed. As will be discussed in the following section, over the past 2-3 years, we have begun exploring whether it is pos‐ sible to exploit these many advantages of MSC as a cellular vehicle for delivering a FVIII gene by testing the ability of FVIII-expressing MSC to correct hemophilia A in a new large

**7. Establishment of a new preclinical model of hemophilia A and success**

A number of animal models have been developed to evaluate new methods of not only treatment of coagulation disorders, but also the prevention and treatment of inhibitor for‐ mation. Transient hemophilic rabbit models induced by infusion of plasma containing in‐ hibitors have been used to evaluate the effect of different bypass products to factor VIII [194], but these models, while valuable for inhibitor studies, do not accurately recapitulate the human disease, precluding their use for gene therapy studies. Fortunately, dog models of hemophilia A with congenital deficiency [195, 196] and mouse models obtained by gene targeting and knockout technology [197] are available to study FVIII function and gene ther‐ apy approaches for treating hemophilia A. Therapeutic benefit has been obtained in numer‐ ous studies using a variety of vector systems in the murine model [121, 122, 198-204], and phenotypic correction of hemophilia A in the dog has been achieved, but has proven to be far more difficult than in mice [205, 206]. Despite promising results in both canine and mur‐ ine models, however, no clinical gene therapy trial has shown phenotypic/clinical improve‐ ment of hemophilia A in human patients. This is in marked contrast to the recent clinical successes with gene therapy for hemophilia B [207]. The reasons for the disparity in the effi‐ cacy of gene therapy for treating hemophilia A versus B is not presently clear. Nonetheless, based on the disappointing results to-date, there are currently no active hemophilia A clini‐ cal gene therapy trials, even though hemophilia A accounts for roughly 80% of all cases of

The difficulties seen thus far translating success in animal models into therapeutic benefit in human patients underscores the importance of preclinical animal models that both precisely mimic the disease process of hemophilia A, and closely parallel normal human immunology and physiology. To this end, between 1979 and 1982, a number of male offspring of a single white alpine ewe at the Swiss Federal Institute of Technology all died several hours postpartum due to severe bleeding from the umbilical cord [208-210]. Daughters and grand‐ daughters of this ewe also gave birth to lambs exhibiting the same pathology. Investigation of the affected animals showed extensive subcutaneous and intramuscular hematomas. Spontaneous hemarthroses were also frequent, leading to reduced locomotion and symp‐

animal model; sheep.

hemophilia.

**with MSC-based treatment**

184 Gene Therapy - Tools and Potential Applications

Unfortunately, due to the expense and effort of maintaining these sheep, the Swiss investi‐ gators allowed the line to die out, saving only a few straws of semen prior to allowing this valuable resource to pass into extinction. We recently used a variety of reproductive tech‐ nologies to successfully re-establish this line of hemophilia A sheep, and fully characterized both the clinical parameters and the precise molecular basis for their disease [211-216]. In similarity to mutations seen in many human patients [217], these animals possess a prema‐ ture stop codon with a frameshift mutation. This is the only animal model of hemophilia A with this clinically relevant mutation-type, providing a unique opportunity to study thera‐ pies in this context. All ten animals to-date have exhibited bleeding from the umbilical cord, prolonged tail and "cuticle" bleeding time, and multiple episodes of severe spontaneous bleeding including hemarthroses, muscle hematomas, and hematuria, all of which have re‐ sponded to human FVIII. Just like human patients with severe hemophilia A, a hallmark symptom in these sheep is repeated spontaneous joint bleeds, which lead to chronic, debili‐ tating arthropathies and reduced mobility. Importantly, chromogenic assays performed in‐ dependently at the BloodCenter of Wisconsin and Emory University revealed undetectable FVIII activity in the circulation of these sheep, explaining their severe, life-threatening phe‐ notype.

In addition to the value of another large animal model of hemophilia A and the uniqueness of the mutation, sheep possess many characteristics that make them an ideal preclinical model for gene therapy. The first of these is the size. Sheep are fairly close in size to humans, weighing roughly 8lbs at birth and 150-200lbs as adults, likely obviating the need for scaleup of cell/vector dose to move from experiments in sheep to trials in humans. This is in marked contrast to mice which are ~2800 times smaller than a typical human patient [218]. Secondly, sheep share many important physiological and developmental characteristics with humans; for example, the pattern of fetal to adult hemoglobin switching, and the natu‐ rally occurring changes in the primary sites of hematopoiesis from yolk sac to fetal liver and finally to the bone marrow near the end of gestation. Thirdly, sheep are outbred, and thus represent a wide spectrum of genetic determinants of the immune response, as do humans. As the immune response to both the vector and the vector–encoded FVIII are likely to play a key role in FVIII inhibitor formation (or lack thereof), this represents an advantage not found in most other models, with the possible exception of the dog, which could conceiva‐ bly be outbred as well to achieve a broader genetic spectrum. In addition, the development of the sheep immune system has been investigated in detail [219-225], making sheep well suited for studying the immunological aspects of gene-based therapies for hemophilia A. Importantly, the large size of the sheep, their long life span (9-12 years), and their relative ease of maintenance and breeding make it possible to conduct long-term studies in relative‐ ly large numbers of animals to fully evaluate the efficacy and safety issues related to gene therapy. For these reasons, we feel that the sheep are a particularly relevant model in which to examine gene and cell-based therapies for hemophilia A. An additional unique advantage to using sheep to study hemophilia A treatment is that in sheep, like human, a large percent‐ age of the vWF is found within platelets rather than free in plasma. This is in contrast to dog (in which vWF circulates free in plasma [226, 227]), and makes the sheep an ideal large ani‐ mal model in which to explore the use of platelet-targeted gene therapy for hemophilia A [126, 228-230].

ciently expanded, the first animal to be transplanted was treated with a dose of hFVIII calcu‐ lated to correct the levels to 200%, to ensure no procedure-related bleeding occurred. The animal was then sedated, and 30x10^6 transduced MSC were transplanted into the perito‐

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Following transplantation, FVIII activity (assessed by chromogenic assay) was undetectable in the circulation, but this animal's clinical picture improved dramatically. All spontaneous bleeding events ceased, and he enjoyed an event-free clinical course, devoid of spontaneous bleeds, enabling us to cease hFVIII infusions. Existing hemarthroses resolved, the animal's joints recovered fully and resumed normal appearance, and he regained normal posture and gait, resuming a normal activity level. To our knowledge, this represents the first report of phenotypic correction of severe hemophilia A in a large animal model following transplan‐ tation of cells engineered to produce FVIII, and the first time that reversal of chronic debili‐

Based on the remarkable clinical improvement we had achieved in this first animal, we transplanted a second animal with 120x10^6 paternal MSC, >95% of which were transduced and expressing pFVIII. We anticipated that by transplanting 4x's the number of cells with roughly 6x's the transduction efficiency, we would achieve pronounced improvement and therapeutic levels of FVIII in the circulation of this animal. In similarity to the first animal, hemarthroses present in this second animal at the time of transplant resolved, and he re‐ sumed normal activity shortly after transplantation. This second animal also became factorindependent following the transplant. These results thus confirm the ability of this MSCbased approach to provide phenotypic correction in this large animal model of hemophilia A. However, just as we had observed in the first animal, no FVIII was detectable in the cir‐ culation of this animal, making the mechanism by which this procedure mediated such pro‐

Despite the pronounced clinical improvement we observed in the first animal, he mounted a rapid and fairly robust immune response to FVIII, in similarity to prior studies performed with hemophilia A mice [237]. Before transplant, this first animal had Bethesda titers against hFVIII of only ~3, yet this lifesaving procedure resulted in a rise in Bethesda titer to ~800 against the vector-encoded pFVIII and nearly 700 to hFVIII. The formation of such high titer inhibitors with cross-reactivity to the human protein was surprising, given the well estab‐ lished ability to successfully use porcine FVIII products in human patients to bypass exist‐ ing anti-hFVIII inhibitors [238-241]. Similarly, despite having no detectable inhibitors prior to transplant, the second animal receiving the higher FVIII-expressing cell dose developed titers of ~150 Bethesda units against the vector-encoded pFVIII following transplantation

Following euthanasia of these animals, we performed a detailed tissue analysis to begin de‐ ciphering the mechanism whereby this novel MSC-based gene delivery produced its pro‐ nounced therapeutic effect at a systemic level. PCR analysis demonstrated readily detectable levels of MSC engraftment in nearly all tissues analyzed, including liver, lymph nodes, in‐ testine, lung, kidney, omentum, and thymus. These molecular analyses thereby proved that it is possible to achieve widespread durable engraftment of MSC following transplantation

neal cavity under ultrasound guidance in the absence of any preconditioning.

tating hemarthroses has been achieved.

nounced clinical improvement uncertain.

which also exhibited cross-reactivity to the human protein.

To experimentally test the ability of MSC to serve as FVIII delivery vehicles and thus treat hemophilia A, we recently tested a novel, non-ablative transplant-based gene therapy in 2 pediatric hemophilia A lambs [110-112]. During the first 3-5 months of life, both these ani‐ mals had received frequent, on-demand infusions of human FVIII for multiple hematomas and chronic, progressive, debilitating hemarthroses of the leg joints which had resulted in severe defects in posture and gait, rendering them nearly immobile. In an ideal situation, one would use autologous cells to deliver a FVIII transgene, and thus avoid any complica‐ tions due to MHC-mismatching. Unfortunately, the severe life-threatening phenotype of the hemophilia A sheep prevented us from collecting bone marrow aspirates to isolate autolo‐ gous cells. We therefore elected to utilize cells from the ram that had sired the two hemo‐ philiac lambs, hoping that, by using paternal (haploidentical) MSC, immunologic incompatibility between the donor and recipient should be minimized sufficiently to allow engraftment, especially given the large body of evidence now accumulating that MSC can be transplanted across allogeneic barriers without eliciting an immune response [163, 164].

Based on our prior work in the fetal sheep model, we knew that the intraperitoneal (IP) transplantation of MSC results in widespread engraftment throughout all of the major or‐ gans [11, 131, 157, 231-233] and durable expression of vector-encoded genes [232-234]. We further reasoned that using the IP route would also have the advantage of enabling the cells to enter the circulation in an almost time-release fashion, after being engulfed by the omen‐ tum and absorbed through the peritoneal lymphatics. Importantly, we also felt that the use of the IP route would enable us to avoid the lung-trapping which hinders the efficient traf‐ ficking of MSC to desired target organs following IV administration, and also poses clinical risks due to emboli formation [235, 236].

Following isolation, MSC were simultaneously transduced with 2 HIV-based lentivectors, the first of which encoded an expression/secretion optimized porcine FVIII (pFVIII) trans‐ gene [112]. We selected a pFVIII transgene for two reasons. First, we had not yet cloned the ovine FVIII cDNA and constructed a B domain-deleted cassette that would fit in a lentivec‐ tor. Secondly, the pFVIII transgene had previously been shown, in human cells, to be ex‐ pressed/secreted at 10-100 times higher levels than human FVIII [120, 121, 237]. We thus felt that these very high levels of expression/secretion might enable us to achieve a therapeutic benefit, even in the event we obtained very low levels of engraftment of the transduced pa‐ ternal MSC. The second lentivector encoded eGFP to facilitate tracking and identification of donor cells in vivo. Combining the 2 vectors unexpectedly resulted in preferential transduc‐ tion with the eGFP vector, such that only about 15% of the MSC were transduced with the pFVIII-encoding vector, as assessed by qPCR. Once the transduced MSC had been suffi‐ ciently expanded, the first animal to be transplanted was treated with a dose of hFVIII calcu‐ lated to correct the levels to 200%, to ensure no procedure-related bleeding occurred. The animal was then sedated, and 30x10^6 transduced MSC were transplanted into the perito‐ neal cavity under ultrasound guidance in the absence of any preconditioning.

therapy. For these reasons, we feel that the sheep are a particularly relevant model in which to examine gene and cell-based therapies for hemophilia A. An additional unique advantage to using sheep to study hemophilia A treatment is that in sheep, like human, a large percent‐ age of the vWF is found within platelets rather than free in plasma. This is in contrast to dog (in which vWF circulates free in plasma [226, 227]), and makes the sheep an ideal large ani‐ mal model in which to explore the use of platelet-targeted gene therapy for hemophilia A

To experimentally test the ability of MSC to serve as FVIII delivery vehicles and thus treat hemophilia A, we recently tested a novel, non-ablative transplant-based gene therapy in 2 pediatric hemophilia A lambs [110-112]. During the first 3-5 months of life, both these ani‐ mals had received frequent, on-demand infusions of human FVIII for multiple hematomas and chronic, progressive, debilitating hemarthroses of the leg joints which had resulted in severe defects in posture and gait, rendering them nearly immobile. In an ideal situation, one would use autologous cells to deliver a FVIII transgene, and thus avoid any complica‐ tions due to MHC-mismatching. Unfortunately, the severe life-threatening phenotype of the hemophilia A sheep prevented us from collecting bone marrow aspirates to isolate autolo‐ gous cells. We therefore elected to utilize cells from the ram that had sired the two hemo‐ philiac lambs, hoping that, by using paternal (haploidentical) MSC, immunologic incompatibility between the donor and recipient should be minimized sufficiently to allow engraftment, especially given the large body of evidence now accumulating that MSC can be transplanted across allogeneic barriers without eliciting an immune response [163, 164].

Based on our prior work in the fetal sheep model, we knew that the intraperitoneal (IP) transplantation of MSC results in widespread engraftment throughout all of the major or‐ gans [11, 131, 157, 231-233] and durable expression of vector-encoded genes [232-234]. We further reasoned that using the IP route would also have the advantage of enabling the cells to enter the circulation in an almost time-release fashion, after being engulfed by the omen‐ tum and absorbed through the peritoneal lymphatics. Importantly, we also felt that the use of the IP route would enable us to avoid the lung-trapping which hinders the efficient traf‐ ficking of MSC to desired target organs following IV administration, and also poses clinical

Following isolation, MSC were simultaneously transduced with 2 HIV-based lentivectors, the first of which encoded an expression/secretion optimized porcine FVIII (pFVIII) trans‐ gene [112]. We selected a pFVIII transgene for two reasons. First, we had not yet cloned the ovine FVIII cDNA and constructed a B domain-deleted cassette that would fit in a lentivec‐ tor. Secondly, the pFVIII transgene had previously been shown, in human cells, to be ex‐ pressed/secreted at 10-100 times higher levels than human FVIII [120, 121, 237]. We thus felt that these very high levels of expression/secretion might enable us to achieve a therapeutic benefit, even in the event we obtained very low levels of engraftment of the transduced pa‐ ternal MSC. The second lentivector encoded eGFP to facilitate tracking and identification of donor cells in vivo. Combining the 2 vectors unexpectedly resulted in preferential transduc‐ tion with the eGFP vector, such that only about 15% of the MSC were transduced with the pFVIII-encoding vector, as assessed by qPCR. Once the transduced MSC had been suffi‐

[126, 228-230].

186 Gene Therapy - Tools and Potential Applications

risks due to emboli formation [235, 236].

Following transplantation, FVIII activity (assessed by chromogenic assay) was undetectable in the circulation, but this animal's clinical picture improved dramatically. All spontaneous bleeding events ceased, and he enjoyed an event-free clinical course, devoid of spontaneous bleeds, enabling us to cease hFVIII infusions. Existing hemarthroses resolved, the animal's joints recovered fully and resumed normal appearance, and he regained normal posture and gait, resuming a normal activity level. To our knowledge, this represents the first report of phenotypic correction of severe hemophilia A in a large animal model following transplan‐ tation of cells engineered to produce FVIII, and the first time that reversal of chronic debili‐ tating hemarthroses has been achieved.

Based on the remarkable clinical improvement we had achieved in this first animal, we transplanted a second animal with 120x10^6 paternal MSC, >95% of which were transduced and expressing pFVIII. We anticipated that by transplanting 4x's the number of cells with roughly 6x's the transduction efficiency, we would achieve pronounced improvement and therapeutic levels of FVIII in the circulation of this animal. In similarity to the first animal, hemarthroses present in this second animal at the time of transplant resolved, and he re‐ sumed normal activity shortly after transplantation. This second animal also became factorindependent following the transplant. These results thus confirm the ability of this MSCbased approach to provide phenotypic correction in this large animal model of hemophilia A. However, just as we had observed in the first animal, no FVIII was detectable in the cir‐ culation of this animal, making the mechanism by which this procedure mediated such pro‐ nounced clinical improvement uncertain.

Despite the pronounced clinical improvement we observed in the first animal, he mounted a rapid and fairly robust immune response to FVIII, in similarity to prior studies performed with hemophilia A mice [237]. Before transplant, this first animal had Bethesda titers against hFVIII of only ~3, yet this lifesaving procedure resulted in a rise in Bethesda titer to ~800 against the vector-encoded pFVIII and nearly 700 to hFVIII. The formation of such high titer inhibitors with cross-reactivity to the human protein was surprising, given the well estab‐ lished ability to successfully use porcine FVIII products in human patients to bypass exist‐ ing anti-hFVIII inhibitors [238-241]. Similarly, despite having no detectable inhibitors prior to transplant, the second animal receiving the higher FVIII-expressing cell dose developed titers of ~150 Bethesda units against the vector-encoded pFVIII following transplantation which also exhibited cross-reactivity to the human protein.

Following euthanasia of these animals, we performed a detailed tissue analysis to begin de‐ ciphering the mechanism whereby this novel MSC-based gene delivery produced its pro‐ nounced therapeutic effect at a systemic level. PCR analysis demonstrated readily detectable levels of MSC engraftment in nearly all tissues analyzed, including liver, lymph nodes, in‐ testine, lung, kidney, omentum, and thymus. These molecular analyses thereby proved that it is possible to achieve widespread durable engraftment of MSC following transplantation in a postnatal setting in a large animal model without the need for preconditioning/ablation, and in the absence of any selective advantage for the donor cells.

are currently not well understood, this observation has raised the exciting prospect of using MSC to treat a wide array of diseases in which inflammation plays a key role such as stroke [87, 88, 92, 248-255], rheumatoid arthritis [256], asthma [257-259] and allergic rhinitis [260],

Mesenchymal Stem Cells as Gene Delivery Vehicles

http://dx.doi.org/10.5772/53240

189

Cancer represents another condition in which there is a selective need for new cells created by the forming tumor, and a chronic state of insult/inflammation within the surrounding tu‐ mor microenvironment. Studies over the last several years have now revealed that MSC have the ability to "sense" this need for cells and the perceived injury to the tissue surround‐ ing the tumor. As a result, both endogenous bone marrow- and adipose-resident MSC, as well as intravenously infused MSC, all appear to have the ability to efficiently migrate to the forming tumor, and contribute to the newly forming tumor "stroma" [191, 262-266]. Clearly, this may not seem ideal, since the MSC could, in fact, provide support to the growing tu‐ mor, potentially worsening the prognosis. Indeed, unraveling the role played by MSC with‐ in the tumor microenvironment is currently an area of active research [191, 192, 262-265, 267-269]. Irrespective of their role in the tumor's health/biology, however, the ability of MSC to selectively traffic to and integrate into the tumor microenvironment can be viewed as a double-edged sword, since this ability has now been recognized to present a very powerful and unique means of selectively delivering anti-cancer gene products to tumor cells in vivo [270-274]. Four of the gene products which have thus far received the most attention are IL-2 [275, 276], IL-12 [277-284], IFN-β [270, 271, 285, 286], and tumor necrosis factor-related apop‐ tosis-inducing ligand (TRAIL) [287-298]. Unfortunately, the utility of these and many other biological agents that could be used for cancer therapy is often limited by both their short half-life in vivo and their pronounced toxicity due to effects on normal, non-malignant cells within the body. Using MSC to deliver these therapeutics promises to solve both of these problems, since the MSC can selectively migrate to the tumor site and release their therapeu‐ tic payload locally. This would be predicted to greatly increase the agent's concentration within the tumor and significantly lower its systemic toxicity. In addition, by genetically modifying the MSC with viral vectors, the engrafted MSC will steadily release the therapeu‐ tic agent, allowing a single administration to result in long-lasting effects. Other studies have now provided evidence that MSC have the ability to not only selectively home to solid tumors [270, 271, 287, 299], but also to actively seek out metastases at sites far removed from the primary site of the tumor [271, 288, 290, 299, 300]. This ability has recently been proven to be of great therapeutic value in the treatment of lung metastases arising from both breast cancer and melanoma in a murine xenograft model [271, 299]. Given the difficulty and fre‐ quent lack of success using traditional approaches such as surgery, radiotherapy, and che‐ motherapeutic agents to treat tumors which are either highly invasive or prone to metastasis, this property of MSC will likely prove to be of great clinical value in the near

One form of cancer for which the use of MSC is receiving a great deal of attention is glio‐ blastoma multiforme (GBM). GBM represents the most common form of malignant glioma. Despite decades of research and many advances in the treatment of this disease with con‐

and both acute and chronic lung injury [261].

future.

Confocal immunofluorescence analysis revealed large numbers of FVIII-expressing MSC within the synovium of the joints which exhibited hemarthrosis at the time of transplant, demonstrating (just as we had hoped/predicted) that the transplanted MSC possessed the intrinsic ability to home to and persist within sites of ongoing injury/inflammation, releasing FVIII locally within the joint, providing an explanation for the dramatic improvement we observed in the animal's joints. This finding is in agreement with prior studies [242], show‐ ing that local delivery of FIX-AAV to the joints of mice with injury-induced hemarthroses led to resolution of the hemarthroses in the absence of any detectable FIX in the circulation. While this finding provides an explanation for the reversal of the joint pathology present in these animals at transplant, it cannot explain the observed systemic benefits such as the ces‐ sation of spontaneous bleeding events.

Confocal analysis also revealed engrafted cells within the small intestine, demonstrating that MSC can still engraft within the intestine following postnatal transplantation, just as we had observed in prior studies in fetal recipients [232]. Given the ease with which proteins secreted from cells within the intestine can enter the circulation, future studies aimed at im‐ proving the levels of engraftment within the intestine have the potential to greatly improve the systemic release of FVIII. In addition to the intestine and hemarthrotic joints, significant levels of engraftment were also seen within the thymus of this animal. While the ability of the transplanted MSC to traffic to the thymus could clearly have important implications for the likelihood of long-term correction with this approach to hemophilia A treatment, addi‐ tional studies are required to determine with which cells within the thymus these MSC are interacting to ascertain the immunologic ramifications of thymic engraftment.

The marked phenotypic improvement and improvement in quality of life we have observed in our studies, to date, in the sheep model thus support the further development of thera‐ peutic strategies for hemophilia A and, perhaps, other coagulation disorders, employing MSC as cellular vehicles to deliver the required transgene.

#### **8. MSC as anti-cancer gene delivery vehicles**

As alluded to earlier, a large number of preclinical animal studies examining the differentia‐ tive potential of MSC isolated from a variety of adult tissues have also highlighted another interesting and clinically valuable characteristic of MSC; their ability to selectively navigate to sites of injury and/or inflammation within the body [192, 193, 243-247]. Once reaching these specific sites, the MSC then mediate repair both by engrafting and generating tissuespecific cells within the injured tissue (but contributing very little if at all to other tissues that are functionally normal [188-190]), and by releasing trophic factors that blunt the in‐ flammatory response and often promote healing by activating the tissue's own endogenous repair mechanisms. While the mechanisms responsible for this trafficking to sites of injury are currently not well understood, this observation has raised the exciting prospect of using MSC to treat a wide array of diseases in which inflammation plays a key role such as stroke [87, 88, 92, 248-255], rheumatoid arthritis [256], asthma [257-259] and allergic rhinitis [260], and both acute and chronic lung injury [261].

in a postnatal setting in a large animal model without the need for preconditioning/ablation,

Confocal immunofluorescence analysis revealed large numbers of FVIII-expressing MSC within the synovium of the joints which exhibited hemarthrosis at the time of transplant, demonstrating (just as we had hoped/predicted) that the transplanted MSC possessed the intrinsic ability to home to and persist within sites of ongoing injury/inflammation, releasing FVIII locally within the joint, providing an explanation for the dramatic improvement we observed in the animal's joints. This finding is in agreement with prior studies [242], show‐ ing that local delivery of FIX-AAV to the joints of mice with injury-induced hemarthroses led to resolution of the hemarthroses in the absence of any detectable FIX in the circulation. While this finding provides an explanation for the reversal of the joint pathology present in these animals at transplant, it cannot explain the observed systemic benefits such as the ces‐

Confocal analysis also revealed engrafted cells within the small intestine, demonstrating that MSC can still engraft within the intestine following postnatal transplantation, just as we had observed in prior studies in fetal recipients [232]. Given the ease with which proteins secreted from cells within the intestine can enter the circulation, future studies aimed at im‐ proving the levels of engraftment within the intestine have the potential to greatly improve the systemic release of FVIII. In addition to the intestine and hemarthrotic joints, significant levels of engraftment were also seen within the thymus of this animal. While the ability of the transplanted MSC to traffic to the thymus could clearly have important implications for the likelihood of long-term correction with this approach to hemophilia A treatment, addi‐ tional studies are required to determine with which cells within the thymus these MSC are

The marked phenotypic improvement and improvement in quality of life we have observed in our studies, to date, in the sheep model thus support the further development of thera‐ peutic strategies for hemophilia A and, perhaps, other coagulation disorders, employing

As alluded to earlier, a large number of preclinical animal studies examining the differentia‐ tive potential of MSC isolated from a variety of adult tissues have also highlighted another interesting and clinically valuable characteristic of MSC; their ability to selectively navigate to sites of injury and/or inflammation within the body [192, 193, 243-247]. Once reaching these specific sites, the MSC then mediate repair both by engrafting and generating tissuespecific cells within the injured tissue (but contributing very little if at all to other tissues that are functionally normal [188-190]), and by releasing trophic factors that blunt the in‐ flammatory response and often promote healing by activating the tissue's own endogenous repair mechanisms. While the mechanisms responsible for this trafficking to sites of injury

interacting to ascertain the immunologic ramifications of thymic engraftment.

MSC as cellular vehicles to deliver the required transgene.

**8. MSC as anti-cancer gene delivery vehicles**

and in the absence of any selective advantage for the donor cells.

sation of spontaneous bleeding events.

188 Gene Therapy - Tools and Potential Applications

Cancer represents another condition in which there is a selective need for new cells created by the forming tumor, and a chronic state of insult/inflammation within the surrounding tu‐ mor microenvironment. Studies over the last several years have now revealed that MSC have the ability to "sense" this need for cells and the perceived injury to the tissue surround‐ ing the tumor. As a result, both endogenous bone marrow- and adipose-resident MSC, as well as intravenously infused MSC, all appear to have the ability to efficiently migrate to the forming tumor, and contribute to the newly forming tumor "stroma" [191, 262-266]. Clearly, this may not seem ideal, since the MSC could, in fact, provide support to the growing tu‐ mor, potentially worsening the prognosis. Indeed, unraveling the role played by MSC with‐ in the tumor microenvironment is currently an area of active research [191, 192, 262-265, 267-269]. Irrespective of their role in the tumor's health/biology, however, the ability of MSC to selectively traffic to and integrate into the tumor microenvironment can be viewed as a double-edged sword, since this ability has now been recognized to present a very powerful and unique means of selectively delivering anti-cancer gene products to tumor cells in vivo [270-274]. Four of the gene products which have thus far received the most attention are IL-2 [275, 276], IL-12 [277-284], IFN-β [270, 271, 285, 286], and tumor necrosis factor-related apop‐ tosis-inducing ligand (TRAIL) [287-298]. Unfortunately, the utility of these and many other biological agents that could be used for cancer therapy is often limited by both their short half-life in vivo and their pronounced toxicity due to effects on normal, non-malignant cells within the body. Using MSC to deliver these therapeutics promises to solve both of these problems, since the MSC can selectively migrate to the tumor site and release their therapeu‐ tic payload locally. This would be predicted to greatly increase the agent's concentration within the tumor and significantly lower its systemic toxicity. In addition, by genetically modifying the MSC with viral vectors, the engrafted MSC will steadily release the therapeu‐ tic agent, allowing a single administration to result in long-lasting effects. Other studies have now provided evidence that MSC have the ability to not only selectively home to solid tumors [270, 271, 287, 299], but also to actively seek out metastases at sites far removed from the primary site of the tumor [271, 288, 290, 299, 300]. This ability has recently been proven to be of great therapeutic value in the treatment of lung metastases arising from both breast cancer and melanoma in a murine xenograft model [271, 299]. Given the difficulty and fre‐ quent lack of success using traditional approaches such as surgery, radiotherapy, and che‐ motherapeutic agents to treat tumors which are either highly invasive or prone to metastasis, this property of MSC will likely prove to be of great clinical value in the near future.

One form of cancer for which the use of MSC is receiving a great deal of attention is glio‐ blastoma multiforme (GBM). GBM represents the most common form of malignant glioma. Despite decades of research and many advances in the treatment of this disease with con‐ ventional surgery, radiotherapy, and chemotherapy, there is no cure, and the current prog‐ nosis is abysmal, with a median survival of only 6-18 months. The failure of current therapies to cure this disease arises predominantly from the highly invasive nature of this cancer and the inability of these agents to effectively target tumor cells which have dissemi‐ nated into the normal parenchyma of the brain, at sites distant from the main tumor mass. Given the ability of MSC to home to tumors and their ability to track to metastases through‐ out the body, gene-modified MSC are receiving a great deal of attention as a possible thera‐ py for GBM. Studies have now shown that MSC migrate through the normal brain parenchyma towards gliomas and appear to possess the uncanny ability to track microscop‐ ic tumor deposits and individual tumor cells which have infiltrated the normal brain paren‐ chyma [276, 282, 289, 290, 301-310]. While these migratory properties are certainly interesting, even more exciting are the dramatic therapeutic benefits these same studies have shown, with reduction in tumor size, and pronounced improvements in survival. It is im‐ portant to note that, in most of these studies, MSC were used as the sole therapy, and defi‐ nite benefits were observed. In the clinical setting, the current plan is to use gene-modified MSC as an adjunct after surgical resection. In this scenario, the vast majority of the tumor mass would be surgically removed, and the MSC would then be transplanted, in the hopes that they would then remove the residual malignant cells at the site of the tumor, and then hunt down and eliminate any invasive tumor cells that have migrated away from the site of the primary tumor. In this context, one would imagine that the therapeutic benefit of the MSC will likely be even more pronounced, since their anti-tumor effects could be focused only on the small number of residual tumor cells that evaded removal during surgery. The remarkable success seen in studies aimed at treating GBM, one of the most devastating forms of cancer, thus serve to highlight the tremendous potential MSC harbor as gene deliv‐ ery vehicles for the treatment of many forms of cancer for which current therapeutic strat‐ egies are ineffective.

**Author details**

**References**

p. 243-72.

1935-45.

p. Abstract 390.

1974. 17(4): p. 331-40.

ence, 1999. 284(5411): p. 143-7.

Circulation, 2004. 109(11): p. 1401-7.

Christopher D. Porada and Graça Almeida-Porada

Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC, USA

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#### **9. Conclusions**

Numerous investigators around the globe have now provided compelling evidence that MSC from a variety of tissues possess a far broader differentiative capacity than anyone would have foreseen at the time Friedenstein originally described his bone marrow-de‐ rived stromal cells. Extrapolating the work thus far on using MSC to deliver FVIII to treat hemophilia A, and the rapidly growing number of studies showing the tremendous po‐ tential of MSC as anti-cancer gene delivery vehicles, and combining this with the relative ease with which MSC can be isolated, propagated in culture, and modified with a variety of viral-based vectors, and their intrinsic ability to seek out sites of injury/inflammation within the body, one can readily see why MSC are widely viewed as being ideally suited not only as cellular therapeutics, but as vehicles to deliver gene therapy vectors to numer‐ ous tissues in the body, thus promising to provide a permanent cure for a diverse range of diseases.

#### **Author details**

ventional surgery, radiotherapy, and chemotherapy, there is no cure, and the current prog‐ nosis is abysmal, with a median survival of only 6-18 months. The failure of current therapies to cure this disease arises predominantly from the highly invasive nature of this cancer and the inability of these agents to effectively target tumor cells which have dissemi‐ nated into the normal parenchyma of the brain, at sites distant from the main tumor mass. Given the ability of MSC to home to tumors and their ability to track to metastases through‐ out the body, gene-modified MSC are receiving a great deal of attention as a possible thera‐ py for GBM. Studies have now shown that MSC migrate through the normal brain parenchyma towards gliomas and appear to possess the uncanny ability to track microscop‐ ic tumor deposits and individual tumor cells which have infiltrated the normal brain paren‐ chyma [276, 282, 289, 290, 301-310]. While these migratory properties are certainly interesting, even more exciting are the dramatic therapeutic benefits these same studies have shown, with reduction in tumor size, and pronounced improvements in survival. It is im‐ portant to note that, in most of these studies, MSC were used as the sole therapy, and defi‐ nite benefits were observed. In the clinical setting, the current plan is to use gene-modified MSC as an adjunct after surgical resection. In this scenario, the vast majority of the tumor mass would be surgically removed, and the MSC would then be transplanted, in the hopes that they would then remove the residual malignant cells at the site of the tumor, and then hunt down and eliminate any invasive tumor cells that have migrated away from the site of the primary tumor. In this context, one would imagine that the therapeutic benefit of the MSC will likely be even more pronounced, since their anti-tumor effects could be focused only on the small number of residual tumor cells that evaded removal during surgery. The remarkable success seen in studies aimed at treating GBM, one of the most devastating forms of cancer, thus serve to highlight the tremendous potential MSC harbor as gene deliv‐ ery vehicles for the treatment of many forms of cancer for which current therapeutic strat‐

Numerous investigators around the globe have now provided compelling evidence that MSC from a variety of tissues possess a far broader differentiative capacity than anyone would have foreseen at the time Friedenstein originally described his bone marrow-de‐ rived stromal cells. Extrapolating the work thus far on using MSC to deliver FVIII to treat hemophilia A, and the rapidly growing number of studies showing the tremendous po‐ tential of MSC as anti-cancer gene delivery vehicles, and combining this with the relative ease with which MSC can be isolated, propagated in culture, and modified with a variety of viral-based vectors, and their intrinsic ability to seek out sites of injury/inflammation within the body, one can readily see why MSC are widely viewed as being ideally suited not only as cellular therapeutics, but as vehicles to deliver gene therapy vectors to numer‐ ous tissues in the body, thus promising to provide a permanent cure for a diverse range

egies are ineffective.

190 Gene Therapy - Tools and Potential Applications

**9. Conclusions**

of diseases.

Christopher D. Porada and Graça Almeida-Porada

Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC, USA

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[259] Nemeth, K., et al., *Bone marrow stromal cells use TGF-beta to suppress allergic responses in a mouse model of ragweed-induced asthma.* Proc Natl Acad Sci U S A, 2010. 107(12): p.

[260] Cho, K.S., et al., *IFATS collection: Immunomodulatory effects of adipose tissue-derived stem*

[261] Iyer, S.S., C. Co, and M. Rojas, *Mesenchymal stem cells and inflammatory lung diseases.*

[262] Kidd, S., et al., *Origins of the tumor microenvironment: quantitative assessment of adipose-*

[263] Klopp, A.H., et al., *Concise review: Dissecting a discrepancy in the literature: do mesenchy‐ mal stem cells support or suppress tumor growth?* Stem Cells, 2011. 29(1): p. 11-9.

[264] Marini, F.C., *The complex love-hate relationship between mesenchymal stromal cells and tu‐*

[265] Martin, F.T., et al., *Potential role of mesenchymal stem cells (MSCs) in the breast tumour microenvironment: stimulation of epithelial to mesenchymal transition (EMT).* Breast Can‐

*cells in an allergic rhinitis mouse model.* Stem Cells, 2009. 27(1): p. 259-65.

*derived and bone marrow-derived stroma.* PLoS One, 2012. 7(2): p. e30563.

*marrow after global cerebral ischemia.* Brain Res, 2010. 1310: p. 8-16.

*mice.* Stem Cells Dev, 2010. March 17. [epub ahead of print].

*cells after cerebral ischemia in rats.* J Neurol Sci, 2001. 189(1-2): p. 49-57.

*tional recovery.* Neurology, 2002. 59(4): p. 514-23.

Thorac Soc, 2008. 5(5): p. 637-67.

Panminerva Med, 2009. 51(1): p. 5-16.

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778-86.

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5652-7.


[282] Ryu, C.H., et al., *Gene therapy of intracranial glioma using interleukin 12-secreting human umbilical cord blood-derived mesenchymal stem cells.* Hum Gene Ther, 2011. 22(6): p. 733-43.

[295] Moniri, M.R., et al., *TRAIL-engineered pancreas-derived mesenchymal stem cells: character‐ ization and cytotoxic effects on pancreatic cancer cells.* Cancer Gene Ther, 2012. 19(9): p.

Mesenchymal Stem Cells as Gene Delivery Vehicles

http://dx.doi.org/10.5772/53240

211

[296] Mueller, L.P., et al., *TRAIL-transduced multipotent mesenchymal stromal cells (TRAIL-MSC) overcome TRAIL resistance in selected CRC cell lines in vitro and in vivo.* Cancer

[297] Sun, X.Y., et al., *MSC(TRAIL)-mediated HepG2 cell death in direct and indirect co-cultures.*

[298] Yang, B., et al., *Dual-targeted antitumor effects against brainstem glioma by intravenous de‐ livery of tumor necrosis factor-related, apoptosis-inducing, ligand-engineered human mesen‐*

[299] Chen, X., et al., *A tumor-selective biotherapy with prolonged impact on established metasta‐ ses based on cytokine gene-engineered MSCs.* Mol Ther, 2008. 16(4): p. 749-56.

[300] Hamada, H., et al., *Mesenchymal stem cells (MSC) as therapeutic cytoreagents for gene*

[301] Nakamizo, A., et al., *Human bone marrow-derived mesenchymal stem cells in the treatment*

[302] Ahmed, A.U., et al., *A comparative study of neural and mesenchymal stem cell-based carri‐ ers for oncolytic adenovirus in a model of malignant glioma.* Mol Pharm, 2011. 8(5): p.

[303] Doucette, T., et al., *Mesenchymal stem cells display tumor-specific tropism in an RCAS/*

[304] Fei, S., et al., *The antitumor effect of mesenchymal stem cells transduced with a lentiviral vector expressing cytosine deaminase in a rat glioma model.* J Cancer Res Clin Oncol, 2012.

[305] Choi, S.A., et al., *Human adipose tissue-derived mesenchymal stem cells: characteristics and therapeutic potential as cellular vehicles for prodrug gene therapy against brainstem gliomas.*

[306] Kim, S.M., et al., *CXC chemokine receptor 1 enhances the ability of human umbilical cord blood-derived mesenchymal stem cells to migrate toward gliomas.* Biochem Biophys Res

[307] Kosaka, H., et al., *Therapeutic effect of suicide gene-transferred mesenchymal stem cells in a*

[308] Park, S.A., et al., *CXCR4-transfected human umbilical cord blood-derived mesenchymal stem cells exhibit enhanced migratory capacity toward gliomas.* Int J Oncol, 2011. 38(1): p.

*rat model of glioma.* Cancer Gene Ther, 2012. 19(8): p. 572-8.

*chymal stem cells.* Neurosurgery, 2009. 65(3): p. 610-24; discussion 624.

652-8.

1559-72.

97-103.

138(2): p. 347-57.

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Commun, 2012. 407(4): p. 741-6.

Gene Ther, 2011. 18(4): p. 229-39.

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*therapy.* Cancer Sci, 2005. 96(3): p. 149-56.

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[282] Ryu, C.H., et al., *Gene therapy of intracranial glioma using interleukin 12-secreting human umbilical cord blood-derived mesenchymal stem cells.* Hum Gene Ther, 2011. 22(6): p.

[283] Seo, S.H., et al., *The effects of mesenchymal stem cells injected via different routes on modi‐*

[284] Zhao, W.H., et al., *[Human umbilical cord mesenchymal stem cells with adenovirus-mediat‐ ed interleukin 12 gene transduction inhibits the growth of ovarian carcinoma cells both in vi‐*

[285] Kidd, S., et al., *Mesenchymal stromal cells alone or expressing interferon-beta suppress pan‐ creatic tumors in vivo, an effect countered by anti-inflammatory treatment.* Cytotherapy,

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[288] Loebinger, M.R., et al., *Mesenchymal stem cell delivery of TRAIL can eliminate metastatic*

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[290] Sasportas, L.S., et al., *Assessment of therapeutic efficacy and fate of engineered human mes‐ enchymal stem cells for cancer therapy.* Proc Natl Acad Sci U S A, 2009. 106(12): p.

[291] Ciavarella, S., et al., *In vitro anti-myeloma activity of TRAIL-expressing adipose-derived*

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*fied IL-12-mediated antitumor activity.* Gene Ther, 2011. 18(5): p. 488-95.

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733-43.

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2320-30.

4822-7.

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[309] Wang, Q., et al., *Mesenchymal stem cells over-expressing PEDF decreased the angiogenesis of gliomas.* Biosci Rep, 2012.

**Chapter 9**

**Cancer Gene Therapy – Key Biological Concepts in the**

**Design of Multifunctional Non-Viral Delivery Systems**

The importance of gene therapy strategies for the treatment of malignancies is highlighted by the fact that there are (at the time of writing) 823 cancer gene therapy clinical trials world‐ wide that are actively, or have yet to begin recruiting patients (www.clinicaltrials.gov - ac‐ cessed November 2012). The potential of the delivery of genetic material for therapeutic purposes has long been recognised, but to this point, has yet to be successfully translated. Strategies that have proved promising in the *in vitro* setting have stumbled when exposed to the complexities of the *in vivo* environment. Classically involving the delivery of plasmid DNA (pDNA) that encodes a therapeutic protein product, the field of gene therapy has evolved to encompass not only delivery of therapeutic DNA, but also micro- (miRNA), short hairpin- (shRNA) and small interfering RNAs (siRNA) and oligodeoxynucleotides (ODNs) [1]. Despite the evolution of the technology for altering the genotype of target cells and tis‐ sues, the problem of overcoming the biological barriers that limit the efficacies of these tech‐ nologies remains. These barriers exist at both systemic and local levels. To date, the only approved nucleic acid-based treatments for clinical use are an antisense ODN for the treat‐ ment of cytomegalovirus retinitis [2], and pegaptanib sodium (Macugen), an RNA aptamer targeted against VEGF-165 and used to treat age-related macular degeneration [3]. This chap‐ ter will focus on the biological barriers faced by non-viral vectors for gene therapy, strategies that have been employed to overcome these barriers, and will conclude by documenting the

state of the art technologies being used to propel non-viral gene therapies forward.

Delivery of genetic material for therapeutic use from virus-like particles has received consid‐ erable attention, and has generated extensive knowledge. The molecular evolution of viruses

> © 2013 McCrudden and McCarthy; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Cian M. McCrudden and Helen O. McCarthy

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54271

**1.1. Non-viral gene therapy**

**1. Introduction**

[310] Yin, J., et al., *hMSC-mediated concurrent delivery of endostatin and carboxylesterase to mouse xenografts suppresses glioma initiation and recurrence.* Mol Ther, 2011. 19(6): p. 1161-9.

### **Cancer Gene Therapy – Key Biological Concepts in the Design of Multifunctional Non-Viral Delivery Systems**

Cian M. McCrudden and Helen O. McCarthy

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54271

#### **1. Introduction**

[309] Wang, Q., et al., *Mesenchymal stem cells over-expressing PEDF decreased the angiogenesis*

[310] Yin, J., et al., *hMSC-mediated concurrent delivery of endostatin and carboxylesterase to*

*mouse xenografts suppresses glioma initiation and recurrence.* Mol Ther, 2011. 19(6): p.

*of gliomas.* Biosci Rep, 2012.

212 Gene Therapy - Tools and Potential Applications

1161-9.

The importance of gene therapy strategies for the treatment of malignancies is highlighted by the fact that there are (at the time of writing) 823 cancer gene therapy clinical trials world‐ wide that are actively, or have yet to begin recruiting patients (www.clinicaltrials.gov - ac‐ cessed November 2012). The potential of the delivery of genetic material for therapeutic purposes has long been recognised, but to this point, has yet to be successfully translated. Strategies that have proved promising in the *in vitro* setting have stumbled when exposed to the complexities of the *in vivo* environment. Classically involving the delivery of plasmid DNA (pDNA) that encodes a therapeutic protein product, the field of gene therapy has evolved to encompass not only delivery of therapeutic DNA, but also micro- (miRNA), short hairpin- (shRNA) and small interfering RNAs (siRNA) and oligodeoxynucleotides (ODNs) [1]. Despite the evolution of the technology for altering the genotype of target cells and tis‐ sues, the problem of overcoming the biological barriers that limit the efficacies of these tech‐ nologies remains. These barriers exist at both systemic and local levels. To date, the only approved nucleic acid-based treatments for clinical use are an antisense ODN for the treat‐ ment of cytomegalovirus retinitis [2], and pegaptanib sodium (Macugen), an RNA aptamer targeted against VEGF-165 and used to treat age-related macular degeneration [3]. This chap‐ ter will focus on the biological barriers faced by non-viral vectors for gene therapy, strategies that have been employed to overcome these barriers, and will conclude by documenting the state of the art technologies being used to propel non-viral gene therapies forward.

#### **1.1. Non-viral gene therapy**

Delivery of genetic material for therapeutic use from virus-like particles has received consid‐ erable attention, and has generated extensive knowledge. The molecular evolution of viruses

© 2013 McCrudden and McCarthy; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

over the aeons has produced DNA-delivering organisms of incomparable efficiency. The use of 'gutted' viruses that lack virulence properties and replicative capacity is the most efficient method of genetic material delivery [4], and modified viruses have been used extensively in gene therapy; commonly employed viruses include adenovirus, retrovirus, vaccinia virus and herpes simplex virus [5]. The allure of viral gene therapy was hindered however, when a clin‐ ical trial patient died four days after receiving adenoviral therapy for treatment of ornithine transcarbamylase deficiency [6]. The negative press that this generated, along with other dis‐ advantages of viral gene therapy (including generation of immune response, possibility of proto-oncogene activation, production costs, and limitations in deliverable gene size) have ne‐ cessitated the generation of alternative gene therapy strategies [7].

were used to reintroduce heparin sulphate 6-O-endosulfatase 1 (HSulf-1) to ovarian SKOV-3 xenografts, which resulted in anti-angiogenesis, induction of apoptosis and suppression of cell proliferation [21], while a PEI-poly(hydroxyethyl glutamine) (PEI-PHEG) copolymer successfully delivered pGL3 pDNA by intratumoural administration to Lewis Lung Carci‐

Cancer Gene Therapy – Key Biological Concepts in the Design of Multifunctional Non-Viral Delivery Systems

http://dx.doi.org/10.5772/54271

215

Cationic peptides that are capable of neutralising, condensing and wrapping pDNA have al‐ so been used as non-viral delivery vehicles [23]. These cell-permeating peptides were de‐ signed to interact with cell membranes similarly to viral fusion proteins. GALA, a synthetic peptide designed to interact with lipid bilayers at an acidic pH, was observed to aid in the delivery of DNA to cells [23]. A derivative, termed KALA, through presence of positively charged lysine residues, is capable of condensing and delivering DNA unaided [24], and im‐ proved gene delivery ten-fold in hepatoma [25] and also in HEK293T and HepG2 cells [26]. The cell-penetrating peptide TAT and fusogenic peptide HA2 were used to improve pDNA delivery by gelatin-silica nanoparticles [27]. Recent developments in the field have seen the development of multi-domain peptidic biomimetic vectors tailored to overcome the various biological barriers that gene delivery vehicles encounter *in vivo*, including degradation by serum nucleases, endosomal entrapment and nuclear localization. One such designer biomi‐ metic vector was used to deliver tumour-related apoptosis inducing ligand (TRAIL) [28] and inducible nitric oxide synthase (iNOS) pDNA to ZR-75-1 breast cancer cells *in vitro* [29].

Non-viral strategies for gene therapy have several advantages over traditional viral ap‐ proaches, including reduced cost and ease of large-scale production, as well as avoidance of the virulence commonly associated with viral delivery. However, non-viral gene delivery systems suffer from lower potency of transfection ability, resultant of lower ability to tra‐

The most fundamental barrier that the human body possesses is its skin. The stratum cor‐ neum is the skin's outermost layer, and provides an imposing barrier to gene delivery [30]; the densely packed cornified cells of this layer protect the body from a range of foreign ma‐ terial. The skin is not a commonly used route for gene therapy approaches, but it is an at‐ tractive route for local targeting of dermatological ailments [31]. However, skin nucleases, and in particular DNAse 2, active at the skin and in the stratum corneum, degrade topicallyapplied nucleic acids [32]. An appropriate and potent delivery mechanism could open the door to gene therapy strategies for the treatment of skin conditions and malignancies, in‐ cluding xeroderma pigmentosum (a cancer-linked disorder that has shown promise in pre‐ clinical gene therapy approaches [33]), when replacement of the defective XPC gene is in

Introduction of micron-sized pores to the skin using minimally-invasive silicone micro‐ needles allowed for the delivery of a 'non-viral gene vector-mimicking' charged fluores‐

verse the various obstacles faced upon administration [7].

**2. Extracellular barriers to gene delivery**

keratinocytes would be therapeutically beneficial [34].

**2.1. The skin**

noma xenografts in C57BL/6 mice [22].

Despite some success when naked DNA has been delivered *in vivo* (naked pDNA has been effectively delivered to the liver in mice and rats [8] by tail vein injection), pDNA for gene therapy is conventionally delivered complexed with materials with suitable physical charac‐ teristics. pDNA's hydrophilicity and anionic nature impair the uncomplexed molecule's passage through the lipophilic plasma membrane [1,9,10]. Non-viral gene therapy strategies usually involve wrapping of the nucleic acid to be delivered in a protective envelope that neutralises the negative charge of the DNA. A range of compounds has been used to envel‐ op pDNA, including cationic lipids, polymers and peptides.

Cationic lipids were among the first compounds complexed with pDNA for non-viral gene delivery. Felgner reported that N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) formed lipid-DNA complexes based on the interaction between the posi‐ tively charged lipid and the negatively charged phosphates of the DNA. The lipoplexes soformed were capable of delivering DNA to cells *in vitro* [11]. Numerous cationic lipids have since been reported to neutralise, condense and encapsulate pDNA, including dioctadecyla‐ midoglycylspermine (DOGS) [12],[1,2-bis(oleoyloxy)-3-(trimethylammonio)propane] (DO‐ TAP) [13] and 3β[N-(N′, N′-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) [14]. Variations on a theme, these lipids behave similarly to innate biological lipids [15]. The ad‐ dition of co-lipids such as cholesterol and dioleoylphosphatidylethanolamine (DOPE) can improve transfection efficiency [16]. Recent developments in the field have seen a 1-palmito‐ yl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPOPC):cholesterol liposome with folate electrostatically-associated used to deliver HSV-tk suicide gene therapy to SCC-VII xeno‐ grafts, which resulted in considerable tumour growth delay [17]. In a magnetofection meth‐ od, intravenous delivery of superparamagnetic iron oxide lipid nanoparticles in combination with an Nd-Fe-B magnet placed externally in the tumour locality resulted in improved IGF-1R shRNA delivery to A549 xenografts [18].

Cationic polymers have been used to condense and deliver genetic material, including poly(l-lysine) (PLL), polyethylenimine (PEI), chitosan and polyamidoamine (PAMAM) [7]. PLL incorporated into a spider silk-based nanoparticle with a tumour-homing peptide was recently reported to deliver a luciferase plasmid to MDA-MB-231 xenografts following intra‐ venous administration [19]. A novel triblockpoly(amido amine)-poly(ethylene glycol)-polyl-lysine (PAMAM-PEG-PLL) nanocarrier successfully delivered Bcl-2 siRNA and elicited knockdown of the same in A2780 ovarian carcinoma cells *in vitro* [20]. Heparin-PEI nanogels were used to reintroduce heparin sulphate 6-O-endosulfatase 1 (HSulf-1) to ovarian SKOV-3 xenografts, which resulted in anti-angiogenesis, induction of apoptosis and suppression of cell proliferation [21], while a PEI-poly(hydroxyethyl glutamine) (PEI-PHEG) copolymer successfully delivered pGL3 pDNA by intratumoural administration to Lewis Lung Carci‐ noma xenografts in C57BL/6 mice [22].

Cationic peptides that are capable of neutralising, condensing and wrapping pDNA have al‐ so been used as non-viral delivery vehicles [23]. These cell-permeating peptides were de‐ signed to interact with cell membranes similarly to viral fusion proteins. GALA, a synthetic peptide designed to interact with lipid bilayers at an acidic pH, was observed to aid in the delivery of DNA to cells [23]. A derivative, termed KALA, through presence of positively charged lysine residues, is capable of condensing and delivering DNA unaided [24], and im‐ proved gene delivery ten-fold in hepatoma [25] and also in HEK293T and HepG2 cells [26]. The cell-penetrating peptide TAT and fusogenic peptide HA2 were used to improve pDNA delivery by gelatin-silica nanoparticles [27]. Recent developments in the field have seen the development of multi-domain peptidic biomimetic vectors tailored to overcome the various biological barriers that gene delivery vehicles encounter *in vivo*, including degradation by serum nucleases, endosomal entrapment and nuclear localization. One such designer biomi‐ metic vector was used to deliver tumour-related apoptosis inducing ligand (TRAIL) [28] and inducible nitric oxide synthase (iNOS) pDNA to ZR-75-1 breast cancer cells *in vitro* [29].

Non-viral strategies for gene therapy have several advantages over traditional viral ap‐ proaches, including reduced cost and ease of large-scale production, as well as avoidance of the virulence commonly associated with viral delivery. However, non-viral gene delivery systems suffer from lower potency of transfection ability, resultant of lower ability to tra‐ verse the various obstacles faced upon administration [7].

#### **2. Extracellular barriers to gene delivery**

#### **2.1. The skin**

over the aeons has produced DNA-delivering organisms of incomparable efficiency. The use of 'gutted' viruses that lack virulence properties and replicative capacity is the most efficient method of genetic material delivery [4], and modified viruses have been used extensively in gene therapy; commonly employed viruses include adenovirus, retrovirus, vaccinia virus and herpes simplex virus [5]. The allure of viral gene therapy was hindered however, when a clin‐ ical trial patient died four days after receiving adenoviral therapy for treatment of ornithine transcarbamylase deficiency [6]. The negative press that this generated, along with other dis‐ advantages of viral gene therapy (including generation of immune response, possibility of proto-oncogene activation, production costs, and limitations in deliverable gene size) have ne‐

Despite some success when naked DNA has been delivered *in vivo* (naked pDNA has been effectively delivered to the liver in mice and rats [8] by tail vein injection), pDNA for gene therapy is conventionally delivered complexed with materials with suitable physical charac‐ teristics. pDNA's hydrophilicity and anionic nature impair the uncomplexed molecule's passage through the lipophilic plasma membrane [1,9,10]. Non-viral gene therapy strategies usually involve wrapping of the nucleic acid to be delivered in a protective envelope that neutralises the negative charge of the DNA. A range of compounds has been used to envel‐

Cationic lipids were among the first compounds complexed with pDNA for non-viral gene delivery. Felgner reported that N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) formed lipid-DNA complexes based on the interaction between the posi‐ tively charged lipid and the negatively charged phosphates of the DNA. The lipoplexes soformed were capable of delivering DNA to cells *in vitro* [11]. Numerous cationic lipids have since been reported to neutralise, condense and encapsulate pDNA, including dioctadecyla‐ midoglycylspermine (DOGS) [12],[1,2-bis(oleoyloxy)-3-(trimethylammonio)propane] (DO‐ TAP) [13] and 3β[N-(N′, N′-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) [14]. Variations on a theme, these lipids behave similarly to innate biological lipids [15]. The ad‐ dition of co-lipids such as cholesterol and dioleoylphosphatidylethanolamine (DOPE) can improve transfection efficiency [16]. Recent developments in the field have seen a 1-palmito‐ yl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPOPC):cholesterol liposome with folate electrostatically-associated used to deliver HSV-tk suicide gene therapy to SCC-VII xeno‐ grafts, which resulted in considerable tumour growth delay [17]. In a magnetofection meth‐ od, intravenous delivery of superparamagnetic iron oxide lipid nanoparticles in combination with an Nd-Fe-B magnet placed externally in the tumour locality resulted in

Cationic polymers have been used to condense and deliver genetic material, including poly(l-lysine) (PLL), polyethylenimine (PEI), chitosan and polyamidoamine (PAMAM) [7]. PLL incorporated into a spider silk-based nanoparticle with a tumour-homing peptide was recently reported to deliver a luciferase plasmid to MDA-MB-231 xenografts following intra‐ venous administration [19]. A novel triblockpoly(amido amine)-poly(ethylene glycol)-polyl-lysine (PAMAM-PEG-PLL) nanocarrier successfully delivered Bcl-2 siRNA and elicited knockdown of the same in A2780 ovarian carcinoma cells *in vitro* [20]. Heparin-PEI nanogels

cessitated the generation of alternative gene therapy strategies [7].

214 Gene Therapy - Tools and Potential Applications

op pDNA, including cationic lipids, polymers and peptides.

improved IGF-1R shRNA delivery to A549 xenografts [18].

The most fundamental barrier that the human body possesses is its skin. The stratum cor‐ neum is the skin's outermost layer, and provides an imposing barrier to gene delivery [30]; the densely packed cornified cells of this layer protect the body from a range of foreign ma‐ terial. The skin is not a commonly used route for gene therapy approaches, but it is an at‐ tractive route for local targeting of dermatological ailments [31]. However, skin nucleases, and in particular DNAse 2, active at the skin and in the stratum corneum, degrade topicallyapplied nucleic acids [32]. An appropriate and potent delivery mechanism could open the door to gene therapy strategies for the treatment of skin conditions and malignancies, in‐ cluding xeroderma pigmentosum (a cancer-linked disorder that has shown promise in pre‐ clinical gene therapy approaches [33]), when replacement of the defective XPC gene is in keratinocytes would be therapeutically beneficial [34].

Introduction of micron-sized pores to the skin using minimally-invasive silicone micro‐ needles allowed for the delivery of a 'non-viral gene vector-mimicking' charged fluores‐ cent nanoparticle [35,36]. A needle-free injection device successfully delivered luciferase pDNA to porcine skin, resulting in higher protein expression than conventional hypoder‐ mic needle administration [37], while subcutaneous melanoma xenografts were targeted for gene therapy using a hybrid electro-microneedle; delivery of 20 µg interleukin-12 pDNA to the skin, followed by eight 50 ms pulses delivering 70V/0.5 cm from the elec‐ trode to improve transfection resulted in a significant improvement in survival of tumourbearing mice [38]. Most recently, researchers from Northwestern University reported the generation of siRNA-carrying nanoparticles (spherical nucleic acid nanoparticle conju‐ gates (SNA-NCs)) that are capable of penetrating nude mouse and human skin, whilst maintaining their RNA interference potential [36]. The nanoparticles comprise gold cores surrounded by a dense shell of highly oriented, covalently immobilised siRNA and could be delivered topically, avoiding the need for disruption of the skin. As the skin is unmo‐ lested, the authors propose that the miniscule nature of the SNA-NCs permit dermal crossing, a theory that is currently under investigation.

of nanoparticles can cause embolization of microvessels, and non delivery of the therapeutic

Cancer Gene Therapy – Key Biological Concepts in the Design of Multifunctional Non-Viral Delivery Systems

http://dx.doi.org/10.5772/54271

217

The differential in ionicity between gene therapy formulation and the extracellular space poses another obstacle for nanoparticles, which can lead to colloidal instability [44]. The is‐ sue of non-specific interaction between nanoparticles and plasma proteins has been ad‐ dressed by the coupling of hydrophilic molecules to the nanoparticle. The most commonly employed candidate is poly(ethylene glycol) (PEG), whose anionicity has led to reduced ag‐ gregation and improved transfection ability [47]. PEG has been incorporated into a myriad of non-viral gene therapy strategies. Recently, polyacridine peptide nanoparticles were PE‐ Gylated and found to persist in the mouse circulation for up to nine hours, compared to non-PEGylated nanoparticles, which were inactive within five minutes [48], while biode‐ gradable dextran nanogels were PEGylated and analysed for their siRNA delivering prow‐ ess [49]. Particulate gene therapies are also subject to entrapment by the mononuclear phagocyte system (reticuloendothelial system - RES), when they are captured and held in the spleen or liver [50], which was responsible for the inactivation of adenoviral vectors that have been used as viral gene therapeutics [51]. Avoidance of non-specific biomolecular in‐ teraction, referred to as 'stealth' [1], is a prerequisite for successful gene delivery. Function‐ alisation of non-viral gene therapies with agents such as PEG to facilitate RES avoidance will

Assuming a gene therapeutic persists in active form in the circulation and the target tissue is reached, extravasation from the circulation is imperative. The architecture of normal vascu‐ lature ensures that transport of macromolecules out of the circulation is difficult. One char‐ acteristic of tumour vasculature, however, is its propensity to leakiness, an attribute that can be exploited by gene therapies. It is unsurprising that siRNA lipoplexes that target RAN GTPase were delivered more effectively, and evoked more impressive anti-tumour effects in highly vascularised xenografts than in xenografts with poorer vascularity [52]. The leaky vessel phenomenon, known as the enhanced permeability and retention (EPR) effect, has been utilised to enable the delivery of pDNA-containing particles in various malignancies [53]. The utilization of EPR will be further discussed below. The angiogenic tumour vascula‐ ture was itself targeted in a murine dorsal air sac assay; siRNA targeting Ago2 was com‐ plexed into cationic liposomes and intravenously administered. The authors successfully delivered the interfering RNA to the angiogenic vessels, and reported tumour regression,

Perhaps the most intimidating vascular obstacle that a gene therapy can face is the bloodbrain barrier, where tight junctions between endothelial cells of the capillaries limit the pas‐ sage of molecules much more than at other capillary sites in the body. One of the most exciting techniques available to the gene therapy researcher is the use of ultrasound-target‐ ed microbubble destruction (UTMD); nucleic acid contained within a gas-filled microbubble is administered, before exposure to ultrasonic waves at a frequency that exceeds the reso‐ nance frequency of the microbubbles, causing their destruction and leading to increased ca‐ pillary and cell membrane permeability [55]. This technology was used to deliver pDNA for

presumed to be resultant of anti-angiogenesis in their model [54].

to target [1].

be discussed in subsequent sections.

#### **2.2. Barriers to systemic gene therapies**

Needle-administered systemic therapeutics bypass the skin, but encounter further extracel‐ lular barriers before reaching their site of action. The various administration routes (intravenous, -muscular, -ocular, -nasal) present their own unique impediments to nucleic acid delivery. Intravenously- [39] and intramuscularly-administered [40] therapies are subject to nuclease degradation from the point of entry. Conversely, naked uncomplexed anti-respira‐ tory syncytial virus (RSV) siRNA was almost as effective as that complexed with TransIT-TKO transfection reagent when nasally-administered in mice [41], suggesting that nasallyadministered gene therapies may not be as prone to nuclease insult. The compartmental nature of the eye, and ease of access to it simplifies avoidance of similar barriers in ocular gene therapy delivery [42]. pDNA complexed with poly(d,l-lactic-co-glycolic) acid (PLGA) and dimethyldioctadecylammonium bromide (DDAB) produced nanoparticles capable of traversing one of the most inhospitable of barriers, the gastric mucus [43]. For simplicity, this chapter will focus on the barriers faced by intravenously delivered therapies, as this route has the potential to target almost all tissues of the body.

The complexing of DNA into lipo- or polyplex nanoparticles in non-viral delivery can effec‐ tively protect the pDNA from nuclease degradation [44] (although, paradoxically, cationic and anionic lipoplexes can hinder pDNA delivery by electroporation [45]). Whilst in the cir‐ culation, however, non-viral agents can be subject to non-specific binding by serum pro‐ teins, which can result in aggregation or dissociation of nanoparticles, resultant of the generally positive charges of the nanoparticles and the negative charge of circulatory pro‐ teins [1]. Positive charge is essential to ensure interaction of the nanoparticle with its target cell, however the mononuclear phagocytic system (MPS) eliminates foreign hydrophobic particles from the circulation [7] by opsonisation. The MPS was neutralised in mice by pretreatment with polyinosinic acid (a synthetic nucleic acid strand) before therapeutic measles virus treatment; this led to competitive inhibition of the scavenging of particles by macro‐ phages, and improved virus delivery to and efficacy at SKOV3 xenografts [46]. Aggregation

of nanoparticles can cause embolization of microvessels, and non delivery of the therapeutic to target [1].

cent nanoparticle [35,36]. A needle-free injection device successfully delivered luciferase pDNA to porcine skin, resulting in higher protein expression than conventional hypoder‐ mic needle administration [37], while subcutaneous melanoma xenografts were targeted for gene therapy using a hybrid electro-microneedle; delivery of 20 µg interleukin-12 pDNA to the skin, followed by eight 50 ms pulses delivering 70V/0.5 cm from the elec‐ trode to improve transfection resulted in a significant improvement in survival of tumourbearing mice [38]. Most recently, researchers from Northwestern University reported the generation of siRNA-carrying nanoparticles (spherical nucleic acid nanoparticle conju‐ gates (SNA-NCs)) that are capable of penetrating nude mouse and human skin, whilst maintaining their RNA interference potential [36]. The nanoparticles comprise gold cores surrounded by a dense shell of highly oriented, covalently immobilised siRNA and could be delivered topically, avoiding the need for disruption of the skin. As the skin is unmo‐ lested, the authors propose that the miniscule nature of the SNA-NCs permit dermal

Needle-administered systemic therapeutics bypass the skin, but encounter further extracel‐ lular barriers before reaching their site of action. The various administration routes (intravenous, -muscular, -ocular, -nasal) present their own unique impediments to nucleic acid delivery. Intravenously- [39] and intramuscularly-administered [40] therapies are subject to nuclease degradation from the point of entry. Conversely, naked uncomplexed anti-respira‐ tory syncytial virus (RSV) siRNA was almost as effective as that complexed with TransIT-TKO transfection reagent when nasally-administered in mice [41], suggesting that nasallyadministered gene therapies may not be as prone to nuclease insult. The compartmental nature of the eye, and ease of access to it simplifies avoidance of similar barriers in ocular gene therapy delivery [42]. pDNA complexed with poly(d,l-lactic-co-glycolic) acid (PLGA) and dimethyldioctadecylammonium bromide (DDAB) produced nanoparticles capable of traversing one of the most inhospitable of barriers, the gastric mucus [43]. For simplicity, this chapter will focus on the barriers faced by intravenously delivered therapies, as this

The complexing of DNA into lipo- or polyplex nanoparticles in non-viral delivery can effec‐ tively protect the pDNA from nuclease degradation [44] (although, paradoxically, cationic and anionic lipoplexes can hinder pDNA delivery by electroporation [45]). Whilst in the cir‐ culation, however, non-viral agents can be subject to non-specific binding by serum pro‐ teins, which can result in aggregation or dissociation of nanoparticles, resultant of the generally positive charges of the nanoparticles and the negative charge of circulatory pro‐ teins [1]. Positive charge is essential to ensure interaction of the nanoparticle with its target cell, however the mononuclear phagocytic system (MPS) eliminates foreign hydrophobic particles from the circulation [7] by opsonisation. The MPS was neutralised in mice by pretreatment with polyinosinic acid (a synthetic nucleic acid strand) before therapeutic measles virus treatment; this led to competitive inhibition of the scavenging of particles by macro‐ phages, and improved virus delivery to and efficacy at SKOV3 xenografts [46]. Aggregation

crossing, a theory that is currently under investigation.

route has the potential to target almost all tissues of the body.

**2.2. Barriers to systemic gene therapies**

216 Gene Therapy - Tools and Potential Applications

The differential in ionicity between gene therapy formulation and the extracellular space poses another obstacle for nanoparticles, which can lead to colloidal instability [44]. The is‐ sue of non-specific interaction between nanoparticles and plasma proteins has been ad‐ dressed by the coupling of hydrophilic molecules to the nanoparticle. The most commonly employed candidate is poly(ethylene glycol) (PEG), whose anionicity has led to reduced ag‐ gregation and improved transfection ability [47]. PEG has been incorporated into a myriad of non-viral gene therapy strategies. Recently, polyacridine peptide nanoparticles were PE‐ Gylated and found to persist in the mouse circulation for up to nine hours, compared to non-PEGylated nanoparticles, which were inactive within five minutes [48], while biode‐ gradable dextran nanogels were PEGylated and analysed for their siRNA delivering prow‐ ess [49]. Particulate gene therapies are also subject to entrapment by the mononuclear phagocyte system (reticuloendothelial system - RES), when they are captured and held in the spleen or liver [50], which was responsible for the inactivation of adenoviral vectors that have been used as viral gene therapeutics [51]. Avoidance of non-specific biomolecular in‐ teraction, referred to as 'stealth' [1], is a prerequisite for successful gene delivery. Function‐ alisation of non-viral gene therapies with agents such as PEG to facilitate RES avoidance will be discussed in subsequent sections.

Assuming a gene therapeutic persists in active form in the circulation and the target tissue is reached, extravasation from the circulation is imperative. The architecture of normal vascu‐ lature ensures that transport of macromolecules out of the circulation is difficult. One char‐ acteristic of tumour vasculature, however, is its propensity to leakiness, an attribute that can be exploited by gene therapies. It is unsurprising that siRNA lipoplexes that target RAN GTPase were delivered more effectively, and evoked more impressive anti-tumour effects in highly vascularised xenografts than in xenografts with poorer vascularity [52]. The leaky vessel phenomenon, known as the enhanced permeability and retention (EPR) effect, has been utilised to enable the delivery of pDNA-containing particles in various malignancies [53]. The utilization of EPR will be further discussed below. The angiogenic tumour vascula‐ ture was itself targeted in a murine dorsal air sac assay; siRNA targeting Ago2 was com‐ plexed into cationic liposomes and intravenously administered. The authors successfully delivered the interfering RNA to the angiogenic vessels, and reported tumour regression, presumed to be resultant of anti-angiogenesis in their model [54].

Perhaps the most intimidating vascular obstacle that a gene therapy can face is the bloodbrain barrier, where tight junctions between endothelial cells of the capillaries limit the pas‐ sage of molecules much more than at other capillary sites in the body. One of the most exciting techniques available to the gene therapy researcher is the use of ultrasound-target‐ ed microbubble destruction (UTMD); nucleic acid contained within a gas-filled microbubble is administered, before exposure to ultrasonic waves at a frequency that exceeds the reso‐ nance frequency of the microbubbles, causing their destruction and leading to increased ca‐ pillary and cell membrane permeability [55]. This technology was used to deliver pDNA for the green fluorescent protein (GFP) reporter gene across the mouse blood-brain barrier [56], and presents new possibilities for overcoming this most daunting of circulatory barriers.

potential; preliminary studies have revealed that phosphonium-based vectors condensed

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219

**Figure 1.** Summary of the extra- and intracellular barriers faced by non-viral gene therapies following systematic deliv‐

Electrostatic interaction between the cationic nanoparticle and the anionic cell membrane that facilitate association between the cell and nanoparticle was assumed to result in endo‐ cytosis of the nanoparticle, although the machinery of internalization appears to be materialand cell type-dependent. Endocytotic access to cells is by pinocytosis in the majority of cases, rather than by phagocytosis [9]. A mechanism of endocytosis was clarified by Payne and colleagues, who followed the intracellular trafficking of PEI- and Lipofectamine™-com‐ plexed nucleic acids in mammalian cells, and reported that endocytosis relied upon cell sur‐ face heparin sulphate proteoglycans (HSPG) and was dependent on dynamin- and flotillin, rather than clathrin- and caveolin-dependent mechanisms [63]. On the other hand, nanopar‐ ticles formed from pDNA complexed with PEG-CK30 were endocytosed after interaction with cell surface nucleolin, a process that was reliant on the activity of lipid rafts [64]. A re‐ cent study reported by a team at the University of Groningen very elegantly showed lasso‐ ing of PEI- and Lipofectamine™-complexed pDNA by syndecan- and actin-rich filaments of HeLa cells, presumed to be filopodia and retraction fibers; the nanoparticles then 'surf' along the filopodia, or the filopodia are retracted toward the cell body, thereby facilitating

ery. Based on [1].

HSPG-mediated endocytosis [65].

DNA at lower charge ratios than corresponding nitrogen-based vectors [62].

#### **2.3. Cellular barriers to gene delivery**

#### *2.3.1. The cell membrane and endocytosis*

Specific targeting of gene vectors to ensure delivery to the target tissue will be discussed in a subsequent section. The nanoparticle's nucleic acid cargo determines the site to which deliv‐ ery is required; plasmid DNA must be delivered to the nucleus to affect transcription, while siRNA need only reach the cytoplasm to interfere with translation [57]. The most elemental impediment to entry into the animal cell is the lipid bilayer membrane. The cell membrane can be breached using physical means in certain circumstances to allow delivery of naked pDNA. These methods include electroporation (local destabilization of the cell membrane using an electric pulse), sonoporation (membrane destabilization using ultrasound) or laser irradiation (introduction of transient pores in the membrane using a lens-focussed laser beam). The application of these methods is limited, however, by inaccessibility to most tis‐ sues [58].

Condensation and neutralization of nucleic acids into nanoparticles abrogates the two fun‐ damental properties of pDNA that preclude its cellular entry, namely its large size and neg‐ ative charge [1]. Particles of excessive size can aggregate and cause embolization of narrow capillaries, as mentioned above. Nanoparticles for gene delivery tend to be sub-200 nm for this reason. However, *in vivo* delivery of fluorescently labelled liposomes of up to 400 nm diameter has been reported [59]. Particle size also appears to dictate the pathway that per‐ forms internalization of complexes; 200 nm particles have entered cells by clathrin-depend‐ ent routes, 300 nm particles by caveolae-mediated pathways [7]. Optimization of the net charge (or zeta/ζ potential) of delivery vehicle/nucleic acid complex of lipid/polymer/ peptide and pDNA complexes is achieved by the electrostatic interaction between the nega‐ tively charged phosphate residues present in the pDNA and the positively charged nitrogen in the vehicle. The net charge of the resultant particle can be increased by increasing the ve‐ hicle (nitrogen) to pDNA (phosphate) ratio (known as N:P ratio) [1]. The negative charge of serum proteins can thwart the therapeutic potential of nanoparticles; this can be overcome by increasing the N:P ratio of complexes above that sufficient to condense the pDNA. Alter‐ ing the net charge of the nanoparticles can significantly alter the array of plasma proteins that interact with the particles. Similar liposomal particles with charges of -9.0, -11.4 and -27.4 were incubated with human plasma, and the interacting proteins were identified; 117 proteins were found bound to particles of all three charges, while 12, 6 and 15 plasma pro‐ teins interacted uniquely with the three particle charge types respectively [60]. Kim and coworkers reported that hyperbranched polysiloxysilane nanoparticles with a moderate positive charge (46 mV) were more efficient gene delivery agents than analogous particles with a high positive charge (64 mV) [61]. Clearly, nanoparticle size and charge are parame‐ ters that require optimization for appropriate cell membrane breaching. Phosphoniumbased vectors (as opposed to nitrogen-based) are also being explored for their gene delivery potential; preliminary studies have revealed that phosphonium-based vectors condensed DNA at lower charge ratios than corresponding nitrogen-based vectors [62].

the green fluorescent protein (GFP) reporter gene across the mouse blood-brain barrier [56], and presents new possibilities for overcoming this most daunting of circulatory barriers.

Specific targeting of gene vectors to ensure delivery to the target tissue will be discussed in a subsequent section. The nanoparticle's nucleic acid cargo determines the site to which deliv‐ ery is required; plasmid DNA must be delivered to the nucleus to affect transcription, while siRNA need only reach the cytoplasm to interfere with translation [57]. The most elemental impediment to entry into the animal cell is the lipid bilayer membrane. The cell membrane can be breached using physical means in certain circumstances to allow delivery of naked pDNA. These methods include electroporation (local destabilization of the cell membrane using an electric pulse), sonoporation (membrane destabilization using ultrasound) or laser irradiation (introduction of transient pores in the membrane using a lens-focussed laser beam). The application of these methods is limited, however, by inaccessibility to most tis‐

Condensation and neutralization of nucleic acids into nanoparticles abrogates the two fun‐ damental properties of pDNA that preclude its cellular entry, namely its large size and neg‐ ative charge [1]. Particles of excessive size can aggregate and cause embolization of narrow capillaries, as mentioned above. Nanoparticles for gene delivery tend to be sub-200 nm for this reason. However, *in vivo* delivery of fluorescently labelled liposomes of up to 400 nm diameter has been reported [59]. Particle size also appears to dictate the pathway that per‐ forms internalization of complexes; 200 nm particles have entered cells by clathrin-depend‐ ent routes, 300 nm particles by caveolae-mediated pathways [7]. Optimization of the net charge (or zeta/ζ potential) of delivery vehicle/nucleic acid complex of lipid/polymer/ peptide and pDNA complexes is achieved by the electrostatic interaction between the nega‐ tively charged phosphate residues present in the pDNA and the positively charged nitrogen in the vehicle. The net charge of the resultant particle can be increased by increasing the ve‐ hicle (nitrogen) to pDNA (phosphate) ratio (known as N:P ratio) [1]. The negative charge of serum proteins can thwart the therapeutic potential of nanoparticles; this can be overcome by increasing the N:P ratio of complexes above that sufficient to condense the pDNA. Alter‐ ing the net charge of the nanoparticles can significantly alter the array of plasma proteins that interact with the particles. Similar liposomal particles with charges of -9.0, -11.4 and -27.4 were incubated with human plasma, and the interacting proteins were identified; 117 proteins were found bound to particles of all three charges, while 12, 6 and 15 plasma pro‐ teins interacted uniquely with the three particle charge types respectively [60]. Kim and coworkers reported that hyperbranched polysiloxysilane nanoparticles with a moderate positive charge (46 mV) were more efficient gene delivery agents than analogous particles with a high positive charge (64 mV) [61]. Clearly, nanoparticle size and charge are parame‐ ters that require optimization for appropriate cell membrane breaching. Phosphoniumbased vectors (as opposed to nitrogen-based) are also being explored for their gene delivery

**2.3. Cellular barriers to gene delivery**

218 Gene Therapy - Tools and Potential Applications

*2.3.1. The cell membrane and endocytosis*

sues [58].

**Figure 1.** Summary of the extra- and intracellular barriers faced by non-viral gene therapies following systematic deliv‐ ery. Based on [1].

Electrostatic interaction between the cationic nanoparticle and the anionic cell membrane that facilitate association between the cell and nanoparticle was assumed to result in endo‐ cytosis of the nanoparticle, although the machinery of internalization appears to be materialand cell type-dependent. Endocytotic access to cells is by pinocytosis in the majority of cases, rather than by phagocytosis [9]. A mechanism of endocytosis was clarified by Payne and colleagues, who followed the intracellular trafficking of PEI- and Lipofectamine™-com‐ plexed nucleic acids in mammalian cells, and reported that endocytosis relied upon cell sur‐ face heparin sulphate proteoglycans (HSPG) and was dependent on dynamin- and flotillin, rather than clathrin- and caveolin-dependent mechanisms [63]. On the other hand, nanopar‐ ticles formed from pDNA complexed with PEG-CK30 were endocytosed after interaction with cell surface nucleolin, a process that was reliant on the activity of lipid rafts [64]. A re‐ cent study reported by a team at the University of Groningen very elegantly showed lasso‐ ing of PEI- and Lipofectamine™-complexed pDNA by syndecan- and actin-rich filaments of HeLa cells, presumed to be filopodia and retraction fibers; the nanoparticles then 'surf' along the filopodia, or the filopodia are retracted toward the cell body, thereby facilitating HSPG-mediated endocytosis [65].

#### *2.3.2. Endosomal escape*

The result of endocytic cellular entry is endosomal entrapment. Endosomes are a range of membrane-bound organelles that include early, late and recycling endosomes that are re‐ sponsible for the short-term storage and sorting of endocytosed materials, including macro‐ molecules and pathogens (including viruses). Once material is endocytosed, it is either evicted from the cell by the recycling endosome, or the complex process of endosome matu‐ ration ensues, late endosomes fuse with lysosomes, and active degradation of endosome car‐ goes occurs [66]. Macromolecules that are unable to escape the endosome are bound for lysosomal degradation.

transfer from vector to nucleus has been reported [73]. Mitotic division temporarily disrupts the nuclear membrane's barrier properties, which can allow pDNA transgene entry [9]. The nuclear pores can be more actively targeted for penetration by the use of nuclear localization signalling (NLS) peptides or DNA targeting sequences (DTS). NLSs are short clusters of ba‐ sic amino acids (such as lysine) that bind to importins, receptors that facilitate cytoplasmnuclear transport [74]. Active transport of macromolecules through nuclear pore complexes causes expansion of the pores to approximately 30 nm in diameter [75]. The nuclear localiza‐ tion peptide SV40 from Simian virus 40 was used to improve the delivery of luciferase genecarrying liposomes to neuroblastoma cells [76], while NLSs from adenovirus E1a, the transcription factor c-myc, mouse FGF3, and the DNA repair protein PARP have all been used to guide transgene delivery to the nucleus [74]. Some of the recently employed nuclear

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As mentioned previously, although viruses are masters of nucleic acid delivery, alternative delivery mechanisms are being sought to avoid the pitfalls associated with viral systems. Fundamentally, viruses remain foreign pathogens, agents that the human body has evolved to protect itself from. Of the commonly employed viral vectors, adenoviral, adenovirus-as‐ sociated vectors and lentivirus vectors all produce immune responses in mice and humans, with antibodies often being produced against both the packaging vector as well as the trans‐ gene product. Exposure to viral particles triggers the adaptive immune response. Pinocyto‐ sis of viral particles by immature dendritic cells elicits maturation of the dendritic cells into mature antigen-presenting cells that present antigens in major histocompatability complexes (MHCs). Activation of T cells by antigen presentation leads to both the destruction of the antigen-presenting cells, and the recruitment and activation of B cells, responsible for anti‐

Attempts to avoid provocation of immunologic responses have been made by the viral gene therapist that include deletion or nullification of viral coding genes and elimination of pathogenic genes, or use of targeting strategies to ensure avoidance of the immune cells. Additionally, pharmacological immunosuppression has been used extensively to avoid the

It is generally accepted that non-viral gene therapy strategies elicit fewer immune responses than their viral counterparts, although certain facets of non-viral complexes mark them as targets for immune system intervention [86]. An early report into immune responses in‐ duced by non-viral gene therapy revealed cytokine induction (TNFα and IL-1β) by PEI/DNA complexes; the extent of immune induction was determined by the route of deliv‐ ery, aerosol proving less detrimental than intravenous [87]. In mice, lipoplex administration evoked complement activation and induction of IFN-γ, TNF-α, IL-6, and IL-12. These effects

envelope penetration strategies are summarised in Table 1.

neutralization of various viral gene therapy strategies [85].

**3.1. DNA-mediated immune responses**

**3. Evading the immune system**

body production [84].

The mechanism of endosomal escape by non-viral vectors is dependent on the complexing material used. Cationic lipids appear to interact with the anionic endosomal membrane, re‐ sulting in ion pair formation and consequent transformation to inverted hexagonal phase (HII), causing disruption of the endosomal membrane. Alternatively, an inversion of the en‐ dosomal membrane as a result of electrostatic interactions has been proposed, which would instigate nucleic acid cargo being deposited in the cytoplasm. When polymeric materials are used to complex pDNA, the polymers themselves absorb protons in the endosome (protonsponge effect), leading to chloride ion influx, increased osmotic pressure and water flow in‐ to the endosome, resulting in endosome rupture [7].

The fusogenic lipid DOPE is frequently used as a co-complexing agent due to its inherent ability to facilitate endosomal escape; conformational change from lipid bilayer to an invert‐ ed hexagonal structure is triggered by the sub-physiological pH of the endosome, and caus‐ es endosomal membrane disruption and escape of nucleic acid cargo [67]. Viral membrane proteins have provided inspiration for the non-viral gene delivery researcher. Influenza vi‐ ruses escape the endosome with the help of hemagluttinin A2 (HA2), while the adenoviral protein, penton, assists adenovirus endosome escape. Glycoprotein H from herpes simplex virus induced 30-fold improvement of transfection by lipoplexes in human cell lines [68]. Conformational changes of these proteins consequential of the acidic environment in the en‐ dosome facilitate viral particle escape from the endosome; the more hydrophobic conforma‐ tion that they adopt at low pH permits membrane fusion and disruption [1].

Synthetic fusogenic peptides are increasingly being used to improve transfection in non-vi‐ ral systems. The conformational status of GALA is responsive to pH, adopting alpha-helical status in acidic environments, and in that sense, mimics the endosomal escape route fav‐ oured by viruses [69]. Derivatives of GALA such as GALAdelE3, YALA [70], and KALA [24] have all shown promise as endosome escaping agents. Similarly, two endosomal escape peptides, INF7 and H5WYG, improved endosome escape of PEG-based vectors by up to 100-fold [71].

#### *2.3.3. Nuclear envelope penetration*

The final obstacle faced by pDNA gene therapies is the nuclear envelope, a barrier punctuat‐ ed with nuclear pores impermeable to molecules greater than 70 kDa, or roughly 10 nm in diameter [72]. Liposomal fusion with the nuclear membrane that facilitates direct cargo transfer from vector to nucleus has been reported [73]. Mitotic division temporarily disrupts the nuclear membrane's barrier properties, which can allow pDNA transgene entry [9]. The nuclear pores can be more actively targeted for penetration by the use of nuclear localization signalling (NLS) peptides or DNA targeting sequences (DTS). NLSs are short clusters of ba‐ sic amino acids (such as lysine) that bind to importins, receptors that facilitate cytoplasmnuclear transport [74]. Active transport of macromolecules through nuclear pore complexes causes expansion of the pores to approximately 30 nm in diameter [75]. The nuclear localiza‐ tion peptide SV40 from Simian virus 40 was used to improve the delivery of luciferase genecarrying liposomes to neuroblastoma cells [76], while NLSs from adenovirus E1a, the transcription factor c-myc, mouse FGF3, and the DNA repair protein PARP have all been used to guide transgene delivery to the nucleus [74]. Some of the recently employed nuclear envelope penetration strategies are summarised in Table 1.

#### **3. Evading the immune system**

*2.3.2. Endosomal escape*

220 Gene Therapy - Tools and Potential Applications

lysosomal degradation.

100-fold [71].

*2.3.3. Nuclear envelope penetration*

to the endosome, resulting in endosome rupture [7].

The result of endocytic cellular entry is endosomal entrapment. Endosomes are a range of membrane-bound organelles that include early, late and recycling endosomes that are re‐ sponsible for the short-term storage and sorting of endocytosed materials, including macro‐ molecules and pathogens (including viruses). Once material is endocytosed, it is either evicted from the cell by the recycling endosome, or the complex process of endosome matu‐ ration ensues, late endosomes fuse with lysosomes, and active degradation of endosome car‐ goes occurs [66]. Macromolecules that are unable to escape the endosome are bound for

The mechanism of endosomal escape by non-viral vectors is dependent on the complexing material used. Cationic lipids appear to interact with the anionic endosomal membrane, re‐ sulting in ion pair formation and consequent transformation to inverted hexagonal phase (HII), causing disruption of the endosomal membrane. Alternatively, an inversion of the en‐ dosomal membrane as a result of electrostatic interactions has been proposed, which would instigate nucleic acid cargo being deposited in the cytoplasm. When polymeric materials are used to complex pDNA, the polymers themselves absorb protons in the endosome (protonsponge effect), leading to chloride ion influx, increased osmotic pressure and water flow in‐

The fusogenic lipid DOPE is frequently used as a co-complexing agent due to its inherent ability to facilitate endosomal escape; conformational change from lipid bilayer to an invert‐ ed hexagonal structure is triggered by the sub-physiological pH of the endosome, and caus‐ es endosomal membrane disruption and escape of nucleic acid cargo [67]. Viral membrane proteins have provided inspiration for the non-viral gene delivery researcher. Influenza vi‐ ruses escape the endosome with the help of hemagluttinin A2 (HA2), while the adenoviral protein, penton, assists adenovirus endosome escape. Glycoprotein H from herpes simplex virus induced 30-fold improvement of transfection by lipoplexes in human cell lines [68]. Conformational changes of these proteins consequential of the acidic environment in the en‐ dosome facilitate viral particle escape from the endosome; the more hydrophobic conforma‐

Synthetic fusogenic peptides are increasingly being used to improve transfection in non-vi‐ ral systems. The conformational status of GALA is responsive to pH, adopting alpha-helical status in acidic environments, and in that sense, mimics the endosomal escape route fav‐ oured by viruses [69]. Derivatives of GALA such as GALAdelE3, YALA [70], and KALA [24] have all shown promise as endosome escaping agents. Similarly, two endosomal escape peptides, INF7 and H5WYG, improved endosome escape of PEG-based vectors by up to

The final obstacle faced by pDNA gene therapies is the nuclear envelope, a barrier punctuat‐ ed with nuclear pores impermeable to molecules greater than 70 kDa, or roughly 10 nm in diameter [72]. Liposomal fusion with the nuclear membrane that facilitates direct cargo

tion that they adopt at low pH permits membrane fusion and disruption [1].

As mentioned previously, although viruses are masters of nucleic acid delivery, alternative delivery mechanisms are being sought to avoid the pitfalls associated with viral systems. Fundamentally, viruses remain foreign pathogens, agents that the human body has evolved to protect itself from. Of the commonly employed viral vectors, adenoviral, adenovirus-as‐ sociated vectors and lentivirus vectors all produce immune responses in mice and humans, with antibodies often being produced against both the packaging vector as well as the trans‐ gene product. Exposure to viral particles triggers the adaptive immune response. Pinocyto‐ sis of viral particles by immature dendritic cells elicits maturation of the dendritic cells into mature antigen-presenting cells that present antigens in major histocompatability complexes (MHCs). Activation of T cells by antigen presentation leads to both the destruction of the antigen-presenting cells, and the recruitment and activation of B cells, responsible for anti‐ body production [84].

Attempts to avoid provocation of immunologic responses have been made by the viral gene therapist that include deletion or nullification of viral coding genes and elimination of pathogenic genes, or use of targeting strategies to ensure avoidance of the immune cells. Additionally, pharmacological immunosuppression has been used extensively to avoid the neutralization of various viral gene therapy strategies [85].

#### **3.1. DNA-mediated immune responses**

It is generally accepted that non-viral gene therapy strategies elicit fewer immune responses than their viral counterparts, although certain facets of non-viral complexes mark them as targets for immune system intervention [86]. An early report into immune responses in‐ duced by non-viral gene therapy revealed cytokine induction (TNFα and IL-1β) by PEI/DNA complexes; the extent of immune induction was determined by the route of deliv‐ ery, aerosol proving less detrimental than intravenous [87]. In mice, lipoplex administration evoked complement activation and induction of IFN-γ, TNF-α, IL-6, and IL-12. These effects


well as eliminating an immune response, evidence exists to suggest that removal of unme‐ thylated CpG motifs can increase the duration of transgene expression [91,92]. PEI-based de‐ livery of CpG-rich pDNA was associated with a reduction in lung compliance, while delivery of CpG-diminished pDNA was not [93]. Furthermore, methylation of CpG motifs in pDNA largely reversed the immunostimulatory activity of lipoplexes and polyplexes in

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Numerous strategies have been investigated to abrogate immune responses upon non-viral gene therapy administration. Liu and colleagues encapsulated various anti-inflammatory agents into DOTAP/pLuciferase liposomes, and termed the resultant complex a 'safeplex'. Safeplexes carrying dexamethasone, prednisone, indomethacin, tetrandrine and gliotoxin in‐ hibited TNFα expression compared to that seen in the absence of anti-inflammatory. Impor‐ tantly, the complexing of dexamethasone into the safeplex did not affect the complex's ability to deliver its pDNA cargo [89]. Delivery of oligonucleotides to inhibit cytokine (NFκB) translation using non-viral carriers has also been proposed as a mechanism of coun‐

Lipid-protamine-DNA complexes (LPDs) were used to deliver a PCR amplicon of the luci‐ ferase gene rather than a bacterial plasmid containing the luciferase gene. Luciferase transla‐ tion was as efficient from the PCR amplicon as from the plasmid when both were complexed with LPD, but the immune response evoked by the PCR fragment complex was three-fold less potent than that evoked by the plasmid complex (determined by TNFα and

The immune response can be avoided by the removal of unnecessary bacterial DNA that contains the immunological CpG motifs, bacterial origin of replication, as well as genes for plasmid antibiotic resistance that are not required for transgene expression. First re‐ ported in 1997, minicircles are gene delivery vehicles that lack prokaryotic nucleic acid, and were produced by the thermo-responsive activity of λ integrase [97]. Minicircles were more potent reporter gene deliverers than their parental pDNA in melanoma and colon carcinoma cell lines by lipofection and electroporation [98]. Minicircles complexed with PEI also delivered the GFP gene more potently than similarly complexed pDNA [99]. The potency of minicircles has been improved by tethering minicircle liposomes to the TetR

A further improvement on the immunologically inert minicircles has recently been mooted. Tightly-wound miniknot vectors are the result of DNA minicircle treatment with DNA top‐ oisomerase II, and are proposed to be more resistant to physical damage (strand breaks) that can linearise (thereby reducing/removing efficacy) pDNA and minicircles. DNA delivery methods such as aerosol inhalation, jet-injection, electroporation, particle bombardment and

It is important to note that immune responses to non-viral gene therapy are not solely resul‐ tant of bacterial CpG motifs. An impressive study from Kyoto University highlighted the

ultrasound DNA transfer can subject DNA to stresses that might cause damage [101].

teracting the host's immune response to non-viral therapies [95].

C57BL/6 mice [94].

IL-12 expression) [96].

nuclear targeting device [100].

**3.2. Carrier-mediated immune responses**

**Table 1.** Recent strategies employed to aid the delivery of non-viral gene therapies to the nucleus.

were independent of N:P ratio or the cationic lipid complexed with the pDNA [88]. Al‐ though observed immune responses tend to be dose-dependent, dose reduction to avoid im‐ mune induction consequently also lessens the transfection ability of the complexes, stressing the narrow therapeutic index of non-viral gene therapies [89].

It is well established that immune responses in non-viral therapies is resultant of the pres‐ ence of unmethylated CpG motifs in the bacterial backbone of the plasmid. In mammals, roughly 75% of CpG motifs are methylated to 5′-methycytosine, whereas in bacteria they are usually unmethylated [89]. Recognition of unmethylated CpG motifs by Toll-like receptor 9 on immune cells causes activation of mitogen-activated protein kinases and NF-κB [90]. As well as eliminating an immune response, evidence exists to suggest that removal of unme‐ thylated CpG motifs can increase the duration of transgene expression [91,92]. PEI-based de‐ livery of CpG-rich pDNA was associated with a reduction in lung compliance, while delivery of CpG-diminished pDNA was not [93]. Furthermore, methylation of CpG motifs in pDNA largely reversed the immunostimulatory activity of lipoplexes and polyplexes in C57BL/6 mice [94].

Numerous strategies have been investigated to abrogate immune responses upon non-viral gene therapy administration. Liu and colleagues encapsulated various anti-inflammatory agents into DOTAP/pLuciferase liposomes, and termed the resultant complex a 'safeplex'. Safeplexes carrying dexamethasone, prednisone, indomethacin, tetrandrine and gliotoxin in‐ hibited TNFα expression compared to that seen in the absence of anti-inflammatory. Impor‐ tantly, the complexing of dexamethasone into the safeplex did not affect the complex's ability to deliver its pDNA cargo [89]. Delivery of oligonucleotides to inhibit cytokine (NFκB) translation using non-viral carriers has also been proposed as a mechanism of coun‐ teracting the host's immune response to non-viral therapies [95].

Lipid-protamine-DNA complexes (LPDs) were used to deliver a PCR amplicon of the luci‐ ferase gene rather than a bacterial plasmid containing the luciferase gene. Luciferase transla‐ tion was as efficient from the PCR amplicon as from the plasmid when both were complexed with LPD, but the immune response evoked by the PCR fragment complex was three-fold less potent than that evoked by the plasmid complex (determined by TNFα and IL-12 expression) [96].

The immune response can be avoided by the removal of unnecessary bacterial DNA that contains the immunological CpG motifs, bacterial origin of replication, as well as genes for plasmid antibiotic resistance that are not required for transgene expression. First re‐ ported in 1997, minicircles are gene delivery vehicles that lack prokaryotic nucleic acid, and were produced by the thermo-responsive activity of λ integrase [97]. Minicircles were more potent reporter gene deliverers than their parental pDNA in melanoma and colon carcinoma cell lines by lipofection and electroporation [98]. Minicircles complexed with PEI also delivered the GFP gene more potently than similarly complexed pDNA [99]. The potency of minicircles has been improved by tethering minicircle liposomes to the TetR nuclear targeting device [100].

A further improvement on the immunologically inert minicircles has recently been mooted. Tightly-wound miniknot vectors are the result of DNA minicircle treatment with DNA top‐ oisomerase II, and are proposed to be more resistant to physical damage (strand breaks) that can linearise (thereby reducing/removing efficacy) pDNA and minicircles. DNA delivery methods such as aerosol inhalation, jet-injection, electroporation, particle bombardment and ultrasound DNA transfer can subject DNA to stresses that might cause damage [101].

#### **3.2. Carrier-mediated immune responses**

were independent of N:P ratio or the cationic lipid complexed with the pDNA [88]. Al‐ though observed immune responses tend to be dose-dependent, dose reduction to avoid im‐ mune induction consequently also lessens the transfection ability of the complexes, stressing

**NLS Sequence Summary Result Ref**

PEG-based vector with DNA binding peptide

Inclusion of NLS peptides in Lipofectamine liposomes for transfection into human and rat mesenchymal stem cells

complexed with pDNA and PEI. Luciferase transfection was assessed in MCF-7 breast cancer

Sequence cloned into promoterless plasmid and microinjected into cytoplasm of MLE-12 cells.

(various molecular weights) and nuclear localization determined

collapses nuclear pore cores allowing macromolecule uptake

pLuciferase

cells

N/A TA was conjugated to PEI

N/A Amphipathic alcohol that

**Table 1.** Recent strategies employed to aid the delivery of non-viral gene therapies to the nucleus.

Up to 15-fold increase in CHO cell transfection

Roughly two-, four- and six-fold enhancement of luciferase expression respectively

130-fold improvement in transfection compared to absence of NLS. Iodination improved nuclear localization

Fluorescent *in situ* hybridization revealed nuclear localization in 25-30% of injected cells compared to control (0%). Specific to alveolar type II

epithelial cells

the nucleus

Low molecular weight PEI/TA efficiently targeted

10 – 100-fold increase in transfection efficiency

Improved Lipofectamine 2000-mediated gene transfection to 293T cells *in vitro*, but was not reproducible *in vivo*

[77]

[78]

[79]

[80]

[81]

[82]

[83]

TAT Ac-GCGYGRKKRRQRRRG-NH2

222 Gene Therapy - Tools and Potential Applications

DPKKKRKV

DPKKKRKV

DPKKKRKVDPKKKRKV DPKKKRKVDPKKKRKV-

I-NLS Iodinated-PKKKRKV Iodinated NLS was

318 nucleotides PCR amplified from genomic

Dexamethasone N/A Polyplexed (PEI) with

DNA

NLS-1 NLS-2 NLS-3

Human surfactant protein C promoter

Triamcinolone acetonide (TA)

Trans-

diol

cyclohexane-1,2-

It is well established that immune responses in non-viral therapies is resultant of the pres‐ ence of unmethylated CpG motifs in the bacterial backbone of the plasmid. In mammals, roughly 75% of CpG motifs are methylated to 5′-methycytosine, whereas in bacteria they are usually unmethylated [89]. Recognition of unmethylated CpG motifs by Toll-like receptor 9 on immune cells causes activation of mitogen-activated protein kinases and NF-κB [90]. As

the narrow therapeutic index of non-viral gene therapies [89].

It is important to note that immune responses to non-viral gene therapy are not solely resul‐ tant of bacterial CpG motifs. An impressive study from Kyoto University highlighted the immune responses that can be generated by liposomes. Using CpG-free pDNA in lipoplex‐ es, the authors demonstrated activation of IFNβ, TNFα and IL-6 in macrophages from TLR9 knockout mice. The extent of the immune response (as determined by *in vitro* cytokine in‐ duction) was dependent on the cationic lipid content of the complex. The reactions elicited by the cationic lipids can be summarised as Lipofectamine 2000 > Lipofectamine Plus > DOTMA/DOPE > DOTMA/cholesterol [102]. The inertness of DOTMA/cholesterol as deliv‐ ery vehicle was supported further *in vivo*, when CpG-free pDNA lipoplexes provoked no IL-6 or IFNβ induction after intravenous injection in mice [103]. The targeting of nucleic acid cargoes to specific cells/tissues (to be discussed shortly) could also remedy the immune re‐ sponse by preventing the transfection of non-target cells, and in particular, the antigen-pre‐ senting cells [85].

Particle size is an important factor for utilizing the EPR effect. Studies have shown that nanoparticles up to 400 nm in diameter can permeate across tumour vessels [59,110]. How‐ ever, circulation times can also play a key role in successful tumour transduction, with a minimum of 6 h required for the EPR effect to occur [111]. The EPR effect has been exploited not only in chemotherapy drug design but also in gene delivery. In one such example, poly‐ glycerolaminne (PG-Amine) dendrimers were complexed with siRNA and delivered intra‐ venously to mice bearing luciferase tagged mammary tumours; after 24 h, there was a 69% reduction in luciferase activity [112]. The authors also reported that there was clear evidence of accumulation of the complexes in the tumours but not in any other organs, which can be attributed to the EPR effect. In order for continuous knockdown of luciferase, it was deter‐

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225

There are however, a number of problems if the EPR effect is the sole mechanism for target‐ ing in cancer gene therapy. One such problem is tumour size. Tumours that are larger than 1 cm in diameter develop hypoxic regions that are characterised by a lack of blood vessels, therefore the EPR effect becomes redundant in these resistant regions. Evans blue dye and albumin were used to generate a synthetic macromolecule delivery of which showed that although selective, in tumours larger than 3 cm, the dye accumulated solely in the peripher‐ al regions and not in the central core [113]. One method to increase the EPR in solid tumours is through the use of nitric oxide donors in combination with macromolecular delivery. En‐ dogenous nitric oxide (NO) has an effect on blood flow, angiogenesis and metastatic poten‐ tial [114-116]. From a therapeutic perspective there are conflicting reports as to whether it is better to enhance or inhibit nitric oxide within tumours [117]. From an enhancement per‐ spective, studies within our own group using iNOS (inducible NO synthase) gene therapy have shown the cytotoxic and radio-chemo sensitizing affects through the generation of µM levels of NO [118,119]. The controlled generation of high levels of nitric oxide as the gene therapy has several anti-cancer advantages, including a genotoxic effect through oxidation, deamination and alkylation of DNA, reduction of the efficiency of DNA repair proteins such as Poly ADP Ribose Polymerase, inhibition of the transcription of hypoxia inducible factor and the anti-apoptotic factor NF-kB which reduces many other pro tumourigenic factors such as MMP1, 3, 9, VEGF, survivin and BCL2 [120]. Derivatives of NO such as peroxyni‐ trite (ONOO) have also been shown to potentiate the EPR effect. Wu and colleagues showed that this was also linked to activation of MMPs, which are known to enhance vascular per‐ meability and angiogenesis through the degradation of matrix proteins [121]. One of the main ways of enhancing the EPR effect is through NO donors, which have been utilised in combination with chemotherapy drugs. For example nitroglycerin has been delivered with vinorelbine and cisplatin in patients with non-small cell lung cancer in a randomised phase II trial. Results showed a response rate of 73% in those patients that received the nitroglycer‐ in plus chemotherapy, compared to 42% in the chemotherapy only arm. The improved ef‐ fects were attributed to the known anti-cancer effects of NO and an improvement in drug delivery to the tumour tissue, i.e. the EPR effect [122]. With respect to gene therapy, the de‐ livery of the iNOS gene would most certainly enhance the EPR effect in solid tumours al‐ though consideration must be given to the amount of NO generated. As gene expression is dependent on successful nuclear transport of the plasmid, it would be very difficult to pre‐

mined that injections would be needed every four days [112].

The continuing evolution of the non-viral gene therapy field has led to the development of transposon-based delivery strategies, including Sleeping Beauty, Tol2, and piggyBac. These systems appear to deliver DNA as efficiently as viruses, and provoke extended transgene product expression, whilst maintaining the low immunogenicity and other risk factors asso‐ ciated with viral gene delivery [104]. It is anticipated that the momentum of non-viral gene therapy research will lead to the development of vehicles and cargoes that will rival the vi‐ ral gene therapy field.

#### **4. Targeting in non-viral systems**

The optimal gene delivery vector will protect its payload from degradation in the circula‐ tion, enable extravasation from the bloodstream, traverse cellular membranes, facilitate en‐ dosomal disruption to deliver the payload to either the cytoplasm, or if necessary, transport to the nucleus. The optimal vector should also be non-immunogenic, as discussed above. Design of such vectors obviously presents a huge challenge. Furthermore, for vectors that accomplish extra- and intracellular barrier and immune system avoidance, there is the add‐ ed layer of complication that targeting presents. Frequently in cancer studies, the payload to be delivered is a therapeutic designed to over-express a protein or knockdown a gene to manifest an anti-cancer effect. In order to spare normal tissue damage, widespread toxicity, and to achieve a clinically viable therapeutic product, targeting has become an essential in the quest for a perfect vector.

#### **4.1. Enhanced permeation and retention effect**

Exploitation of the tumour microenvironment presents an obvious option in the targeting strategies employed by many delivery systems. The enhanced permeation and retention ef‐ fect (EPR), which was mentioned briefly above, is a phenomenon whereby there is defective architecture in blood vessels, extensive angiogenesis, increased vascular permeability and an impaired function of the mononuclear phagocytic system [105-107]. The consequences of these tumour-specific physiological changes is that macromolecules > 40 kDa selectively 'leak' out of the blood vessels and extravasate into the interstitial tumour tissue [108,109]. Particle size is an important factor for utilizing the EPR effect. Studies have shown that nanoparticles up to 400 nm in diameter can permeate across tumour vessels [59,110]. How‐ ever, circulation times can also play a key role in successful tumour transduction, with a minimum of 6 h required for the EPR effect to occur [111]. The EPR effect has been exploited not only in chemotherapy drug design but also in gene delivery. In one such example, poly‐ glycerolaminne (PG-Amine) dendrimers were complexed with siRNA and delivered intra‐ venously to mice bearing luciferase tagged mammary tumours; after 24 h, there was a 69% reduction in luciferase activity [112]. The authors also reported that there was clear evidence of accumulation of the complexes in the tumours but not in any other organs, which can be attributed to the EPR effect. In order for continuous knockdown of luciferase, it was deter‐ mined that injections would be needed every four days [112].

immune responses that can be generated by liposomes. Using CpG-free pDNA in lipoplex‐ es, the authors demonstrated activation of IFNβ, TNFα and IL-6 in macrophages from TLR9 knockout mice. The extent of the immune response (as determined by *in vitro* cytokine in‐ duction) was dependent on the cationic lipid content of the complex. The reactions elicited by the cationic lipids can be summarised as Lipofectamine 2000 > Lipofectamine Plus > DOTMA/DOPE > DOTMA/cholesterol [102]. The inertness of DOTMA/cholesterol as deliv‐ ery vehicle was supported further *in vivo*, when CpG-free pDNA lipoplexes provoked no IL-6 or IFNβ induction after intravenous injection in mice [103]. The targeting of nucleic acid cargoes to specific cells/tissues (to be discussed shortly) could also remedy the immune re‐ sponse by preventing the transfection of non-target cells, and in particular, the antigen-pre‐

The continuing evolution of the non-viral gene therapy field has led to the development of transposon-based delivery strategies, including Sleeping Beauty, Tol2, and piggyBac. These systems appear to deliver DNA as efficiently as viruses, and provoke extended transgene product expression, whilst maintaining the low immunogenicity and other risk factors asso‐ ciated with viral gene delivery [104]. It is anticipated that the momentum of non-viral gene therapy research will lead to the development of vehicles and cargoes that will rival the vi‐

The optimal gene delivery vector will protect its payload from degradation in the circula‐ tion, enable extravasation from the bloodstream, traverse cellular membranes, facilitate en‐ dosomal disruption to deliver the payload to either the cytoplasm, or if necessary, transport to the nucleus. The optimal vector should also be non-immunogenic, as discussed above. Design of such vectors obviously presents a huge challenge. Furthermore, for vectors that accomplish extra- and intracellular barrier and immune system avoidance, there is the add‐ ed layer of complication that targeting presents. Frequently in cancer studies, the payload to be delivered is a therapeutic designed to over-express a protein or knockdown a gene to manifest an anti-cancer effect. In order to spare normal tissue damage, widespread toxicity, and to achieve a clinically viable therapeutic product, targeting has become an essential in

Exploitation of the tumour microenvironment presents an obvious option in the targeting strategies employed by many delivery systems. The enhanced permeation and retention ef‐ fect (EPR), which was mentioned briefly above, is a phenomenon whereby there is defective architecture in blood vessels, extensive angiogenesis, increased vascular permeability and an impaired function of the mononuclear phagocytic system [105-107]. The consequences of these tumour-specific physiological changes is that macromolecules > 40 kDa selectively 'leak' out of the blood vessels and extravasate into the interstitial tumour tissue [108,109].

senting cells [85].

224 Gene Therapy - Tools and Potential Applications

ral gene therapy field.

the quest for a perfect vector.

**4.1. Enhanced permeation and retention effect**

**4. Targeting in non-viral systems**

There are however, a number of problems if the EPR effect is the sole mechanism for target‐ ing in cancer gene therapy. One such problem is tumour size. Tumours that are larger than 1 cm in diameter develop hypoxic regions that are characterised by a lack of blood vessels, therefore the EPR effect becomes redundant in these resistant regions. Evans blue dye and albumin were used to generate a synthetic macromolecule delivery of which showed that although selective, in tumours larger than 3 cm, the dye accumulated solely in the peripher‐ al regions and not in the central core [113]. One method to increase the EPR in solid tumours is through the use of nitric oxide donors in combination with macromolecular delivery. En‐ dogenous nitric oxide (NO) has an effect on blood flow, angiogenesis and metastatic poten‐ tial [114-116]. From a therapeutic perspective there are conflicting reports as to whether it is better to enhance or inhibit nitric oxide within tumours [117]. From an enhancement per‐ spective, studies within our own group using iNOS (inducible NO synthase) gene therapy have shown the cytotoxic and radio-chemo sensitizing affects through the generation of µM levels of NO [118,119]. The controlled generation of high levels of nitric oxide as the gene therapy has several anti-cancer advantages, including a genotoxic effect through oxidation, deamination and alkylation of DNA, reduction of the efficiency of DNA repair proteins such as Poly ADP Ribose Polymerase, inhibition of the transcription of hypoxia inducible factor and the anti-apoptotic factor NF-kB which reduces many other pro tumourigenic factors such as MMP1, 3, 9, VEGF, survivin and BCL2 [120]. Derivatives of NO such as peroxyni‐ trite (ONOO) have also been shown to potentiate the EPR effect. Wu and colleagues showed that this was also linked to activation of MMPs, which are known to enhance vascular per‐ meability and angiogenesis through the degradation of matrix proteins [121]. One of the main ways of enhancing the EPR effect is through NO donors, which have been utilised in combination with chemotherapy drugs. For example nitroglycerin has been delivered with vinorelbine and cisplatin in patients with non-small cell lung cancer in a randomised phase II trial. Results showed a response rate of 73% in those patients that received the nitroglycer‐ in plus chemotherapy, compared to 42% in the chemotherapy only arm. The improved ef‐ fects were attributed to the known anti-cancer effects of NO and an improvement in drug delivery to the tumour tissue, i.e. the EPR effect [122]. With respect to gene therapy, the de‐ livery of the iNOS gene would most certainly enhance the EPR effect in solid tumours al‐ though consideration must be given to the amount of NO generated. As gene expression is dependent on successful nuclear transport of the plasmid, it would be very difficult to pre‐ dict and indeed control. With NO gene therapy, it is therefore essential to target the expres‐ sion of the gene to the target tissue to gain the maximum therapeutic benefit.

pression than transferrin-free nanoparticles and a 'shielding' effect to bypass organs such as

Cancer Gene Therapy – Key Biological Concepts in the Design of Multifunctional Non-Viral Delivery Systems

http://dx.doi.org/10.5772/54271

227

Identification of overexpressed receptors can also lead to the development tumour target‐ ing peptides. Using phage display methods, the T7 peptide (HAIYPRH) was identified and shown to specifically bind to the human transferrin receptor, with competitive stud‐ ies indicating that T7 bound at a different site to transferrin [128]. This T7 peptide has re‐ cently been utilised for targeted co-delivery of the chemotherapy drug doxorubicin (DOX) together with the human TRAIL gene (Tumour necrosis factor Related Apoptosis-Induc‐ ing Ligand) to target gliomas which are known to overexpress the transferrin receptor [129]. DOX was conjugated with a pH linker (for endosomal release) to T7-modified den‐ digraft poly-L-Lysine dendrimers which then condensed the pORF-hTRAIL DNA [129]. *In vitro* and *in vivo* evaluations revealed targeting via the transferrin receptor and accumula‐ tion of the nanoparticles in gliomas following systemic delivery with a synergistic effect. In addition, the targeted T7 nanoparticles induced much less off site toxicity while induc‐

Other targeting ligands of note include the epidermal growth factor receptor that is upregu‐ lated in a number of solid tumours such as breast, prostate, colorectal, and ovarian [130]. Al‐ though some anti-cancer strategies are designed to prevent EGFR activation via small molecule inhibitors such as gefitinib or antibodies such as Cetuximab [131], an alternative is to exploit the differential expression of EGFR. Thiol functionalisation to attach the mouse EGF ligand to PEGylated branched PEI (25 kDa) has shown excellent *in vivo* targeting to hepatocellular carcinoma. Biodistribution studies illustrate quite clearly that there is signifi‐ cantly more expression of the luciferase gene in both Huh-7 and HepG2 HCC tumours com‐ pared to other organs following intravenous injection of the complexes [132]. The authors also found that any distribution of the DNA to the liver was exclusively in the Kupffer cells

The EGF-PEG-PEI system has also been used to selectively deliver synthetic double strand‐ ed RNA (poly IC) [133]. Typically dsRNA is found in virally infected cells and an associated response involves the induction of apoptosis and recruitment of inflammatory cytokines [134,135]. Delivery of poly IC with PEI25-PEG-EGF killed up to 85% of EGFR-over-express‐ ing glioblastoma multiform cells *in vitro* via apoptosis after 1 hour [133]. This cytotoxic effect was significantly enhanced when the PEI was partially replaced with a PEI-Mellitin conju‐ gate, which improved endosomal disruption, enabling greater delivery of the dsRNA to the cytoplasm. In addition, the intratumoural delivery of (poly IC) PEI-PEG-EGF+PEI-Mel com‐ plexes completely eradicated the intracranial tumours for more than 1 year [133]. Further studies have revealed that with further formulation of the delivery vehicle (Linear PEI-PEG 2 kDa-EGF), systemic delivery of poly IC can significantly reduce A431 tumour growth *in vivo* [136]. Similar to transferrin, polypeptides for EGFR have been isolated and used effec‐ tively in cancer gene therapy. Phage display revealed an 11 amino acid sequence (YHWY‐ GYTPQNVI) termed GE11 that has shown specificity to the EGFR after both *in vitro* and *in vivo* studies [137]. Furthermore, when the GE11 peptide was conjugated to PEI and com‐ pared with EGF-PEI, it was found that the latter enhanced mitogenic activity, which is clear‐

the lung and target the tumour [127].

ing a significant anti-tumour effect [129].

and not the epithelial cells, indicative of degradation.

Another problem in EPR targeting comes about with what is commonly termed the 'PEG di‐ lemma'. This is essentially a trade-off between circulation time and efficacy of nucleic acid de‐ livery. Many nanoparticles are PEGylated to increase circulation time, avoid clearance by the reticuloendothelial system (RES) and evade an immune response. As previously stated, if the EPR effect is to be exploited in solid tumours, a long circulation time is needed and PEG repre‐ sents a possible solution. However, the physiochemical properties of many delivery systems are altered when PEG is introduced; this is particularly the case when the cargo is nucleic acids such as siRNA or DNA. In order for nucleic acids to be successful they must be deliv‐ ered to the correct intracellular destination. PEG not only reduces the overall charge of the nanoparticles, which in turn lowers the cellular uptake, but also impairs disruption of the en‐ dosome. Therefore if PEGylation is to be used to enable EPR targeting, novel systems must be developed that can overcome the intracellular barriers to effective nucleic acid delivery.

#### **4.2. Targeting ligands**

One method of targeting is via the incorporation of targeting ligands that bind to cell-sur‐ face receptors. This approach is dependent upon possession of the knowledge of which re‐ ceptor or combinations of receptors are hyperactivated on the cancer cell surface. One such example is the asialoglycoprotein receptor (ASGPr) which, although present on the surface of normal hepatocytes, is overexpressed in hepatocarcinoma cells. The ligand asialofetuin has been attached to a novel lipopolymeric nanoparticle to deliver the immunostimulatory IL-12 cytokine in the treatment of hepatocellular carcinoma. Following intratumoural ad‐ ministration of the targeted nanoparticles, the authors showed survival in 75% of mice treat‐ ed with targeted nanoparticles compared to 38% in the non-targeted nanoparticles. This indicates that the presence of the ASGPr targeting ligand improves intracellular internaliza‐ tion via receptor-mediated endocytosis. Following systemic delivery of either nanoparticle type, luciferase expression in the liver and lungs was assessed. Luciferase expression was 10-fold higher in the livers of those mice that received targeted nanoparticles. However, there was also gene expression in the lung with no significant differences between targeted and non-targeted nanoparticles which indicates that further formulations may be necessary, and that evaluation of gene expression in all the organs is necessary to confirm appropriate targeting [123].

Another useful targeting ligand for cancer gene therapy is transferrin. Transferrin is overex‐ pressed in many malignancies including breast, bladder and lung [124-126]. The differential expression of the transferrin receptor and its extracellular location make it an ideal target for systemic targeting. Systemic delivery of transferrin covalently linked to polyethylenimine has not only shown effective tumour targeting *in vivo*, but it can also shield the positive charge of the nanoparticles [127]. Studies by Kircheis showed that a lower molecular weight of PEI was less toxic and that the incorporation of 25% of the negatively charged lipophilic transferrin ligand gave an almost neutral zeta potential with a significant reduction in ag‐ gregation of erythrocytes. *In vivo* this translated into lower toxicity, one log greater gene ex‐ pression than transferrin-free nanoparticles and a 'shielding' effect to bypass organs such as the lung and target the tumour [127].

dict and indeed control. With NO gene therapy, it is therefore essential to target the expres‐

Another problem in EPR targeting comes about with what is commonly termed the 'PEG di‐ lemma'. This is essentially a trade-off between circulation time and efficacy of nucleic acid de‐ livery. Many nanoparticles are PEGylated to increase circulation time, avoid clearance by the reticuloendothelial system (RES) and evade an immune response. As previously stated, if the EPR effect is to be exploited in solid tumours, a long circulation time is needed and PEG repre‐ sents a possible solution. However, the physiochemical properties of many delivery systems are altered when PEG is introduced; this is particularly the case when the cargo is nucleic acids such as siRNA or DNA. In order for nucleic acids to be successful they must be deliv‐ ered to the correct intracellular destination. PEG not only reduces the overall charge of the nanoparticles, which in turn lowers the cellular uptake, but also impairs disruption of the en‐ dosome. Therefore if PEGylation is to be used to enable EPR targeting, novel systems must be developed that can overcome the intracellular barriers to effective nucleic acid delivery.

One method of targeting is via the incorporation of targeting ligands that bind to cell-sur‐ face receptors. This approach is dependent upon possession of the knowledge of which re‐ ceptor or combinations of receptors are hyperactivated on the cancer cell surface. One such example is the asialoglycoprotein receptor (ASGPr) which, although present on the surface of normal hepatocytes, is overexpressed in hepatocarcinoma cells. The ligand asialofetuin has been attached to a novel lipopolymeric nanoparticle to deliver the immunostimulatory IL-12 cytokine in the treatment of hepatocellular carcinoma. Following intratumoural ad‐ ministration of the targeted nanoparticles, the authors showed survival in 75% of mice treat‐ ed with targeted nanoparticles compared to 38% in the non-targeted nanoparticles. This indicates that the presence of the ASGPr targeting ligand improves intracellular internaliza‐ tion via receptor-mediated endocytosis. Following systemic delivery of either nanoparticle type, luciferase expression in the liver and lungs was assessed. Luciferase expression was 10-fold higher in the livers of those mice that received targeted nanoparticles. However, there was also gene expression in the lung with no significant differences between targeted and non-targeted nanoparticles which indicates that further formulations may be necessary, and that evaluation of gene expression in all the organs is necessary to confirm appropriate

Another useful targeting ligand for cancer gene therapy is transferrin. Transferrin is overex‐ pressed in many malignancies including breast, bladder and lung [124-126]. The differential expression of the transferrin receptor and its extracellular location make it an ideal target for systemic targeting. Systemic delivery of transferrin covalently linked to polyethylenimine has not only shown effective tumour targeting *in vivo*, but it can also shield the positive charge of the nanoparticles [127]. Studies by Kircheis showed that a lower molecular weight of PEI was less toxic and that the incorporation of 25% of the negatively charged lipophilic transferrin ligand gave an almost neutral zeta potential with a significant reduction in ag‐ gregation of erythrocytes. *In vivo* this translated into lower toxicity, one log greater gene ex‐

sion of the gene to the target tissue to gain the maximum therapeutic benefit.

**4.2. Targeting ligands**

226 Gene Therapy - Tools and Potential Applications

targeting [123].

Identification of overexpressed receptors can also lead to the development tumour target‐ ing peptides. Using phage display methods, the T7 peptide (HAIYPRH) was identified and shown to specifically bind to the human transferrin receptor, with competitive stud‐ ies indicating that T7 bound at a different site to transferrin [128]. This T7 peptide has re‐ cently been utilised for targeted co-delivery of the chemotherapy drug doxorubicin (DOX) together with the human TRAIL gene (Tumour necrosis factor Related Apoptosis-Induc‐ ing Ligand) to target gliomas which are known to overexpress the transferrin receptor [129]. DOX was conjugated with a pH linker (for endosomal release) to T7-modified den‐ digraft poly-L-Lysine dendrimers which then condensed the pORF-hTRAIL DNA [129]. *In vitro* and *in vivo* evaluations revealed targeting via the transferrin receptor and accumula‐ tion of the nanoparticles in gliomas following systemic delivery with a synergistic effect. In addition, the targeted T7 nanoparticles induced much less off site toxicity while induc‐ ing a significant anti-tumour effect [129].

Other targeting ligands of note include the epidermal growth factor receptor that is upregu‐ lated in a number of solid tumours such as breast, prostate, colorectal, and ovarian [130]. Al‐ though some anti-cancer strategies are designed to prevent EGFR activation via small molecule inhibitors such as gefitinib or antibodies such as Cetuximab [131], an alternative is to exploit the differential expression of EGFR. Thiol functionalisation to attach the mouse EGF ligand to PEGylated branched PEI (25 kDa) has shown excellent *in vivo* targeting to hepatocellular carcinoma. Biodistribution studies illustrate quite clearly that there is signifi‐ cantly more expression of the luciferase gene in both Huh-7 and HepG2 HCC tumours com‐ pared to other organs following intravenous injection of the complexes [132]. The authors also found that any distribution of the DNA to the liver was exclusively in the Kupffer cells and not the epithelial cells, indicative of degradation.

The EGF-PEG-PEI system has also been used to selectively deliver synthetic double strand‐ ed RNA (poly IC) [133]. Typically dsRNA is found in virally infected cells and an associated response involves the induction of apoptosis and recruitment of inflammatory cytokines [134,135]. Delivery of poly IC with PEI25-PEG-EGF killed up to 85% of EGFR-over-express‐ ing glioblastoma multiform cells *in vitro* via apoptosis after 1 hour [133]. This cytotoxic effect was significantly enhanced when the PEI was partially replaced with a PEI-Mellitin conju‐ gate, which improved endosomal disruption, enabling greater delivery of the dsRNA to the cytoplasm. In addition, the intratumoural delivery of (poly IC) PEI-PEG-EGF+PEI-Mel com‐ plexes completely eradicated the intracranial tumours for more than 1 year [133]. Further studies have revealed that with further formulation of the delivery vehicle (Linear PEI-PEG 2 kDa-EGF), systemic delivery of poly IC can significantly reduce A431 tumour growth *in vivo* [136]. Similar to transferrin, polypeptides for EGFR have been isolated and used effec‐ tively in cancer gene therapy. Phage display revealed an 11 amino acid sequence (YHWY‐ GYTPQNVI) termed GE11 that has shown specificity to the EGFR after both *in vitro* and *in vivo* studies [137]. Furthermore, when the GE11 peptide was conjugated to PEI and com‐ pared with EGF-PEI, it was found that the latter enhanced mitogenic activity, which is clear‐ ly undesirable in the cancer environment. The authors indicate that due to this lack of mitogenic activity, the GE11 ligand is safer *in vivo*, and delivery of the luciferase gene intra‐ venously revealed an 18-fold increase in luciferase expression in human hepatoma SMMC-7721 tumours compared to non-targeted PEI [137].

which has an affinity level of 22 pmol/L [143]. The increased affinity translated into a 4-fold in‐ crease in tumour uptake of ZHER2:342 four hours post-injection with clear contrast in imaging and stability at least up to 24 h post-injection. With respect to the first generation EGFR affi‐ bodies, the affinity was in the 150 nM range [148] which is sub optimal for effective systemic targeting. A similar one step maturation procedure for the EGFR affibody showed that affini‐ ty could be significantly improved whereby ZEGFR1907 had a *Kd* of 5.4 nM. Furthermore, there was significant uptake of the indium-111-labeled affibody ZEGFR1907 in A431 tumours and

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229

Translational applications of these 2nd generation affibodies to date include therapeutic tools for diagnostic and imaging purposes to aid in the identification of molecular drug targets and for the stratification of cancer patient populations. However these highly stable, selec‐ tive proteins are undoubtedly going to have a huge role in the advancement of targeted non-viral systems in cancer gene therapy. Recently a peptide chimera was designed that consisted of the cell penetrating peptide TAT (T), the DNA condensing motif Mu and the HER2 affibody (AF). The position of the AF was critical to ensure targeting functionality given that the affibody must be able to fold properly. Prior to synthesis, ITASSER software was employed to predict functionality based upon peptide design with a linker between TAT-MU and AF that was helical and should therefore ensure stability of the domains [149]. Studies with the purified recombinant TAT-Mu-AF showed that this ternary complex could condense DNA, confer protection from degradation by DNase I and offer stability in serum [150]. Using GFP DNA complexed at a 1:8:2 ratio, there was little uptake of the complexes in the HER2-null MDA-MB-231 cell line and green fluorescence in the HER2-expressing MDA-MB-453, SK-OV-3 and SK-BR-3 cell lines that was proportional to HER2 receptor density. Furthermore, the complexes were non-toxic and functional when injected intra-tumourally into the HER2 positive MDA-MB-453 breast tumours *in vivo* [150]. Unfortunately these ter‐ nary complexes as yet have not been administered intravenously, which is the ultimate test of functionality, given the range of extracellular barriers previously discussed. Nevertheless, this ternary peptide system holds a lot of promise in the next generation of targeted peptide/

PEGylated liposomes have also been synthesised conjugated to the ZEGFR:1907 affibody with a cysteine residue at the C-terminus to form sterically stabilised affibody liposomes (SAL) [151]. Although the SAL system was loaded with the drug mitoxantrone (MTO), other mac‐ romolecules such as nucleic acids could be applied to this system. The MTO-SAL nanoparti‐ cles were tested for cytotoxicity on EGFR-expressing A431 and MDA-MB-468 cell lines with MCF-7 as a negative control. Results indicated that the MTO-SAL nanoparticles had no ef‐ fect on the viability of the EGFR-negative MCF-7 cell line (IC50 value > 100 µM, compared with an IC50 of 18 µm for MTO alone), while the EGFR-expressing A431 (2.8 µM) and MDA-MB-468 (6.8 µM) cell lines were as sensitive to MTO-SAL nanoparticles as they were to MTO only (IC50 1.3 µM and 3 µM respectively) [151]. Taken together, these data suggest that SAL specifically delivered its MTO payload to the EGFR-expressing cells, and that EGFR-null cells were protected from MTO-induced cytotoxicity by the SAL vehicle. These studies illustrate that cysteine-modified affibodies can be targeting ligands on liposomal de‐

EGFR-expressing organs *in vivo* compared to the non-EGFR-affibodyZtaq [144].

protein delivery systems for non-viral gene therapy.

The fibronectin attachment protein of mycobacterium has also been utilised as a targeting ligand to the fibronectin molecule on epithelial cell membranes [138]. The Fab receptor was conjugated to chitosan-DNA nanoparticles and delivered via an air jet nebuliser to enhance gene expression in the lung epithelium. Again using the luciferase reporter gene, studies re‐ vealed that there was a 16-fold increase in gene expression over the non-targeted chitosan nanoparticles [138]. Another example of exploitation of differential expression is in glioma brain capillary endothelial cells that have an upregulation of the lipoprotein receptor-related protein-1. The angiopep-2 peptide ligand (TFFYGGSRGKRNNFKTEEY) has been success‐ fully conjugated to a polyamidoaminedendrimer (PAMAM) with a PEG spacer and studies showed that cellular uptake of the nanoparticles was targeting ligand dose-dependent, and that targeting to the brain was achieved following intravenous delivery [139]. The angiopep targeting system has also been utilised to achieve a therapeutic efficacy in the delivery of PAMAM-PEG-Angiopep/pORF-TRAIL to glial tumours [140]. The administration of these modified nanoparticles yielded an average survival time of 69 days compared to 30 days in the parental PAMAM-PEG/pORF-TRAIL nanoparticle-receiving mice [140].

There are of course numerous examples of systemic targeted delivery employing such li‐ gands. The message that is apparent from all of these studies is that if there is enough infor‐ mation on the expression of a certain receptor, then the incorporation of its targeting ligand into cationic non-viral systems can significantly enhance tumour-targeted accumulation of the nucleic acid.

#### **4.3. Affibody targeting**

Affibodies are small stable alpha helical proteins that lack disulphide bonds, have a low mo‐ lecular weight and are essentially designed to mimic the action of antibodies. An original affibody protein scaffold is used as a template from which combinatorial phage libraries can be generated and subsequently ligand-specific affibodies can be selected from using phage display technology. Such protein scaffolds have been generated from bacterial surface recep‐ tors such as the IgG binding domains of staphylococcal protein A (SPA). The 58 amino acid Z domain from staphylococcal protein A (SPA) is one such scaffold that has been used as a template for ligand specific affibodies [141,142].

With respect to cancer targeting, high affinity affibodies have been generated for Human Epi‐ dermal Growth Factor Receptor 2 [143], Epidermal Growth Factor Receptor [144], Insulin-like Growth Factor-1 Receptor [145] and Platelet Derived Growth Factor Receptor β [146]. Using radio-labelling, all of the affibodies have been shown to accumulate in tumours *in vivo* with an impressive level of specificity following systemic delivery. The affinity of the affibodies is an important factor and ideally should be in the nanomolar range for effective targeting. For ex‐ ample, the affinity levels of affibody ZHER2:4 are 50 nmol/L [147] whereas using a one step affin‐ ity maturation process, Orlova and colleagues were able to generate the ZHER2:342 affibody which has an affinity level of 22 pmol/L [143]. The increased affinity translated into a 4-fold in‐ crease in tumour uptake of ZHER2:342 four hours post-injection with clear contrast in imaging and stability at least up to 24 h post-injection. With respect to the first generation EGFR affi‐ bodies, the affinity was in the 150 nM range [148] which is sub optimal for effective systemic targeting. A similar one step maturation procedure for the EGFR affibody showed that affini‐ ty could be significantly improved whereby ZEGFR1907 had a *Kd* of 5.4 nM. Furthermore, there was significant uptake of the indium-111-labeled affibody ZEGFR1907 in A431 tumours and EGFR-expressing organs *in vivo* compared to the non-EGFR-affibodyZtaq [144].

ly undesirable in the cancer environment. The authors indicate that due to this lack of mitogenic activity, the GE11 ligand is safer *in vivo*, and delivery of the luciferase gene intra‐ venously revealed an 18-fold increase in luciferase expression in human hepatoma

The fibronectin attachment protein of mycobacterium has also been utilised as a targeting ligand to the fibronectin molecule on epithelial cell membranes [138]. The Fab receptor was conjugated to chitosan-DNA nanoparticles and delivered via an air jet nebuliser to enhance gene expression in the lung epithelium. Again using the luciferase reporter gene, studies re‐ vealed that there was a 16-fold increase in gene expression over the non-targeted chitosan nanoparticles [138]. Another example of exploitation of differential expression is in glioma brain capillary endothelial cells that have an upregulation of the lipoprotein receptor-related protein-1. The angiopep-2 peptide ligand (TFFYGGSRGKRNNFKTEEY) has been success‐ fully conjugated to a polyamidoaminedendrimer (PAMAM) with a PEG spacer and studies showed that cellular uptake of the nanoparticles was targeting ligand dose-dependent, and that targeting to the brain was achieved following intravenous delivery [139]. The angiopep targeting system has also been utilised to achieve a therapeutic efficacy in the delivery of PAMAM-PEG-Angiopep/pORF-TRAIL to glial tumours [140]. The administration of these modified nanoparticles yielded an average survival time of 69 days compared to 30 days in

the parental PAMAM-PEG/pORF-TRAIL nanoparticle-receiving mice [140].

the nucleic acid.

**4.3. Affibody targeting**

template for ligand specific affibodies [141,142].

There are of course numerous examples of systemic targeted delivery employing such li‐ gands. The message that is apparent from all of these studies is that if there is enough infor‐ mation on the expression of a certain receptor, then the incorporation of its targeting ligand into cationic non-viral systems can significantly enhance tumour-targeted accumulation of

Affibodies are small stable alpha helical proteins that lack disulphide bonds, have a low mo‐ lecular weight and are essentially designed to mimic the action of antibodies. An original affibody protein scaffold is used as a template from which combinatorial phage libraries can be generated and subsequently ligand-specific affibodies can be selected from using phage display technology. Such protein scaffolds have been generated from bacterial surface recep‐ tors such as the IgG binding domains of staphylococcal protein A (SPA). The 58 amino acid Z domain from staphylococcal protein A (SPA) is one such scaffold that has been used as a

With respect to cancer targeting, high affinity affibodies have been generated for Human Epi‐ dermal Growth Factor Receptor 2 [143], Epidermal Growth Factor Receptor [144], Insulin-like Growth Factor-1 Receptor [145] and Platelet Derived Growth Factor Receptor β [146]. Using radio-labelling, all of the affibodies have been shown to accumulate in tumours *in vivo* with an impressive level of specificity following systemic delivery. The affinity of the affibodies is an important factor and ideally should be in the nanomolar range for effective targeting. For ex‐ ample, the affinity levels of affibody ZHER2:4 are 50 nmol/L [147] whereas using a one step affin‐ ity maturation process, Orlova and colleagues were able to generate the ZHER2:342 affibody

SMMC-7721 tumours compared to non-targeted PEI [137].

228 Gene Therapy - Tools and Potential Applications

Translational applications of these 2nd generation affibodies to date include therapeutic tools for diagnostic and imaging purposes to aid in the identification of molecular drug targets and for the stratification of cancer patient populations. However these highly stable, selec‐ tive proteins are undoubtedly going to have a huge role in the advancement of targeted non-viral systems in cancer gene therapy. Recently a peptide chimera was designed that consisted of the cell penetrating peptide TAT (T), the DNA condensing motif Mu and the HER2 affibody (AF). The position of the AF was critical to ensure targeting functionality given that the affibody must be able to fold properly. Prior to synthesis, ITASSER software was employed to predict functionality based upon peptide design with a linker between TAT-MU and AF that was helical and should therefore ensure stability of the domains [149]. Studies with the purified recombinant TAT-Mu-AF showed that this ternary complex could condense DNA, confer protection from degradation by DNase I and offer stability in serum [150]. Using GFP DNA complexed at a 1:8:2 ratio, there was little uptake of the complexes in the HER2-null MDA-MB-231 cell line and green fluorescence in the HER2-expressing MDA-MB-453, SK-OV-3 and SK-BR-3 cell lines that was proportional to HER2 receptor density. Furthermore, the complexes were non-toxic and functional when injected intra-tumourally into the HER2 positive MDA-MB-453 breast tumours *in vivo* [150]. Unfortunately these ter‐ nary complexes as yet have not been administered intravenously, which is the ultimate test of functionality, given the range of extracellular barriers previously discussed. Nevertheless, this ternary peptide system holds a lot of promise in the next generation of targeted peptide/ protein delivery systems for non-viral gene therapy.

PEGylated liposomes have also been synthesised conjugated to the ZEGFR:1907 affibody with a cysteine residue at the C-terminus to form sterically stabilised affibody liposomes (SAL) [151]. Although the SAL system was loaded with the drug mitoxantrone (MTO), other mac‐ romolecules such as nucleic acids could be applied to this system. The MTO-SAL nanoparti‐ cles were tested for cytotoxicity on EGFR-expressing A431 and MDA-MB-468 cell lines with MCF-7 as a negative control. Results indicated that the MTO-SAL nanoparticles had no ef‐ fect on the viability of the EGFR-negative MCF-7 cell line (IC50 value > 100 µM, compared with an IC50 of 18 µm for MTO alone), while the EGFR-expressing A431 (2.8 µM) and MDA-MB-468 (6.8 µM) cell lines were as sensitive to MTO-SAL nanoparticles as they were to MTO only (IC50 1.3 µM and 3 µM respectively) [151]. Taken together, these data suggest that SAL specifically delivered its MTO payload to the EGFR-expressing cells, and that EGFR-null cells were protected from MTO-induced cytotoxicity by the SAL vehicle. These studies illustrate that cysteine-modified affibodies can be targeting ligands on liposomal de‐ livery vehicles. Furthermore, by ensuring that receptor-mediated endocytosis occurs via the affibodies, a protective effect is conferred on non-expressing receptor tissue which is highly attractive for the delivery of cytotoxic nucleic acids.

Insulin-like Growth Factor 2 (IGF2) is involved in cellular proliferation and differentiation, but is also overexpressed in a variety of tumours such as bladder carcinoma [157]. IGF2 has a total of four promoters with P3 and P4 promoters responsible for IGF2 expression during foetal and tumour development [158]. P3 and P4 have been utilized to drive expression of the cytotoxic Diphtheria Toxin A gene both as a single promoter system and a dual promot‐ er construct termed P4-DTA-P3-DTA [159]. Part of the rationale for this was related to the differential activation of both P3 and P4 regulatory sequences in human tumours, so a dual system would ensure induction of DTA in a larger population of tumours. Using PEI as the delivery vehicle, bladder carcinoma studies have revealed that P4-DTA-P3-DTA was superi‐ or *in vitro* and *in vivo* in both heterotropic and orthotopic bladder tumour models [159]. Sim‐ ilar studies have also been performed in glioma models utilising the cancer-specific H19 promoter in tandem with the P4-IGF2 promoter to selectively control DTA expression [160]. These dual systems have to-date focused on accessible tumours where intratumoural injec‐ tion would suffice, but only systemic delivery of such systems will fully validate the tran‐

Cancer Gene Therapy – Key Biological Concepts in the Design of Multifunctional Non-Viral Delivery Systems

http://dx.doi.org/10.5772/54271

231

Many cancers have the propensity to metastasize to bone, and such tumours acquire osteomi‐ metic characteristics in order to adapt and thrive in the local bone environment. Disseminat‐ ed bone deposits are resistant to conventional therapies and are particularly difficult to target. Osteocalcin is the most abundant noncollagenous bone matrix protein and is involved in the regulation of bone formation and resorption [161-163]. Osteocalcin is also overe-xpressed in a range of cancers including ovarian, lung, brain, breast and prostate [164-166]. The transcrip‐ tion factor largely responsible for activating the osteocalcin promoter is the master transcrip‐ tion factor RUNX2. RUNX2 is also highly expressed in tumours that metastasise to bone, and therefore widespread activation of the human osteocalcin (hOC) promoter should be ach‐ ieved, regardless of the heterogeneous tumour microenvironment. The hOC promoter has been utilized to drive inducible nitric oxide synthase (iNOS) expression [167,168]. Commer‐ cially available liposomes were used as a delivery vehicle for the hOC-iNOS construct. This resulted in exquisite specificity for androgen-independent prostate cancer cells *in vitro,* cou‐ pled with cytotoxicity comparable to that of constitutively expressed iNOS. *In vivo* data also confirmed the potency of hOC-iNOS gene therapy in a mouse xenograft model of human prostate (PC-3) cancer. Multiple intra-tumoural injections slowed tumour growth dramatical‐ ly and led to some complete responses. On average, tumour growth was delayed by 59 days compared to vector only controls. This data from these studies supports the premise that tu‐ mour-specific promoters can effectively drive iNOS monotherapy giving long term tumour control. Future work within this group is now focused on systemic delivery of hOC-iNOS gene therapy. The hOC promoter has also been delivered systemically to control expression of TK in a replication-defective adenovirus (Ad-hOC-TK) and early viral genes in a replication competent adenovirus (Ad-hOC-E1) [169]. The authors found that vitamins C and D3 signifi‐ cantly increased the activity of the hOC promoter and that triple therapy with Ad-hOC-E1, vi‐ tamin D3 and vitamin C resulted in complete regression in 38% of renal cell carcinomas *in vivo*

scriptional control afforded by these promoters.

following a single intravenous injection [169].

Another example of the use of the ZHER2:342 affibody [143] is in a multifunctional biopolymer system that comprises several discrete functions [152]. This system consists of a fusogenic peptide (FP) sequence H5WYG [153], a DNA-condensing and endosomolytic domain (DCE) with repeating sequences of arginine and histidine, a M9 nuclear localization domain (NLS) [154] and a C-terminal ZHER2:342 affibody [143]. What is particularly striking about this deliv‐ ery system is that the authors have designed it taking into account all of the intracellular barriers, and with the use of discrete motifs, have attempted to overcome each hurdle to suc‐ cessful gene delivery. Engineered within this delivery system is also cathepsin D enzyme substrate (CS) to enable cleavage of the targeting motif from the rest of the vector in late en‐ dosomes [152]. The DNA sequence for FP-(DCE)3-NLS-CS-TM was cloned into an inducible expression system and the recombinant biopolymer was expressed and extracted using af‐ finity and size exclusion chromatography [152]. The functionality of each discrete motif was proven and competitive inhibitor binding and transfection studies clearly indicated that the affibody ensured receptor-mediated endocytosis *in vitro* [152]. Transfection efficiency of 21% was achieved in the SKOV-3 HER2-expressing cell line, while efficiencies of only 0.1 and 2% were achieved in the non-expressing PC-3 and MDA-MB-231 prostate and breast cancer cell lines, respectively. *In vivo* delivery and evaluation of the immune response are critical for the future development of such smart biopolymer systems. Nevertheless, this study illus‐ trates that high affinity affibodies can be functional in recombinant delivery vectors, thus enabling receptor targeting to occur.

#### **4.4. Transcriptional targeting**

Of course it may not be necessary to have a targeted delivery system to achieve expression of a desired gene in a particular tissue. Many tumours have a differential expression of a particu‐ lar transcription factor that can be exploited and used to restrict gene expression to a particu‐ lar site. Several promoters that are either tissue- or tumour-specific have been developed that can circumvent the issues surrounding targeting delivery systems. For example the differen‐ tial expression in telomerase activity between tumour and normal tissue together with the identification of the minimal components necessary for telomerase activity has enabled the use of the human telomerase reverse transcriptase and the template containing telomerase (hTERT and hTER) promoters to control gene expression. Studies by Dufès et al showed that systemic delivery of polypropylenimedendrimers complexed with a TNFalpha-expressing plasmid under the control of hTER and hTERT gave regression of solid carcinomas in xeno‐ grafts with 100% survival with no obvious signs of toxicity [155]. The hTERT promoter has al‐ so been used in a dual reporter system with the human alpha fetoprotein (hAFP) promoter to drive expression of MicroRNA-26a (MiR-26a), a known tumour suppressor downregulated in hepatocellular carcinoma (HCC) [156]. The dual promoter system significantly increased MiR-26a expression and reduced viability *in vitro* and *in vivo* compared to single promoter or constitutively driven MiR-26a constructs in the HCC cell lines [156].

Insulin-like Growth Factor 2 (IGF2) is involved in cellular proliferation and differentiation, but is also overexpressed in a variety of tumours such as bladder carcinoma [157]. IGF2 has a total of four promoters with P3 and P4 promoters responsible for IGF2 expression during foetal and tumour development [158]. P3 and P4 have been utilized to drive expression of the cytotoxic Diphtheria Toxin A gene both as a single promoter system and a dual promot‐ er construct termed P4-DTA-P3-DTA [159]. Part of the rationale for this was related to the differential activation of both P3 and P4 regulatory sequences in human tumours, so a dual system would ensure induction of DTA in a larger population of tumours. Using PEI as the delivery vehicle, bladder carcinoma studies have revealed that P4-DTA-P3-DTA was superi‐ or *in vitro* and *in vivo* in both heterotropic and orthotopic bladder tumour models [159]. Sim‐ ilar studies have also been performed in glioma models utilising the cancer-specific H19 promoter in tandem with the P4-IGF2 promoter to selectively control DTA expression [160]. These dual systems have to-date focused on accessible tumours where intratumoural injec‐ tion would suffice, but only systemic delivery of such systems will fully validate the tran‐ scriptional control afforded by these promoters.

livery vehicles. Furthermore, by ensuring that receptor-mediated endocytosis occurs via the affibodies, a protective effect is conferred on non-expressing receptor tissue which is highly

Another example of the use of the ZHER2:342 affibody [143] is in a multifunctional biopolymer system that comprises several discrete functions [152]. This system consists of a fusogenic peptide (FP) sequence H5WYG [153], a DNA-condensing and endosomolytic domain (DCE) with repeating sequences of arginine and histidine, a M9 nuclear localization domain (NLS) [154] and a C-terminal ZHER2:342 affibody [143]. What is particularly striking about this deliv‐ ery system is that the authors have designed it taking into account all of the intracellular barriers, and with the use of discrete motifs, have attempted to overcome each hurdle to suc‐ cessful gene delivery. Engineered within this delivery system is also cathepsin D enzyme substrate (CS) to enable cleavage of the targeting motif from the rest of the vector in late en‐ dosomes [152]. The DNA sequence for FP-(DCE)3-NLS-CS-TM was cloned into an inducible expression system and the recombinant biopolymer was expressed and extracted using af‐ finity and size exclusion chromatography [152]. The functionality of each discrete motif was proven and competitive inhibitor binding and transfection studies clearly indicated that the affibody ensured receptor-mediated endocytosis *in vitro* [152]. Transfection efficiency of 21% was achieved in the SKOV-3 HER2-expressing cell line, while efficiencies of only 0.1 and 2% were achieved in the non-expressing PC-3 and MDA-MB-231 prostate and breast cancer cell lines, respectively. *In vivo* delivery and evaluation of the immune response are critical for the future development of such smart biopolymer systems. Nevertheless, this study illus‐ trates that high affinity affibodies can be functional in recombinant delivery vectors, thus

Of course it may not be necessary to have a targeted delivery system to achieve expression of a desired gene in a particular tissue. Many tumours have a differential expression of a particu‐ lar transcription factor that can be exploited and used to restrict gene expression to a particu‐ lar site. Several promoters that are either tissue- or tumour-specific have been developed that can circumvent the issues surrounding targeting delivery systems. For example the differen‐ tial expression in telomerase activity between tumour and normal tissue together with the identification of the minimal components necessary for telomerase activity has enabled the use of the human telomerase reverse transcriptase and the template containing telomerase (hTERT and hTER) promoters to control gene expression. Studies by Dufès et al showed that systemic delivery of polypropylenimedendrimers complexed with a TNFalpha-expressing plasmid under the control of hTER and hTERT gave regression of solid carcinomas in xeno‐ grafts with 100% survival with no obvious signs of toxicity [155]. The hTERT promoter has al‐ so been used in a dual reporter system with the human alpha fetoprotein (hAFP) promoter to drive expression of MicroRNA-26a (MiR-26a), a known tumour suppressor downregulated in hepatocellular carcinoma (HCC) [156]. The dual promoter system significantly increased MiR-26a expression and reduced viability *in vitro* and *in vivo* compared to single promoter or

constitutively driven MiR-26a constructs in the HCC cell lines [156].

attractive for the delivery of cytotoxic nucleic acids.

230 Gene Therapy - Tools and Potential Applications

enabling receptor targeting to occur.

**4.4. Transcriptional targeting**

Many cancers have the propensity to metastasize to bone, and such tumours acquire osteomi‐ metic characteristics in order to adapt and thrive in the local bone environment. Disseminat‐ ed bone deposits are resistant to conventional therapies and are particularly difficult to target. Osteocalcin is the most abundant noncollagenous bone matrix protein and is involved in the regulation of bone formation and resorption [161-163]. Osteocalcin is also overe-xpressed in a range of cancers including ovarian, lung, brain, breast and prostate [164-166]. The transcrip‐ tion factor largely responsible for activating the osteocalcin promoter is the master transcrip‐ tion factor RUNX2. RUNX2 is also highly expressed in tumours that metastasise to bone, and therefore widespread activation of the human osteocalcin (hOC) promoter should be ach‐ ieved, regardless of the heterogeneous tumour microenvironment. The hOC promoter has been utilized to drive inducible nitric oxide synthase (iNOS) expression [167,168]. Commer‐ cially available liposomes were used as a delivery vehicle for the hOC-iNOS construct. This resulted in exquisite specificity for androgen-independent prostate cancer cells *in vitro,* cou‐ pled with cytotoxicity comparable to that of constitutively expressed iNOS. *In vivo* data also confirmed the potency of hOC-iNOS gene therapy in a mouse xenograft model of human prostate (PC-3) cancer. Multiple intra-tumoural injections slowed tumour growth dramatical‐ ly and led to some complete responses. On average, tumour growth was delayed by 59 days compared to vector only controls. This data from these studies supports the premise that tu‐ mour-specific promoters can effectively drive iNOS monotherapy giving long term tumour control. Future work within this group is now focused on systemic delivery of hOC-iNOS gene therapy. The hOC promoter has also been delivered systemically to control expression of TK in a replication-defective adenovirus (Ad-hOC-TK) and early viral genes in a replication competent adenovirus (Ad-hOC-E1) [169]. The authors found that vitamins C and D3 signifi‐ cantly increased the activity of the hOC promoter and that triple therapy with Ad-hOC-E1, vi‐ tamin D3 and vitamin C resulted in complete regression in 38% of renal cell carcinomas *in vivo* following a single intravenous injection [169].

Targeting to a desired tissue is quite often the stumbling block to systemic cancer gene ther‐ apy. For the delivery of DNA, targeting can be achieved through the use of promoter se‐ quences. The success of this method of targeting is reliant upon prior knowledge of a difference in transcription factor expression between the target and normal tissue. A deliv‐ ery system could therefore be designed to condense the DNA, traverse cell membranes, dis‐ rupt endosomes and actively transport the payload to the nucleus without the added biophysical complications of having an external targeting motif. Such a delivery system would in theory deliver the DNA to all tissue, but the DNA would only be transcribed and translated where the desired transcription factor is present, namely the target tissue.

#### **5. Multifunctional delivery**

An understanding of the key biological barriers is critical to the success of a multifunctional delivery vehicle. Perhaps one of the most multifaceted delivery vehicles is the Multifunc‐ tional Envelope-type Nano Device MEND system [170]. The authors describe this as a 'pro‐ grammed packaging' system whereby each part of the system is designed to carry out a specific function in a time-controlled manner. In this system the nucleic acid is condensed with a cationic polymer, wrapped in a lipid envelope which is then functionalised with PEG or other targeting ligands [170]. It is quite clear that PEGylated MEND did have a longer circulation time and was not rapidly cleared from the liver. These are ideal extracellular de‐ livery characteristics, but unfortunately this translated into a much lower gene expression. Therefore circumvention of the 'PEG dilemma' could be achieved via the attachment of tar‐ geting ligands to receptors that are known to be over-expressed on tumour cells coupled with the attachment of a cleavable PEG that exploits either intracellular or tumour-specific characteristics. Figure 2 contains a representation of a MEND that highlights some of the nu‐ cleic acids that have been delivered and functionalisation strategies that have been used.

**Figure 2.** Simplified amalgamation of multifunctional envelope-type nano devices (MENDs) that have been employed for non-viral gene therapy development. pDNA cargoes encoding proteins such as luciferase [173-175] and GFP [176] have been delivered, as well as siRNA targeting luciferase [177,178] and ACTB [179]. MEND polycations are generally PLL [174,178] or protamine [173,175,176]. Lipid envelopes usually comprise DOTAP, DOPE and cholesterol [177,179,180], but can also include CHEMS [174,178]. Tetra-lamellar MEND envelopes comprise DOPE/cholesterol in‐ ner and DOPE/phosphatidic acid outer layers [176]. Functionalisation of MENDS with GALA/short GALA [179], STR-R8 [174,176,178], PEG and MMP-cleavable PEG [170,173,177] and sugar-lipid conjugates [175] have all been reported.

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233

The fundamental limitation associated with non-viral gene therapies is their low transfec‐ tion ability, compared with viral systems [1]. pDNA condensed using poly-L-lysine and in‐ corporated into a MEND that comprised DOPE, cholesterylhemisuccinate and an octaarginine (R8) peptide (DOPE/CHEMS/STR-R8) transfected HeLa and A549 cells as efficiently as an adenovirus vector. Moreover, the parity of transfection efficiency was ach‐ ieved without negatively impacting cell viability, as was the case with adenovirus and Lipo‐ fectamine™, and evoked its therapeutic benefit following transdermal delivery in mice [181]. The R8 peptide has been used similarly to deliver proteins directly to cells [182].

A tetra-lamellar MEND (T-MEND) was nano-engineered that envelops the cationically-con‐ densed pDNA in distinct functional layers to target the distinct barrier membranes faced by a nanoparticle. The pDNA-containing core was wrapped in a nucleus-fusogenic lipid mem‐ brane, which was in turn wrapped in an endosome-fusogenic lipid membrane that was

Based on [181].

The avoidance of an immune response to non-viral strategies can also be greatly improved by use of MENDs as delivery devices. Delivery of pDNA to mice by a MEND resulted in differential expression of almost 1600 genes; PEGylation of the MEND reversed the altered expression of many of these genes. Gene Ontology analysis revealed that in general, the up‐ regulated genes were associated with "immune response," "response to biotic stimulus," "defence response," and related processes. The expression of IL-6, but not IFNα (commonly activated cytokines, as discussed above), were lower in the PEGylated MEND group com‐ pared with the non-PEGylated [171]. PEGylation has been shown to limit endosomal escape of gene delivery complexes [172]; inclusion of GALA in the MEND facilitated endosomal es‐ cape, and diminished the previously elevated IFNα levels [171]. A MEND functionalised with a PEG-peptide-DOPE conjugate (PPD) was stable in the systemic circulation after intra‐ venous delivery (thereby benefiting from PEG's stabilising characteristic), while it also po‐ tently delivered its pDNA cargo to HT1080 fibrosarcoma cells (MMP-rich), but not to HEK293 human embryonic kidney cells (MMP-deficient), thereby avoiding PEG's limiting characteristic [173]. Cleavage of PEG by MMPs facilitates the targeting of tumour tissues which are high in MMPs.

Cancer Gene Therapy – Key Biological Concepts in the Design of Multifunctional Non-Viral Delivery Systems http://dx.doi.org/10.5772/54271 233

Targeting to a desired tissue is quite often the stumbling block to systemic cancer gene ther‐ apy. For the delivery of DNA, targeting can be achieved through the use of promoter se‐ quences. The success of this method of targeting is reliant upon prior knowledge of a difference in transcription factor expression between the target and normal tissue. A deliv‐ ery system could therefore be designed to condense the DNA, traverse cell membranes, dis‐ rupt endosomes and actively transport the payload to the nucleus without the added biophysical complications of having an external targeting motif. Such a delivery system would in theory deliver the DNA to all tissue, but the DNA would only be transcribed and

translated where the desired transcription factor is present, namely the target tissue.

An understanding of the key biological barriers is critical to the success of a multifunctional delivery vehicle. Perhaps one of the most multifaceted delivery vehicles is the Multifunc‐ tional Envelope-type Nano Device MEND system [170]. The authors describe this as a 'pro‐ grammed packaging' system whereby each part of the system is designed to carry out a specific function in a time-controlled manner. In this system the nucleic acid is condensed with a cationic polymer, wrapped in a lipid envelope which is then functionalised with PEG or other targeting ligands [170]. It is quite clear that PEGylated MEND did have a longer circulation time and was not rapidly cleared from the liver. These are ideal extracellular de‐ livery characteristics, but unfortunately this translated into a much lower gene expression. Therefore circumvention of the 'PEG dilemma' could be achieved via the attachment of tar‐ geting ligands to receptors that are known to be over-expressed on tumour cells coupled with the attachment of a cleavable PEG that exploits either intracellular or tumour-specific characteristics. Figure 2 contains a representation of a MEND that highlights some of the nu‐ cleic acids that have been delivered and functionalisation strategies that have been used.

The avoidance of an immune response to non-viral strategies can also be greatly improved by use of MENDs as delivery devices. Delivery of pDNA to mice by a MEND resulted in differential expression of almost 1600 genes; PEGylation of the MEND reversed the altered expression of many of these genes. Gene Ontology analysis revealed that in general, the up‐ regulated genes were associated with "immune response," "response to biotic stimulus," "defence response," and related processes. The expression of IL-6, but not IFNα (commonly activated cytokines, as discussed above), were lower in the PEGylated MEND group com‐ pared with the non-PEGylated [171]. PEGylation has been shown to limit endosomal escape of gene delivery complexes [172]; inclusion of GALA in the MEND facilitated endosomal es‐ cape, and diminished the previously elevated IFNα levels [171]. A MEND functionalised with a PEG-peptide-DOPE conjugate (PPD) was stable in the systemic circulation after intra‐ venous delivery (thereby benefiting from PEG's stabilising characteristic), while it also po‐ tently delivered its pDNA cargo to HT1080 fibrosarcoma cells (MMP-rich), but not to HEK293 human embryonic kidney cells (MMP-deficient), thereby avoiding PEG's limiting characteristic [173]. Cleavage of PEG by MMPs facilitates the targeting of tumour tissues

**5. Multifunctional delivery**

232 Gene Therapy - Tools and Potential Applications

which are high in MMPs.

**Figure 2.** Simplified amalgamation of multifunctional envelope-type nano devices (MENDs) that have been employed for non-viral gene therapy development. pDNA cargoes encoding proteins such as luciferase [173-175] and GFP [176] have been delivered, as well as siRNA targeting luciferase [177,178] and ACTB [179]. MEND polycations are generally PLL [174,178] or protamine [173,175,176]. Lipid envelopes usually comprise DOTAP, DOPE and cholesterol [177,179,180], but can also include CHEMS [174,178]. Tetra-lamellar MEND envelopes comprise DOPE/cholesterol in‐ ner and DOPE/phosphatidic acid outer layers [176]. Functionalisation of MENDS with GALA/short GALA [179], STR-R8 [174,176,178], PEG and MMP-cleavable PEG [170,173,177] and sugar-lipid conjugates [175] have all been reported. Based on [181].

The fundamental limitation associated with non-viral gene therapies is their low transfec‐ tion ability, compared with viral systems [1]. pDNA condensed using poly-L-lysine and in‐ corporated into a MEND that comprised DOPE, cholesterylhemisuccinate and an octaarginine (R8) peptide (DOPE/CHEMS/STR-R8) transfected HeLa and A549 cells as efficiently as an adenovirus vector. Moreover, the parity of transfection efficiency was ach‐ ieved without negatively impacting cell viability, as was the case with adenovirus and Lipo‐ fectamine™, and evoked its therapeutic benefit following transdermal delivery in mice [181]. The R8 peptide has been used similarly to deliver proteins directly to cells [182].

A tetra-lamellar MEND (T-MEND) was nano-engineered that envelops the cationically-con‐ densed pDNA in distinct functional layers to target the distinct barrier membranes faced by a nanoparticle. The pDNA-containing core was wrapped in a nucleus-fusogenic lipid mem‐ brane, which was in turn wrapped in an endosome-fusogenic lipid membrane that was modified with a high density of octa-arginine. Upon endocytosis into the cell, the T-MEND's outermost membrane fuses with the endosomal membrane, releasing the nucleus-fusogenic lipid membrane-bound pDNA core from the endosome into the cytoplasm. The nuclear membrane is then overcome by fusion of the inner nucleus-fusogenic lipid membrane with the nuclear membrane, facilitating transport of the pDNA core into the nucleus. Despite the complexity of the particles, the fully-formed T-MEND produced particles of 163 nm diame‐ ter, and zeta potential of 54.5mV. Unsurprisingly, the T-MEND facilitated impressive pLuci‐ ferase delivery *in vitro* [176]. This exciting T-MEND was further functionalised by addition of fusogenic KALA to the outer and inner membranes, which improved transfection 20-fold [183]. To the authors' knowledge, systemic delivery of multi-layered MENDs is yet to be re‐ ported. Caution must be advised, as promising *in vitro* findings do not always translate into impressive *in vivo* developments.

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#### **6. Conclusion**

It is apparent that the field of non-viral gene delivery is making significant progress in the quest for the ideal gene delivery vehicle. What is also evident is that the most successful sys‐ tems are designed to overcome many biological barriers and as a consequence the tradition‐ al single function systems are now rendered obsolete. Viruses are nature's perfect delivery vehicle and provide the inspiration to many non-viral gene therapy researchers in the de‐ sign of state of the art multi-faceted vehicles. Through a greater understanding and appreci‐ ation of the biological barriers to systemic gene delivery, non- viral gene therapy researchers are on the cusp of creating a variety of highly efficient vehicles that will revolutionise cancer gene therapy.

#### **Author details**

Cian M. McCrudden and Helen O. McCarthy

School of Pharmacy, Queen's University Belfast, Northern Ireland, UK

#### **References**


[3] Ng EW, Shima DT, Calias P, Cunningham ET,Jr, Guyer DR, Adamis AP. Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nat Rev Drug Discov 2006 Feb;5(2):123-132.

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It is apparent that the field of non-viral gene delivery is making significant progress in the quest for the ideal gene delivery vehicle. What is also evident is that the most successful sys‐ tems are designed to overcome many biological barriers and as a consequence the tradition‐ al single function systems are now rendered obsolete. Viruses are nature's perfect delivery vehicle and provide the inspiration to many non-viral gene therapy researchers in the de‐ sign of state of the art multi-faceted vehicles. Through a greater understanding and appreci‐ ation of the biological barriers to systemic gene delivery, non- viral gene therapy researchers are on the cusp of creating a variety of highly efficient vehicles that will revolutionise cancer

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**6. Conclusion**

gene therapy.

**Author details**

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Cian M. McCrudden and Helen O. McCarthy

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School of Pharmacy, Queen's University Belfast, Northern Ireland, UK


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**Chapter 10**

**Gene Therapy Based on Fragment C of**

Ana C. Calvo, Pilar Zaragoza and Rosario Osta

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52896

**1. Introduction**

dystrophies among others.

**Tetanus Toxin in ALS: A Promising Neuroprotective**

Neurodegenerative diseases cover a wide range of neurogenetic disorders including Amyo‐ trophic Lateral Sclerosis (ALS), Alzheimer's disease (AD), Huntington's disease (HD), the spinocerebellar ataxias, inherited prion diseases, the inherited neuropathies, and muscular

In particular, ALS belongs to the group of motor neuron diseases, involving the loss of cortex, brainstem, and spinal cord motor neurons that result in muscle paralysis [1]. Motor neurons, which are localized in the brain, brainstem and spinal cord, behave as a crucial links between the nervous system and the voluntary muscles of the body, as they let synaptic signals travel from upper motor neurons in the brain to lower motor neurons in the spinal cord and finally to muscles. In accordance with the revised El Escorial criteria [2], both the upper motor neu‐ rons and the lower motor neurons degenerate or die in ALS, and as a consequence the com‐ munication between neuron and muscle is lost, prompting the progressive muscle weakening and the appearance of fasciculations. In the later stages of the disease, patients become para‐

lyzed although the disease usually does not impair a person's mind or intelligence.

Nowadays, the cause of ALS and its early manifestations still remain to be elucidated. The pathophysiological mechanisms that prompt the neurodegenerative process in both familial (FALS) and sporadic (SALS) ALS are unknown. However, there is growing evidence that the pathogenic process involved in ALS are multifactorial and include oxidative stress, glu‐ tamate excitotoxicity, mitochondrial dysfunction, axonal transport systems and dysfunction of glial cells, yielding the damage of critical proteins and organelles in the motor neuron triggering the neurodegeneration [3]. Due to the fact that FALS and SALS share clinical and

> © 2013 Calvo et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Strategy for the Bench to the Bedside Approach**

