**Challenges in Advancing the Field of Cancer Gene Therapy: An Overview of the Multi-Functional Nanocarriers**

Azam Bolhassani and Tayebeh Saleh

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54862

## **1. Introduction**

Recent developments in molecular biology and cell biology have led to the discovery of novel genes and proteins having therapeutic potentials for various diseases including cancers. Based on these findings, novel categories of therapeutic biomacromolecules in‐ cluding genes, small interfering RNA (siRNAs), antisense oligonucleic acids, bioactive proteins and peptides have been developed. These macromolecules can be more advanta‐ geous than small-molecular-weight therapeutic agents in terms of their specificity and high potency to the target molecules [Nakase et al., 2010]. Gene therapy is the newest therapeutic strategy for treating human diseases. The basic idea of gene therapy is a gene or gene product that can be selectively delivered to a specific cell/tissue with mini‐ mal toxicity. This product can inhibit the expression of a specific defective gene or ex‐ press a normal gene. Efficient and safe delivery is one of the key issues for the clinical application of nucleic acids as therapeutic agents [Du et al., 2010]. The goal of the Phar‐ maceutical Industry is to have a gene therapy medical product that can be delivered sys‐ temically. *In vivo* gene therapies have focused on viral vectors for gene delivery and have had marginal clinical successes. Major disadvantage of these delivery systems is the integration of some viral vectors into human chromosomes of normal tissue. There are four issues to be solved before cancer gene therapy will be successful: 1) Identification of key target genes critical for the disease pathology and progression; 2) Determination of the correct therapeutic gene to inhibit disease progression; 3) Optimal trans-gene expres‐ sion for suppressing the target gene; and 4) Delivery of therapeutic product to the target tissue at an efficient dose [Scanlon, 2004].

© 2013 Bolhassani and Saleh; 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. © 2013 The Author(s). Licensee InTech. This chapter is 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.

Delivery is one of the most difficult challenges facing the gene therapy field. It is needed to use an efficient transfer system that can stabilize, transduce and express a transgene in the target tissue. Recently, non-viral technologies have been widely used as a signifi‐ cant alternative for gene delivery. The non-viral delivery systems have reduced adverse immune responses, are easier to manufacture and can be produced for the pharmaceuti‐ cal industry in large quantities. The current progresses of gene therapy are further fo‐ cused on synthesized nano-particle technologies. Some of these new chemical compositions are cationic molecules such as polymers, lipids and peptides [Scanlon, 2004; Gao et al., 2007].

**2.1. History**

In 1972, Friedmann and Roblin published a paper in Science entitled as "Gene therapy for human genetic disease" [Friedmann and Roblin, 1972; Rogers, 1970]. The first approved gene therapy case in the United States took place in 1990, at the National Institute of Health. It was performed on a four year old girl with a genetic defect associated to an im‐ mune system deficiency. The effects were only temporary, but successful [Blaese et al., 1995]. In addition, sickle cell disease was successfully treated in mice [Fisher, 1995]. At the same time, the researchers were able to create tiny liposomes 25 nanometers (nm) that can carry therapeutic DNA through pores in the nuclear membrane [www. newscientist.com, 2002]. In 1992, Claudio Bordignon performed the first procedure of gene therapy using hematopoietic stem cells as vectors to deliver genes aimed to correct hereditary diseases [Abbott, 1992]. In 2002, this work led to the publication of the first successful gene thera‐ py treatment for adenosine deaminase-deficiency (SCID) [Cavazzana-Calvo et al., 2004]. In 2003, a Los Angeles research team inserted genes into the brain using liposomes coated in a polymer called polyethylene glycol (PEG). The transfer of genes into the brain is a sig‐ nificant achievement because viral vectors are too big to get across the blood–brain barri‐ er. This method had potential for treating Parkinson's disease. At the same time, RNA interference or gene silencing was considered as a new way to treat Huntington's disease [www. newscientist.com, 2003]. In 2006, scientists at the National Institutes of Health have successfully treated metastatic melanoma in two patients using killer T cells genetically re‐ targeted to attack the cancer cells. For the first time, this study demonstrated that gene therapy can be effective in treating cancer. In November 2006, Preston Nix reported on VRX496, a gene-based immunotherapy for the treatment of human immunodeficiency vi‐ rus (HIV) that uses a lentiviral vector for delivery of an antisense gene against the HIV

Challenges in Advancing the Field of Cancer Gene Therapy: An Overview of the Multi-Functional Nanocarriers

http://dx.doi.org/10.5772/54862

199

Leber's congenital amaurosis is an inherited blinding disease caused by mutations in the RPE65 gene. In 2007-2008, the world's first gene therapy trial was announced for inherited retinal disease. They determined the safety of the sub-retinal delivery of recombinant adenoassociated virus (AAV) carrying RPE65 gene and found the positive results in vision and without the apparent side-effects [www. news.bbc.co.uk, 2007]. In 2009, the researchers were succeeded at arresting a fatal brain disease, adrenoleukodystrophy, using a vector derived from HIV to deliver the gene for the missing enzyme [Kaiser, 2009]. A paper published in 2010, deals with gene therapy for a form of achromatopsia in dogs. Achromatopsia, or complete color blindness, is presented as an ideal model to develop gene therapy directed to cone photoreceptors [Komáromy et al., 2010]. In 2011, a study carried out using genetically modified T cells to fight the chronic lymphocytic leukemia (CLL) disease. Moreover, Human HGF plasmid DNA therapy of cardiomyocytes was examined as a potential treatment for coronary artery disease as well as myocardial infarction [www. nature.com, 2011; Yang et al., 2008; Hahn et al., 2011]. However, most of the approved European and United States gene therapy protocols are for cancer (~ 66%), in contrast to monogenetic diseases (~ 11%) and cardiovascular diseases (~ 8%). The focus of cancer gene therapy has been on melanoma, prostate and ovarian

envelope [Levine et al., 2006; www.eurekalert.org, 2009].

cancer and leukemia [Scanlon, 2004].

However, for successful clinical trial, ideal non-viral vectors should be degradable into low molecular weight components, in response to biological stimuli, for lowered cytotoxicity and effective systemic clearance. They should also be efficient in overcoming extracellular and intracellular barriers, tissue/cell-targeted for specific accumulations and multi-functional for synergistic therapeutic and diagnostic outcomes. Recently, a broad range of different stimuliresponsive strategies (virus-mimicking gene delivery systems) has been employed to develop non-viral nucleic acid carriers that efficiently enhance multiple extracellular and intracellular gene delivery pathways by altering their physico-chemical properties in response to a variety of extra- and intra-cellular stimuli (e.g., pH, redox potential, and enzyme), as well as external triggers (e.g., light) [Zhu and Torchilin, 2012].

Therefore, numerous challenges remain to be overcome before gene therapy becomes available as a safe and effective treatment option. For instance, the cationic molecules (e.g., polymers, lipids and peptides) have the potential to be systemically delivered but selectivity for the target tissue needs to be validated. The best method for delivering genes will depend on the type of tissue targeted [Scanlon, 2004]. Recently, the development of virus-mimicking, multi-func‐ tional gene delivery systems is considered to be a potent strategy in the future, in particular for intravenous administration. This chapter summarizes the challenges for cancer gene therapy as well as addressing the advances in the multi-functional nano-carriers as a potent non-viral delivery system.

## **2. Gene therapy**

Medicine has a long history of treating patients with cell therapies (i.e., blood transfusions) and protein therapies (i.e., growth factors and cytokines). Gene therapies are the newest therapeutic strategy for treating human diseases especially cancer [Scanlon, 2004]. This technology has been used to develop new strategies for killing cells selectively or inhibiting their growth. The field of cancer gene therapy comprises a range of technologies from direct attack on tumor cells to inducing the immune response to tumor antigens [McCormick, 2001]. However, there are serious doubts about gene therapy; for example, short-lived nature of gene therapy, immune response to a foreign object, problems with viral vectors and insertional mutagenesis inducing a tumor [Korthof, 1999].

#### **2.1. History**

Delivery is one of the most difficult challenges facing the gene therapy field. It is needed to use an efficient transfer system that can stabilize, transduce and express a transgene in the target tissue. Recently, non-viral technologies have been widely used as a signifi‐ cant alternative for gene delivery. The non-viral delivery systems have reduced adverse immune responses, are easier to manufacture and can be produced for the pharmaceuti‐ cal industry in large quantities. The current progresses of gene therapy are further fo‐ cused on synthesized nano-particle technologies. Some of these new chemical compositions are cationic molecules such as polymers, lipids and peptides [Scanlon,

However, for successful clinical trial, ideal non-viral vectors should be degradable into low molecular weight components, in response to biological stimuli, for lowered cytotoxicity and effective systemic clearance. They should also be efficient in overcoming extracellular and intracellular barriers, tissue/cell-targeted for specific accumulations and multi-functional for synergistic therapeutic and diagnostic outcomes. Recently, a broad range of different stimuliresponsive strategies (virus-mimicking gene delivery systems) has been employed to develop non-viral nucleic acid carriers that efficiently enhance multiple extracellular and intracellular gene delivery pathways by altering their physico-chemical properties in response to a variety of extra- and intra-cellular stimuli (e.g., pH, redox potential, and enzyme), as well as external

Therefore, numerous challenges remain to be overcome before gene therapy becomes available as a safe and effective treatment option. For instance, the cationic molecules (e.g., polymers, lipids and peptides) have the potential to be systemically delivered but selectivity for the target tissue needs to be validated. The best method for delivering genes will depend on the type of tissue targeted [Scanlon, 2004]. Recently, the development of virus-mimicking, multi-func‐ tional gene delivery systems is considered to be a potent strategy in the future, in particular for intravenous administration. This chapter summarizes the challenges for cancer gene therapy as well as addressing the advances in the multi-functional nano-carriers as a potent

Medicine has a long history of treating patients with cell therapies (i.e., blood transfusions) and protein therapies (i.e., growth factors and cytokines). Gene therapies are the newest therapeutic strategy for treating human diseases especially cancer [Scanlon, 2004]. This technology has been used to develop new strategies for killing cells selectively or inhibiting their growth. The field of cancer gene therapy comprises a range of technologies from direct attack on tumor cells to inducing the immune response to tumor antigens [McCormick, 2001]. However, there are serious doubts about gene therapy; for example, short-lived nature of gene therapy, immune response to a foreign object, problems with viral vectors and insertional

2004; Gao et al., 2007].

198 Novel Gene Therapy Approaches

non-viral delivery system.

**2. Gene therapy**

triggers (e.g., light) [Zhu and Torchilin, 2012].

mutagenesis inducing a tumor [Korthof, 1999].

In 1972, Friedmann and Roblin published a paper in Science entitled as "Gene therapy for human genetic disease" [Friedmann and Roblin, 1972; Rogers, 1970]. The first approved gene therapy case in the United States took place in 1990, at the National Institute of Health. It was performed on a four year old girl with a genetic defect associated to an im‐ mune system deficiency. The effects were only temporary, but successful [Blaese et al., 1995]. In addition, sickle cell disease was successfully treated in mice [Fisher, 1995]. At the same time, the researchers were able to create tiny liposomes 25 nanometers (nm) that can carry therapeutic DNA through pores in the nuclear membrane [www. newscientist.com, 2002]. In 1992, Claudio Bordignon performed the first procedure of gene therapy using hematopoietic stem cells as vectors to deliver genes aimed to correct hereditary diseases [Abbott, 1992]. In 2002, this work led to the publication of the first successful gene thera‐ py treatment for adenosine deaminase-deficiency (SCID) [Cavazzana-Calvo et al., 2004]. In 2003, a Los Angeles research team inserted genes into the brain using liposomes coated in a polymer called polyethylene glycol (PEG). The transfer of genes into the brain is a sig‐ nificant achievement because viral vectors are too big to get across the blood–brain barri‐ er. This method had potential for treating Parkinson's disease. At the same time, RNA interference or gene silencing was considered as a new way to treat Huntington's disease [www. newscientist.com, 2003]. In 2006, scientists at the National Institutes of Health have successfully treated metastatic melanoma in two patients using killer T cells genetically re‐ targeted to attack the cancer cells. For the first time, this study demonstrated that gene therapy can be effective in treating cancer. In November 2006, Preston Nix reported on VRX496, a gene-based immunotherapy for the treatment of human immunodeficiency vi‐ rus (HIV) that uses a lentiviral vector for delivery of an antisense gene against the HIV envelope [Levine et al., 2006; www.eurekalert.org, 2009].

Leber's congenital amaurosis is an inherited blinding disease caused by mutations in the RPE65 gene. In 2007-2008, the world's first gene therapy trial was announced for inherited retinal disease. They determined the safety of the sub-retinal delivery of recombinant adenoassociated virus (AAV) carrying RPE65 gene and found the positive results in vision and without the apparent side-effects [www. news.bbc.co.uk, 2007]. In 2009, the researchers were succeeded at arresting a fatal brain disease, adrenoleukodystrophy, using a vector derived from HIV to deliver the gene for the missing enzyme [Kaiser, 2009]. A paper published in 2010, deals with gene therapy for a form of achromatopsia in dogs. Achromatopsia, or complete color blindness, is presented as an ideal model to develop gene therapy directed to cone photoreceptors [Komáromy et al., 2010]. In 2011, a study carried out using genetically modified T cells to fight the chronic lymphocytic leukemia (CLL) disease. Moreover, Human HGF plasmid DNA therapy of cardiomyocytes was examined as a potential treatment for coronary artery disease as well as myocardial infarction [www. nature.com, 2011; Yang et al., 2008; Hahn et al., 2011]. However, most of the approved European and United States gene therapy protocols are for cancer (~ 66%), in contrast to monogenetic diseases (~ 11%) and cardiovascular diseases (~ 8%). The focus of cancer gene therapy has been on melanoma, prostate and ovarian cancer and leukemia [Scanlon, 2004].

#### **2.2. The challenges of gene therapy**

Recently, the use of gene therapy in medicine using plasmid DNA (pDNA), oligodeoxynu‐ cleotide (ODN) or small interfering RNA (siRNA) represents a promising new approach for treating a variety of genetic and acquired diseases (e.g., cancer). To date, more than 1000 different gene-therapy clinical trials for the treatment of many different diseases are in progress worldwide, but, the success with gene therapy has been limited. This lack of success can be assigned to difficulties related to the effective delivery of nucleic acids into target cells [Mastrobattista et al., 2006]. Naked DNA is unable to efficiently cross cellular barriers by passive diffusion because of its large size, strong negative charge, hydrophilicity and suscept‐ ibility to nuclease attack. Hence, the major challenge for *in vitro* DNA delivery is designing suitable vectors which can protect DNA and efficiently deliver it to the targeted sites in the cell. Successful application of such therapeutic vector-DNA formulations *in vivo* requires them to be stable during circulation in blood, resistant against rapid metabolic clearance and efficiently targeted to the appropriate tissue/cell [Mann et al., 2008].

core is released into cytoplasmic space; e) the core is transferred to the nucleus and viral gene expression progresses. Due to this very efficient cell-entry mechanism, the transfection

Challenges in Advancing the Field of Cancer Gene Therapy: An Overview of the Multi-Functional Nanocarriers

http://dx.doi.org/10.5772/54862

201

However, there are several problems to overcome before this therapy can be successful in clinical settings such as a) Enhanced lytic properties of these viruses; b) Improved yields with better manufacturing procedures of production for clinical studies; c) Systemic delivery; d) limited DNA-carrying capacity; e) lack of target-cell specificity; f) immunogenicity and h) for some viral vectors, insertional mutagenesis [Scanlon, 2004; Mastrobattista et al., 2006].

There are some non-viral technologies that offer several advantages over the viral meth‐ odologies. Non-viral delivery systems have reduced adverse immune responses (relative‐ ly safe), are easier to manufacture, can be produced for the pharmaceutical industry in large quantities and modified by the incorporation of ligands for targeting to specific cell types [Scanlon, 2004; Mastrobattista et al., 2006]. Chemically synthesized nanoparticles constitute a new technology and offer several new strategies for successful systemic gene therapy delivery. Synthetic gene-delivery systems consist of a self-assembling complex of DNA with positively charged molecules (for example, polymers, peptides, lipids or their combinations). These complexes are small in size (40-150 nm) and usually have a net positive surface charge, which enables adsorption-mediated cell binding and internaliza‐ tion [Mastrobattista et al., 2006]. Some of these new chemical compositions are polymers containing either DNA/stearyl polylysine-coated lipids or peptoids (DNA coated with glycine oligomers) or cationic molecules (DNA/combined with positively charged B-cy‐ clodextrin/adamantane and PEG). These molecules have been shown to be effective in cancer-related angiogenesis. These new agents have the potential to be systemically de‐ livered, but their selectivity for the target tissue needs to be validated [Scanlon, 2004]. However, the levels of gene expression and the transfection efficiency mediated by syn‐

thetic vectors are low compared to viral vectors [Mastrobattista et al., 2006].

are already in Phase I clinical studies [Scanlon, 2004].

One of the opportunities for gene therapy is to combine therapeutic genes with a cell to overcome the delivery to target tissues. The advantages of cell delivery of therapeutic products include minimal immune response; tissue directed therapy; selectivity and improved potency of the product. However, there are several problems to be solved: a) Determining optimal transduction of cells; b) Gene-transformed cells will require a selective growth advantage over defective cells to re-populate the host; c) DNA repair genes action (minimized mutations in the gene-transformed cells); d) Genomic stability (for optimal gene expression); e) Determining cell type for therapy (e.g., embryonic stem cells or activated, differentiated cells); f) Incorpo‐ ration of a safety mechanism (*i.e.* a suicide gene) to destroy the gene-transformed cells if a problem arises. In addition, other cell therapies are currently being developed using bacteria, such as modified *Salmonella*, for gene delivery in cancer patients. These modified bacterial cells

efficiencies of viral vectors remain uniqe [Sasaki et al., 2008].

**3.2. Non-viral delivery**

**3.3. Cell delivery**

### **3. Delivery systems in gene therapy**

#### **3.1. Viral delivery**

Delivery is one of the most difficult challenges facing the gene therapy field. An efficient transfer system has not yet been found to stabilize, transduce and express a transgene in the target tissue. Limitations of the present vector technologies have slowed the progress of clinical gene therapy [Scanlon, 2004]. Various viruses, such as *influenza* viruses and *adenoviruses*, can efficiently deliver genes into the nucleus via sophisticated mechanisms. Despite the potent immunogenicity of viral vectors, their developed cell entry mechanism and high transfection efficiency in both dividing and non-dividing cells is desirable [Wagner, 2011; Mastrobattista et al., 2006]. All the viral gene strategies used to date have significant delivery limitations. The best method for delivering genes may depend on the type of targeted tissue. There are some promising delivery technologies for viral therapies, including the use of replication competent viruses. *Adenoviruses*, *herpes simplex virus* and *Newcastle disease virus* have all been modified for replication competent properties in human tumor cells. This has been one of the most popular areas in gene therapy and offers promises for treating cancer, especially when combined with chemotherapy [Scanlon, 2004]. Recently, the viral vectors have been developed into gene vectors and have provided convincing successes in gene therapy. Viruses have developed mechanisms to survive in the extracellular environment, attach to cells, cross cellular mem‐ branes, steal intracellular transport systems and subsequently deliver their genomes into the appropriate sub-cellular compartment (e.g., cytosol or nucleus) [Wagner, 2011; Sasaki et al., 2008]. For example, *influenza viruses* infect cells in a multi-step process: a) the virus binds to a receptor on the cell surface mediated by hemagglutinin (HA) protein; b) the virus invades via receptor-mediated endocytosis; c) the internalized virus is trafficked to a late endosome; d) the acidic endosomal environment induces membrane fusion between the virus and endosome, which is brought about by a conformational change of HA and the ribonucleoprotein complex core is released into cytoplasmic space; e) the core is transferred to the nucleus and viral gene expression progresses. Due to this very efficient cell-entry mechanism, the transfection efficiencies of viral vectors remain uniqe [Sasaki et al., 2008].

However, there are several problems to overcome before this therapy can be successful in clinical settings such as a) Enhanced lytic properties of these viruses; b) Improved yields with better manufacturing procedures of production for clinical studies; c) Systemic delivery; d) limited DNA-carrying capacity; e) lack of target-cell specificity; f) immunogenicity and h) for some viral vectors, insertional mutagenesis [Scanlon, 2004; Mastrobattista et al., 2006].

#### **3.2. Non-viral delivery**

**2.2. The challenges of gene therapy**

200 Novel Gene Therapy Approaches

Recently, the use of gene therapy in medicine using plasmid DNA (pDNA), oligodeoxynu‐ cleotide (ODN) or small interfering RNA (siRNA) represents a promising new approach for treating a variety of genetic and acquired diseases (e.g., cancer). To date, more than 1000 different gene-therapy clinical trials for the treatment of many different diseases are in progress worldwide, but, the success with gene therapy has been limited. This lack of success can be assigned to difficulties related to the effective delivery of nucleic acids into target cells [Mastrobattista et al., 2006]. Naked DNA is unable to efficiently cross cellular barriers by passive diffusion because of its large size, strong negative charge, hydrophilicity and suscept‐ ibility to nuclease attack. Hence, the major challenge for *in vitro* DNA delivery is designing suitable vectors which can protect DNA and efficiently deliver it to the targeted sites in the cell. Successful application of such therapeutic vector-DNA formulations *in vivo* requires them to be stable during circulation in blood, resistant against rapid metabolic clearance and

Delivery is one of the most difficult challenges facing the gene therapy field. An efficient transfer system has not yet been found to stabilize, transduce and express a transgene in the target tissue. Limitations of the present vector technologies have slowed the progress of clinical gene therapy [Scanlon, 2004]. Various viruses, such as *influenza* viruses and *adenoviruses*, can efficiently deliver genes into the nucleus via sophisticated mechanisms. Despite the potent immunogenicity of viral vectors, their developed cell entry mechanism and high transfection efficiency in both dividing and non-dividing cells is desirable [Wagner, 2011; Mastrobattista et al., 2006]. All the viral gene strategies used to date have significant delivery limitations. The best method for delivering genes may depend on the type of targeted tissue. There are some promising delivery technologies for viral therapies, including the use of replication competent viruses. *Adenoviruses*, *herpes simplex virus* and *Newcastle disease virus* have all been modified for replication competent properties in human tumor cells. This has been one of the most popular areas in gene therapy and offers promises for treating cancer, especially when combined with chemotherapy [Scanlon, 2004]. Recently, the viral vectors have been developed into gene vectors and have provided convincing successes in gene therapy. Viruses have developed mechanisms to survive in the extracellular environment, attach to cells, cross cellular mem‐ branes, steal intracellular transport systems and subsequently deliver their genomes into the appropriate sub-cellular compartment (e.g., cytosol or nucleus) [Wagner, 2011; Sasaki et al., 2008]. For example, *influenza viruses* infect cells in a multi-step process: a) the virus binds to a receptor on the cell surface mediated by hemagglutinin (HA) protein; b) the virus invades via receptor-mediated endocytosis; c) the internalized virus is trafficked to a late endosome; d) the acidic endosomal environment induces membrane fusion between the virus and endosome, which is brought about by a conformational change of HA and the ribonucleoprotein complex

efficiently targeted to the appropriate tissue/cell [Mann et al., 2008].

**3. Delivery systems in gene therapy**

**3.1. Viral delivery**

There are some non-viral technologies that offer several advantages over the viral meth‐ odologies. Non-viral delivery systems have reduced adverse immune responses (relative‐ ly safe), are easier to manufacture, can be produced for the pharmaceutical industry in large quantities and modified by the incorporation of ligands for targeting to specific cell types [Scanlon, 2004; Mastrobattista et al., 2006]. Chemically synthesized nanoparticles constitute a new technology and offer several new strategies for successful systemic gene therapy delivery. Synthetic gene-delivery systems consist of a self-assembling complex of DNA with positively charged molecules (for example, polymers, peptides, lipids or their combinations). These complexes are small in size (40-150 nm) and usually have a net positive surface charge, which enables adsorption-mediated cell binding and internaliza‐ tion [Mastrobattista et al., 2006]. Some of these new chemical compositions are polymers containing either DNA/stearyl polylysine-coated lipids or peptoids (DNA coated with glycine oligomers) or cationic molecules (DNA/combined with positively charged B-cy‐ clodextrin/adamantane and PEG). These molecules have been shown to be effective in cancer-related angiogenesis. These new agents have the potential to be systemically de‐ livered, but their selectivity for the target tissue needs to be validated [Scanlon, 2004]. However, the levels of gene expression and the transfection efficiency mediated by syn‐ thetic vectors are low compared to viral vectors [Mastrobattista et al., 2006].

#### **3.3. Cell delivery**

One of the opportunities for gene therapy is to combine therapeutic genes with a cell to overcome the delivery to target tissues. The advantages of cell delivery of therapeutic products include minimal immune response; tissue directed therapy; selectivity and improved potency of the product. However, there are several problems to be solved: a) Determining optimal transduction of cells; b) Gene-transformed cells will require a selective growth advantage over defective cells to re-populate the host; c) DNA repair genes action (minimized mutations in the gene-transformed cells); d) Genomic stability (for optimal gene expression); e) Determining cell type for therapy (e.g., embryonic stem cells or activated, differentiated cells); f) Incorpo‐ ration of a safety mechanism (*i.e.* a suicide gene) to destroy the gene-transformed cells if a problem arises. In addition, other cell therapies are currently being developed using bacteria, such as modified *Salmonella*, for gene delivery in cancer patients. These modified bacterial cells are already in Phase I clinical studies [Scanlon, 2004].

## **4. The challenges of nucleic acid delivery**

There are many biological barriers for efficient gene delivery that need to be overcome [Mastrobattista et al., 2006]. This inefficiency of gene delivery is primarily a result of the inability of these vectors to overcome the numerous barriers encountered between the site of administration and localization in the cell nucleus. This series of barriers to efficient non-viral gene delivery is thought to include: a) the physical and chemical stability of DNA and its delivery vehicle in the extracellular space, b) cellular uptake by endocytosis, c) escape from the endosomal compartments prior to trafficking to lysosomes, d) cytosolic transport and e) nuclear localization of the plasmid for transcription. In addition to these physical and chemical obstacles, biological barriers, such as immunogenic responses to the vector itself as well as immune stimulation by certain DNA sequences containing a central un-methylated CpG motif, are present. The studies have shown that it is possible to minimize biological barriers by optimizing the plasmid sequence, thus physical and chemical barriers appear to be the negative factors to successful non-viral gene delivery [Wiethoff and Middaugh, 2003]. These barriers are briefly described as following:

in a variety of cellular processes, including differentiation, adhesion and migration. More recently they have been found to mediate the binding and internalization of several vi‐ ruses, including HIV-1, HSV-2, AAV-2, and *adenovirus*. The composition of heparin sul‐ fate proteoglycans (HSPGs) includes a protein core with one or more attached

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In general, binding of nano-carrier to the cell surfaces is not, a problem if the gene carriers have a net positive surface charge, which readily induces adsorption onto negatively charged cell membranes. However, this form of binding is random and does not allow restricted delivery to target cells (e.g., tumor cells) [Mastrobattista et al., 2006]. Several additional strategies have been attempted to increase the specificity of DNA binding to cells and induce targeting to particular cell types [Wiethoff and Middaugh, 2003]. Active targeting can be achieved by the functionalization of NPs with ligands such as antibodies, peptides, nucleic acids (aptamers),

Since many chemotherapeutic drugs and particularly gene therapeutics, would benefit from intracellular targeting, nano-carrier systems can be designed for receptor-mediated cell uptake, intracellular drug protection and intracellular target delivery [van Vlerken et al., 2007]. Once inside the cell, several intracellular barriers need to be crossed before the foreign DNA can be transcribed and translated [Mastrobattista et al., 2006]. These barriers contain endosomal escape, cytosolic transport of DNA and nuclear localization of plasmid DNA that

The endocytic pathway is one of the uptake mechanisms of cells. In general, non-viral nano vectors have been developed to mimic the receptor-mediated cell entry mechanism of viruses and the main mechanism of internalization was confirmed to be endocytosis. This pathway is composed of vesicles known as endosomes with an internal pH around 5 that mature in a unidirectional manner from early endosomes to late endosomes before fusing with intracellular organelles called lysosomes which contain certain digestive enzymes. Thus, particles entering the cells via the endocytic pathway become entrapped in endosomes and eventually end up in the lysosome, where active enzymatic degradation processes take place [Varkouhi et al., 2011]. The entrapment of internalized DNA carriers in endocytic compartments prevents further intracellular transport towards the nucleus and will often result in degradation of the carrier and its associated DNA in the endosomal/lysosomal compartments [Mastrobattista et al., 2006]. While many viruses have evolved quite efficient systems for endosomal release, the situation is different for non-viral vectors, where in many cases the lack of endosomal escape is a major obstacle for efficient biological delivery, implying that more efficient methods for endosomal release would lead to improvements in designing synthetic transfection systems. In contrast to synthetic vectors, viral vectors are known to be efficient both for *in vitro* and *in*

glycosaminoglycans [Wiethoff and Middaugh, 2003].

carbohydrates and small molecules [Gu et al., 2007].

described briefly in below:

*vivo* applications [Varkouhi et al., 2011].

*4.3.1. Endosomal escape*

**4.3. Intracellular trafficking of non-viral gene delivery systems**

#### **4.1. Stability in extracellular compartments**

The stability of non-viral delivery systems in the extracellular milieu, such as intercellular or intravascular spaces, is related to the chemical stability of the DNA as well as the physical stability of the delivery system [Wiethoff and Middaugh, 2003]. In extracellular environment, the carrier is exposed to blood components such as nucleases. This can result in premature destabilization with simultaneous release and degradation of the plasmid DNA. The half-life of naked plasmid DNA in blood is on the order of minutes. Therefore, carrier-mediated protection during transport through the blood circulation is a prerequisite to make the DNA inaccessible to degradative enzymes [Mastrobattista et al., 2006]. Condensing the DNA with a variety of polycations or by complexing with polymers that bind to DNA protects it from degradation [Wiethoff and Middaugh, 2003].

#### **4.2. Cellular association of DNA**

Irrespective of the injection route, the gene carriers should be able to bind to cells for al‐ lowing cellular uptake [Mastrobattista et al., 2006]. Association of DNA with the cell sur‐ face is typically very low in the absence of any delivery agent as an immediate consequence of the relatively high negative charge density of both the DNA and the cell surface. Polycations have been shown to substantially increase the cellular association of DNA by neutralization of the DNA negative charge, with the charge ratio of the com‐ plex modulating the extent of this contact. The degree of cellular association has also been shown to be related to the colloidal stability of these delivery systems, with those that aggregate often manifesting a greater degree of cellular association *in vitro*. The as‐ sociation of non-viral gene delivery systems containing either cationic lipids or polymers is thought to be mediated by interactions with cell surface heparin sulfate proteoglycans (HSPGs). These proteoglycans are ubiquitous to the surface of all cells and are involved in a variety of cellular processes, including differentiation, adhesion and migration. More recently they have been found to mediate the binding and internalization of several vi‐ ruses, including HIV-1, HSV-2, AAV-2, and *adenovirus*. The composition of heparin sul‐ fate proteoglycans (HSPGs) includes a protein core with one or more attached glycosaminoglycans [Wiethoff and Middaugh, 2003].

In general, binding of nano-carrier to the cell surfaces is not, a problem if the gene carriers have a net positive surface charge, which readily induces adsorption onto negatively charged cell membranes. However, this form of binding is random and does not allow restricted delivery to target cells (e.g., tumor cells) [Mastrobattista et al., 2006]. Several additional strategies have been attempted to increase the specificity of DNA binding to cells and induce targeting to particular cell types [Wiethoff and Middaugh, 2003]. Active targeting can be achieved by the functionalization of NPs with ligands such as antibodies, peptides, nucleic acids (aptamers), carbohydrates and small molecules [Gu et al., 2007].

#### **4.3. Intracellular trafficking of non-viral gene delivery systems**

Since many chemotherapeutic drugs and particularly gene therapeutics, would benefit from intracellular targeting, nano-carrier systems can be designed for receptor-mediated cell uptake, intracellular drug protection and intracellular target delivery [van Vlerken et al., 2007]. Once inside the cell, several intracellular barriers need to be crossed before the foreign DNA can be transcribed and translated [Mastrobattista et al., 2006]. These barriers contain endosomal escape, cytosolic transport of DNA and nuclear localization of plasmid DNA that described briefly in below:

#### *4.3.1. Endosomal escape*

**4. The challenges of nucleic acid delivery**

202 Novel Gene Therapy Approaches

are briefly described as following:

**4.1. Stability in extracellular compartments**

degradation [Wiethoff and Middaugh, 2003].

**4.2. Cellular association of DNA**

There are many biological barriers for efficient gene delivery that need to be overcome [Mastrobattista et al., 2006]. This inefficiency of gene delivery is primarily a result of the inability of these vectors to overcome the numerous barriers encountered between the site of administration and localization in the cell nucleus. This series of barriers to efficient non-viral gene delivery is thought to include: a) the physical and chemical stability of DNA and its delivery vehicle in the extracellular space, b) cellular uptake by endocytosis, c) escape from the endosomal compartments prior to trafficking to lysosomes, d) cytosolic transport and e) nuclear localization of the plasmid for transcription. In addition to these physical and chemical obstacles, biological barriers, such as immunogenic responses to the vector itself as well as immune stimulation by certain DNA sequences containing a central un-methylated CpG motif, are present. The studies have shown that it is possible to minimize biological barriers by optimizing the plasmid sequence, thus physical and chemical barriers appear to be the negative factors to successful non-viral gene delivery [Wiethoff and Middaugh, 2003]. These barriers

The stability of non-viral delivery systems in the extracellular milieu, such as intercellular or intravascular spaces, is related to the chemical stability of the DNA as well as the physical stability of the delivery system [Wiethoff and Middaugh, 2003]. In extracellular environment, the carrier is exposed to blood components such as nucleases. This can result in premature destabilization with simultaneous release and degradation of the plasmid DNA. The half-life of naked plasmid DNA in blood is on the order of minutes. Therefore, carrier-mediated protection during transport through the blood circulation is a prerequisite to make the DNA inaccessible to degradative enzymes [Mastrobattista et al., 2006]. Condensing the DNA with a variety of polycations or by complexing with polymers that bind to DNA protects it from

Irrespective of the injection route, the gene carriers should be able to bind to cells for al‐ lowing cellular uptake [Mastrobattista et al., 2006]. Association of DNA with the cell sur‐ face is typically very low in the absence of any delivery agent as an immediate consequence of the relatively high negative charge density of both the DNA and the cell surface. Polycations have been shown to substantially increase the cellular association of DNA by neutralization of the DNA negative charge, with the charge ratio of the com‐ plex modulating the extent of this contact. The degree of cellular association has also been shown to be related to the colloidal stability of these delivery systems, with those that aggregate often manifesting a greater degree of cellular association *in vitro*. The as‐ sociation of non-viral gene delivery systems containing either cationic lipids or polymers is thought to be mediated by interactions with cell surface heparin sulfate proteoglycans (HSPGs). These proteoglycans are ubiquitous to the surface of all cells and are involved The endocytic pathway is one of the uptake mechanisms of cells. In general, non-viral nano vectors have been developed to mimic the receptor-mediated cell entry mechanism of viruses and the main mechanism of internalization was confirmed to be endocytosis. This pathway is composed of vesicles known as endosomes with an internal pH around 5 that mature in a unidirectional manner from early endosomes to late endosomes before fusing with intracellular organelles called lysosomes which contain certain digestive enzymes. Thus, particles entering the cells via the endocytic pathway become entrapped in endosomes and eventually end up in the lysosome, where active enzymatic degradation processes take place [Varkouhi et al., 2011]. The entrapment of internalized DNA carriers in endocytic compartments prevents further intracellular transport towards the nucleus and will often result in degradation of the carrier and its associated DNA in the endosomal/lysosomal compartments [Mastrobattista et al., 2006]. While many viruses have evolved quite efficient systems for endosomal release, the situation is different for non-viral vectors, where in many cases the lack of endosomal escape is a major obstacle for efficient biological delivery, implying that more efficient methods for endosomal release would lead to improvements in designing synthetic transfection systems. In contrast to synthetic vectors, viral vectors are known to be efficient both for *in vitro* and *in vivo* applications [Varkouhi et al., 2011].

#### *4.3.2. Cytosolic transport of DNA*

After endosomal escape, DNA must traverse the cytosol to access the nucleus. Those DNA carriers that manage to escape the endosomal compartments are then challenged by the complex environment of the cytosol, which contains many filamentous struc‐ tures that hinder the free diffusion of large particles such as DNA carriers. Dissocia‐ tion of the carrier at this stage might be required to allow further transport of the plasmid DNA molecules. However, several studies have found evidence that plasmid DNA is largely immobile in the cytosol and is rapidly degraded by cytosolic nucleases [Mastrobattista et al., 2006]. Diffusion of DNA in the cytoplasm has been found to be substantially less than that observed in dilute solution. For DNA >2000 base pairs in length, the diffusion co-efficient in the cytosol is <1% of that in water, suggesting a substantial diffusional barrier. This decreased mobility has been ascribed to molecular crowding of the plasmid, but may also reflect an increased viscosity of the cytoplasm. As expected, the diffusion coefficient of DNA in the cytoplasm is inversely related to the size of the plasmid, suggesting smaller plasmids may be more desirable. No evi‐ dence for active transport of DNA in the cytoplasm has been reported. In addition to the considerable diffusional barrier for DNA in the cytosol, the presence of calciumsensitive cytosolic nucleases pose a significant metabolic barrier. Micro-injection of DNA into the cytoplasm results in significant degradation of the DNA with a half-life of 50-90 min [Wiethoff and Middaugh, 2003].

which explains the increased levels of transfection in dividing cells as compared to their growth-arrested counterparts. However, as most cells do not divide or divide only slowly, an active transport mechanism is needed to carry the DNA from the cytosol into the nucleus

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An important obstacle to effective non-viral gene delivery is the cytotoxicity of the delivery vectors [Wiethoff and Middaugh, 2003]. Inside the nucleus, the transgene encoded on the plasmid vector should be expressed to establish therapeutic levels of recombinant proteins within the affected cell. This requires gene transcription regulatory elements such as promoters and enhancers, to drive the expression of the transgene in mammalian cells. Viral promoters are often used because of their strong transcriptional activation. However, their constitutive nature does not allow control over the level of transgene expression. For the expression of proteins with a narrow therapeutic window, tight control over the level of transgene expres‐ sion is essential. In addition, the introduction of foreign DNA into mammalian cells can induce a profound immune response, presumably triggered by differences in the degree of methyla‐ tion of the foreign DNA compared with the mammalian genome [Mastrobattista et al., 2006]. Induction of innate immune responses by un-methylated CpG sequences in plasmid DNA is perhaps a more serious concern because these species have been demonstrated to greatly reduce the efficiency of non-viral gene delivery. This immunotoxicity is thought to be related to the efficient transfection of immune cells because polycation/DNA complexes evoke a considerably greater immune response than DNA or cationic vector alone. Initial observations suggest that delivery systems that are phagocytosed, due to their large size, may promote a greater immune response. Removal of CpG motifs from DNA containing the transgene has proven a successful means of improving gene expression. The methods involved in producing vectors without these motifs are not insignificant, and currently present a major limitation to their widespread usage. It has therefore been proposed that avoidance of transfection of immune cells either by specific targeting to particular cell types or by manipulation of the physicochemical properties of the delivery systems is necessary for significant improvements

Currently used pharmaceutical nanocarriers, such as liposomes, micelles, nanoemulsions, polymeric nano-particles and many others demonstrate a broad variety of useful properties including: a) longevity in the blood allowing for their accumulation in pathological areas; b) specific targeting to certain disease sites due to various targeting ligands attached to the surface of the nanocarriers; c) enhanced intracellular penetration with the help of surface-attached cellpenetrating molecules; d) contrast properties due to the carrier loading with various contrast materials allowing for direct carrier visualization *in vivo*; e) stimuli-sensitivity allowing for drug release from the carriers under certain physiological conditions and etc. Some of those pharmaceutical carriers have already made their way into clinic, while others are still under

[Mastrobattista et al., 2006].

**4.4. Toxicity of non-viral delivery system**

in non-viral gene delivery [Wiethoff and Middaugh, 2003].

**5. Multi-functional nanocarriers**

#### *4.3.3. Nuclear localization of plasmid DNA*

Ultimately, delivery of DNA to the nucleus must occur for transcription of the transgene to take place. The mechanism of DNA nuclear translocation and whether the DNA is still associated with the delivery system are not fully understood but appear to depend on the type of delivery vehicle employed. At least three possible routes exist for DNA transport to the nucleus. The DNA can pass into the nucleus through nuclear pores, it can become physically associated with chromatin during mitosis when the nuclear envelope breaks down or it could traverse the nuclear envelope. Of these three possibilities, the latter seems impossible and without experimental support. Nuclear pores are embedded in the nuclear envelope at fairly high surface densities (3000-4000/nucleus) and exist in at least two conformational states. The closed state permits the passive diffusion of molecules of < 9 nm in diameter, whereas the open state facilitates transport of particles < 26 nm. This latter state could certainly assist the ''threading'' of supercoiled plasmid through the nuclear pore but not passage of typical nonviral gene delivery complexes [Wiethoff and Middaugh, 2003]. Small molecules (< 40 kDa) can diffuse freely through the pores of the nuclear pore complexes (NPC), whereas larger mole‐ cules and particles (up to 40 nm in size) can only be imported through the NPC by an active transport mechanism [Mastrobattista et al., 2006]. In a few cases, collapsed particles of < 30 nm have been produced, which could presumably enter the nucleus by this mechanism. The second and perhaps quite widespread mechanism by which DNA is thought to gain access to the nucleus is by association with nuclear material on breakdown of the nuclear envelope during mitosis [Wiethoff and Middaugh, 2003]. In this case, the nuclear barrier breaks down, which explains the increased levels of transfection in dividing cells as compared to their growth-arrested counterparts. However, as most cells do not divide or divide only slowly, an active transport mechanism is needed to carry the DNA from the cytosol into the nucleus [Mastrobattista et al., 2006].

#### **4.4. Toxicity of non-viral delivery system**

*4.3.2. Cytosolic transport of DNA*

204 Novel Gene Therapy Approaches

of 50-90 min [Wiethoff and Middaugh, 2003].

*4.3.3. Nuclear localization of plasmid DNA*

After endosomal escape, DNA must traverse the cytosol to access the nucleus. Those DNA carriers that manage to escape the endosomal compartments are then challenged by the complex environment of the cytosol, which contains many filamentous struc‐ tures that hinder the free diffusion of large particles such as DNA carriers. Dissocia‐ tion of the carrier at this stage might be required to allow further transport of the plasmid DNA molecules. However, several studies have found evidence that plasmid DNA is largely immobile in the cytosol and is rapidly degraded by cytosolic nucleases [Mastrobattista et al., 2006]. Diffusion of DNA in the cytoplasm has been found to be substantially less than that observed in dilute solution. For DNA >2000 base pairs in length, the diffusion co-efficient in the cytosol is <1% of that in water, suggesting a substantial diffusional barrier. This decreased mobility has been ascribed to molecular crowding of the plasmid, but may also reflect an increased viscosity of the cytoplasm. As expected, the diffusion coefficient of DNA in the cytoplasm is inversely related to the size of the plasmid, suggesting smaller plasmids may be more desirable. No evi‐ dence for active transport of DNA in the cytoplasm has been reported. In addition to the considerable diffusional barrier for DNA in the cytosol, the presence of calciumsensitive cytosolic nucleases pose a significant metabolic barrier. Micro-injection of DNA into the cytoplasm results in significant degradation of the DNA with a half-life

Ultimately, delivery of DNA to the nucleus must occur for transcription of the transgene to take place. The mechanism of DNA nuclear translocation and whether the DNA is still associated with the delivery system are not fully understood but appear to depend on the type of delivery vehicle employed. At least three possible routes exist for DNA transport to the nucleus. The DNA can pass into the nucleus through nuclear pores, it can become physically associated with chromatin during mitosis when the nuclear envelope breaks down or it could traverse the nuclear envelope. Of these three possibilities, the latter seems impossible and without experimental support. Nuclear pores are embedded in the nuclear envelope at fairly high surface densities (3000-4000/nucleus) and exist in at least two conformational states. The closed state permits the passive diffusion of molecules of < 9 nm in diameter, whereas the open state facilitates transport of particles < 26 nm. This latter state could certainly assist the ''threading'' of supercoiled plasmid through the nuclear pore but not passage of typical nonviral gene delivery complexes [Wiethoff and Middaugh, 2003]. Small molecules (< 40 kDa) can diffuse freely through the pores of the nuclear pore complexes (NPC), whereas larger mole‐ cules and particles (up to 40 nm in size) can only be imported through the NPC by an active transport mechanism [Mastrobattista et al., 2006]. In a few cases, collapsed particles of < 30 nm have been produced, which could presumably enter the nucleus by this mechanism. The second and perhaps quite widespread mechanism by which DNA is thought to gain access to the nucleus is by association with nuclear material on breakdown of the nuclear envelope during mitosis [Wiethoff and Middaugh, 2003]. In this case, the nuclear barrier breaks down, An important obstacle to effective non-viral gene delivery is the cytotoxicity of the delivery vectors [Wiethoff and Middaugh, 2003]. Inside the nucleus, the transgene encoded on the plasmid vector should be expressed to establish therapeutic levels of recombinant proteins within the affected cell. This requires gene transcription regulatory elements such as promoters and enhancers, to drive the expression of the transgene in mammalian cells. Viral promoters are often used because of their strong transcriptional activation. However, their constitutive nature does not allow control over the level of transgene expression. For the expression of proteins with a narrow therapeutic window, tight control over the level of transgene expres‐ sion is essential. In addition, the introduction of foreign DNA into mammalian cells can induce a profound immune response, presumably triggered by differences in the degree of methyla‐ tion of the foreign DNA compared with the mammalian genome [Mastrobattista et al., 2006]. Induction of innate immune responses by un-methylated CpG sequences in plasmid DNA is perhaps a more serious concern because these species have been demonstrated to greatly reduce the efficiency of non-viral gene delivery. This immunotoxicity is thought to be related to the efficient transfection of immune cells because polycation/DNA complexes evoke a considerably greater immune response than DNA or cationic vector alone. Initial observations suggest that delivery systems that are phagocytosed, due to their large size, may promote a greater immune response. Removal of CpG motifs from DNA containing the transgene has proven a successful means of improving gene expression. The methods involved in producing vectors without these motifs are not insignificant, and currently present a major limitation to their widespread usage. It has therefore been proposed that avoidance of transfection of immune cells either by specific targeting to particular cell types or by manipulation of the physicochemical properties of the delivery systems is necessary for significant improvements in non-viral gene delivery [Wiethoff and Middaugh, 2003].

## **5. Multi-functional nanocarriers**

Currently used pharmaceutical nanocarriers, such as liposomes, micelles, nanoemulsions, polymeric nano-particles and many others demonstrate a broad variety of useful properties including: a) longevity in the blood allowing for their accumulation in pathological areas; b) specific targeting to certain disease sites due to various targeting ligands attached to the surface of the nanocarriers; c) enhanced intracellular penetration with the help of surface-attached cellpenetrating molecules; d) contrast properties due to the carrier loading with various contrast materials allowing for direct carrier visualization *in vivo*; e) stimuli-sensitivity allowing for drug release from the carriers under certain physiological conditions and etc. Some of those pharmaceutical carriers have already made their way into clinic, while others are still under preclinical development. However, the combination of pharmaceutical nanocarriers with several mentioned abilities, are rare. For example, long-circulating immunoliposomes capable of prolonged residence in the blood and specific target recognition represent one of few examples of this kind. At the same time, the engineering of multi-functional pharmaceutical nanocarriers combinig several useful properties in one particle can significantly enhance the efficacy of many therapeutic and diagnostic protocols [Torchilin, 2006].

**6. Different types of nanocarrier systems**

stimuli-responsive release properties [Jabr-Milane et al., 2008].

circulation and achieve passive targeted delivery [Jabr-Milane et al., 2008].

Recently, nanoparticulate systems have been also developed for therapeutic gene delivery. Some researchers have developed glutathione-responsive nanoparticles for the delivery of plasmid DNA. The nanocarrier platform consisted of thiolated gelatin which was synthe‐ sized using 2-iminothiolane to covalently modify type B gelatin (pI ~ 4.5). The release pro‐ files of cross-linked and non-cross-linked thiolated gelatin nanoparticles and gelatin nanoparticles (control) loaded with fluorescein isothiocyanate-conjugated dextran (FITCdextran) were assessed in the presence of different glutathione concentrations (from 0.1 mM to 5 mM). A higher percentage of FITC–dextran was released from non-crosslinked nanoparticles compared to the cross-linked nanoparticles. The rate of FITC–dextran release from thiolated gelatin nanoparticles enhanced with increasing concentrations of gluta‐

Over the last eight years, a laboratory at Northeastern University has developed an array of multi-functional nanocarriers for the delivery of genes, drugs and imaging modalities. These flexible platforms consist of polymeric and lipid systems that combine different modalities and

Challenges in Advancing the Field of Cancer Gene Therapy: An Overview of the Multi-Functional Nanocarriers

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The diagnostic and/or therapeutic objectives of a multi-functional nanocarrier system deter‐ mine the design of the formulation. A review of the literature shows that there are many different types of nanocarrier formulations for the diagnosis, imaging and treatment of a wide spectrum of diseases. These multi-functional carriers share three main design compo‐ nents: platform (core) material, encapsulated payload/biologically active agents and target‐ ing/surface properties. Nanocarrier platforms can be categorized as organic-based, inorganic-based or a hybrid combination. Organic nano-platforms include polymeric nano‐ carriers, lipid-based nanocarriers (e.g., liposomes and nanoemulsions), dendrimers and car‐ bon-based nanocarriers (e.g., fullerenes and carbon nanotubes) [Jabr-Milane et al., 2008]. Inorganic nano-platforms include metallic nanostructures, silica nanoparticles and quan‐ tum dots. An example of a hybrid platform is colloidal gold encapsulated in liposomes or superparamagnetic iron oxide particles encapsulated in polymeric nanoparticles. Selection of the core material is highly dependent on the properties of the biologically active agents. Inherent and dynamic properties of the agents such as therapeutic index, lipophilicity, charge and size should be considered. When combining therapeutic agents with each other, with an imaging/diagnostic modality, or with energy delivery, the interaction of system components (i.e., synergy, quenching, enhanced toxicity) and release kinetics should also be considered. Surface properties are the third design component of multi-functional nano‐ carriers. A common surface modification technique that decreases reticuloendothelial sys‐ tem (RES) clearance is the physical or covalent attachment of PEG chains to the nanocarrier platform. Since tumor microvasculature is known to be highly fenestrated, colloidal parti‐ cles can accumulate by the enhanced permeability and retention (EPR) effect. PEG surface modification increases circulatory residence time, which increases the probability of accu‐ mulation at the target. Block co-polymers of poly (ethylene oxide) (PEO) and poly (propy‐ lene oxide) (PPO) (e.g., Pluronics®) have also been used as surface conjugates to enhance

The use of cationic lipids and cationic polymers as transfection vectors for efficient intracellular delivery of DNA was suggested in 1987. Complexes between cationic lipids (such as Lipofec‐ tin®, an equimolar mixture of N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride–DOTMA and dioleoyl phosphatidylethanolamine –DOPE) and DNA (lipoplexes) and complexes between cationic polymers, such as PEI and DNA (polyplexes) are formed because of strong electrostatic interactions between the positively charged carrier and nega‐ tively charged DNA. A slight net positive charge of formed lipoplexes and polyplexes is believed to facilitate their interaction with negatively charged cells and improve transfection efficiency. Endocytosis (including the receptor mediated endocytosis) was repeatedly con‐ firmed as the main mechanism of lipoplex/polyplex internalization by cells [Torchilin, 2006]. Of special importance is the fact that despite of endocytosis-mediated uptake lipolexes and polyplexes, DNA does not end in lysosomes but releases in the cytoplasm due to the destabi‐ lization of the endosomal membrane provoked by lipid or polymeric component of the complexes. In particular, lipoplexes fuse with the endosomal membrane when they contain a fusogenic lipid, dioleylphosphatidylethanolamine (DOPE), which easily undergoes the transition from bilayer to hexagonal phase facilitating the fusion. In case of polyplexes, which cannot directly destabilize the endosomal membrane, the mechanism of DNA escape from endosomes, is associated with the ability of polymers, strongly protonate under the acidic pH inside endosome and create a charge gradient ultimately provoking a water influx and endosomal swelling and disintegration. In both cases, DNA-containing complexes when released into the cytosol, dissociate allowing for nuclear entry of free DNA. Nuclear translo‐ cation of the plasmid DNA is relatively inefficient because of the barrier function of the nuclear membrane and small size of nuclear pores (25 nm), as well as DNA degrades fast under the action of cytoplasmic nucleases. It was estimated that only 0.1% of plasmids undergo nuclear translocation from the cytosol. The attachment of nuclear localization sequences to plasmid DNA may significantly enhance its nuclear translocation and transfection efficiency. New approaches in using multifunctional carriers for DNA delivery include the application of bimetallic nano-rods that can simultaneously bind compacted DNA plasmid and targeting ligands in a spatially defined manner [Torchilin, 2006].

DNA–lipid amphiphiles self-assemble into novel "DNAsomes"-liposome-like core-shell structures with subunits composed of branched DNA-lipid hybrid molecules. These DNA‐ somes can be precisely modified over a wide range in terms of both size and surface charge. More importantly, DNAsome is a natural carrier of small interfering RNA (siRNA) due to DNA-RNA base-pairing, enabling efficient co-delivery of drugs and siRNA. The DNAsome represents a universal multi-functional drug vector for simultaneous delivery of drugs, tracer dyes, or antibodies, along with genes, siRNA or antisense nucleic acids [Roh et al., 2011].

## **6. Different types of nanocarrier systems**

preclinical development. However, the combination of pharmaceutical nanocarriers with several mentioned abilities, are rare. For example, long-circulating immunoliposomes capable of prolonged residence in the blood and specific target recognition represent one of few examples of this kind. At the same time, the engineering of multi-functional pharmaceutical nanocarriers combinig several useful properties in one particle can significantly enhance the

The use of cationic lipids and cationic polymers as transfection vectors for efficient intracellular delivery of DNA was suggested in 1987. Complexes between cationic lipids (such as Lipofec‐ tin®, an equimolar mixture of N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride–DOTMA and dioleoyl phosphatidylethanolamine –DOPE) and DNA (lipoplexes) and complexes between cationic polymers, such as PEI and DNA (polyplexes) are formed because of strong electrostatic interactions between the positively charged carrier and nega‐ tively charged DNA. A slight net positive charge of formed lipoplexes and polyplexes is believed to facilitate their interaction with negatively charged cells and improve transfection efficiency. Endocytosis (including the receptor mediated endocytosis) was repeatedly con‐ firmed as the main mechanism of lipoplex/polyplex internalization by cells [Torchilin, 2006]. Of special importance is the fact that despite of endocytosis-mediated uptake lipolexes and polyplexes, DNA does not end in lysosomes but releases in the cytoplasm due to the destabi‐ lization of the endosomal membrane provoked by lipid or polymeric component of the complexes. In particular, lipoplexes fuse with the endosomal membrane when they contain a fusogenic lipid, dioleylphosphatidylethanolamine (DOPE), which easily undergoes the transition from bilayer to hexagonal phase facilitating the fusion. In case of polyplexes, which cannot directly destabilize the endosomal membrane, the mechanism of DNA escape from endosomes, is associated with the ability of polymers, strongly protonate under the acidic pH inside endosome and create a charge gradient ultimately provoking a water influx and endosomal swelling and disintegration. In both cases, DNA-containing complexes when released into the cytosol, dissociate allowing for nuclear entry of free DNA. Nuclear translo‐ cation of the plasmid DNA is relatively inefficient because of the barrier function of the nuclear membrane and small size of nuclear pores (25 nm), as well as DNA degrades fast under the action of cytoplasmic nucleases. It was estimated that only 0.1% of plasmids undergo nuclear translocation from the cytosol. The attachment of nuclear localization sequences to plasmid DNA may significantly enhance its nuclear translocation and transfection efficiency. New approaches in using multifunctional carriers for DNA delivery include the application of bimetallic nano-rods that can simultaneously bind compacted DNA plasmid and targeting

DNA–lipid amphiphiles self-assemble into novel "DNAsomes"-liposome-like core-shell structures with subunits composed of branched DNA-lipid hybrid molecules. These DNA‐ somes can be precisely modified over a wide range in terms of both size and surface charge. More importantly, DNAsome is a natural carrier of small interfering RNA (siRNA) due to DNA-RNA base-pairing, enabling efficient co-delivery of drugs and siRNA. The DNAsome represents a universal multi-functional drug vector for simultaneous delivery of drugs, tracer dyes, or antibodies, along with genes, siRNA or antisense nucleic acids [Roh et al., 2011].

efficacy of many therapeutic and diagnostic protocols [Torchilin, 2006].

206 Novel Gene Therapy Approaches

ligands in a spatially defined manner [Torchilin, 2006].

Over the last eight years, a laboratory at Northeastern University has developed an array of multi-functional nanocarriers for the delivery of genes, drugs and imaging modalities. These flexible platforms consist of polymeric and lipid systems that combine different modalities and stimuli-responsive release properties [Jabr-Milane et al., 2008].

The diagnostic and/or therapeutic objectives of a multi-functional nanocarrier system deter‐ mine the design of the formulation. A review of the literature shows that there are many different types of nanocarrier formulations for the diagnosis, imaging and treatment of a wide spectrum of diseases. These multi-functional carriers share three main design compo‐ nents: platform (core) material, encapsulated payload/biologically active agents and target‐ ing/surface properties. Nanocarrier platforms can be categorized as organic-based, inorganic-based or a hybrid combination. Organic nano-platforms include polymeric nano‐ carriers, lipid-based nanocarriers (e.g., liposomes and nanoemulsions), dendrimers and car‐ bon-based nanocarriers (e.g., fullerenes and carbon nanotubes) [Jabr-Milane et al., 2008]. Inorganic nano-platforms include metallic nanostructures, silica nanoparticles and quan‐ tum dots. An example of a hybrid platform is colloidal gold encapsulated in liposomes or superparamagnetic iron oxide particles encapsulated in polymeric nanoparticles. Selection of the core material is highly dependent on the properties of the biologically active agents. Inherent and dynamic properties of the agents such as therapeutic index, lipophilicity, charge and size should be considered. When combining therapeutic agents with each other, with an imaging/diagnostic modality, or with energy delivery, the interaction of system components (i.e., synergy, quenching, enhanced toxicity) and release kinetics should also be considered. Surface properties are the third design component of multi-functional nano‐ carriers. A common surface modification technique that decreases reticuloendothelial sys‐ tem (RES) clearance is the physical or covalent attachment of PEG chains to the nanocarrier platform. Since tumor microvasculature is known to be highly fenestrated, colloidal parti‐ cles can accumulate by the enhanced permeability and retention (EPR) effect. PEG surface modification increases circulatory residence time, which increases the probability of accu‐ mulation at the target. Block co-polymers of poly (ethylene oxide) (PEO) and poly (propy‐ lene oxide) (PPO) (e.g., Pluronics®) have also been used as surface conjugates to enhance circulation and achieve passive targeted delivery [Jabr-Milane et al., 2008].

Recently, nanoparticulate systems have been also developed for therapeutic gene delivery. Some researchers have developed glutathione-responsive nanoparticles for the delivery of plasmid DNA. The nanocarrier platform consisted of thiolated gelatin which was synthe‐ sized using 2-iminothiolane to covalently modify type B gelatin (pI ~ 4.5). The release pro‐ files of cross-linked and non-cross-linked thiolated gelatin nanoparticles and gelatin nanoparticles (control) loaded with fluorescein isothiocyanate-conjugated dextran (FITCdextran) were assessed in the presence of different glutathione concentrations (from 0.1 mM to 5 mM). A higher percentage of FITC–dextran was released from non-crosslinked nanoparticles compared to the cross-linked nanoparticles. The rate of FITC–dextran release from thiolated gelatin nanoparticles enhanced with increasing concentrations of gluta‐ thione. Glutathione in the media enhanced the release of FITC-dextran from thiolated gela‐ tin nanoparticles by about 40%, while only a 20% enhancement was seen with gelatin nanoparticles [Jabr-Milane et al., 2008]. After establishing the rapid, stimuli-responsive re‐ lease profile of these particles, the thiolated gelatin nanoparticles were loaded with plas‐ mid DNA expressing green fluorescent protein (GFP). Upon incubation of the formulations with murine fibroblast cells, fluorescence imaging revealed transfection and protein expres‐ sion after 6 h continuing as long as 96 h. Flow cytometry indicated that the cross-linked thiolated gelatin nanoparticles had the highest transfection efficiency. These nanocarriers are capable of rapid DNA delivery in response to intracellular glutathione. This nanocarri‐ er platform was further modified to develop an anti-angiogenic gene therapy for the treat‐ ment of cancer. This platform consisted of PEG-modified thiolated gelatin nanoparticles loaded with plasmid DNA encoding the soluble form of the extracellular domain of vascu‐ lar endothelial growth factor (VEGF) receptor-1 (sFlt-1). VEGF receptor over-expression in cancer is associated with neo-vascularization; sFlt-1 was selected as it blocks the VEGF re‐ ceptor and the associated signal cascade [Jabr-Milane et al., 2008]. The PEG modified thio‐ lated gelatin nanoparticles showed superior *in vitro* transfection in human breast adenocarcinoma cells when compared to plain gelatin nanoparticles, PEG-modified gelatin nanoparticles, thiolated gelatin nanoparticles, Lipofectin–plasmid DNA complexes and naked plasmid. *In vivo* evaluation of the formulation in *nu/nu* mice bearing orthotopic hu‐ man breast adenocarcinoma (MDA-MB-435) xenografts established transfection and expres‐ sion of sFlt-1 as assessed by ELISA, western blot analysis, tumor volume, microvessel density and immunostaining. PEG-modified thiolated gelatin nanoparticles were effective in transfection with sFlt-1 expressing plasmid DNA *in vivo* and showed significant suppres‐ sion of tumor growth in MDAMB-435 tumor-bearing mice. The expressed sFlt-1 was able to suppress angiogenesis [Jabr-Milane et al., 2008]. As such, PEG-modified thiolated gelatin nanoparticles are a viable platform for the delivery of therapeutic DNA to tumor mass. In addition, a system was developed for oral gene delivery. The nanoparticles-in-microsphere oral system (NiMOS) consists of type B gelatin nanoparticles encapsulated in Poly-ε-capro‐ lactone (PCL) microspheres. Based on the successful transfection results with sFlt-1, type B gelatin nanoparticles were selected to encapsulate and deliver DNA, while PCL was select‐ ed to protect the nanoparticles from degradation in the stomach and deliver the particles to the intestine, where PCL is degraded by lipases. To evaluate the biodistribution of NiMOS, the researchers encapsulated 111In-radiolabeled gelatin nanoparticles in PCL microspheres, orally administered the formulation to fasted Winstar rats, harvested the tissues at differ‐ ent time points and compared the results to 111In-radiolabeled gelatin nanoparticles. The gelatin nanoparticles showed immediate and high accumulation in the large intestine whereas NiMOS accumulation was initially high in the stomach (after 1 h) [Jabr-Milane et al., 2008]. It then transferred predominately to the large intestine after 2 h. To explore the effect of this biodistribution profile on transfection, the formulations were loaded with re‐ porter plasmids expressing β-galactosidase (CMV-βgal) or expressing GFP. DNA loaded NiMOS were orally administered to fasted Winstar rats at an oral dose of 100 μg plasmid DNA [Jabr-Milane et al., 2008].

Five days after administration, the rats were sacrificed and the GI tract was harvested for analysis. The results were compared to unloaded NiMOS formulations, naked plasmid and loaded gelatin nanoparticles. Transgene expression was evident in the small and large intestines with both reporter plasmids although GFP expression was more prominent with the loaded NiMOS formulation relative to the controls. Similar results were obtained in studies with Balb/c mice. Clearly, NiMOS is a promising system for targeted delivery of therapeutic

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An artificial gene-delivery vector, with a multi-component architecture is able to overcoming all the barriers, in which each component performs a different task in a planned fashion. The design of multi-functional nano-carrier is a multidisciplinary task that requires a profound understanding of the physico-chemical mechanisms that drive the assembly of such nanoparticles. One among many prerequisites for a successful carrier system for nucleic acids is high stability in the extracellular environment. In addition, the biological processes that elicit the cellular uptake and intracellular processing accompanied by an efficient release of the cargo in the intracellular compartment of these gene delivery systems have to be well understood. A promising strategy to create such an interactive delivery system is to exploit the various biological stimuli. With greater understanding of physiological differences between normal and disease tissues and advances in material design, there is an opportunity to develop nanocarrier systems for target-specific drug and gene delivery that will respond to the local stimuli [Qiao et al., 2010; Mastrobattista et al., 2006]. At present, many examples of versatile, selfassembling nano-particles for the delivery of DNA can be found in literature and this number is continuously growing. In this section, emphasis is placed on the functional components that are needed for effective gene delivery and also the biological stimuli such as pH and redox potential for the synthesis of multi-functional intelligent delivery systems. Briefly, Table 1

The artificial virus should preferably be constructed from materials that are biocompatible and biodegradable to prevent carrier-induced toxicities and the accumulation of carrier compo‐ nents in the body. In general, lipids are well tolerated. Synthetic polymers, on the other hand, have shown to induce some cytotoxicity *in vitro* and *in vivo.* It is difficult to predict, however, which polymer will be cytotoxic and which not on the basis of the structure of the cationic polymer. In general, low-molecular-mass cationic polymers are less toxic than high-molecularmass polymers. Peptides derived from L-amino acids are inherently biodegradable. However, when proteins or peptides contain large numbers of positively charged or exposed hydro‐ phobic amino acids, they can destabilize biological membranes and thereby cause cytotoxicity

DNA to the GI tract [Jabr-Milane et al., 2008].

indicates these functional carriers.

[Mastrobattista et al., 2006].

**7.1. Bio-compatible, bio-degradable nano-carriers**

**7. Design of multi-functional nanocarrier system**

Five days after administration, the rats were sacrificed and the GI tract was harvested for analysis. The results were compared to unloaded NiMOS formulations, naked plasmid and loaded gelatin nanoparticles. Transgene expression was evident in the small and large intestines with both reporter plasmids although GFP expression was more prominent with the loaded NiMOS formulation relative to the controls. Similar results were obtained in studies with Balb/c mice. Clearly, NiMOS is a promising system for targeted delivery of therapeutic DNA to the GI tract [Jabr-Milane et al., 2008].

## **7. Design of multi-functional nanocarrier system**

thione. Glutathione in the media enhanced the release of FITC-dextran from thiolated gela‐ tin nanoparticles by about 40%, while only a 20% enhancement was seen with gelatin nanoparticles [Jabr-Milane et al., 2008]. After establishing the rapid, stimuli-responsive re‐ lease profile of these particles, the thiolated gelatin nanoparticles were loaded with plas‐ mid DNA expressing green fluorescent protein (GFP). Upon incubation of the formulations with murine fibroblast cells, fluorescence imaging revealed transfection and protein expres‐ sion after 6 h continuing as long as 96 h. Flow cytometry indicated that the cross-linked thiolated gelatin nanoparticles had the highest transfection efficiency. These nanocarriers are capable of rapid DNA delivery in response to intracellular glutathione. This nanocarri‐ er platform was further modified to develop an anti-angiogenic gene therapy for the treat‐ ment of cancer. This platform consisted of PEG-modified thiolated gelatin nanoparticles loaded with plasmid DNA encoding the soluble form of the extracellular domain of vascu‐ lar endothelial growth factor (VEGF) receptor-1 (sFlt-1). VEGF receptor over-expression in cancer is associated with neo-vascularization; sFlt-1 was selected as it blocks the VEGF re‐ ceptor and the associated signal cascade [Jabr-Milane et al., 2008]. The PEG modified thio‐ lated gelatin nanoparticles showed superior *in vitro* transfection in human breast adenocarcinoma cells when compared to plain gelatin nanoparticles, PEG-modified gelatin nanoparticles, thiolated gelatin nanoparticles, Lipofectin–plasmid DNA complexes and naked plasmid. *In vivo* evaluation of the formulation in *nu/nu* mice bearing orthotopic hu‐ man breast adenocarcinoma (MDA-MB-435) xenografts established transfection and expres‐ sion of sFlt-1 as assessed by ELISA, western blot analysis, tumor volume, microvessel density and immunostaining. PEG-modified thiolated gelatin nanoparticles were effective in transfection with sFlt-1 expressing plasmid DNA *in vivo* and showed significant suppres‐ sion of tumor growth in MDAMB-435 tumor-bearing mice. The expressed sFlt-1 was able to suppress angiogenesis [Jabr-Milane et al., 2008]. As such, PEG-modified thiolated gelatin nanoparticles are a viable platform for the delivery of therapeutic DNA to tumor mass. In addition, a system was developed for oral gene delivery. The nanoparticles-in-microsphere oral system (NiMOS) consists of type B gelatin nanoparticles encapsulated in Poly-ε-capro‐ lactone (PCL) microspheres. Based on the successful transfection results with sFlt-1, type B gelatin nanoparticles were selected to encapsulate and deliver DNA, while PCL was select‐ ed to protect the nanoparticles from degradation in the stomach and deliver the particles to the intestine, where PCL is degraded by lipases. To evaluate the biodistribution of NiMOS, the researchers encapsulated 111In-radiolabeled gelatin nanoparticles in PCL microspheres, orally administered the formulation to fasted Winstar rats, harvested the tissues at differ‐ ent time points and compared the results to 111In-radiolabeled gelatin nanoparticles. The gelatin nanoparticles showed immediate and high accumulation in the large intestine whereas NiMOS accumulation was initially high in the stomach (after 1 h) [Jabr-Milane et al., 2008]. It then transferred predominately to the large intestine after 2 h. To explore the effect of this biodistribution profile on transfection, the formulations were loaded with re‐ porter plasmids expressing β-galactosidase (CMV-βgal) or expressing GFP. DNA loaded NiMOS were orally administered to fasted Winstar rats at an oral dose of 100 μg plasmid

DNA [Jabr-Milane et al., 2008].

208 Novel Gene Therapy Approaches

An artificial gene-delivery vector, with a multi-component architecture is able to overcoming all the barriers, in which each component performs a different task in a planned fashion. The design of multi-functional nano-carrier is a multidisciplinary task that requires a profound understanding of the physico-chemical mechanisms that drive the assembly of such nanoparticles. One among many prerequisites for a successful carrier system for nucleic acids is high stability in the extracellular environment. In addition, the biological processes that elicit the cellular uptake and intracellular processing accompanied by an efficient release of the cargo in the intracellular compartment of these gene delivery systems have to be well understood. A promising strategy to create such an interactive delivery system is to exploit the various biological stimuli. With greater understanding of physiological differences between normal and disease tissues and advances in material design, there is an opportunity to develop nanocarrier systems for target-specific drug and gene delivery that will respond to the local stimuli [Qiao et al., 2010; Mastrobattista et al., 2006]. At present, many examples of versatile, selfassembling nano-particles for the delivery of DNA can be found in literature and this number is continuously growing. In this section, emphasis is placed on the functional components that are needed for effective gene delivery and also the biological stimuli such as pH and redox potential for the synthesis of multi-functional intelligent delivery systems. Briefly, Table 1 indicates these functional carriers.

#### **7.1. Bio-compatible, bio-degradable nano-carriers**

The artificial virus should preferably be constructed from materials that are biocompatible and biodegradable to prevent carrier-induced toxicities and the accumulation of carrier compo‐ nents in the body. In general, lipids are well tolerated. Synthetic polymers, on the other hand, have shown to induce some cytotoxicity *in vitro* and *in vivo.* It is difficult to predict, however, which polymer will be cytotoxic and which not on the basis of the structure of the cationic polymer. In general, low-molecular-mass cationic polymers are less toxic than high-molecularmass polymers. Peptides derived from L-amino acids are inherently biodegradable. However, when proteins or peptides contain large numbers of positively charged or exposed hydro‐ phobic amino acids, they can destabilize biological membranes and thereby cause cytotoxicity [Mastrobattista et al., 2006].


Polymeric nano-particles offer significant advantages over other nano-carrier platforms primarily since a remarkable flexibility in polymer matrices allows for tailoring of the nanoparticle properties to meet the specific planed need. Other advantages of polymeric nanoparticles include ease of production, ease of surface modification, encapsulation efficiency of the payload, payload protection, large area-to-volume, slow or fast polymer degradation and stimuli-responsive polymer erosion for temporal control over the release of drugs and feasibility of scale-up and manufacturing. Some examples of the most commonly used polymers for nano-carriers include the synthetic polymers such as poly (D, L-lactide-coglyco‐ lide) (PLGA), poly (L-lactic acid) (PLL), poly (epsiloncaprolactone) (PCL), poly (alkylcyanoa‐ crylates) and natural polymers such as gelatin, chitosan and hyaluronic acid [van Vlerken et

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A prerequisite for every systemic nucleic-acid delivery system is stability in the blood stream prior to reaching its target cell. Therefore, the carrier must prevent the premature release of its load. Among the different delivery systems, nano-sized drug carriers are receiving consider‐ able attention. Through precise selection of candidate therapeutics and appropriate function‐ alization of the nano-carrier systems, it is possible to develop fairly sophisticated multifunctional systems that can provide optimized anticancer therapy, a function that imparts particular use in intracellular delivery and sub-cellular localization of drugs. Reversible nucleic acid condensation by cationic proteins is a common natural process, e.g., in packaging of whole mammalian genomes into chromatin, or RNA into organelles. Compaction is also a key function of viral cores for protection against the degradative environment during infection. Nucleic acids as macromolecules are subjected to a variety of environmental factors such as pH or enzymes (e.g., nucleases) that can degrade or destroy them. Complexation of nucleic acids by cationic polymers or lipids is a widely used method to reduce their sizes and prevent

Reversibility is important; the delivered nucleic acid has to be accessible for subsequent tran‐ scription [Wagner, 2011]. Polyplexes are non-viral vectors consisting of DNA and polycations have shown potential in systemic targeted gene delivery in various animal models, but suffer from far lower gene transfer efficiency than viral vectors. To improve efficiency, researchers are trying to engineer synthetic vectors with virus-like qualities. For successful condensation of DNA into virus size particles, an excess of polycations is necessary, which results in a positive surface charge. The positive surface charge on particles has been shown to be generally advanta‐ geous for cell uptake [Walker et al., 2005]. Polyionic interactions, hydrogen bonding and hydro‐ phobic interactions control the condensation of nucleic acids. In electrostatic complexes of plasmid DNA (pDNA) with polycations such as polylysine (pLys) or polyethylenimine (PEI), neutralization of approximately 10,000 negative phosphate charges of one pDNA molecule by approximately 100 polycation molecules results in compaction into "polyplexes" with sizes of 20 to > 100 nm (depending on aggregation events). The polymer/ pDNA core may be regarded as the engine of the delivery vehicle; for efficient and specific delivery, like in natural viruses, addition‐

al domains for cell entry and endosomal escape are required [Fig.1A & B; Wagner, 2011].

al., 2007; Mastrobattista et al., 2006].

their destruction by nucleases.

**7.2. Packaging nucleic acids into compact nano-particles**

**Table 1.** The use of different formulations with specific functions for designing of multi-functional nanocarrier systems

Polymeric nano-particles offer significant advantages over other nano-carrier platforms primarily since a remarkable flexibility in polymer matrices allows for tailoring of the nanoparticle properties to meet the specific planed need. Other advantages of polymeric nanoparticles include ease of production, ease of surface modification, encapsulation efficiency of the payload, payload protection, large area-to-volume, slow or fast polymer degradation and stimuli-responsive polymer erosion for temporal control over the release of drugs and feasibility of scale-up and manufacturing. Some examples of the most commonly used polymers for nano-carriers include the synthetic polymers such as poly (D, L-lactide-coglyco‐ lide) (PLGA), poly (L-lactic acid) (PLL), poly (epsiloncaprolactone) (PCL), poly (alkylcyanoa‐ crylates) and natural polymers such as gelatin, chitosan and hyaluronic acid [van Vlerken et al., 2007; Mastrobattista et al., 2006].

#### **7.2. Packaging nucleic acids into compact nano-particles**

**Function Examples Reference**

(L-lactic acid),

Synthetic polymers:

Poly (epiloncaprolactone), Poly (alkylcyanoacrylates) Natural polymers:

Polyethyleimine (PEI), Polylysine (pLys)

Polyvinylalcohol,

factor, Vitamin

compartment: Influenza HA2 peptide,

GALA peptide,

melittin,

PEG [PEGylated polyplexes]

Monoclonal antibody, Affibody, Aptamer, Oligopeptides, Growth

T-domain of the diphtheria toxin,

Cytosolic un-packaging: Reduction-

SV40 from the large tumor antigen

M9 from nuclear ribonucleoprotein

**Table 1.** The use of different formulations with specific functions for designing of multi-functional nanocarrier

pH-sensitive polymers: PEI

sensitive polyplexes Nuclear Import:

Simian virus 40,

Long-circulation of nanocarrier Polysaccharides, Polyacrylamide,

Intracellular delivery Escape from the endosomal

Poly (D, L-lactide-coglycolide), Poly

van Vlerken et al., 2007; Mastrobattista

Wagner, 2011; Walker et al., 2005

Wang et al., 2011; Wagner, 2011; Mastrobattista et al., 2006; Gu et al., 2007; van Vlerken et al., 2007; Peer et al., 2007; Alexis et al., 2008; Farokhzad et al., 2006; Zhang et al., 2001; Askoxylakis et al., 2005; Soudy et al., 2011; Kim et al., 2006; Ogris et al.,

Eliyahu et al., 2005; Shim and Kwon, 2012; Vercauteren et al., 2012; Du et al., 2010; Schaffer et al., 2000; Bauhuber et al., 2009

van Vlerken et al., 2007; Mastrobattista

et al., 2006

et al., 2006

2003

Gelatin, Chitosan, Hyaluronic acid

Bio-compatible, biodegradable

210 Novel Gene Therapy Approaches

Packaging nucleic acids into compact nanoparticles

Targeting molecules for the development of targeted NPs

systems

nanocarrier

A prerequisite for every systemic nucleic-acid delivery system is stability in the blood stream prior to reaching its target cell. Therefore, the carrier must prevent the premature release of its load. Among the different delivery systems, nano-sized drug carriers are receiving consider‐ able attention. Through precise selection of candidate therapeutics and appropriate function‐ alization of the nano-carrier systems, it is possible to develop fairly sophisticated multifunctional systems that can provide optimized anticancer therapy, a function that imparts particular use in intracellular delivery and sub-cellular localization of drugs. Reversible nucleic acid condensation by cationic proteins is a common natural process, e.g., in packaging of whole mammalian genomes into chromatin, or RNA into organelles. Compaction is also a key function of viral cores for protection against the degradative environment during infection. Nucleic acids as macromolecules are subjected to a variety of environmental factors such as pH or enzymes (e.g., nucleases) that can degrade or destroy them. Complexation of nucleic acids by cationic polymers or lipids is a widely used method to reduce their sizes and prevent their destruction by nucleases.

Reversibility is important; the delivered nucleic acid has to be accessible for subsequent tran‐ scription [Wagner, 2011]. Polyplexes are non-viral vectors consisting of DNA and polycations have shown potential in systemic targeted gene delivery in various animal models, but suffer from far lower gene transfer efficiency than viral vectors. To improve efficiency, researchers are trying to engineer synthetic vectors with virus-like qualities. For successful condensation of DNA into virus size particles, an excess of polycations is necessary, which results in a positive surface charge. The positive surface charge on particles has been shown to be generally advanta‐ geous for cell uptake [Walker et al., 2005]. Polyionic interactions, hydrogen bonding and hydro‐ phobic interactions control the condensation of nucleic acids. In electrostatic complexes of plasmid DNA (pDNA) with polycations such as polylysine (pLys) or polyethylenimine (PEI), neutralization of approximately 10,000 negative phosphate charges of one pDNA molecule by approximately 100 polycation molecules results in compaction into "polyplexes" with sizes of 20 to > 100 nm (depending on aggregation events). The polymer/ pDNA core may be regarded as the engine of the delivery vehicle; for efficient and specific delivery, like in natural viruses, addition‐ al domains for cell entry and endosomal escape are required [Fig.1A & B; Wagner, 2011].

effective, fueling its wide-spread use. PEG has a general structure of HO–(CH2CH2O)n– CH2CH2–OH, including a polyether backbone that is chemically inert, with terminal hydroxyl groups that can be activated for conjugation to different types of polymers and drugs. PEG offers the advantage that it is non-toxic and non-immunogenic, leading to approval by the United States Food and Drug Administration (FDA) for internal use in humans and inclusion in the list of inactive ingredients for oral and parenteral applications [Fig. 1 A & B; van Vlerken et al., 2007]. The protective (stealth) action of PEG is mainly due to the formation of a dense, hydrophilic cloud of long flexible chains on the surface of the colloidal particle that reduces the hydrophobic interactions with the RES. The chemically anchored PEG chains can undergo spatial conformations, thus preventing the opsonization of particles by the macrophages of the RES, which leads to preferential accumulation in the liver and spleen. PEG surface modification, therefore, enhances the circulation time of molecules and colloidal particles in the blood. The mechanism of steric hindrance by the PEG modified surface has been thor‐ oughly examined. The water molecules form a structured shell through hydrogen bonding to the ether oxygen molecules of PEG. The tightly bound water forms a hydrated film around the particle and prevents the protein interactions. In addition, PEG surface modification may also increase the hydrodynamic size of the particle decreasing its clearance, a process that is dependent on the molecular size as well as particle volume. Ultimately, this helps in greatly increasing circulation half-life of the particles [van Vlerken et al., 2007]. In cancer therapy, PEGylated polyplexes with elongated plasma circulation may take advantage of the "enhanced permeability and retention" (EPR) effect. Long-term circulating nano-particles can extravasate and passively accumulate at tumor sites due to the leakiness of tumor vessels and ineffective

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lymphatic efflux ("passive tumor targeting") [Wagner, 2011].

**7.4. Targeting molecules for the development of targeted NPs**

efficiency [Wang et al., 2011].

Appropriate packaging of nucleic acids and the use of PEG as a shield can help the complex survive within the circulation without being degraded or taken up by the mononuclear phagocyte system MPS. However, the next challenge for a PEGylated gene complex is to specifically target to cells or tissue of interest. By taking advantage of increased expression levels of receptors or antigens in diseased conditions, such as cancer, gene complexes can be targeted using specific ligands, such as antibodies, peptides, proteins, small molecules and RNA aptamer that recognize and bind to the cells of interest, resulting in high transfection

Viruses are optimized for surviving in the relevant body fluids. Their surface is decorated with ligands for attachment to their target cell surface receptors. Often they use more than one receptor type for intracellular uptake into host cells. In design of synthetic nano-particles, such multivalent recognition and cell uptake mechanisms for nucleic acid delivery can be utilized [Wagner, 2011]. A simultaneous effect of the surface modification of gene carriers are that the positive surface charges are shielded, which significantly reduces the non-specific adsorption onto cell membranes. This enables targeting of the gene carriers towards specific cell types by conjugating ligands to the hydrophilic coat around the gene carrier that specifically bind internalizing cell-surface receptors. In this way, delivery of the transgene and subsequent

**Figure 1. A) Schematic model of the pH-sensitive polyplexes:** Covalent attachment of active targeting ligands e.g., peptides, proteins, aptamers and small molecules to PEG; Formation of the polycation- pDNA polyplex through charge-charge interaction; Complex shielded at physiological pH and deshielded at acidic pH. **B) pH-reversible pEGy‐ lated lipopolyplexes:** Formation of complex by mixing DNA and PEI (polyplex); Incubation of the cationic polyplexes with liposomes (liposome-Pyridylhydrazone-PEG); Shielding in extracellular compartments and deshielding in acidic medium (e.g., endosomes).

#### **7.3. Long-circulation of nanocarrier**

Both naked DNA and lipoplexes have showed rapid hepatic clearance during systemic administration. The liver elimination of lipoplexes was due to phagocytosis by Kupffer cells. Absence of any hydrophilic surface group on the particles, may lead to their interaction with plasma proteins, opsonization and removal from the circulation. The mononuclear phagocytic system (MPS) plays a key role in systemic removal of hydrophobic particles. In addition to biodegradability and biocompatibility, the non-viral carrier should be 'invisible' to the innate and acquired immune system of the patient in order to prevent unwanted immune reactions against the carrier and, consequently, rapid clearance of carriers from the blood circulation after intravenous administration. This can be achieved by adding a hydrophilic coat around the carrier. The coat can consist of a lipid bilayer or hydrophilic polymers grafted onto the surface of the carriers [Mastrobattista et al., 2006]. Among several strategies to impart particles with stealth-shielding, including surface modification with polysaccharides, polyacrylamide, and polyvinyl alcohol, surface modification with PEG and PEG co-polymers proved to be most effective, fueling its wide-spread use. PEG has a general structure of HO–(CH2CH2O)n– CH2CH2–OH, including a polyether backbone that is chemically inert, with terminal hydroxyl groups that can be activated for conjugation to different types of polymers and drugs. PEG offers the advantage that it is non-toxic and non-immunogenic, leading to approval by the United States Food and Drug Administration (FDA) for internal use in humans and inclusion in the list of inactive ingredients for oral and parenteral applications [Fig. 1 A & B; van Vlerken et al., 2007]. The protective (stealth) action of PEG is mainly due to the formation of a dense, hydrophilic cloud of long flexible chains on the surface of the colloidal particle that reduces the hydrophobic interactions with the RES. The chemically anchored PEG chains can undergo spatial conformations, thus preventing the opsonization of particles by the macrophages of the RES, which leads to preferential accumulation in the liver and spleen. PEG surface modification, therefore, enhances the circulation time of molecules and colloidal particles in the blood. The mechanism of steric hindrance by the PEG modified surface has been thor‐ oughly examined. The water molecules form a structured shell through hydrogen bonding to the ether oxygen molecules of PEG. The tightly bound water forms a hydrated film around the particle and prevents the protein interactions. In addition, PEG surface modification may also increase the hydrodynamic size of the particle decreasing its clearance, a process that is dependent on the molecular size as well as particle volume. Ultimately, this helps in greatly increasing circulation half-life of the particles [van Vlerken et al., 2007]. In cancer therapy, PEGylated polyplexes with elongated plasma circulation may take advantage of the "enhanced permeability and retention" (EPR) effect. Long-term circulating nano-particles can extravasate and passively accumulate at tumor sites due to the leakiness of tumor vessels and ineffective lymphatic efflux ("passive tumor targeting") [Wagner, 2011].

#### **7.4. Targeting molecules for the development of targeted NPs**

**Figure 1. A) Schematic model of the pH-sensitive polyplexes:** Covalent attachment of active targeting ligands e.g., peptides, proteins, aptamers and small molecules to PEG; Formation of the polycation- pDNA polyplex through charge-charge interaction; Complex shielded at physiological pH and deshielded at acidic pH. **B) pH-reversible pEGy‐ lated lipopolyplexes:** Formation of complex by mixing DNA and PEI (polyplex); Incubation of the cationic polyplexes with liposomes (liposome-Pyridylhydrazone-PEG); Shielding in extracellular compartments and deshielding in acidic

Both naked DNA and lipoplexes have showed rapid hepatic clearance during systemic administration. The liver elimination of lipoplexes was due to phagocytosis by Kupffer cells. Absence of any hydrophilic surface group on the particles, may lead to their interaction with plasma proteins, opsonization and removal from the circulation. The mononuclear phagocytic system (MPS) plays a key role in systemic removal of hydrophobic particles. In addition to biodegradability and biocompatibility, the non-viral carrier should be 'invisible' to the innate and acquired immune system of the patient in order to prevent unwanted immune reactions against the carrier and, consequently, rapid clearance of carriers from the blood circulation after intravenous administration. This can be achieved by adding a hydrophilic coat around the carrier. The coat can consist of a lipid bilayer or hydrophilic polymers grafted onto the surface of the carriers [Mastrobattista et al., 2006]. Among several strategies to impart particles with stealth-shielding, including surface modification with polysaccharides, polyacrylamide, and polyvinyl alcohol, surface modification with PEG and PEG co-polymers proved to be most

medium (e.g., endosomes).

212 Novel Gene Therapy Approaches

**7.3. Long-circulation of nanocarrier**

Appropriate packaging of nucleic acids and the use of PEG as a shield can help the complex survive within the circulation without being degraded or taken up by the mononuclear phagocyte system MPS. However, the next challenge for a PEGylated gene complex is to specifically target to cells or tissue of interest. By taking advantage of increased expression levels of receptors or antigens in diseased conditions, such as cancer, gene complexes can be targeted using specific ligands, such as antibodies, peptides, proteins, small molecules and RNA aptamer that recognize and bind to the cells of interest, resulting in high transfection efficiency [Wang et al., 2011].

Viruses are optimized for surviving in the relevant body fluids. Their surface is decorated with ligands for attachment to their target cell surface receptors. Often they use more than one receptor type for intracellular uptake into host cells. In design of synthetic nano-particles, such multivalent recognition and cell uptake mechanisms for nucleic acid delivery can be utilized [Wagner, 2011]. A simultaneous effect of the surface modification of gene carriers are that the positive surface charges are shielded, which significantly reduces the non-specific adsorption onto cell membranes. This enables targeting of the gene carriers towards specific cell types by conjugating ligands to the hydrophilic coat around the gene carrier that specifically bind internalizing cell-surface receptors. In this way, delivery of the transgene and subsequent expression can be restricted to target cells. Several different types of targeting ligands have been used for this purpose, including peptides, antibodies and vitamins. If targeting ligands are directed towards internalizing receptors, receptor binding will lead to receptor-mediated endocytosis of the targeted gene carriers, as they are small enough (< 200-300 nm). This route of uptake is to be preferred as it ensures intracellular accumulation of gene carriers in a receptor-specific way [Mastrobattista et al., 2006]. While it has been demonstrated that PEG surface modification of nano-carriers causes a greater accumulation of drug at the tumor-site by passive targeting, active targeting of the carrier can help in selection of the target cell-type within the tumor site and internalization of the nano-particles to a greater extent inside the target cells. Active targeting can be achieved by the functionalization of NPs with ligands such as proteins (mainly antibodies and their fragments), nucleic acids (aptamers), or other receptor ligands (peptides, carbohydrates and vitamins) [Gu et al., 2007]. Regardless of the targeting moiety, the principle outcome is essentially the same, mainly improved tumor cell recognition, improved tumor cell uptake, and reduced recognition at non-specific sites. PEG surface modification provides an advantage whereby the terminal groups of PEG can be functional‐ ized to reactive groups for covalent coupling. Most commonly, PEG is functionalized to reactive carboxylic acids, amine, or sulfhydryl groups, allowing for efficient covalent attach‐ ment of the wide variety of targeting ligands by amide bonding or disulfide bridge formation [van Vlerken et al., 2007]. In this section, we describe some classes of targeting molecules.

affibody molecules are considerably higher compared with the corresponding antibodies. The binding pocket of an affibody is composed of 13 amino acids, which can be randomized to bind a variety of targets. In contrast to monoclonal antibody, affibody has following advan‐ tages as a targeting ligand. First, the small size of affibody (MW: 6 kDa) guarantees its tissue/ cell penetration ability. Second, its functional end groups for chemical conjugation are distanced from its binding site. Moreover, affibody has a robust structure, and can be easily synthesized in a large-scale manner. All of these advantages make the affibody a valuable ligand for targeted drug delivery [Alexis et al., 2008; Manjappa et al., 2011]. Recently, anti-HER2 affibody was also employed as a targeting ligand for nano-scaled drug delivery systems. Alexis et al. conjugated the anti-HER2 affibody to poly-(D, L-lactic acid)-poly (ethylene glycol) maleimide (PLA-PEG-Mal) copolymer for targeted delivery to cells that over-express the

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A novel class of molecules, referred to as nucleic acid ligands (aptamers), has been devel‐ oped to generate targeting agents. Aptamers are short single-stranded DNA or RNA oligo‐ nucleotides or modified DNA or RNA oligonucleotides that fold by intramolecular interaction into unique conformations with ligand-binding characteristics. Like antibodies, aptamers can be prepared to bind target antigens with high specificity and affinity. The use of aptamers as targeting molecules has several potential advantages over antibodies. Ap‐ tamers with high affinity for a target can be prepared through *in vitro* selection; a process called systemic evolution of ligands by exponential enrichment (SELEX) [Chai et al., 2011]. Conjugating aptamers to nanoparticles has shown to result in more efficient targeted thera‐ peutics or selective diagnostics than non-targeted NPs. Farokhzad et al have developed NP-aptamer (NP-Apt) conjugates that target the prostate specific membrane antigen (PSMA), a transmembrane protein that is up-regulated in a variety of cancers, using the A10 aptamer [Farokhzad et al., 2004]. This formulation has been further evaluated *in vivo* in a tumor model of LNCaP prostate cancer cells, which express PSMA antigens, and has been shown to regress tumor size effectively following a single intra-tumor injection over a

Recently, a number of tumor homing peptides have been reported that specifically target cancer cells and show promising results for tumor targeted drug delivery. Peptides, being smaller than other targeting ligands, have excellent tissue penetration properties and can be easily conjugated to drugs and oligonucleotides by chemical synthesis. Peptides are nearly invisible to the immune system and are not taken up in the reticuloendothelial system like antibodies and so are expected to cause minimal or no side effects to bone marrow, liver, and spleen [Gu et al., 2007]. For example, Cilengitide® is a cyclic arginine-glycine-aspartic acid (RGD) peptide that binds to the cell adhesion integrin αvβ3 on endothelial cells results in increased intracellular drug delivery in different murine tumor models and is currently in phase II clinical trials for the treatment of non-small cell lung cancer and pancreatic cancer.

HER-2 antigen [Alexis et al., 2008].

109-day study [Farokhzad et al., 2006].

*7.4.4. Oligopeptide-based targeting molecules*

*7.4.3. Aptamer targeting molecules*

#### *7.4.1. Monoclonal antibodies*

Monoclonal antibodies (mAb) are the first and are still the preferred class of targeting mole‐ cules. The mAb Rituximab was approved by the FDA for treating B-cell lymphoma in 1997. Another successful therapeutic mAb is Trastuzumab (Herceptin), an anti-HER2 mAb which binds to ErbB2 receptors and was approved by the FDA for treating breast cancer in 1998. Cetuximab, which binds to epidermal growth factor receptors (EGFR), was approved for treating colorectal cancer in 2004 and head/neck cancer in 2006. Bevacizumab, a tumor angiogenesis inhibitor that binds to vascular endothelial growth factor (VEGF), was approved for treating colorectal cancer in 2004. Trastuzumab and rituximab have been conjugated to poly (lactic acid) (PLA) NPs resulting in conjugates that exhibit a six-fold increase in the rate of particle uptake compared with similar particles lacking the mAb targeting. Today, over 200 delivery systems based on antibodies or their fragments are in preclinical and clinical trials. Antibodies may be used in their native state or as fragments for targeting [Imai and Takaoka, 2006; Peer et al., 2007].

#### *7.4.2. Affibodies as targeting ligands*

In recent times, a novel class of small molecules called "affibodies," which can be considered antibody mimics, have been examined for targeting. Affibodies are a class of polypeptide ligands that are potential candidates for tissue-specific targeting of drug-encapsulated controlled release polymeric nanoparticles. Affibody molecules are relatively small proteins (6-8 kDa) that offer the advantage of being extremely stable, highly soluble, and readily expressed in bacterial systems or produced by peptide synthesis. The binding affinities of affibody molecules are considerably higher compared with the corresponding antibodies. The binding pocket of an affibody is composed of 13 amino acids, which can be randomized to bind a variety of targets. In contrast to monoclonal antibody, affibody has following advan‐ tages as a targeting ligand. First, the small size of affibody (MW: 6 kDa) guarantees its tissue/ cell penetration ability. Second, its functional end groups for chemical conjugation are distanced from its binding site. Moreover, affibody has a robust structure, and can be easily synthesized in a large-scale manner. All of these advantages make the affibody a valuable ligand for targeted drug delivery [Alexis et al., 2008; Manjappa et al., 2011]. Recently, anti-HER2 affibody was also employed as a targeting ligand for nano-scaled drug delivery systems. Alexis et al. conjugated the anti-HER2 affibody to poly-(D, L-lactic acid)-poly (ethylene glycol) maleimide (PLA-PEG-Mal) copolymer for targeted delivery to cells that over-express the HER-2 antigen [Alexis et al., 2008].

#### *7.4.3. Aptamer targeting molecules*

expression can be restricted to target cells. Several different types of targeting ligands have been used for this purpose, including peptides, antibodies and vitamins. If targeting ligands are directed towards internalizing receptors, receptor binding will lead to receptor-mediated endocytosis of the targeted gene carriers, as they are small enough (< 200-300 nm). This route of uptake is to be preferred as it ensures intracellular accumulation of gene carriers in a receptor-specific way [Mastrobattista et al., 2006]. While it has been demonstrated that PEG surface modification of nano-carriers causes a greater accumulation of drug at the tumor-site by passive targeting, active targeting of the carrier can help in selection of the target cell-type within the tumor site and internalization of the nano-particles to a greater extent inside the target cells. Active targeting can be achieved by the functionalization of NPs with ligands such as proteins (mainly antibodies and their fragments), nucleic acids (aptamers), or other receptor ligands (peptides, carbohydrates and vitamins) [Gu et al., 2007]. Regardless of the targeting moiety, the principle outcome is essentially the same, mainly improved tumor cell recognition, improved tumor cell uptake, and reduced recognition at non-specific sites. PEG surface modification provides an advantage whereby the terminal groups of PEG can be functional‐ ized to reactive groups for covalent coupling. Most commonly, PEG is functionalized to reactive carboxylic acids, amine, or sulfhydryl groups, allowing for efficient covalent attach‐ ment of the wide variety of targeting ligands by amide bonding or disulfide bridge formation [van Vlerken et al., 2007]. In this section, we describe some classes of targeting molecules.

Monoclonal antibodies (mAb) are the first and are still the preferred class of targeting mole‐ cules. The mAb Rituximab was approved by the FDA for treating B-cell lymphoma in 1997. Another successful therapeutic mAb is Trastuzumab (Herceptin), an anti-HER2 mAb which binds to ErbB2 receptors and was approved by the FDA for treating breast cancer in 1998. Cetuximab, which binds to epidermal growth factor receptors (EGFR), was approved for treating colorectal cancer in 2004 and head/neck cancer in 2006. Bevacizumab, a tumor angiogenesis inhibitor that binds to vascular endothelial growth factor (VEGF), was approved for treating colorectal cancer in 2004. Trastuzumab and rituximab have been conjugated to poly (lactic acid) (PLA) NPs resulting in conjugates that exhibit a six-fold increase in the rate of particle uptake compared with similar particles lacking the mAb targeting. Today, over 200 delivery systems based on antibodies or their fragments are in preclinical and clinical trials. Antibodies may be used in their native state or as fragments for targeting [Imai and Takaoka,

In recent times, a novel class of small molecules called "affibodies," which can be considered antibody mimics, have been examined for targeting. Affibodies are a class of polypeptide ligands that are potential candidates for tissue-specific targeting of drug-encapsulated controlled release polymeric nanoparticles. Affibody molecules are relatively small proteins (6-8 kDa) that offer the advantage of being extremely stable, highly soluble, and readily expressed in bacterial systems or produced by peptide synthesis. The binding affinities of

*7.4.1. Monoclonal antibodies*

214 Novel Gene Therapy Approaches

2006; Peer et al., 2007].

*7.4.2. Affibodies as targeting ligands*

A novel class of molecules, referred to as nucleic acid ligands (aptamers), has been devel‐ oped to generate targeting agents. Aptamers are short single-stranded DNA or RNA oligo‐ nucleotides or modified DNA or RNA oligonucleotides that fold by intramolecular interaction into unique conformations with ligand-binding characteristics. Like antibodies, aptamers can be prepared to bind target antigens with high specificity and affinity. The use of aptamers as targeting molecules has several potential advantages over antibodies. Ap‐ tamers with high affinity for a target can be prepared through *in vitro* selection; a process called systemic evolution of ligands by exponential enrichment (SELEX) [Chai et al., 2011]. Conjugating aptamers to nanoparticles has shown to result in more efficient targeted thera‐ peutics or selective diagnostics than non-targeted NPs. Farokhzad et al have developed NP-aptamer (NP-Apt) conjugates that target the prostate specific membrane antigen (PSMA), a transmembrane protein that is up-regulated in a variety of cancers, using the A10 aptamer [Farokhzad et al., 2004]. This formulation has been further evaluated *in vivo* in a tumor model of LNCaP prostate cancer cells, which express PSMA antigens, and has been shown to regress tumor size effectively following a single intra-tumor injection over a 109-day study [Farokhzad et al., 2006].

#### *7.4.4. Oligopeptide-based targeting molecules*

Recently, a number of tumor homing peptides have been reported that specifically target cancer cells and show promising results for tumor targeted drug delivery. Peptides, being smaller than other targeting ligands, have excellent tissue penetration properties and can be easily conjugated to drugs and oligonucleotides by chemical synthesis. Peptides are nearly invisible to the immune system and are not taken up in the reticuloendothelial system like antibodies and so are expected to cause minimal or no side effects to bone marrow, liver, and spleen [Gu et al., 2007]. For example, Cilengitide® is a cyclic arginine-glycine-aspartic acid (RGD) peptide that binds to the cell adhesion integrin αvβ3 on endothelial cells results in increased intracellular drug delivery in different murine tumor models and is currently in phase II clinical trials for the treatment of non-small cell lung cancer and pancreatic cancer. Despite the success mentioned, RGD-targeted therapy still encounters many challenges. First challenge is the limitation associated with the non-specific adhesive nature of the RGD-integrin targeting system. Integrins are extracellular receptors that are not only expressed on cancer cells but also on nearly all epithelial cells and are therefore not cancer specific. Recent devel‐ opment of phage display screening methods has successfully isolated peptide ligands with high specificity and affinity to cell-surface hormone receptors (LHRH receptors, somatostatin receptors) and tumor vasculature antigens [Gu et al., 2007]. One of those is a dodecapeptide identified through phage display by Zhang *et al*, referred to as peptide p160. Peptide p160 displays high affinity for the human breast cancer cell lines MDA-MB-435 and MCF-7 *in vitro* with very little affinity for primary endothelial HUVEC cells [Zhang et al., 2001]. Fur‐ thermore, *in vivo* bio-distribution experiments in tumor-bearing mice, p160 showed a higher uptake in tumors than in organs such as heart, liver, lung and kidney. Relative to the RGD-4C peptide, p160 showed high accumulation in tumor versus normal organs [Askoxylakis et al., 2005; Soudy et al., 2011; Zhang et al., 2001]. Despite the potential of peptide p160 as a potent tumor homing peptide, its applicability would be largely hindered by its instability toward proteases. To overcome this, peptides have to be chemically modified so that their blood clearance is minimized in comparison with their rate of uptake at the target sites. The most common strategies used to increase peptide proteolytic stability include introduction of D- or un-natural amino acids and peptide cyclization. In a recent study, Soudy *et al* have developed analogues of cancer targeting peptide p160 to improve proteolytic stability and maintain specific affinity for breast cancer cells. These analogues are potentially safe with minimal cellular toxicity and are efficient targeting moieties for specific drug delivery to breast cancer cells [Soudy et al., 2011].

linked PEG-PEI was developed for tumor-selective gene delivery. The surface charge of the complexes was shielded by either PEG or a higher density of linked Tf to block undesired nonspecific interactions with blood components, followed by selective targeting to tumor cells. This approach resulted in a 100-fold higher gene expression in tumor cells compared with

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One challenge with targeting receptors whose expression correlates with metabolic rate, such as folate and Tf, is that these receptors are also expressed in fast growing healthy cells such as fibroblasts, epithelial and endothelial cells. Therefore, NP delivery system needs to be further

Non-viral vectors have to overcome multiple intracellular barriers subsequently after cellular uptake usually via endocytosis. After cellular internalization, a critical intracellular obstacle to non-viral gene delivery is degradation in the endosome/lysosome where nucleic acids are easi‐ ly degraded by a mildly acidic pH and acid activated enzymes. The ability of non-viral vectors to release the nucleic acids in their intracellular targets, while surviving through low pH and di‐ gestive processes and avoiding unwanted premature decomplexation, is a key efficiency-deter‐ mining requirement. The most widely believed theory in designing non-viral vectors for efficient endosomal escape is employing the "proton sponge effect" [Eliyahu et al., 2005]. Im‐ portantly, intracellular targets, where the nucleic acids are released from the vector, determine the overall gene delivery efficiency because the action sites are different for nucleic acid types. For example, plasmid DNA must be localized in the nucleus, where gene expression is initiated by transcription. However, nuclear entry is one of the major obstacles to non-viral gene delivery although nuclear translocation of plasmid DNA is crucial to achieve desired transfection effi‐ ciency. Moreover, the nuclear translocation mechanism of plasmid DNA in the cytoplasm has not yet been fully elucidated. Passive diffusion of macromolecules (e.g., plasmid DNA) in the cytoplasm is restricted by a complex network of microtubules, proteins, and various sub-cellu‐ lar organelles. One explanation is the nuclear localization of plasmid DNA during cellular mito‐ sis when the nuclear envelope disassembles. For non-dividing cells, the transport of polyplexes into the nucleus occurs only via an active transport mechanism through a nuclear pore complex that prevents molecules larger than 40 kDa from passively diffusing into the nucleus. In con‐ trast to plasmid DNA delivery, it is obvious that transnuclear localization of siRNA should be prevented to achieve efficient RNA interference (RNAi) since siRNA acts in the cytosol, where it targets mRNA with matching sequences [Shim and Kwon, 2012]. As nano-medicines often re‐ quire delivery of their therapeutic payload to specific sub-cellular locations, knowledge about intracellular trafficking might prove useful for the control of the intracellular processing of

refined and tuned to increase tumor selectivity [Gu et al., 2007; Peer et al., 2007].

nano-particles and lead to optimization of their design [Vercauteren et al., 2012].

Following internalization of peptide-DNA condensates by endocytosis, the polyplex must be able to escape the endosome so that the DNA can be delivered to the nucleus for gene expres‐ sion. After endocytosis, entrapment of the vector within the acidified vesicles of the endosomal/

*7.5.1. Escape from the endosomal compartment*

other tissues [Ogris et al., 2003].

**7.5. Intracellular delivery**

#### *7.4.5. Growth factor or vitamin-based targeting molecules*

Growth factor or vitamin interactions with cancer cells represent a commonly used targeting strategy, as cancer cells over-express the receptors for nutrition to maintain their fast-growing metabolism. Epidermal growth factor (EGF) has been shown to block and reduce tumor expression of the EGF receptor, which is over-expressed in a variety of tumor cells such as breast and tongue cancer [Peer et al., 2007].

One of the most extensively studied small molecule targeting moieties for drug delivery is folic acid (folate). The high-affinity vitamin is a commonly used ligand for cancer targeting because folate receptors (FRs) are frequently over-expressed in a range of tumor cells. It has been used as a targeting moiety combined with a wide array of drug delivery vehicles including lipo‐ somes, protein toxins, polymeric NPs, linear polymers, and dendrimers to deliver drugs selectively into cancer cells using FR-mediated endocytosis [Gu et al., 2007]. The folic acid-PEGylated PEI polyplex was evaluated as a gene carrier, and successfully delivered siRNA and pDNA to tumour cells [Kim et al., 2006].

Transferrin (Tf), an iron-binding glycoprotein interacts with Tf receptors (TfRs), which are overexpressed on a variety of tumor cells (including pancreatic, colon, lung, and bladder cancer) owing to increased metabolic rates. Direct coupling of these targeting agents to nanocarriers has improved intracellular delivery and therapeutic outcome in animal models. Tflinked PEG-PEI was developed for tumor-selective gene delivery. The surface charge of the complexes was shielded by either PEG or a higher density of linked Tf to block undesired nonspecific interactions with blood components, followed by selective targeting to tumor cells. This approach resulted in a 100-fold higher gene expression in tumor cells compared with other tissues [Ogris et al., 2003].

One challenge with targeting receptors whose expression correlates with metabolic rate, such as folate and Tf, is that these receptors are also expressed in fast growing healthy cells such as fibroblasts, epithelial and endothelial cells. Therefore, NP delivery system needs to be further refined and tuned to increase tumor selectivity [Gu et al., 2007; Peer et al., 2007].

#### **7.5. Intracellular delivery**

Despite the success mentioned, RGD-targeted therapy still encounters many challenges. First challenge is the limitation associated with the non-specific adhesive nature of the RGD-integrin targeting system. Integrins are extracellular receptors that are not only expressed on cancer cells but also on nearly all epithelial cells and are therefore not cancer specific. Recent devel‐ opment of phage display screening methods has successfully isolated peptide ligands with high specificity and affinity to cell-surface hormone receptors (LHRH receptors, somatostatin receptors) and tumor vasculature antigens [Gu et al., 2007]. One of those is a dodecapeptide identified through phage display by Zhang *et al*, referred to as peptide p160. Peptide p160 displays high affinity for the human breast cancer cell lines MDA-MB-435 and MCF-7 *in vitro* with very little affinity for primary endothelial HUVEC cells [Zhang et al., 2001]. Fur‐ thermore, *in vivo* bio-distribution experiments in tumor-bearing mice, p160 showed a higher uptake in tumors than in organs such as heart, liver, lung and kidney. Relative to the RGD-4C peptide, p160 showed high accumulation in tumor versus normal organs [Askoxylakis et al., 2005; Soudy et al., 2011; Zhang et al., 2001]. Despite the potential of peptide p160 as a potent tumor homing peptide, its applicability would be largely hindered by its instability toward proteases. To overcome this, peptides have to be chemically modified so that their blood clearance is minimized in comparison with their rate of uptake at the target sites. The most common strategies used to increase peptide proteolytic stability include introduction of D- or un-natural amino acids and peptide cyclization. In a recent study, Soudy *et al* have developed analogues of cancer targeting peptide p160 to improve proteolytic stability and maintain specific affinity for breast cancer cells. These analogues are potentially safe with minimal cellular toxicity and are efficient targeting moieties for specific drug delivery to breast cancer

Growth factor or vitamin interactions with cancer cells represent a commonly used targeting strategy, as cancer cells over-express the receptors for nutrition to maintain their fast-growing metabolism. Epidermal growth factor (EGF) has been shown to block and reduce tumor expression of the EGF receptor, which is over-expressed in a variety of tumor cells such as

One of the most extensively studied small molecule targeting moieties for drug delivery is folic acid (folate). The high-affinity vitamin is a commonly used ligand for cancer targeting because folate receptors (FRs) are frequently over-expressed in a range of tumor cells. It has been used as a targeting moiety combined with a wide array of drug delivery vehicles including lipo‐ somes, protein toxins, polymeric NPs, linear polymers, and dendrimers to deliver drugs selectively into cancer cells using FR-mediated endocytosis [Gu et al., 2007]. The folic acid-PEGylated PEI polyplex was evaluated as a gene carrier, and successfully delivered siRNA

Transferrin (Tf), an iron-binding glycoprotein interacts with Tf receptors (TfRs), which are overexpressed on a variety of tumor cells (including pancreatic, colon, lung, and bladder cancer) owing to increased metabolic rates. Direct coupling of these targeting agents to nanocarriers has improved intracellular delivery and therapeutic outcome in animal models. Tf-

cells [Soudy et al., 2011].

216 Novel Gene Therapy Approaches

*7.4.5. Growth factor or vitamin-based targeting molecules*

breast and tongue cancer [Peer et al., 2007].

and pDNA to tumour cells [Kim et al., 2006].

Non-viral vectors have to overcome multiple intracellular barriers subsequently after cellular uptake usually via endocytosis. After cellular internalization, a critical intracellular obstacle to non-viral gene delivery is degradation in the endosome/lysosome where nucleic acids are easi‐ ly degraded by a mildly acidic pH and acid activated enzymes. The ability of non-viral vectors to release the nucleic acids in their intracellular targets, while surviving through low pH and di‐ gestive processes and avoiding unwanted premature decomplexation, is a key efficiency-deter‐ mining requirement. The most widely believed theory in designing non-viral vectors for efficient endosomal escape is employing the "proton sponge effect" [Eliyahu et al., 2005]. Im‐ portantly, intracellular targets, where the nucleic acids are released from the vector, determine the overall gene delivery efficiency because the action sites are different for nucleic acid types. For example, plasmid DNA must be localized in the nucleus, where gene expression is initiated by transcription. However, nuclear entry is one of the major obstacles to non-viral gene delivery although nuclear translocation of plasmid DNA is crucial to achieve desired transfection effi‐ ciency. Moreover, the nuclear translocation mechanism of plasmid DNA in the cytoplasm has not yet been fully elucidated. Passive diffusion of macromolecules (e.g., plasmid DNA) in the cytoplasm is restricted by a complex network of microtubules, proteins, and various sub-cellu‐ lar organelles. One explanation is the nuclear localization of plasmid DNA during cellular mito‐ sis when the nuclear envelope disassembles. For non-dividing cells, the transport of polyplexes into the nucleus occurs only via an active transport mechanism through a nuclear pore complex that prevents molecules larger than 40 kDa from passively diffusing into the nucleus. In con‐ trast to plasmid DNA delivery, it is obvious that transnuclear localization of siRNA should be prevented to achieve efficient RNA interference (RNAi) since siRNA acts in the cytosol, where it targets mRNA with matching sequences [Shim and Kwon, 2012]. As nano-medicines often re‐ quire delivery of their therapeutic payload to specific sub-cellular locations, knowledge about intracellular trafficking might prove useful for the control of the intracellular processing of nano-particles and lead to optimization of their design [Vercauteren et al., 2012].

#### *7.5.1. Escape from the endosomal compartment*

Following internalization of peptide-DNA condensates by endocytosis, the polyplex must be able to escape the endosome so that the DNA can be delivered to the nucleus for gene expres‐ sion. After endocytosis, entrapment of the vector within the acidified vesicles of the endosomal/ lysosomal system is a critical barrier to non-viral gene delivery systems. Unfortunately, the en‐ vironment of the lysosomal interior is harmful for nucleic acid (NA) integrity, unless the carrier offers sufficient protection against the degradation by the acid hydrolases [Vercauteren et al., 2012]. Viruses and some pathogenic bacteria have pH-sensitive surface proteins that change conformation in mildly acidic environments such as in endosomes, and exhibit membrane-dis‐ ruptive (fusogenic or endosomolytic) properties. Synthetic fusogenic peptides that mimic the sequences of these natural proteins have been confirmed to increase cytoplasmic gene delivery [Du et al., 2010]. Since escape from the endosomes is essential for efficient NA therapy, much at‐ tention has been paid to this issue, resulting in a variety of endosomolytic carriers. For example, cationic and pH-responsive lipids can be added to phospholipid carriers to assist in releasing the NAs into the cytoplasm by destabilizing the endosomal lipid bilayer in the acidic environ‐ ment of the endolysosomes. Another approach is based on the coupling of fusogenic peptides to the carriers, which undergo conformational changes after their exposure to the decreasing en‐ dosomal pH values, exposing hydrophobic faces of the fusion peptide which destabilizes the endosomal membrane. Examples of these endosome-disruptive peptides are the influenza HA2 peptide, melittin, the T-domain of the diphtheria toxin or the GALA peptide [Vercauteren et al., 2012]. In an effort to translate the proton sponge activity to gene delivery peptides, histi‐ dine has been added to peptide sequences. The imidazole group of histidine has a pKa of ~ 6.0, therefore allowing it to become protonated in the acidic environment of the endosome. At phys‐ iological pH the histidines will remain neutrally charged, thereby imparting selective mem‐ brane disruption in the acidic endosome. In the past two decades, synthetic pH-sensitive polymers as endosomolytic agents have attracted great interest due to their low or non immu‐ nogenicity, which is a concern of using fusogenic proteins or peptides. Polymers such as PEI contain several secondary amines that are easily protonated in the acidic environment of the en‐ dosome. As protons are pumped in, PEI absorbs the protons leading to endosomal swelling and membrane disruption. In general, these smart polymers have both hydrophobic parts and weakly acidic groups, which afford them pH-dependent endosomolytic properties. At physio‐ logical pH, the polymers have little endosomolytic activity but undergo a conformational change at endosomal pH and show membrane-disruptive properties. This provides the poly‐ mers with reduced toxicity to the other biomembranes at neutral pH, but with the ability to facil‐ itate endosomal escape [Fig. 1A; Du et al., 2010].

the hydrolysis of an ester or amide bond have been widely used as gene carriers with decreased cytotoxicities, it is difficult to control the degradation occurring in the cytoplasm where free siRNA should be released to take action. Since the reduction potential in the cytoplasm is much higher (100 fold) than in the extracellular environments, a promising strategy to create an interactive delivery system is to exploit the redox gradient between the extra- and intra-cellular compartments, reduction-sensitive polyplexes are considered to be superior degradable

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The nuclear envelope that separates the cell's genetic material from the surrounding cytoplasm represents a physical barrier for nuclear import of macromolecules such as pDNA. The nuclear envelope contains openings in the form of nuclear pore complexes, which allow free diffusion of molecules up to 50 kDa, corresponding to a hydrodynamic diameter of approximately 10 nm. There are several lines of evidence showing that nuclear import is a rate-limiting step for transfection of pDNA. Nuclear import of pDNA may be more challenging for transfection of non-dividing cells. Indeed, non-dividing cells showed a 90% lower expression level as compared to actively dividing cells. Strategies for nuclear import of genes have been developed following further elucidation of the endogenous nuclear import machinery [Wang et al., 2011]. In nature, large molecules with sizes up to 30 nm in diameter that require trans-nuclear transport contain nuclear localization signals (NLS) that are recognized by nuclear transport receptors like importins or transportins, and the whole complex is thereafter actively trans‐ ported through NPCs [Vercauteren et al., 2012]. The nuclear localization sequence is a major player that shuttles protein–plasmid complexes through the nuclear pore. NLS-mediated active nuclear translocation involves a process starting from its interaction with cytoplasmic importins to binding of the NLS to the nuclear pore complex and the passage through the pore. Identification of the NLSs, such as SV40 from the larger tumor antigen Simian virus 40 and M9 from nuclear ribonucleoprotein, enabled design of non-viral gene vectors with nuclear

candidates, especially for siRNA delivery [Bauhuber et al., 2009; Du et al., 2010].

**8. Designing switchable nanosystems for medical application**

To enhance therapeutic efficacy while minimizing side effects, a large number of nanomate‐ rial based platforms have been developed that allow simple delivery of genes. Based on im‐ portant earlier work in the field of liposomal gene delivery and inorganic nanomaterials, the last decade has brought a broad array of new and improved nanoscale carrier plat‐ forms such as biodegradable and non-degradable polymers, dendrimers, carbon nano‐ tubes, metallic and organic nanoparticles, quantum dots, nanogels or peptidic nanoparticles. Ideally, gene vectors should be capable of self-assembly with nucleic acids and accommodate with any type of nucleic acid or their combination. They should also tar‐ get cells of interest, escape from endosomes and/or transport into nuclei. A viable gene vec‐ tor for systemic delivery needs to minimize toxicity and phagocytosis and avoid nonspecific interactions and self-aggregation. For potential gene delivery applications, ideal

*7.5.3. Nuclear import*

targeting properties [Wang et al., 2011].

#### *7.5.2. Cytosolic un-packaging*

The final step of the gene delivery process, un-packaging of the polyplex, can limit the efficiency of gene delivery and expression. For *in vivo* polyplex gene delivery, the polycation condenses the DNA to protect it and facilitate its entry and passage through target cells. However, once inside the nucleus, in order to be processed by RNA transcription complexes, the DNA may first need to dissociate from the polycation. Strong binding of the polycations to the nucleic acids may limit the intracellular un-packing of the polyplexes which is necessary for an efficient transfection. Although some viruses have evolved highly specific and intensive mechanisms for uncoating within the cell, a synthetic polycation may not release DNA with similar high efficiency [Schaffer et al., 2000]. One of the solutions to these problems is to use degradable polycations. Although the degradable polycations whose degradation is based on the hydrolysis of an ester or amide bond have been widely used as gene carriers with decreased cytotoxicities, it is difficult to control the degradation occurring in the cytoplasm where free siRNA should be released to take action. Since the reduction potential in the cytoplasm is much higher (100 fold) than in the extracellular environments, a promising strategy to create an interactive delivery system is to exploit the redox gradient between the extra- and intra-cellular compartments, reduction-sensitive polyplexes are considered to be superior degradable candidates, especially for siRNA delivery [Bauhuber et al., 2009; Du et al., 2010].

### *7.5.3. Nuclear import*

lysosomal system is a critical barrier to non-viral gene delivery systems. Unfortunately, the en‐ vironment of the lysosomal interior is harmful for nucleic acid (NA) integrity, unless the carrier offers sufficient protection against the degradation by the acid hydrolases [Vercauteren et al., 2012]. Viruses and some pathogenic bacteria have pH-sensitive surface proteins that change conformation in mildly acidic environments such as in endosomes, and exhibit membrane-dis‐ ruptive (fusogenic or endosomolytic) properties. Synthetic fusogenic peptides that mimic the sequences of these natural proteins have been confirmed to increase cytoplasmic gene delivery [Du et al., 2010]. Since escape from the endosomes is essential for efficient NA therapy, much at‐ tention has been paid to this issue, resulting in a variety of endosomolytic carriers. For example, cationic and pH-responsive lipids can be added to phospholipid carriers to assist in releasing the NAs into the cytoplasm by destabilizing the endosomal lipid bilayer in the acidic environ‐ ment of the endolysosomes. Another approach is based on the coupling of fusogenic peptides to the carriers, which undergo conformational changes after their exposure to the decreasing en‐ dosomal pH values, exposing hydrophobic faces of the fusion peptide which destabilizes the endosomal membrane. Examples of these endosome-disruptive peptides are the influenza HA2 peptide, melittin, the T-domain of the diphtheria toxin or the GALA peptide [Vercauteren et al., 2012]. In an effort to translate the proton sponge activity to gene delivery peptides, histi‐ dine has been added to peptide sequences. The imidazole group of histidine has a pKa of ~ 6.0, therefore allowing it to become protonated in the acidic environment of the endosome. At phys‐ iological pH the histidines will remain neutrally charged, thereby imparting selective mem‐ brane disruption in the acidic endosome. In the past two decades, synthetic pH-sensitive polymers as endosomolytic agents have attracted great interest due to their low or non immu‐ nogenicity, which is a concern of using fusogenic proteins or peptides. Polymers such as PEI contain several secondary amines that are easily protonated in the acidic environment of the en‐ dosome. As protons are pumped in, PEI absorbs the protons leading to endosomal swelling and membrane disruption. In general, these smart polymers have both hydrophobic parts and weakly acidic groups, which afford them pH-dependent endosomolytic properties. At physio‐ logical pH, the polymers have little endosomolytic activity but undergo a conformational change at endosomal pH and show membrane-disruptive properties. This provides the poly‐ mers with reduced toxicity to the other biomembranes at neutral pH, but with the ability to facil‐

The final step of the gene delivery process, un-packaging of the polyplex, can limit the efficiency of gene delivery and expression. For *in vivo* polyplex gene delivery, the polycation condenses the DNA to protect it and facilitate its entry and passage through target cells. However, once inside the nucleus, in order to be processed by RNA transcription complexes, the DNA may first need to dissociate from the polycation. Strong binding of the polycations to the nucleic acids may limit the intracellular un-packing of the polyplexes which is necessary for an efficient transfection. Although some viruses have evolved highly specific and intensive mechanisms for uncoating within the cell, a synthetic polycation may not release DNA with similar high efficiency [Schaffer et al., 2000]. One of the solutions to these problems is to use degradable polycations. Although the degradable polycations whose degradation is based on

itate endosomal escape [Fig. 1A; Du et al., 2010].

*7.5.2. Cytosolic un-packaging*

218 Novel Gene Therapy Approaches

The nuclear envelope that separates the cell's genetic material from the surrounding cytoplasm represents a physical barrier for nuclear import of macromolecules such as pDNA. The nuclear envelope contains openings in the form of nuclear pore complexes, which allow free diffusion of molecules up to 50 kDa, corresponding to a hydrodynamic diameter of approximately 10 nm. There are several lines of evidence showing that nuclear import is a rate-limiting step for transfection of pDNA. Nuclear import of pDNA may be more challenging for transfection of non-dividing cells. Indeed, non-dividing cells showed a 90% lower expression level as compared to actively dividing cells. Strategies for nuclear import of genes have been developed following further elucidation of the endogenous nuclear import machinery [Wang et al., 2011]. In nature, large molecules with sizes up to 30 nm in diameter that require trans-nuclear transport contain nuclear localization signals (NLS) that are recognized by nuclear transport receptors like importins or transportins, and the whole complex is thereafter actively trans‐ ported through NPCs [Vercauteren et al., 2012]. The nuclear localization sequence is a major player that shuttles protein–plasmid complexes through the nuclear pore. NLS-mediated active nuclear translocation involves a process starting from its interaction with cytoplasmic importins to binding of the NLS to the nuclear pore complex and the passage through the pore. Identification of the NLSs, such as SV40 from the larger tumor antigen Simian virus 40 and M9 from nuclear ribonucleoprotein, enabled design of non-viral gene vectors with nuclear targeting properties [Wang et al., 2011].

## **8. Designing switchable nanosystems for medical application**

To enhance therapeutic efficacy while minimizing side effects, a large number of nanomate‐ rial based platforms have been developed that allow simple delivery of genes. Based on im‐ portant earlier work in the field of liposomal gene delivery and inorganic nanomaterials, the last decade has brought a broad array of new and improved nanoscale carrier plat‐ forms such as biodegradable and non-degradable polymers, dendrimers, carbon nano‐ tubes, metallic and organic nanoparticles, quantum dots, nanogels or peptidic nanoparticles. Ideally, gene vectors should be capable of self-assembly with nucleic acids and accommodate with any type of nucleic acid or their combination. They should also tar‐ get cells of interest, escape from endosomes and/or transport into nuclei. A viable gene vec‐ tor for systemic delivery needs to minimize toxicity and phagocytosis and avoid nonspecific interactions and self-aggregation. For potential gene delivery applications, ideal gene carriers need to combine both the targeting property and the stimulus responsiveness to enhance the bioavailability of the gene as well as to reduce the side effects. Therefore, designing stimulus-responsive nanoparticles for programmed gene delivery, which release the gene on arrival at the targeted site, is highly desired. Stimulus-responsive nanoparticles produce physical or chemical changes when subjected to external signals, including varia‐ tions of macromolecular structures, solubility, surface properties, swelling and dissociation [Lehner et al., 2012]. Stimulus-responsive nanoparticles can be classified based on the type of stimulus as internally and externally controllable materials. Internal stimuli (e.g. activa‐ tion by pH, redox potential, enzymes) might be controlled by a molecular mechanism high‐ ly specific for a disease and therefore improve on targeting properties. However, absolutely disease-specific internal molecular triggers are difficult to find for certain diseases. External stimuli like light, ultrasound, electromagnetic fields or ionizing radiation have the advant‐ age of being focusable on certain body areas. This may be a significant advantage where a target cell is strongly involved in pathogenesis at one location (e.g., cancer stem cells in a cancer tissue), but of vital importance in other locations (e.g., stem cells in the bone mar‐ row). A key challenge in externally controlled nanomaterials is tissue penetration and avoidance of undesired tissue damage in the radiation path from radiation source to target tissue. The ease of temporal control in external stimuli may represent a particular advant‐ age for certain applications [Lehner et al., 2012]. Herein, we focus on the use of internal and biological stimuli that can be used to incorporate switch functionality into such nanocarri‐ ers and describe the clinical experience with various nanosize carrier systems as a basis for the design of new, improved, functional and "intelligent" nanosystems for gene delivery.

[Ganta et al., 2008]. This behavior can be utilized for the preparation of stimuli responsive drug or gene-delivery systems, which can exploit the biochemical properties at the contaminated site for targeted delivery. Cellular components such as the cytoplasm, endosomes, lysosomes, endoplasmic reticulum, golgi bodies, mitochondria and nuclei are known to maintain their own characteristic pH values. It is well known that the lower pH values are found in endo‐ somes (5.5-6.0) and lysosomes (4.5-5.5). On the basis of these discoveries, various pH/acidsensitive polymers have been developed as carriers for pDNA, ODN or siRNA delivery [Du

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This part focuses on the design and synthesis of polymeric carriers that can condense a large dose of therapeutic nucleic acids into particles that can sense the differences in envi‐ ronmental pH. The pH-modulated or self-regulated polymers are designed to use these pH differences by incorporating appropriate structural or functional features into the basic scaffold of the polymers to improve the efficacy of gene delivery. In general, polycations (PCs), such as PEI or dendrimers (polyplexes) or with cationic lipids (lipoplexes) with posi‐ tively charged surfaces are preferred for gene delivery in cell culture [Du et al., 2010]. Al‐ though, these positively charged particles are favorable for gene transfer efficiency *in vitro*, they are problematic for systemic gene targeting. Upon systemic application of positively charged cationic lipid based formulations (CL), polycations and lipopolyplexes may result in significant toxicity and/or poor efficiency due to plasma protein binding, interaction with blood cells or activation of the complement system and therefore has limited their ap‐ plication for *in vivo* uses via systemic administration [Du et al., 2010; Walker et al., 2005]. In general, the toxicity of cationic polymers increases with molecular weight, branched poly‐ mer morphology, and cationic charge density. A common approach for masking the sur‐ face charge of polyplexes is to coat particles with a hydrophilic polymer such as polyethylene glycol (PEG). Shielding the cationic surface by PEGylation and tailoring cati‐ onic density and polymer morphology have been popularly exploited to reduce the cyto‐ toxicity [Shim and Kwon, 2012]. PEGylation of polyplexes prevents their aggregation, lowers toxicity, increases circulation time and improves systemic targeted gene transfer. Unfortunately, at the same time as shielding improves the properties of polyplexes for sys‐ temic application it appears to reduce its cell transfection activity due to two important bar‐ riers such as reduced cellular uptake and inadequate release of the transported gene at the target cells. The cellular uptake has been enhanced by attaching targeting ligands to the polyplexes, however, the transfection efficiency of the targeted PEG vectors often still does not reach the level of the uncoated gene vectors. This suggests that the stable shield may al‐ so hinder intracellular gene transfer steps following endosomal uptake. Therefore, it is clear that to develop an optimal nonviral system for systemic application it must have a more dynamic character [Du et al., 2010] Its surface charge must be neutralized during circula‐ tion but after reaching its target cell, the cationic surface charge should be re-exposed for efficient gene transfer. In order to solve these conflicting issues, pH-sensitive reversible shielding or masking strategies have been developed through pH-sensitive PEGylation of lipid- or polymer-based carriers as the PEG shield intended to be removable in intracellular

et al., 2010; Ganta et al., 2008].

*8.1.1. Low pH-sensitive reversible shielding/masking*

#### **8.1. pH differences for stimuli-responsive delivery**

The main property that is the basis of utilization of pH responsive polymers in gene delivery is the significant change in pH value within the cellular compartments. There are numerous pH gradients in physiological and pathological processes [Du et al., 2010]. The pH profile of pathological tissues, such as inflammation, infection and cancer, is significantly different from that of the normal tissue. The pH at systemic sites of infections, primary tumors, and meta‐ stasized tumors is lower than the pH of normal tissue. The pH, surface charge and density of low density lipoprotein receptors are the factors that show notable differences among the normal and tumor tissues. All these properties are known to influence the drugs' physico‐ chemical properties and are exploited for enhanced delivery to the target site. The extracellular pH values of solid tumor are in general slightly lower than in blood or other normal tissues. Since tumors proliferate very rapidly, the vasculature of tumor is often insufficient to supply enough nutritional and oxygen needs for the expanding population of tumor cells. This results in difference in metabolic environment between the various solid tumors and the surrounding normal tissue. The insufficient oxygen in tumor leads to hypoxia and causes production of lactic acid and hydrolysis of ATP in an energy-deficient environment contributes to an acidic microenvironment, which has been found in many tumors. Most of the solid tumors have lower extracellular pH (6.5) than the surrounding tissues (pH= 7.5). The pH is compartmen‐ talized in tumor tissue into an intracellular component (pHi), which is similar in tumor and normal tissues and an extracellular component (pHe), which is relatively acidic in tumors [Ganta et al., 2008]. This behavior can be utilized for the preparation of stimuli responsive drug or gene-delivery systems, which can exploit the biochemical properties at the contaminated site for targeted delivery. Cellular components such as the cytoplasm, endosomes, lysosomes, endoplasmic reticulum, golgi bodies, mitochondria and nuclei are known to maintain their own characteristic pH values. It is well known that the lower pH values are found in endo‐ somes (5.5-6.0) and lysosomes (4.5-5.5). On the basis of these discoveries, various pH/acidsensitive polymers have been developed as carriers for pDNA, ODN or siRNA delivery [Du et al., 2010; Ganta et al., 2008].

#### *8.1.1. Low pH-sensitive reversible shielding/masking*

gene carriers need to combine both the targeting property and the stimulus responsiveness to enhance the bioavailability of the gene as well as to reduce the side effects. Therefore, designing stimulus-responsive nanoparticles for programmed gene delivery, which release the gene on arrival at the targeted site, is highly desired. Stimulus-responsive nanoparticles produce physical or chemical changes when subjected to external signals, including varia‐ tions of macromolecular structures, solubility, surface properties, swelling and dissociation [Lehner et al., 2012]. Stimulus-responsive nanoparticles can be classified based on the type of stimulus as internally and externally controllable materials. Internal stimuli (e.g. activa‐ tion by pH, redox potential, enzymes) might be controlled by a molecular mechanism high‐ ly specific for a disease and therefore improve on targeting properties. However, absolutely disease-specific internal molecular triggers are difficult to find for certain diseases. External stimuli like light, ultrasound, electromagnetic fields or ionizing radiation have the advant‐ age of being focusable on certain body areas. This may be a significant advantage where a target cell is strongly involved in pathogenesis at one location (e.g., cancer stem cells in a cancer tissue), but of vital importance in other locations (e.g., stem cells in the bone mar‐ row). A key challenge in externally controlled nanomaterials is tissue penetration and avoidance of undesired tissue damage in the radiation path from radiation source to target tissue. The ease of temporal control in external stimuli may represent a particular advant‐ age for certain applications [Lehner et al., 2012]. Herein, we focus on the use of internal and biological stimuli that can be used to incorporate switch functionality into such nanocarri‐ ers and describe the clinical experience with various nanosize carrier systems as a basis for the design of new, improved, functional and "intelligent" nanosystems for gene delivery.

The main property that is the basis of utilization of pH responsive polymers in gene delivery is the significant change in pH value within the cellular compartments. There are numerous pH gradients in physiological and pathological processes [Du et al., 2010]. The pH profile of pathological tissues, such as inflammation, infection and cancer, is significantly different from that of the normal tissue. The pH at systemic sites of infections, primary tumors, and meta‐ stasized tumors is lower than the pH of normal tissue. The pH, surface charge and density of low density lipoprotein receptors are the factors that show notable differences among the normal and tumor tissues. All these properties are known to influence the drugs' physico‐ chemical properties and are exploited for enhanced delivery to the target site. The extracellular pH values of solid tumor are in general slightly lower than in blood or other normal tissues. Since tumors proliferate very rapidly, the vasculature of tumor is often insufficient to supply enough nutritional and oxygen needs for the expanding population of tumor cells. This results in difference in metabolic environment between the various solid tumors and the surrounding normal tissue. The insufficient oxygen in tumor leads to hypoxia and causes production of lactic acid and hydrolysis of ATP in an energy-deficient environment contributes to an acidic microenvironment, which has been found in many tumors. Most of the solid tumors have lower extracellular pH (6.5) than the surrounding tissues (pH= 7.5). The pH is compartmen‐ talized in tumor tissue into an intracellular component (pHi), which is similar in tumor and normal tissues and an extracellular component (pHe), which is relatively acidic in tumors

**8.1. pH differences for stimuli-responsive delivery**

220 Novel Gene Therapy Approaches

This part focuses on the design and synthesis of polymeric carriers that can condense a large dose of therapeutic nucleic acids into particles that can sense the differences in envi‐ ronmental pH. The pH-modulated or self-regulated polymers are designed to use these pH differences by incorporating appropriate structural or functional features into the basic scaffold of the polymers to improve the efficacy of gene delivery. In general, polycations (PCs), such as PEI or dendrimers (polyplexes) or with cationic lipids (lipoplexes) with posi‐ tively charged surfaces are preferred for gene delivery in cell culture [Du et al., 2010]. Al‐ though, these positively charged particles are favorable for gene transfer efficiency *in vitro*, they are problematic for systemic gene targeting. Upon systemic application of positively charged cationic lipid based formulations (CL), polycations and lipopolyplexes may result in significant toxicity and/or poor efficiency due to plasma protein binding, interaction with blood cells or activation of the complement system and therefore has limited their ap‐ plication for *in vivo* uses via systemic administration [Du et al., 2010; Walker et al., 2005]. In general, the toxicity of cationic polymers increases with molecular weight, branched poly‐ mer morphology, and cationic charge density. A common approach for masking the sur‐ face charge of polyplexes is to coat particles with a hydrophilic polymer such as polyethylene glycol (PEG). Shielding the cationic surface by PEGylation and tailoring cati‐ onic density and polymer morphology have been popularly exploited to reduce the cyto‐ toxicity [Shim and Kwon, 2012]. PEGylation of polyplexes prevents their aggregation, lowers toxicity, increases circulation time and improves systemic targeted gene transfer. Unfortunately, at the same time as shielding improves the properties of polyplexes for sys‐ temic application it appears to reduce its cell transfection activity due to two important bar‐ riers such as reduced cellular uptake and inadequate release of the transported gene at the target cells. The cellular uptake has been enhanced by attaching targeting ligands to the polyplexes, however, the transfection efficiency of the targeted PEG vectors often still does not reach the level of the uncoated gene vectors. This suggests that the stable shield may al‐ so hinder intracellular gene transfer steps following endosomal uptake. Therefore, it is clear that to develop an optimal nonviral system for systemic application it must have a more dynamic character [Du et al., 2010] Its surface charge must be neutralized during circula‐ tion but after reaching its target cell, the cationic surface charge should be re-exposed for efficient gene transfer. In order to solve these conflicting issues, pH-sensitive reversible shielding or masking strategies have been developed through pH-sensitive PEGylation of lipid- or polymer-based carriers as the PEG shield intended to be removable in intracellular endosomes or in the slightly acidic extracellular microenvironment of tumors. Lipoplexes and polyplexes containing a pH cleavable PEG shield were found to be less effective in gene transfer than the corresponding stably shielded particles. There is a strategy to over‐ come this challenge. The neutralizing shield is attached to the DNA polyplex core via an acid-labile linkage forming a shielded particle for systemic circulation. Chemical linkages that may display pH-dependent hydrolytic degradation, once internalized into endosomal and lysosomal compartments include acetal-ketal linkage, vinyl ether, orthoesters, and hy‐ drazones. Under the acidic environment of the endosomal compartment, these linkages un‐ dergo acid-induced hydrolysis and thereby trigger deshielding of the polyplex core. Therefore, the acid-labile bioreversible shielding polymer exerts a significant impact on the outcome of transfection efficiency [Du et al., 2010].

Recently, Sethuraman *et al*. have developed pH-Responsive Sulfonamide/PEI nanoparticles that effectively target the acidic extracellular matrix of tumors, which shows a sharp pH profile, was able to shield positively charged complexes at physiological pH of 7.4. The pH sensitive polymer was able to detach from the complex when the pH environment decreased to pH= 6.6. The polymeric nanoparticle formed through electrostatic attraction is designed in such a way that the final particle is neutral. The polyplexes formed by PEI and pDNA were coated electrostatically with an ultra pH-sensitive diblock copolymer, poly (methacryloyl sulfadime‐ thoxine)-b-PEG. The central idea of this design is that when the particles experience a decrease in pH as they extravasate into tumor tissue due to enhanced permeability and retention effect, the sulfonamide groups would lose their charge and get detached from the carrier complex. Most of the carriers developed so far do not have the high sensitivity that is required to respond to such small differences in pH between tumors and normal tissues. This is because the carboxylic acid based polymers show transitions in about one pH unit which is very broad, and that transition is much below the physiological and tumor pH range, whereas the poly(methacryloyl sulfadimethoxine) (PSD)-*block*-PEG PSD*b*-PEG polymer shows transition within 0.2 pH units between the physiological and tumor pH. These sulfonamide polymers are able to distinguish the small difference in pH between normal and tumor tissues and hence

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has remarkable potential in drug targeting to tumor areas [Sethuraman et al., 2008].

systems are major strategies being exploited to facilitate endosomal escape.

swells and ruptures endosomal membrane [Eliyahu et al., 2005].

Following internalization of lipoplexes or polyplexes via the endocytic pathway, endosomal entrapment and subsequent lysosomal degradation are a major blockage that limits the efficiency of gene delivery. After cellular uptake of the gene carriers, the carrier should escape from the endosomal compartment in order to reach the cytosol and then, the nucleus. This requires dissociation of the internalized carrier from the receptors that triggered the internal‐ ization process. In addition, the membranes of the endosomes should be destabilized to allow translocation of the carriers into the cytosol [Mastrobattista et al., 2006]. Identification of endosomolytic or fusogenic components and their integration into non-viral gene delivery

In the case of polyplexes, PEI and polyamidoamine (PAMAM) are two representative cationic polymers with a high efficiency of gene transfer due in part to their capability to facilitate endosomal escape. A "proton sponge effect" provides a sound explanation for the intrinsic endosomolytic activity. Upon PEI-based or PAMAM-based polyplex entry into acidic endo‐ somes, the polymer behaves as a sponge that absorbs protons as a result of protonation of the polymer-containing amine groups (primary, secondary and tertiary). Accumulation of protons subsequently drives an influx of counter chloride ion into endosomes, leading to increased osmotic pressure and subsequent flow of water into the endosomal interior and eventually

PEIs are available in a wide range of molecular weights (MW) from 423 Da to 800 kDa and with different branching degree (from linear to branched). Generally, high MW, branched PEIs have high transfection efficiency but also high toxicity due to high cationic charge. By contrast, low MW PEIs are less toxic but less efficient as gene delivery agents. Many efforts have been

*8.1.2. pH-dependent endosomolytic polymers*

Walker and colleagues reported reversible shielding of polyplexes with pH-triggered de‐ shielding properties, enabled by conjugating PEG to the polycations poly-L-lysine (PLL) and PEI via a pH-sensitive hydrazone bond (PC-HZN-PEG) of acyl hydrazides or 2-pyridyl hydrazines. The reversibility of the hydrazone bond within conjugates was determined at physiological and endosomal acidic pH, identifying suitable linker systems for endosomal deshielding. The polyplexes with the acid-sensitive linkages showed much higher plasmid gene delivery efficiencies (1-2 orders of magnitude) than those with stable linkages, both *in vitro* and *in vivo* [Walker et al., 2005].

Sawant *et al*. demonstrated that the use of a lowered pH-degradable PEG-Hz-PE produced particles (polyethylene glycol-phosphatidylethanolamine conjugates) with transfection activity sensitive to changes in pH, which has a promise for site-specific transfection of tumor cells *in vivo*. In this study, the encapsulation of PEI-PE/DNA complexes into pH-sensitive micelle-like PEG-Hz-PE coat increased the stability of DNA in complete medium and increased transfection efficiency by being responsive to changes in pH. *In vivo*, the PEG2000-Hz-PE is expected to shield the PEI-PE/DNA complex in the systemic circulation and expose the complex only at the tumor sites where the pH is slightly acidic and can facilitate the removal of the PEG coat [Sawant et al., 2012].

Murthy *et al*. synthesized 'encrypted' polymeric carrier that consisted of hydrophobic, membrane disruptive methacrylate polymers onto which hydrophilic PEG chains have been grafted through acid-degradable acetal linkages; a *p*-aminobenzaldehyde acetal linkage demonstrated a suitable hydrolysis profile at endosomal pH [Murthy et al., 2003].

In 2007, Knorr *et al*. synthesized a new PEGylation reagent containing *p*-piperazinobenzalde‐ hyde acetal linkage and a maleimide moiety which can be coupled to thiol-functionalized compounds. For reversible shielding of polyplexes, PEG-acetal-maleimide (MAL) was conjugated to PEI. At 37<sup>о</sup>C, the PEG-acetal-PEI conjugate were found to be shielded and stable. In contrast, at endosomal pH, the particles were deshielded and aggregated within 0.5 h. The reversibly shielded (PEG-acetal-PEI) polyplexes were found to have approximately 10-fold enhanced gene transfer efficiency than stable shielded polyplexes when tested on the two different cell lines, Renca-EGFR cells and K562 cells [Knorr et al., 2007].

Recently, Sethuraman *et al*. have developed pH-Responsive Sulfonamide/PEI nanoparticles that effectively target the acidic extracellular matrix of tumors, which shows a sharp pH profile, was able to shield positively charged complexes at physiological pH of 7.4. The pH sensitive polymer was able to detach from the complex when the pH environment decreased to pH= 6.6. The polymeric nanoparticle formed through electrostatic attraction is designed in such a way that the final particle is neutral. The polyplexes formed by PEI and pDNA were coated electrostatically with an ultra pH-sensitive diblock copolymer, poly (methacryloyl sulfadime‐ thoxine)-b-PEG. The central idea of this design is that when the particles experience a decrease in pH as they extravasate into tumor tissue due to enhanced permeability and retention effect, the sulfonamide groups would lose their charge and get detached from the carrier complex. Most of the carriers developed so far do not have the high sensitivity that is required to respond to such small differences in pH between tumors and normal tissues. This is because the carboxylic acid based polymers show transitions in about one pH unit which is very broad, and that transition is much below the physiological and tumor pH range, whereas the poly(methacryloyl sulfadimethoxine) (PSD)-*block*-PEG PSD*b*-PEG polymer shows transition within 0.2 pH units between the physiological and tumor pH. These sulfonamide polymers are able to distinguish the small difference in pH between normal and tumor tissues and hence has remarkable potential in drug targeting to tumor areas [Sethuraman et al., 2008].

#### *8.1.2. pH-dependent endosomolytic polymers*

endosomes or in the slightly acidic extracellular microenvironment of tumors. Lipoplexes and polyplexes containing a pH cleavable PEG shield were found to be less effective in gene transfer than the corresponding stably shielded particles. There is a strategy to over‐ come this challenge. The neutralizing shield is attached to the DNA polyplex core via an acid-labile linkage forming a shielded particle for systemic circulation. Chemical linkages that may display pH-dependent hydrolytic degradation, once internalized into endosomal and lysosomal compartments include acetal-ketal linkage, vinyl ether, orthoesters, and hy‐ drazones. Under the acidic environment of the endosomal compartment, these linkages un‐ dergo acid-induced hydrolysis and thereby trigger deshielding of the polyplex core. Therefore, the acid-labile bioreversible shielding polymer exerts a significant impact on the

Walker and colleagues reported reversible shielding of polyplexes with pH-triggered de‐ shielding properties, enabled by conjugating PEG to the polycations poly-L-lysine (PLL) and PEI via a pH-sensitive hydrazone bond (PC-HZN-PEG) of acyl hydrazides or 2-pyridyl hydrazines. The reversibility of the hydrazone bond within conjugates was determined at physiological and endosomal acidic pH, identifying suitable linker systems for endosomal deshielding. The polyplexes with the acid-sensitive linkages showed much higher plasmid gene delivery efficiencies (1-2 orders of magnitude) than those with stable linkages, both *in*

Sawant *et al*. demonstrated that the use of a lowered pH-degradable PEG-Hz-PE produced particles (polyethylene glycol-phosphatidylethanolamine conjugates) with transfection activity sensitive to changes in pH, which has a promise for site-specific transfection of tumor cells *in vivo*. In this study, the encapsulation of PEI-PE/DNA complexes into pH-sensitive micelle-like PEG-Hz-PE coat increased the stability of DNA in complete medium and increased transfection efficiency by being responsive to changes in pH. *In vivo*, the PEG2000-Hz-PE is expected to shield the PEI-PE/DNA complex in the systemic circulation and expose the complex only at the tumor sites where the pH is slightly acidic and can facilitate the removal

Murthy *et al*. synthesized 'encrypted' polymeric carrier that consisted of hydrophobic, membrane disruptive methacrylate polymers onto which hydrophilic PEG chains have been grafted through acid-degradable acetal linkages; a *p*-aminobenzaldehyde acetal linkage

In 2007, Knorr *et al*. synthesized a new PEGylation reagent containing *p*-piperazinobenzalde‐ hyde acetal linkage and a maleimide moiety which can be coupled to thiol-functionalized compounds. For reversible shielding of polyplexes, PEG-acetal-maleimide (MAL) was conjugated to PEI. At 37<sup>о</sup>C, the PEG-acetal-PEI conjugate were found to be shielded and stable. In contrast, at endosomal pH, the particles were deshielded and aggregated within 0.5 h. The reversibly shielded (PEG-acetal-PEI) polyplexes were found to have approximately 10-fold enhanced gene transfer efficiency than stable shielded polyplexes when tested on the two

demonstrated a suitable hydrolysis profile at endosomal pH [Murthy et al., 2003].

different cell lines, Renca-EGFR cells and K562 cells [Knorr et al., 2007].

outcome of transfection efficiency [Du et al., 2010].

*vitro* and *in vivo* [Walker et al., 2005].

222 Novel Gene Therapy Approaches

of the PEG coat [Sawant et al., 2012].

Following internalization of lipoplexes or polyplexes via the endocytic pathway, endosomal entrapment and subsequent lysosomal degradation are a major blockage that limits the efficiency of gene delivery. After cellular uptake of the gene carriers, the carrier should escape from the endosomal compartment in order to reach the cytosol and then, the nucleus. This requires dissociation of the internalized carrier from the receptors that triggered the internal‐ ization process. In addition, the membranes of the endosomes should be destabilized to allow translocation of the carriers into the cytosol [Mastrobattista et al., 2006]. Identification of endosomolytic or fusogenic components and their integration into non-viral gene delivery systems are major strategies being exploited to facilitate endosomal escape.

In the case of polyplexes, PEI and polyamidoamine (PAMAM) are two representative cationic polymers with a high efficiency of gene transfer due in part to their capability to facilitate endosomal escape. A "proton sponge effect" provides a sound explanation for the intrinsic endosomolytic activity. Upon PEI-based or PAMAM-based polyplex entry into acidic endo‐ somes, the polymer behaves as a sponge that absorbs protons as a result of protonation of the polymer-containing amine groups (primary, secondary and tertiary). Accumulation of protons subsequently drives an influx of counter chloride ion into endosomes, leading to increased osmotic pressure and subsequent flow of water into the endosomal interior and eventually swells and ruptures endosomal membrane [Eliyahu et al., 2005].

PEIs are available in a wide range of molecular weights (MW) from 423 Da to 800 kDa and with different branching degree (from linear to branched). Generally, high MW, branched PEIs have high transfection efficiency but also high toxicity due to high cationic charge. By contrast, low MW PEIs are less toxic but less efficient as gene delivery agents. Many efforts have been directed towards creating PEI derivatives combining higher transfection efficacy and good biocompatibility. One approach to reduce the cytotoxicity, biodegradable polyethylenimine with imine linkages as acid-labile moieties were synthesized and investigated for pDNA delivery. The half-life of the acid-labile PEI was 1.1 h at pH= 4.5 and 118 h at pH= 7.4, suggesting that the acid-labile PEI may be rapidly degraded into non-toxic low molecular weight PEI in acidic endosome. Acid-labile PEIs showed close transfection efficiency to PEI 25KDa, but much less toxicity due to the degradation of acid-labile linkage. Therefore, the acid-labile PEIs may be useful for the development of a non-toxic polymeric gene carrier [Kim et al., 2005].

particles of 160 nm size and a zeta potential of +7 mV. Pyridylhydrazone-based Chol-PEG was included in the liposomes for shielding in extracellular compartments and dynamic deshield‐ ing in acidic conditions such as in endosomes. In addition, this cholesterol-PEG derivative contained also a pyridyldithio moiety to provide the possibility of coupling thiol-functional‐

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The endosome-escape potential of poly-histidine increases their use for delivery of nucleic acids. The imidazole ring within histidine is a major component. Under the action of an acidic endosomal interior, the weak basic nature of the imidazole ring with pKa around 6 allows its protonation and acquires cationic charges which trigger the destabilization of en‐ dosomal membranes. Accumulation of histidine residues within endosomes could elicit a proton sponge effect and destroy endosomes as a result of their increased osmolarity. Both chemistry conjugation and genetic engineering have produced a series of histidine-rich pol‐ ymers and peptides as well as lipids with imidazole, imidazolinium or imidazolium polar heads. These histidylated carriers have been used to deliver nucleic acids including pDNA, mRNA or siRNA duplex *in vitro* and *in vivo* with increased transfection efficiency [Midoux

The histidine-rich peptide H5WYG is a derivative of the N-terminal sequence of the HA-2 subunit of the *influenza virus* hemagglutinin in which five of the amino acids have been re‐ placed with histidine residues. H5WYG is able to selectively destabilize membranes at a slightly acidic pH as the histidine residues are protonated. An anionic derivative of this peptide, E5WYG, in which the histidines are replaced by glutamic acid residues, is com‐ pletely ineffective at membrane permeabilization at a pH= 6.8, while H5WYG can disrupt 50% of cells at pH= 6.8 and 97% of cells at pH= 6.2 within 15 minutes. H5WYG was also able to retain its activity in the presence of serum, making it possible for use *in vivo*. The most well-known non-viral DNA condensing agent is poly-L-lysine. However, PLL only exhibits modest transfection when used alone and requires the addition of an endosomolyt‐ ic agent such as chloroquine or a fusogenic peptide to allow for release into the cytoplasm. To increase the transfection efficiency of polylysine without the addition of membrane-dis‐ rupting agents, a histidine-substituted polylysine can be constructed that is able to become

In an attempt to facilitate endosome escape, many strategies have been developed to mimic the viral mechanism for endosome destabilization. Viruses have acquired efficient solutions for escaping from the maturating acidifying endosomes. For example, glycoproteins of enveloped viruses contain hidden fusion peptides which are exposed after endocytosis, to trigger fusion of the viral with the endosomal membrane [Wagner, 2011]. It is well-established that the influenza virus utilizes the pH-sensitive membrane-destabilizing Hemagglutinin protein (HA2) displayed on the viral coat to disrupt the endosomal membrane and enter the cytoplasm. Hemagglutinin and other fusion proteins are characterized by their unique ability to switch from an ionized and hydrophilic conformation at physiologic pH to a hydrophobic and membrane-active one in response to acidic endosomal pH gradients, which destabilizes the endosomal membrane leading to leakage of endosomal contents into the cytoplasm [Lin et al., 2010]. At neutral pH, the HA2 subunit adapts a non-helical conformation due to charge

ized compounds, such as receptor ligands [Fig. 1B.; Nie et al., 2011].

et al., 2007; Martin and Rice, 2007].

cationic at endosomal pH [Martin and Rice, 2007].

The cationic lipids have been shown to destabilize the endosomal membrane. Addition of dioleoyl phosphatidylethanolamine (DOPE) or cholesteryl hemisuccinate (CHEMS) to nonviral vectors affected the pH-sensitivity of the formulations. The mechanism behind the phenomenon, is that electrical interaction between cationic lipids and anion endosomal membranes results in the formation of ion-pairs that promote the formation of the inverted hexagonal (HII) phase and disrupt endosomal membrane. Many preparations of lipoplexes contain DOPE as a helper lipid for fusogenic functionality. The ability of DOPE to destabilize endosomal membranes is based on its propensity to acquire an inverted hexagonal phase (HII). DOPE has a small cross-section head group and a large hydrocarbon area that favors a nonbilayer structure with a cone shape that facilitates the destabilization of endosomal membranes and gene transfection. The pH-responsive component, CHEMS, being negatively charged at neutral pH stabilizes the bilayer structure, however, at acidic pH it becomes protonated and loses its stabilization property [Ma et al., 2007; Wu and Zhao, 2007].

Another approach to reduce the cytotoxicity of PEI is modification with hydrophobic moi‐ eties, such as lipids. Since lipids are the main component of cell membrane, modification with hydrophobic moieties may result in additional hydrophobic interaction between poly‐ plexes and cell membranes, which in turn would facilitate the delivery of a payload into cells. Various hydrophobic modifications have been tried, including modification with cho‐ lesterol, myristate, dodecyl iodide, hexadecyl iodide, palmitic acid, oleic acid, stearic acid, and phosphatidylcholine. In a recent study, Sawant *et al*. developed the synthesis and char‐ acterization of a PEI-DOPE conjugate, which was explored for gene delivery *in vitro*. The modification with DOPE strongly increased the transfection efficiency of low molecular weight PEI-1.8 kDa without any negative effects on its low cytotoxicity. The PEI-PE conju‐ gate was synthesized by reacting a phospholipid with low molecular weight PEI (PEI-1.8). It was assumed that a PEI-PE conjugate would condense DNA due to the electrostatic in‐ teraction between polycationic PEI moieties, while the lipid moieties would help to in‐ crease cell interaction of the complexes and facilitate their incorporation into lipid-based micellar systems via hydrophobic interactions [Sawant et al., 2012].

In order to take the advantages of both polycationic polymers and liposomes, Nie *et al.* have constructed programmed lipopolyplexes, featuring well compacted DNA by PEI and liposome complexing. Liposomes contain the helper lipid DOPE and a pH-cleavable PEG-hydrazonecholesterol conjugate for shielding. Lipopolyplexes composed of DNA condensed with PEI, phospholipids including dioleoyl phosphatidyl ethanolamine (DOPE) and pH-labile ω-2 pyridyldithio polyethylene glycol α-succinimidylester (OPSS)-PEG-HZN-Chol and yielded particles of 160 nm size and a zeta potential of +7 mV. Pyridylhydrazone-based Chol-PEG was included in the liposomes for shielding in extracellular compartments and dynamic deshield‐ ing in acidic conditions such as in endosomes. In addition, this cholesterol-PEG derivative contained also a pyridyldithio moiety to provide the possibility of coupling thiol-functional‐ ized compounds, such as receptor ligands [Fig. 1B.; Nie et al., 2011].

directed towards creating PEI derivatives combining higher transfection efficacy and good biocompatibility. One approach to reduce the cytotoxicity, biodegradable polyethylenimine with imine linkages as acid-labile moieties were synthesized and investigated for pDNA delivery. The half-life of the acid-labile PEI was 1.1 h at pH= 4.5 and 118 h at pH= 7.4, suggesting that the acid-labile PEI may be rapidly degraded into non-toxic low molecular weight PEI in acidic endosome. Acid-labile PEIs showed close transfection efficiency to PEI 25KDa, but much less toxicity due to the degradation of acid-labile linkage. Therefore, the acid-labile PEIs may

224 Novel Gene Therapy Approaches

be useful for the development of a non-toxic polymeric gene carrier [Kim et al., 2005].

loses its stabilization property [Ma et al., 2007; Wu and Zhao, 2007].

micellar systems via hydrophobic interactions [Sawant et al., 2012].

The cationic lipids have been shown to destabilize the endosomal membrane. Addition of dioleoyl phosphatidylethanolamine (DOPE) or cholesteryl hemisuccinate (CHEMS) to nonviral vectors affected the pH-sensitivity of the formulations. The mechanism behind the phenomenon, is that electrical interaction between cationic lipids and anion endosomal membranes results in the formation of ion-pairs that promote the formation of the inverted hexagonal (HII) phase and disrupt endosomal membrane. Many preparations of lipoplexes contain DOPE as a helper lipid for fusogenic functionality. The ability of DOPE to destabilize endosomal membranes is based on its propensity to acquire an inverted hexagonal phase (HII). DOPE has a small cross-section head group and a large hydrocarbon area that favors a nonbilayer structure with a cone shape that facilitates the destabilization of endosomal membranes and gene transfection. The pH-responsive component, CHEMS, being negatively charged at neutral pH stabilizes the bilayer structure, however, at acidic pH it becomes protonated and

Another approach to reduce the cytotoxicity of PEI is modification with hydrophobic moi‐ eties, such as lipids. Since lipids are the main component of cell membrane, modification with hydrophobic moieties may result in additional hydrophobic interaction between poly‐ plexes and cell membranes, which in turn would facilitate the delivery of a payload into cells. Various hydrophobic modifications have been tried, including modification with cho‐ lesterol, myristate, dodecyl iodide, hexadecyl iodide, palmitic acid, oleic acid, stearic acid, and phosphatidylcholine. In a recent study, Sawant *et al*. developed the synthesis and char‐ acterization of a PEI-DOPE conjugate, which was explored for gene delivery *in vitro*. The modification with DOPE strongly increased the transfection efficiency of low molecular weight PEI-1.8 kDa without any negative effects on its low cytotoxicity. The PEI-PE conju‐ gate was synthesized by reacting a phospholipid with low molecular weight PEI (PEI-1.8). It was assumed that a PEI-PE conjugate would condense DNA due to the electrostatic in‐ teraction between polycationic PEI moieties, while the lipid moieties would help to in‐ crease cell interaction of the complexes and facilitate their incorporation into lipid-based

In order to take the advantages of both polycationic polymers and liposomes, Nie *et al.* have constructed programmed lipopolyplexes, featuring well compacted DNA by PEI and liposome complexing. Liposomes contain the helper lipid DOPE and a pH-cleavable PEG-hydrazonecholesterol conjugate for shielding. Lipopolyplexes composed of DNA condensed with PEI, phospholipids including dioleoyl phosphatidyl ethanolamine (DOPE) and pH-labile ω-2 pyridyldithio polyethylene glycol α-succinimidylester (OPSS)-PEG-HZN-Chol and yielded The endosome-escape potential of poly-histidine increases their use for delivery of nucleic acids. The imidazole ring within histidine is a major component. Under the action of an acidic endosomal interior, the weak basic nature of the imidazole ring with pKa around 6 allows its protonation and acquires cationic charges which trigger the destabilization of en‐ dosomal membranes. Accumulation of histidine residues within endosomes could elicit a proton sponge effect and destroy endosomes as a result of their increased osmolarity. Both chemistry conjugation and genetic engineering have produced a series of histidine-rich pol‐ ymers and peptides as well as lipids with imidazole, imidazolinium or imidazolium polar heads. These histidylated carriers have been used to deliver nucleic acids including pDNA, mRNA or siRNA duplex *in vitro* and *in vivo* with increased transfection efficiency [Midoux et al., 2007; Martin and Rice, 2007].

The histidine-rich peptide H5WYG is a derivative of the N-terminal sequence of the HA-2 subunit of the *influenza virus* hemagglutinin in which five of the amino acids have been re‐ placed with histidine residues. H5WYG is able to selectively destabilize membranes at a slightly acidic pH as the histidine residues are protonated. An anionic derivative of this peptide, E5WYG, in which the histidines are replaced by glutamic acid residues, is com‐ pletely ineffective at membrane permeabilization at a pH= 6.8, while H5WYG can disrupt 50% of cells at pH= 6.8 and 97% of cells at pH= 6.2 within 15 minutes. H5WYG was also able to retain its activity in the presence of serum, making it possible for use *in vivo*. The most well-known non-viral DNA condensing agent is poly-L-lysine. However, PLL only exhibits modest transfection when used alone and requires the addition of an endosomolyt‐ ic agent such as chloroquine or a fusogenic peptide to allow for release into the cytoplasm. To increase the transfection efficiency of polylysine without the addition of membrane-dis‐ rupting agents, a histidine-substituted polylysine can be constructed that is able to become cationic at endosomal pH [Martin and Rice, 2007].

In an attempt to facilitate endosome escape, many strategies have been developed to mimic the viral mechanism for endosome destabilization. Viruses have acquired efficient solutions for escaping from the maturating acidifying endosomes. For example, glycoproteins of enveloped viruses contain hidden fusion peptides which are exposed after endocytosis, to trigger fusion of the viral with the endosomal membrane [Wagner, 2011]. It is well-established that the influenza virus utilizes the pH-sensitive membrane-destabilizing Hemagglutinin protein (HA2) displayed on the viral coat to disrupt the endosomal membrane and enter the cytoplasm. Hemagglutinin and other fusion proteins are characterized by their unique ability to switch from an ionized and hydrophilic conformation at physiologic pH to a hydrophobic and membrane-active one in response to acidic endosomal pH gradients, which destabilizes the endosomal membrane leading to leakage of endosomal contents into the cytoplasm [Lin et al., 2010]. At neutral pH, the HA2 subunit adapts a non-helical conformation due to charge repulsion arising from ionization of glutamic and aspartic acid residues. Within the interior of the acidic endosomal compartment, however, the HA2 subunit transitions into a stable helical secondary structure due to the protonation of glutamic and aspartic acids. The hydrophobic and hydrophilic faces of the helical conformation favor endosomal membrane destabilization.

assemble into organized membrane-enclosed structures has been mimicked by amphiphilic co-polymers. The combination of a hydrophobic monomer and an ionizable co-monomer that has a more hydrophilic nature is one of the interesting strategies that have been adopted frequently for pH-responsive gene delivery. A change in pH and subsequent adjustment in the net charge causes the phase transformation depending on the hydrophobic and hydrophilic balance of the copolymer. These synthetic amphiphiles form assemblies that are remarkably similar to biological analogues, such as vesicles. Polymeric amphiphiles have much higher molecular weights than phospholipids and can self-assemble into more entangled membranes, imparting improved mechanical properties to the final structure [Park et al., 2010]. Polymer vesicles or polymersomes can combine several different polymeric compositions provided that they have the correct hydrophile/hydrophobe ratio. Particularly interesting for biomedical applications, are those copolymers that combine hydrophobic blocks with the non-antigenic properties of either PEG or biomimetic poly (2-(methacryloyloxy) ethyl phosphorylcholine) (PMPC). Indeed, the macromolecular nature of such polymersomes offers several advantages as compared to low-molecular-mass amphiphilic systems such as liposomes. PEG or PMPC polymersomes can be decorated by denser and higher molecular mass hydrophilic polymeric corona, with consequent longer circulation times than more traditional delivery systems such as stealth liposomes and other nanoparticles. Typical examples are the copolymers of methyl methacrylate (MMA) with methacrylic acid (MAc) or dimethylaminoethyl methacrylate (DMAEMA). The MMA is the hydrophobic section while MAc is the hydrophilic part of the chains. MAc is more hydrophilic at high pH when the carboxylic groups (COOH) are depro‐ tonated, but becomes more hydrophobic when the carboxylic groups are protonated. The phase change occurs around the pKa value of the carboxylic groups, which is around 4.5-5.5. The copolymers of MMA with DMAEMA, which are hydrophilic at low pH, when the amino groups are protonated, but more hydrophobic when the amino groups are deprotonated. The key feature of these polymers is their ability to directly enhance the intracellular delivery of DNA, by destabilizing biological membranes in response to pH changes within the vesicular

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These copolymers are characterized by their unique ability to "sense" the changes in environ‐ ment pH where they undergo a change from a hydrophilic, stealth-like conformation at physiologic pH to a hydrophobic and membrane-destabilizing one in response to acidic endosomal pH gradients [Lin et al., 2010]. Poly(ethylacrylic acid) (PEAA) is the first reported polymer to display a pH-dependent disruption of synthetic lipid vesicles at acidic pH= 6.3 or lower. The family of poly (alkyl acrylic acid) like poly (methyl acrylic acid) (PMAA), poly (ethyl acrylic acid) (PEAA), poly (propyl acrylic acid) (PPAA), and poly (butyl acrylic acid) (PBAA) were provided with pH-dependent, membrane destabilizing activities. These polymers are hydrophilic and stealth-like at physiological pH, but become membrane-destabilizing after uptake into the endosomal compartment where they enhance the release of therapeutic cargo

Diblock copolymers PMPC-PDPA as biomimetic polymersomes were used for gene delivery made of pH-sensitive poly (2-(methacryloyloxy) ethyl-phosphorylcholine)-co-poly (2- (diisopropylamino) ethyl-methacrylate) (PMPC-PDPA) diblock copolymers. The high bio‐

compartment [Park et al., 2010].

into the cytoplasm [Lin et al., 2010].

Endocytosed non-enveloped viruses such as *rhinovirus* or *adenovirus* expose lytic domains which directly disrupt the endosomal membrane, either (in case of *rhinovirus*) generating a pore large enough for crossing of the viral RNA strand into the cytoplasm or (in case of *adenovirus*) disrupting the whole endosome. Such lytic domains have been utilized in artificial settings as synthetic peptides for endosomal escape of polyplexes [Wagner, 2011].

Synthetic peptides mimicking a virus's fusogenic peptides have also been designed for delivery of nucleic acids. The amphipathic peptide, GALA, was synthesized with 30 amino acid residues with a repeated amino acid sequence (e.g., glutamic acid-alanine-leucinealanine) that demonstrated pH sensitive fusogenic properties. A GALA peptide with a formulation based on DNA/cationic liposome/ Transferrin (Tf) complexes induced enhanced gene transfection. It is assumed that Tf-triggered internalization of the complexes by receptormediated endocytosis and the GALA peptide promoted endosomal destabilization and release of the genetic material into the cytoplasm [Park et al., 2010].

Modification of multifunctional envelope-type nano-devices (MENDs) with GALA peptide facilitating endosomal escape, leads to the enhanced transfection efficiency of pDNA and siRNA duplex *in vitro* and *in vivo*. To mimic envelope-type virus-like delivery systems, Sasaki *et al*. developed an artificial nanocarrier system termed as a MEND, which consists of a condensed DNA nanoparticle and lipid envelope which is further equipped with Tf, Choles‐ terol-GALA (Chol-GALA), or PEG-GALA to achieve target specificity and controlled intra‐ cellular trafficking, especially endosomal escape. As GALA can show fusogenic activity only at acidic pH, the direct fusion of MEND with the plasma membrane is feasible only after internalization. The Tf-MEND introduced with Chol-GALA or PEG-GALA showed a ten-fold higher transfection than that displayed by Tf-MEND. However, the simultaneous introduction of Chol-GALA and PEG-GALA enhanced the transfection efficiency more than 100-fold as compared to Tf-MEND mediated transfection. Chol-GALA and PEG-GALA operated synerg‐ istically to destabilize the envelope and endosomal membranes, respectively. Chol-GALA interacted with the envelope membrane whereas PEG-GALA penetrated into the endosomal membrane, which could destabilize the membranes and induce fusion [Sasaki et al., 2008].

A cationic counterpart of GALA is the peptide KALA which is formed by substitution of the alanine of GALA with lysine and a decrease in content of glutamic acid. KALA was the first designed peptide that could bind DNA, destabilize membranes, and mediate significant gene delivery. KALA undergoes a pH-dependent amphipathic α-helix to random coil conforma‐ tional change, when the environmental pH decreased from 7.5 to 5.0. KALA can deliver both ODN and pDNA into cells [Park et al., 2010].

Several groups have focused their efforts on the development of synthetic, polymeric carriers that mimic the endosomolytic properties of fusogenic proteins and enhance the cytoplasmic delivery of therapeutic macromolecules. Recently, the ability of natural phospholipids to selfrepulsion arising from ionization of glutamic and aspartic acid residues. Within the interior of the acidic endosomal compartment, however, the HA2 subunit transitions into a stable helical secondary structure due to the protonation of glutamic and aspartic acids. The hydrophobic and hydrophilic faces of the helical conformation favor endosomal membrane destabilization.

Endocytosed non-enveloped viruses such as *rhinovirus* or *adenovirus* expose lytic domains which directly disrupt the endosomal membrane, either (in case of *rhinovirus*) generating a pore large enough for crossing of the viral RNA strand into the cytoplasm or (in case of *adenovirus*) disrupting the whole endosome. Such lytic domains have been utilized in artificial

Synthetic peptides mimicking a virus's fusogenic peptides have also been designed for delivery of nucleic acids. The amphipathic peptide, GALA, was synthesized with 30 amino acid residues with a repeated amino acid sequence (e.g., glutamic acid-alanine-leucinealanine) that demonstrated pH sensitive fusogenic properties. A GALA peptide with a formulation based on DNA/cationic liposome/ Transferrin (Tf) complexes induced enhanced gene transfection. It is assumed that Tf-triggered internalization of the complexes by receptormediated endocytosis and the GALA peptide promoted endosomal destabilization and release

Modification of multifunctional envelope-type nano-devices (MENDs) with GALA peptide facilitating endosomal escape, leads to the enhanced transfection efficiency of pDNA and siRNA duplex *in vitro* and *in vivo*. To mimic envelope-type virus-like delivery systems, Sasaki *et al*. developed an artificial nanocarrier system termed as a MEND, which consists of a condensed DNA nanoparticle and lipid envelope which is further equipped with Tf, Choles‐ terol-GALA (Chol-GALA), or PEG-GALA to achieve target specificity and controlled intra‐ cellular trafficking, especially endosomal escape. As GALA can show fusogenic activity only at acidic pH, the direct fusion of MEND with the plasma membrane is feasible only after internalization. The Tf-MEND introduced with Chol-GALA or PEG-GALA showed a ten-fold higher transfection than that displayed by Tf-MEND. However, the simultaneous introduction of Chol-GALA and PEG-GALA enhanced the transfection efficiency more than 100-fold as compared to Tf-MEND mediated transfection. Chol-GALA and PEG-GALA operated synerg‐ istically to destabilize the envelope and endosomal membranes, respectively. Chol-GALA interacted with the envelope membrane whereas PEG-GALA penetrated into the endosomal membrane, which could destabilize the membranes and induce fusion [Sasaki et al., 2008].

A cationic counterpart of GALA is the peptide KALA which is formed by substitution of the alanine of GALA with lysine and a decrease in content of glutamic acid. KALA was the first designed peptide that could bind DNA, destabilize membranes, and mediate significant gene delivery. KALA undergoes a pH-dependent amphipathic α-helix to random coil conforma‐ tional change, when the environmental pH decreased from 7.5 to 5.0. KALA can deliver both

Several groups have focused their efforts on the development of synthetic, polymeric carriers that mimic the endosomolytic properties of fusogenic proteins and enhance the cytoplasmic delivery of therapeutic macromolecules. Recently, the ability of natural phospholipids to self-

settings as synthetic peptides for endosomal escape of polyplexes [Wagner, 2011].

of the genetic material into the cytoplasm [Park et al., 2010].

226 Novel Gene Therapy Approaches

ODN and pDNA into cells [Park et al., 2010].

assemble into organized membrane-enclosed structures has been mimicked by amphiphilic co-polymers. The combination of a hydrophobic monomer and an ionizable co-monomer that has a more hydrophilic nature is one of the interesting strategies that have been adopted frequently for pH-responsive gene delivery. A change in pH and subsequent adjustment in the net charge causes the phase transformation depending on the hydrophobic and hydrophilic balance of the copolymer. These synthetic amphiphiles form assemblies that are remarkably similar to biological analogues, such as vesicles. Polymeric amphiphiles have much higher molecular weights than phospholipids and can self-assemble into more entangled membranes, imparting improved mechanical properties to the final structure [Park et al., 2010]. Polymer vesicles or polymersomes can combine several different polymeric compositions provided that they have the correct hydrophile/hydrophobe ratio. Particularly interesting for biomedical applications, are those copolymers that combine hydrophobic blocks with the non-antigenic properties of either PEG or biomimetic poly (2-(methacryloyloxy) ethyl phosphorylcholine) (PMPC). Indeed, the macromolecular nature of such polymersomes offers several advantages as compared to low-molecular-mass amphiphilic systems such as liposomes. PEG or PMPC polymersomes can be decorated by denser and higher molecular mass hydrophilic polymeric corona, with consequent longer circulation times than more traditional delivery systems such as stealth liposomes and other nanoparticles. Typical examples are the copolymers of methyl methacrylate (MMA) with methacrylic acid (MAc) or dimethylaminoethyl methacrylate (DMAEMA). The MMA is the hydrophobic section while MAc is the hydrophilic part of the chains. MAc is more hydrophilic at high pH when the carboxylic groups (COOH) are depro‐ tonated, but becomes more hydrophobic when the carboxylic groups are protonated. The phase change occurs around the pKa value of the carboxylic groups, which is around 4.5-5.5. The copolymers of MMA with DMAEMA, which are hydrophilic at low pH, when the amino groups are protonated, but more hydrophobic when the amino groups are deprotonated. The key feature of these polymers is their ability to directly enhance the intracellular delivery of DNA, by destabilizing biological membranes in response to pH changes within the vesicular compartment [Park et al., 2010].

These copolymers are characterized by their unique ability to "sense" the changes in environ‐ ment pH where they undergo a change from a hydrophilic, stealth-like conformation at physiologic pH to a hydrophobic and membrane-destabilizing one in response to acidic endosomal pH gradients [Lin et al., 2010]. Poly(ethylacrylic acid) (PEAA) is the first reported polymer to display a pH-dependent disruption of synthetic lipid vesicles at acidic pH= 6.3 or lower. The family of poly (alkyl acrylic acid) like poly (methyl acrylic acid) (PMAA), poly (ethyl acrylic acid) (PEAA), poly (propyl acrylic acid) (PPAA), and poly (butyl acrylic acid) (PBAA) were provided with pH-dependent, membrane destabilizing activities. These polymers are hydrophilic and stealth-like at physiological pH, but become membrane-destabilizing after uptake into the endosomal compartment where they enhance the release of therapeutic cargo into the cytoplasm [Lin et al., 2010].

Diblock copolymers PMPC-PDPA as biomimetic polymersomes were used for gene delivery made of pH-sensitive poly (2-(methacryloyloxy) ethyl-phosphorylcholine)-co-poly (2- (diisopropylamino) ethyl-methacrylate) (PMPC-PDPA) diblock copolymers. The high bio‐ compatibility features are ascribed to PMPC residue whereas the PDPA block imparts pH responsiveness. These diblock copolymers exist as stable vesicles at physiological pH but these vesicles rapidly dissociate at around pH= 5-6 to form unimers. The phase transition of vesicles to unimers takes place because of protonation of the tertiary amine groups of PDPA chains, which transforms hydrophobic PDPA chains into a hydrophilic entity from physiological pH to mild acidic conditions. pH-responsive PMPC-PDPA vesicles release their contents upon exposure to the low pH (5.5) media in endosomes or lysosomes [Lomas et al., 2007].

ternary complexes to achieve cell sensitization through simultaneous siRNA and drug de‐ livery from a single carrier. These results show that knockdown of *plk1* results in sensiti‐ zation of multi-drug resistant cells to doxorubicin, and this combination of gene silencing and small molecule drug delivery may prove useful to achieve potent therapeutic effects

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Acid-degradable and targetable polyion complex (PIC) micelles increased the gene silencing in hepatoma cells. This multi-functional carrier was synthesized by assembling lactosylated-PEG-siRNA conjugates via acid-labile β-thiopropionate linkages into PIC micelles through the mixing with poly (L-lysine). The lactosylated-PEG-siRNA/PLL polyplexes were successfully transported into hepatoma cells in a receptor-mediated manner, releasing hundreds of active siRNA molecules into the cellular interior responding to the pH decrease in the endosomal compartment. This carrier exhibited almost 100 times enhancement in gene silencing activity

One of the several micro-environmental features, which have been widely exploited for improving the efficiency of nucleic acid delivery, is the redox potential gradient existing between extracellular environment and various subcellular organelles in normal as well as pathological states. The existence of a high redox potential gradient between oxidizing extracellular space and the reducing environment of subcellular organelles has been exploited mainly by incorporating a disulfide bond(s) into the structure of the delivery vectors to provide them with a capability to release the therapeutic nucleotides selectively in the subcellular reducing space. The original interest in gene delivery systems controlled by redox potential gradients was guided by the need to transiently enhance stability of the vectors during the

The design of reduction-sensitive polymers and conjugates usually involves incorporation of disulfide linkage(s) in the main chain, at the side chain or the cross-linker in the struc‐ ture of the polymers of either linear or branched structure. There are only a few examples of polyplexes where the disulfide bonds are associated with the nucleic acids [Soundara Manickam and Oupický, 2006]. Reduction-sensitive polymers and conjugates are character‐ ized by an excellent stability in the circulation and in extracellular fluids, whereas they are prone to rapid degradation under a reductive environment present in intracellular com‐ partments such as the cytoplasm and the cell nucleus at a time scale from minutes to hours, through thiol-disulfide exchange reactions. This quick-response chemical degradation be‐ havior is distinct from common hydrolytically degradable polymers such as aliphatic poly‐ esters and polycarbonates in which the ester and carbonate bonds usually exhibit gradual degradation kinetics inside body with degradation times ranging from days to weeks/or to months. This remarkable feature renders them extremely for the controlled cytoplasmic de‐ livery of a variety of bioactive molecules including DNA, siRNA, antisense oligonucleotide

Disulfide bonds present in the structure of polyplexes are readily reduced in the reducing intra‐ cellular environment, while largely preserved in the predominantly oxidizing extracellular

and facilitating the practical utility of siRNA therapeutics [Oishi et al., 2005].

[Benoit et al., 2010].

**8.2. Redox-responsive nanocarriers**

delivery [Cheng et al., 2011; Meng et al., 2009].

(ODN), proteins, drugs, etc [Du et al., 2010].

Comb-like diblock copolymers with a robust membrane-destabilizing activity in response to a mildly acidic pH in the endosome were also developed. Acid-cleavable hydrazone linker has also been frequently utilized to achieve the facilitated release of nucleic acids from polyplexes. The first pH-sensitive block was the copolymer of ethyl acrylic acid (EAA) and hydrophobic methacrylate, and the second block was the copolymer of hexyl methacrylate (HMA) and trimethyl aminoethyl methacrylate (TMAEMA) grafted with Nacryloxy succinimide or β-benzyl L-aspartate N-carboxy-anhydride via acid-cleavable hy‐ drazone linkages. These comb-like polymers exhibited a high concentration-dependent hemolytic activity in acidic solutions and degraded into smaller fragments via acid-hydrol‐ ysis of hydrazone linkages, resulting in minimized toxicity and facilitated elimination by renal excretion *in vivo*. These polymers formed siRNA complexing polyplexes that were stable even in the presence of serum and nucleases, and efficiently silenced GAPDH ex‐ pression in MCF-7 breast cancer cells *in vitro* [Lin et al., 2010].

#### *8.1.3. Low pH-sensitive siRNA/ODN–polymer conjugates*

Small interfering RNA (siRNA)-based therapies have great potential for the treatment of diseases such as cancer, but an effective delivery strategy for siRNA is unclear. Benoit *et al*. developed a ternary pH responsive endosomolythic complex for the delivery of siRNA in order to sensitize drug-resistant ovarian cancer cells to doxorubicin. The electrostatic complexes were self-assembled by cationic micelles used as a nucleating core, siRNA and a pH-responsive endosomolytic polymer. Cationic micelles were formed from diblock co‐ polymers of dimethylaminoethyl methacrylate (pDMAEMA) and butyl methacrylate (pDbB). The hydrophobic butyl core mediated micelle formation while the positively charged pDMAEMA corona enabled siRNA condensation. To enhance cytosolic delivery through endosomal release, a pH-responsive copolymer of poly (styrene-*alt*-maleic anhy‐ dride) (pSMA) was electrostatically complexed with the positively charged siRNA/micelle to form a ternary complex. Complexes exhibited size (30-105 nm) and charge (slightly pos‐ itive) properties and mediated uptake in > 70% of ovarian cancer cells after 1 h of incuba‐ tion [Benoit et al., 2010]. The optimized formulation of the resulting ternary nano-vector were used to deliver siRNA against polo-like kinase 1 (*plk1*), a gene up-regulated in many cancers and increased doxorubicin sensitivity in the drug-resistant ovarian cancer cells. Sensitization occurred through a p53 signaling pathway, as indicated by caspase 3/7 upregulation following *plk1* knockdown and doxorubicin treatment, and this effect could be abrogated using a p53 inhibitor. To demonstrate the potential for dual delivery from this polymer system, micelle cores were subsequently loaded with doxorubicin and utilized in ternary complexes to achieve cell sensitization through simultaneous siRNA and drug de‐ livery from a single carrier. These results show that knockdown of *plk1* results in sensiti‐ zation of multi-drug resistant cells to doxorubicin, and this combination of gene silencing and small molecule drug delivery may prove useful to achieve potent therapeutic effects [Benoit et al., 2010].

Acid-degradable and targetable polyion complex (PIC) micelles increased the gene silencing in hepatoma cells. This multi-functional carrier was synthesized by assembling lactosylated-PEG-siRNA conjugates via acid-labile β-thiopropionate linkages into PIC micelles through the mixing with poly (L-lysine). The lactosylated-PEG-siRNA/PLL polyplexes were successfully transported into hepatoma cells in a receptor-mediated manner, releasing hundreds of active siRNA molecules into the cellular interior responding to the pH decrease in the endosomal compartment. This carrier exhibited almost 100 times enhancement in gene silencing activity and facilitating the practical utility of siRNA therapeutics [Oishi et al., 2005].

#### **8.2. Redox-responsive nanocarriers**

compatibility features are ascribed to PMPC residue whereas the PDPA block imparts pH responsiveness. These diblock copolymers exist as stable vesicles at physiological pH but these vesicles rapidly dissociate at around pH= 5-6 to form unimers. The phase transition of vesicles to unimers takes place because of protonation of the tertiary amine groups of PDPA chains, which transforms hydrophobic PDPA chains into a hydrophilic entity from physiological pH to mild acidic conditions. pH-responsive PMPC-PDPA vesicles release their contents upon

Comb-like diblock copolymers with a robust membrane-destabilizing activity in response to a mildly acidic pH in the endosome were also developed. Acid-cleavable hydrazone linker has also been frequently utilized to achieve the facilitated release of nucleic acids from polyplexes. The first pH-sensitive block was the copolymer of ethyl acrylic acid (EAA) and hydrophobic methacrylate, and the second block was the copolymer of hexyl methacrylate (HMA) and trimethyl aminoethyl methacrylate (TMAEMA) grafted with Nacryloxy succinimide or β-benzyl L-aspartate N-carboxy-anhydride via acid-cleavable hy‐ drazone linkages. These comb-like polymers exhibited a high concentration-dependent hemolytic activity in acidic solutions and degraded into smaller fragments via acid-hydrol‐ ysis of hydrazone linkages, resulting in minimized toxicity and facilitated elimination by renal excretion *in vivo*. These polymers formed siRNA complexing polyplexes that were stable even in the presence of serum and nucleases, and efficiently silenced GAPDH ex‐

Small interfering RNA (siRNA)-based therapies have great potential for the treatment of diseases such as cancer, but an effective delivery strategy for siRNA is unclear. Benoit *et al*. developed a ternary pH responsive endosomolythic complex for the delivery of siRNA in order to sensitize drug-resistant ovarian cancer cells to doxorubicin. The electrostatic complexes were self-assembled by cationic micelles used as a nucleating core, siRNA and a pH-responsive endosomolytic polymer. Cationic micelles were formed from diblock co‐ polymers of dimethylaminoethyl methacrylate (pDMAEMA) and butyl methacrylate (pDbB). The hydrophobic butyl core mediated micelle formation while the positively charged pDMAEMA corona enabled siRNA condensation. To enhance cytosolic delivery through endosomal release, a pH-responsive copolymer of poly (styrene-*alt*-maleic anhy‐ dride) (pSMA) was electrostatically complexed with the positively charged siRNA/micelle to form a ternary complex. Complexes exhibited size (30-105 nm) and charge (slightly pos‐ itive) properties and mediated uptake in > 70% of ovarian cancer cells after 1 h of incuba‐ tion [Benoit et al., 2010]. The optimized formulation of the resulting ternary nano-vector were used to deliver siRNA against polo-like kinase 1 (*plk1*), a gene up-regulated in many cancers and increased doxorubicin sensitivity in the drug-resistant ovarian cancer cells. Sensitization occurred through a p53 signaling pathway, as indicated by caspase 3/7 upregulation following *plk1* knockdown and doxorubicin treatment, and this effect could be abrogated using a p53 inhibitor. To demonstrate the potential for dual delivery from this polymer system, micelle cores were subsequently loaded with doxorubicin and utilized in

exposure to the low pH (5.5) media in endosomes or lysosomes [Lomas et al., 2007].

pression in MCF-7 breast cancer cells *in vitro* [Lin et al., 2010].

*8.1.3. Low pH-sensitive siRNA/ODN–polymer conjugates*

228 Novel Gene Therapy Approaches

One of the several micro-environmental features, which have been widely exploited for improving the efficiency of nucleic acid delivery, is the redox potential gradient existing between extracellular environment and various subcellular organelles in normal as well as pathological states. The existence of a high redox potential gradient between oxidizing extracellular space and the reducing environment of subcellular organelles has been exploited mainly by incorporating a disulfide bond(s) into the structure of the delivery vectors to provide them with a capability to release the therapeutic nucleotides selectively in the subcellular reducing space. The original interest in gene delivery systems controlled by redox potential gradients was guided by the need to transiently enhance stability of the vectors during the delivery [Cheng et al., 2011; Meng et al., 2009].

The design of reduction-sensitive polymers and conjugates usually involves incorporation of disulfide linkage(s) in the main chain, at the side chain or the cross-linker in the struc‐ ture of the polymers of either linear or branched structure. There are only a few examples of polyplexes where the disulfide bonds are associated with the nucleic acids [Soundara Manickam and Oupický, 2006]. Reduction-sensitive polymers and conjugates are character‐ ized by an excellent stability in the circulation and in extracellular fluids, whereas they are prone to rapid degradation under a reductive environment present in intracellular com‐ partments such as the cytoplasm and the cell nucleus at a time scale from minutes to hours, through thiol-disulfide exchange reactions. This quick-response chemical degradation be‐ havior is distinct from common hydrolytically degradable polymers such as aliphatic poly‐ esters and polycarbonates in which the ester and carbonate bonds usually exhibit gradual degradation kinetics inside body with degradation times ranging from days to weeks/or to months. This remarkable feature renders them extremely for the controlled cytoplasmic de‐ livery of a variety of bioactive molecules including DNA, siRNA, antisense oligonucleotide (ODN), proteins, drugs, etc [Du et al., 2010].

Disulfide bonds present in the structure of polyplexes are readily reduced in the reducing intra‐ cellular environment, while largely preserved in the predominantly oxidizing extracellular space. The intracellular reduction of disulfide bonds is most likely mediated by small redox mol‐ ecules like glutathione (GSH) and thioredoxin, either alone or with the help of redox enzymes. Glutathione tripeptide (g-glutamyl-cysteinyl-glycine; GSH) is the most abundant intracellular sulfhydryl present in millimolar concentrations inside the cell but only in micromolar concentra‐ tions in the blood plasma and GSH/glutathione disulfide (GSSG) is the major redox couple in ani‐ mal cells [Meng et al., 2009]. Glutathione has multiple direct and indirect functions in many critical cellular processes like synthesis of proteins and DNA, amino acid transport, enzyme ac‐ tivity, metabolism and protection of cells. Glutathione also serves as a reductant by functioning to destroy the free radicals, hydrogen peroxide and other peroxides. It also functions as a storage form of cysteine. The intracellular glutathione concentration is an additive function of both the oxidized (GSSG) and the reduced forms (GSH) of glutathione. The glutathione redox ratio is maintained and determined by the activity of glutathione reductase, NADPH concentrations and transaldolase activity. The redox state of the GSH/GSSG couple is often used as an indicator of the overall redox environment of the cell. The large difference in reducing potential between the intracellular and extracellular milieu may be exploited for triggered intracellular delivery of a variety of bioactive molecules including DNA, siRNA, antisense oligonucleotide (ODN), pro‐ teins and low molecular weight drugs. Furthermore, of particular interest is that tumor tissues are highly reducing and hypoxic compared with normal tissues, with at least 4-fold higher con‐ centrations of GSH in the tumor tissues over normal tissues, rendering the reducible bioconju‐ gates valuable for tumor-specific drug and gene delivery [Meng et al., 2009]. Some examples are mentioned for the use of redox responsive nanocarrier in below:

release of nucleic acids in these vectors relied on cleavage of the RPC in the reduced intracel‐ lular environment, eliminating the toxicity associated with high molecular weight polymers. However these polymers rely on chloroquine or cationic lipids to enhance endosomal escape

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Low molecular weight DNA condensing polypeptides were developed by substituting one to four lysine residues of Cys-Trp-Lys18 (CWK18) with cysteine groups. These polypeptides could spontaneously oxidize to form interpeptide disulfide crosslinks after binding to plasmid DNA, resulting in small stabilized DNA condensates. These reversibly cross-linked polypep‐ tide DNA condensates were 5-60-fold more potent at mediating gene expression in HepG2 and COS-7 cells as compared to the un-crosslinked alkylated CWK18 (AlkCWK18) DNA condensates [Fig. 2 A; McKenzie et al., 2000]. In another study, in order to improve the endosomolytic properties, Histidine containing reducible polycations based on CH6K3H6C monomers (His6 RPCs) was developed, which are highly effective DNA transfection agents, provided sufficient buffering capacity and enhanced *in vitro* gene expression with rapid unpackaging following reduction in the cytoplasm without aid of chloroquine, an agent that

High molecular weight polypeptides containing disulfide bonds in the backbone were synthesized by an oxidative copolymerization of a histidine-rich peptide (HRP) and a nuclear localization sequence (NLS) peptide derived from the importin α-binding SV40 T antigen sequence. The synthetic approach allowed an easy synthesis of reducible copolypeptides (rCPP) with different relative contents of the HRP and NLS sequences. The rCPPs were synthesized by DMSO-mediated oxidative polycondensation. To combine two functional peptides into a single polymeric carrier, multiblock reducible copolypeptides were synthe‐ sized by randomly connecting HRP and NLS peptides via disulfide bonds into a linear polypeptide chain. Mild oxidation of the terminal Cys residues with DMSO was used. The copolypeptides show minimal cytotoxicity and transfection activity comparable to or better

Other carriers, such as chitosan (deacetylated chitin) were also stabilized via disulfide bonds. For this purpose, the primary amino groups of low-molecular-weight chitosan were thiolated with 2-iminothiolane. These modified polymers, such as chitosanthiobutylami‐ dines, were mixed with pDNA to form coacervates in the nanometer range [Bauhuber et al., 2009]. In a recent study, Ho *et al*. modified chitosan (CS) with extending arms consist‐ ing of disulfide spacers and arginine (Arg) residues (CS–SS–Arg) as a novel non-viral carri‐ er for gene delivery. Cleavage of disulfide spacers by glutathione (GSH) and dithiothreitol due to thiol-disulfide exchange reactions indicates that CS–SS–Arg is likely reducible in cy‐ toplasm. The CS–SS–Arg was allowed to condense GFP DNA to form self-organized nano‐ particles with a diameter of 130 nm and zeta potential of 35 mV. The DNA was released from CS–SS–Arg/ DNA nanoparticles over time in the presence of GSH and the results sug‐ gest that the Arg-rich bioreducible CS–SS–Arg/ DNA nanoparticles are promising as a car‐

A dilemma in non-viral nucleic acid delivery is demonstrated by considering the polymeric transfection agent PEI, which is often referred to as the gold standard for polymer-based

and mediate transfection [Read et al., 2003].

promotes endosomal escape [Stevenson et al., 2008].

than control PEI polyplexes [Manickam and Oupický, 2006].

rier for gene delivery [Ho et al., 2011].

#### *8.2.1. The use of responsive sensitive nanocarrier for the stabilization of the carrier and decrease the cytotoxicity*

A prerequisite for every systemic nucleic-acid delivery system is stability in the blood stream prior to reaching its target cell. Therefore, the carrier must prevent the premature release of its load. In order to enhance the stability of the delivery system, either the surface of the carriers was crosslinked, or the low-molecular-weight materials were sulfhydryl polymerized. Pepti‐ des containing many lysines and histidines were used for the complexation of nucleic acids, while cysteines were introduced to obtain stable and reductively degradable carriers. A series of different vehicles was built from sequences of many lysines, tryptophan, and a variable num‐ ber of cysteines. All peptides obtained were able to condense DNA into particles, and exhibited increased stability after disulfide formation. The disulfide crosslinked carriers conveyed 5- to 60-fold higher gene expression as compared to their non-crosslinked analogs. The degree of gene expression was dependent on the number of incorporated cysteines, with a maximum for terminally inserted cysteines. The modification of the e-amino groups of pLL with 3-(2-aminoe‐ thyldithio) propionyl residues or their crosslinking with Dimethyl 3,3-dithiobispropionimi‐ date (DTBP) comprises another approach utilizing reductively degradable gene carriers. These molecules were able to complex nucleotides into particles that were stable under physiological conditions but disintegrated upon treatment with GSH [Meng et al., 2009].

Read *et al*. prepared gene delivery vectors based on reducible polycations (RPCs) by oxidative polycondensation of the peptide Cys-Lys10-Cys and used to condense nucleic acids. The release of nucleic acids in these vectors relied on cleavage of the RPC in the reduced intracel‐ lular environment, eliminating the toxicity associated with high molecular weight polymers. However these polymers rely on chloroquine or cationic lipids to enhance endosomal escape and mediate transfection [Read et al., 2003].

space. The intracellular reduction of disulfide bonds is most likely mediated by small redox mol‐ ecules like glutathione (GSH) and thioredoxin, either alone or with the help of redox enzymes. Glutathione tripeptide (g-glutamyl-cysteinyl-glycine; GSH) is the most abundant intracellular sulfhydryl present in millimolar concentrations inside the cell but only in micromolar concentra‐ tions in the blood plasma and GSH/glutathione disulfide (GSSG) is the major redox couple in ani‐ mal cells [Meng et al., 2009]. Glutathione has multiple direct and indirect functions in many critical cellular processes like synthesis of proteins and DNA, amino acid transport, enzyme ac‐ tivity, metabolism and protection of cells. Glutathione also serves as a reductant by functioning to destroy the free radicals, hydrogen peroxide and other peroxides. It also functions as a storage form of cysteine. The intracellular glutathione concentration is an additive function of both the oxidized (GSSG) and the reduced forms (GSH) of glutathione. The glutathione redox ratio is maintained and determined by the activity of glutathione reductase, NADPH concentrations and transaldolase activity. The redox state of the GSH/GSSG couple is often used as an indicator of the overall redox environment of the cell. The large difference in reducing potential between the intracellular and extracellular milieu may be exploited for triggered intracellular delivery of a variety of bioactive molecules including DNA, siRNA, antisense oligonucleotide (ODN), pro‐ teins and low molecular weight drugs. Furthermore, of particular interest is that tumor tissues are highly reducing and hypoxic compared with normal tissues, with at least 4-fold higher con‐ centrations of GSH in the tumor tissues over normal tissues, rendering the reducible bioconju‐ gates valuable for tumor-specific drug and gene delivery [Meng et al., 2009]. Some examples are

*8.2.1. The use of responsive sensitive nanocarrier for the stabilization of the carrier and decrease the*

A prerequisite for every systemic nucleic-acid delivery system is stability in the blood stream prior to reaching its target cell. Therefore, the carrier must prevent the premature release of its load. In order to enhance the stability of the delivery system, either the surface of the carriers was crosslinked, or the low-molecular-weight materials were sulfhydryl polymerized. Pepti‐ des containing many lysines and histidines were used for the complexation of nucleic acids, while cysteines were introduced to obtain stable and reductively degradable carriers. A series of different vehicles was built from sequences of many lysines, tryptophan, and a variable num‐ ber of cysteines. All peptides obtained were able to condense DNA into particles, and exhibited increased stability after disulfide formation. The disulfide crosslinked carriers conveyed 5- to 60-fold higher gene expression as compared to their non-crosslinked analogs. The degree of gene expression was dependent on the number of incorporated cysteines, with a maximum for terminally inserted cysteines. The modification of the e-amino groups of pLL with 3-(2-aminoe‐ thyldithio) propionyl residues or their crosslinking with Dimethyl 3,3-dithiobispropionimi‐ date (DTBP) comprises another approach utilizing reductively degradable gene carriers. These molecules were able to complex nucleotides into particles that were stable under physiological

Read *et al*. prepared gene delivery vectors based on reducible polycations (RPCs) by oxidative polycondensation of the peptide Cys-Lys10-Cys and used to condense nucleic acids. The

mentioned for the use of redox responsive nanocarrier in below:

conditions but disintegrated upon treatment with GSH [Meng et al., 2009].

*cytotoxicity*

230 Novel Gene Therapy Approaches

Low molecular weight DNA condensing polypeptides were developed by substituting one to four lysine residues of Cys-Trp-Lys18 (CWK18) with cysteine groups. These polypeptides could spontaneously oxidize to form interpeptide disulfide crosslinks after binding to plasmid DNA, resulting in small stabilized DNA condensates. These reversibly cross-linked polypep‐ tide DNA condensates were 5-60-fold more potent at mediating gene expression in HepG2 and COS-7 cells as compared to the un-crosslinked alkylated CWK18 (AlkCWK18) DNA condensates [Fig. 2 A; McKenzie et al., 2000]. In another study, in order to improve the endosomolytic properties, Histidine containing reducible polycations based on CH6K3H6C monomers (His6 RPCs) was developed, which are highly effective DNA transfection agents, provided sufficient buffering capacity and enhanced *in vitro* gene expression with rapid unpackaging following reduction in the cytoplasm without aid of chloroquine, an agent that promotes endosomal escape [Stevenson et al., 2008].

High molecular weight polypeptides containing disulfide bonds in the backbone were synthesized by an oxidative copolymerization of a histidine-rich peptide (HRP) and a nuclear localization sequence (NLS) peptide derived from the importin α-binding SV40 T antigen sequence. The synthetic approach allowed an easy synthesis of reducible copolypeptides (rCPP) with different relative contents of the HRP and NLS sequences. The rCPPs were synthesized by DMSO-mediated oxidative polycondensation. To combine two functional peptides into a single polymeric carrier, multiblock reducible copolypeptides were synthe‐ sized by randomly connecting HRP and NLS peptides via disulfide bonds into a linear polypeptide chain. Mild oxidation of the terminal Cys residues with DMSO was used. The copolypeptides show minimal cytotoxicity and transfection activity comparable to or better than control PEI polyplexes [Manickam and Oupický, 2006].

Other carriers, such as chitosan (deacetylated chitin) were also stabilized via disulfide bonds. For this purpose, the primary amino groups of low-molecular-weight chitosan were thiolated with 2-iminothiolane. These modified polymers, such as chitosanthiobutylami‐ dines, were mixed with pDNA to form coacervates in the nanometer range [Bauhuber et al., 2009]. In a recent study, Ho *et al*. modified chitosan (CS) with extending arms consist‐ ing of disulfide spacers and arginine (Arg) residues (CS–SS–Arg) as a novel non-viral carri‐ er for gene delivery. Cleavage of disulfide spacers by glutathione (GSH) and dithiothreitol due to thiol-disulfide exchange reactions indicates that CS–SS–Arg is likely reducible in cy‐ toplasm. The CS–SS–Arg was allowed to condense GFP DNA to form self-organized nano‐ particles with a diameter of 130 nm and zeta potential of 35 mV. The DNA was released from CS–SS–Arg/ DNA nanoparticles over time in the presence of GSH and the results sug‐ gest that the Arg-rich bioreducible CS–SS–Arg/ DNA nanoparticles are promising as a car‐ rier for gene delivery [Ho et al., 2011].

A dilemma in non-viral nucleic acid delivery is demonstrated by considering the polymeric transfection agent PEI, which is often referred to as the gold standard for polymer-based gene carriers due to the relatively high transfection efficacy of its polyplexes. Unfortunate‐ ly, efficacy and adverse reactions seem to be strongly associated with the use of PEI. A popular strategy to reduce the toxicity of polyplexes is to use low MW polycations or cati‐ onic monomers that are less cytolytic, and crosslink them with agents that can be cleaved or activated by the intracellular environment. Numerous reports describe the synthesis of carrier systems containing disulfide bonds that allow for a compaction and protection of the nucleic acid in the extracellular environment accompanied by reduced toxicity due to intracellular polymer degradation. Peng *et al*. prepared thiolated PEI (800 Da) to further ox‐ idize into disulfide cross-linked PEI (PEI-SS), with average molecular weights of 7.1, 8.0 and 8.4 kDa depending on the degree of thiolation. Those PEI-SS had lower cytotoxicities and higher transgene expressions compared with that of the 25-kDa branched PEI (bPEI) [Peng et al., 2008].

Kang *et al*. synthesized reducible polycations (RPC) that degrade due to changes in the in‐ tracellular reduction potential from low molecular weight (MW) bPEI 0.8 kDa via thiola‐ tion and oxidation. In this study, 2-iminothiolane was used to create thiol groups from primary amines. In this approach, a primary amine reacts with 2-iminothiolane to yield a thiol group and an amidine moiety. A procedure based on the use of 2-iminothiolane was employed in order to avoid problems associated with the use of DTBP. As a result, unlike other thiolation methods, by converting primary amines to amidine groups, the number of positive charges in the final product at a neutral pH was preserved. Moreover, the newly generated amidines contribute to the electrostatic condensation of nucleic acids. The cyto‐ toxicity of RPC-bPEI 0.8 kDa was 8-19 times less than that of the gold standard of polymer‐ ic transfection reagents, bPEI 25 kDa. In general, the toxicity of High MW (HMW) PEI is greater than that of Low MW (LMW) PEI because HMW PEI interacts more effectively with components that are essential for cell survival such as intracellular membranes, vital pro‐ teins, and nucleic acids than its LMW counterpart. Thus, the reduced cytotoxicity of HMWRPC-bPEI 0.8 kDa may be due to the degradation of the polymer in the intracellular

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In another study, Son *et al* developed a multi-functional gene carrier based on thiolated low molecular weight bPEI with functional moieties for cytoplasm-sensitive reduction, tumor targeting, and prolonged circulation in blood. The bPEI was modified with α-maleimide-ω-N-hydroxysuccinimide ester polyethylene glycol (MAL-PEG-NHS, MW: 5000) and cyclic NGR peptide for enhanced blood compatibility and tumor targeting ability. The resulting polymer (bPEI-SS-PEG-cNGR) exhibited DNA condensing capacity, a reducing property, and im‐ proved tumor targeting, suggesting that the multifunctional polymer constitutes a promising

DOPE and N-[2-methoxypoly (ethylene glycol)-α-aminocarbonylethyl-dithiopropionate] formed a liposome that was covalently coupled to distearoylphosphatidylethanolamine (mPEG-SS-DSPE). The incorporation of the mPEG-SS-DSPE stabilized these liposomes at low pH, but the stabilizing effect was quickly reversed when the carrier was incubated with either DTT or cell-free extracts. Under these conditions, the disulfides were reduced, and thus the protecting PEG chains were cleaved, and the vehicle became degradable at low pH, releasing its load. Disulfides were used for the formation of copolymers consisting of bPEI 25 kDa and PEG of 20 or 30 kDa. The connection of PEG via DSPE led to a 125% increase in blood levels

Polyion complex (PIC) micelles are self-assembling particles with a core-shell structure formed by complexation between a pair of oppositely charged polymers having hydrophil‐ ic PEG segments. They can be utilized as gene delivery vectors with high water-solubility and colloidal stability, employing negatively charged genetic materials and cationic poly‐ mers. One approach to enhance the stability of PIC micelles is glutathione (GSH)-sensitive stabilization of PIC micelles with the core cross-linked through disulfide bonds, composed of antisense oligonucleotide and thiolated PEG-PLL block copolymer. The micelles showed

non-viral vector for cytoplasm- and tumor-specificities [Son et al., 2010].

and decreased hemolysis compared to bPEI 25 kDa [Bauhuber et al., 2009].

environment after cellular uptake [Kang et al., 2011].

*8.2.2. Disulfides for the attachment of a shielding moiety*

**Figure 2. A) Cross-linked peptide-DNA condensates:** Formation of peptide-DNA condensates through ionic binding of the peptide to the pDNA followed by interpeptide oxidation; Stabilization of the DNA condensates by reversible disulfide bonds. B) **A novel stimuli-sensitive liposome**: This carrier composes of cationic peptides (e.g., Arginine oc‐ tamer) and detachable coat (e.g., PEG) for the intracellular gene or drug delivery

Kang *et al*. synthesized reducible polycations (RPC) that degrade due to changes in the in‐ tracellular reduction potential from low molecular weight (MW) bPEI 0.8 kDa via thiola‐ tion and oxidation. In this study, 2-iminothiolane was used to create thiol groups from primary amines. In this approach, a primary amine reacts with 2-iminothiolane to yield a thiol group and an amidine moiety. A procedure based on the use of 2-iminothiolane was employed in order to avoid problems associated with the use of DTBP. As a result, unlike other thiolation methods, by converting primary amines to amidine groups, the number of positive charges in the final product at a neutral pH was preserved. Moreover, the newly generated amidines contribute to the electrostatic condensation of nucleic acids. The cyto‐ toxicity of RPC-bPEI 0.8 kDa was 8-19 times less than that of the gold standard of polymer‐ ic transfection reagents, bPEI 25 kDa. In general, the toxicity of High MW (HMW) PEI is greater than that of Low MW (LMW) PEI because HMW PEI interacts more effectively with components that are essential for cell survival such as intracellular membranes, vital pro‐ teins, and nucleic acids than its LMW counterpart. Thus, the reduced cytotoxicity of HMWRPC-bPEI 0.8 kDa may be due to the degradation of the polymer in the intracellular environment after cellular uptake [Kang et al., 2011].

In another study, Son *et al* developed a multi-functional gene carrier based on thiolated low molecular weight bPEI with functional moieties for cytoplasm-sensitive reduction, tumor targeting, and prolonged circulation in blood. The bPEI was modified with α-maleimide-ω-N-hydroxysuccinimide ester polyethylene glycol (MAL-PEG-NHS, MW: 5000) and cyclic NGR peptide for enhanced blood compatibility and tumor targeting ability. The resulting polymer (bPEI-SS-PEG-cNGR) exhibited DNA condensing capacity, a reducing property, and im‐ proved tumor targeting, suggesting that the multifunctional polymer constitutes a promising non-viral vector for cytoplasm- and tumor-specificities [Son et al., 2010].

#### *8.2.2. Disulfides for the attachment of a shielding moiety*

gene carriers due to the relatively high transfection efficacy of its polyplexes. Unfortunate‐ ly, efficacy and adverse reactions seem to be strongly associated with the use of PEI. A popular strategy to reduce the toxicity of polyplexes is to use low MW polycations or cati‐ onic monomers that are less cytolytic, and crosslink them with agents that can be cleaved or activated by the intracellular environment. Numerous reports describe the synthesis of carrier systems containing disulfide bonds that allow for a compaction and protection of the nucleic acid in the extracellular environment accompanied by reduced toxicity due to intracellular polymer degradation. Peng *et al*. prepared thiolated PEI (800 Da) to further ox‐ idize into disulfide cross-linked PEI (PEI-SS), with average molecular weights of 7.1, 8.0 and 8.4 kDa depending on the degree of thiolation. Those PEI-SS had lower cytotoxicities and higher transgene expressions compared with that of the 25-kDa branched PEI (bPEI)

**Figure 2. A) Cross-linked peptide-DNA condensates:** Formation of peptide-DNA condensates through ionic binding of the peptide to the pDNA followed by interpeptide oxidation; Stabilization of the DNA condensates by reversible disulfide bonds. B) **A novel stimuli-sensitive liposome**: This carrier composes of cationic peptides (e.g., Arginine oc‐

tamer) and detachable coat (e.g., PEG) for the intracellular gene or drug delivery

[Peng et al., 2008].

232 Novel Gene Therapy Approaches

DOPE and N-[2-methoxypoly (ethylene glycol)-α-aminocarbonylethyl-dithiopropionate] formed a liposome that was covalently coupled to distearoylphosphatidylethanolamine (mPEG-SS-DSPE). The incorporation of the mPEG-SS-DSPE stabilized these liposomes at low pH, but the stabilizing effect was quickly reversed when the carrier was incubated with either DTT or cell-free extracts. Under these conditions, the disulfides were reduced, and thus the protecting PEG chains were cleaved, and the vehicle became degradable at low pH, releasing its load. Disulfides were used for the formation of copolymers consisting of bPEI 25 kDa and PEG of 20 or 30 kDa. The connection of PEG via DSPE led to a 125% increase in blood levels and decreased hemolysis compared to bPEI 25 kDa [Bauhuber et al., 2009].

Polyion complex (PIC) micelles are self-assembling particles with a core-shell structure formed by complexation between a pair of oppositely charged polymers having hydrophil‐ ic PEG segments. They can be utilized as gene delivery vectors with high water-solubility and colloidal stability, employing negatively charged genetic materials and cationic poly‐ mers. One approach to enhance the stability of PIC micelles is glutathione (GSH)-sensitive stabilization of PIC micelles with the core cross-linked through disulfide bonds, composed of antisense oligonucleotide and thiolated PEG-PLL block copolymer. The micelles showed sufficient colloidal stability due to the PEG shell and core cross-linking. Moreover, ODN entrapped in the micelles also displayed highly increased stability against nuclease, com‐ pared to that in the micelles without cross-linking. Release of ODN from the dissociated micelles at intracellular GSH concentration suggested the potential for intracellular ODN delivery. It is reported that PEG-peptide (PEG-SS-Cys-Trp-Lys(18): PEG-SS-CWK18) having disulfide cross-linker displayed much enhanced gene transfer efficiency compared to PEGpeptide having non-reducible cross-linker (PEG-VS-CWK18) when complexed with plas‐ mid DNA [Kim and Kim, 2011].

Nevertheless, there has been limited success to date in the development of PICs for siR‐ NA delivery. In general, PICs from monomeric siRNA, which has a short structure com‐ pared to pDNA, lack stability under the physiological condition; therefore, substantial stabilization of PICs is essential to successful siRNA delivery. Meanwhile, after PICs inter‐ nalize and reach the cytoplasm, they are required to efficiently release siRNA to exert its gene silencing effect. To complete such variable properties, considerable efforts have been dedicated to the design and chemical modification of polycations [Yu-Lin et al., 2011]. Takemoto *et al*. reported a new class of chemically modified siRNA, i.e., siRNA-grafted poly (aspartic acid) [PAsp (-SS-siRNA)], for PIC based siRNA delivery. PAsp (-SS-siRNA) consists of a backbone of a poly (aspartic acid) [PAsp] derivative and grafted siRNAs via a disulfide linkage. The siRNA-grafted polymer formed stable PICs due to its larger num‐ bers and higher density of anionic charges compared with monomeric siRNA, leading to effective internalization by cultured cells. Following the endosomal escape of the PIC, the disulfide linkage of the siRNA-grafted polymer allowed efficient siRNA release from the PIC under intracellular reductive conditions. Consequently, the PIC from the siRNA-graft‐ ed polymer showed a potent gene silencing effect without cytotoxicity or immunogenici‐ ty, demonstrating a promising feature of the siRNA-grafted polymer to construct the PIC-

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The conjugation of nucleic acids including siRNA and antisense oligodeoxynucleotide to polymer such as PEG and hyaluronic acid through a disulfide bond represents a approach to construct GSH-responsive gene delivery systems [Cheng et al., 2011]. Lee *et al*. explored the potential possibility of hyaluronic acid (HA) as a biocompatible, biodegradable, and non-cytotoxic material for delivery of siRNA. Nano-sized HA hydrogels, called HA nano‐ gels, were prepared for target-specific intracellular delivery of siRNA to HA receptor over-expressing cancer cells. HA nanogels crosslinked with disulfide linkages were pre‐ pared by an inverse emulsion method. An aqueous phase containing thiol functionalized HA and siRNA was emulsified in an oil phase under ultrasonication, thus generating self cross-linked HA nanogels encapsulating anti-green fluorescent protein (GFP) siRNA. Re‐ lease profiles of siRNA from HA nanogels were studied in response to various reductive conditions that could cleave the disulfide linkages of HA nanogels to varying extents. The HA/siRNA nanogels were readily taken up by HA receptor positive cells (HCT-116 cells) having HA-specific CD44 receptors on the surface [Lee et al., 2007]. Kam *et al*. conjugated various biological molecules, including oligonucleotides and siRNA, to phospholipid–PEG functionalized single-walled carbon nanotube (SWNT) via cleavable disulfide linkage. Due to the presence of a PEG linker, these SWNT conjugates form highly stable suspensions in aqueous solutions including physiological buffers. The SWNT carriers mediated efficient delivery and release of DNA and siRNA inside cells. Gene silencing experiments using HeLa cells displayed a two-fold higher silencing efficiency compared to lipofectamine at the same siRNA concentration. This is ascribed to a high surface area of SWNT for effi‐ cient siRNA cargo loading, high intracellular transporting ability of SWNT, and high de‐ gree of endosome/lysosome escape owing to the disulfide approach [Kam et al., 2005;

based nanocarrier for *in vivo* siRNA delivery [Takemoto et al., 2010].

Meng et al., 2009].

#### *8.2.3. Disulfides for the attachment of a targeting moiety*

For a successful accumulation of the nucleic acid in the target cell, the delivery system requires the attachment of a specific "recognition element". However, since many targeting moieties are rather large, they hinder effective unpacking of the gene vector. Thus, inside cells, it is very important to remove them to facilitate the disassembly of the load. Here, disulfides can be quite useful, as they can be cleaved at the cell surface during cellular entry or in the cytosol [Bauhuber et al., 2009]. The frequency of sulfhydryl occurrence in Antibodies/proteins or other molecules is usually low as compared to other groups like amines or carboxylates. The use of sulfhydryl reactive chemistries thus can restrict modification to only a limited number of sites within a target molecule. N-Succinimidyl 3-(2-pyridyldithio) propionate (SPDP) is one of the most popular heterobi-functional cross-linking agents. The NHS ester end of SPDP reacts with amine groups to form an amide linkage, while the 2-pyridyldithiol group at the other end can react with sulfhydryl residues to form a disulfide linkage. The reagent is also useful in creating sulfhydryls in proteins and other molecules. Once modified with SPDP, a protein can be treated with DTT (or other disulfide reducing agents, to release the pyridine-2-thione leaving group and form the free sulfhydryl [Manjappa et al., 2011].

Iminothiolane (Traut's reagent) can react with primary amines in a ring-opening reac‐ tion that regenerates the free sulfhydryl. An example is the thiolation of antibody us‐ ing Traut's reagent in the preparation of immunoliposomes. It is an excellent thiolation reagent for use in the preparation of immunotoxins. It has also been used to modify and introduce sulfhydryls into oligosaccharides from asparagines linked glycans [Man‐ jappa et al., 2011].

#### *8.2.4. Disulfide bonds to enhance the intracellular release of the nucleic acid*

SiRNA has generated great interest as a research tool and a therapeutic agent because of its ability to efficiently silence specific genes by the mechanism of RNA interference [Vi‐ dugirienė et al., 2007]. However, siRNA cannot penetrate the cellular membrane alone and can be easily degraded by RNase; therefore, effective siRNA carriers are needed for siRNA-based therapies. In this regard, poly ion complexes (PICs) formed from nucleic acids and oppositely charged polycations have been widely studied as a promising nucle‐ ic acids carrier because of the variety of chemical designs of polycations. PICs protect nu‐ cleic acids from enzymatic degradation and show facilitated cellular uptake. Thus, PICs have demonstrated to be useful for plasmid DNA (pDNA) delivery *in vitro* and *in vivo*. Nevertheless, there has been limited success to date in the development of PICs for siR‐ NA delivery. In general, PICs from monomeric siRNA, which has a short structure com‐ pared to pDNA, lack stability under the physiological condition; therefore, substantial stabilization of PICs is essential to successful siRNA delivery. Meanwhile, after PICs inter‐ nalize and reach the cytoplasm, they are required to efficiently release siRNA to exert its gene silencing effect. To complete such variable properties, considerable efforts have been dedicated to the design and chemical modification of polycations [Yu-Lin et al., 2011]. Takemoto *et al*. reported a new class of chemically modified siRNA, i.e., siRNA-grafted poly (aspartic acid) [PAsp (-SS-siRNA)], for PIC based siRNA delivery. PAsp (-SS-siRNA) consists of a backbone of a poly (aspartic acid) [PAsp] derivative and grafted siRNAs via a disulfide linkage. The siRNA-grafted polymer formed stable PICs due to its larger num‐ bers and higher density of anionic charges compared with monomeric siRNA, leading to effective internalization by cultured cells. Following the endosomal escape of the PIC, the disulfide linkage of the siRNA-grafted polymer allowed efficient siRNA release from the PIC under intracellular reductive conditions. Consequently, the PIC from the siRNA-graft‐ ed polymer showed a potent gene silencing effect without cytotoxicity or immunogenici‐ ty, demonstrating a promising feature of the siRNA-grafted polymer to construct the PICbased nanocarrier for *in vivo* siRNA delivery [Takemoto et al., 2010].

sufficient colloidal stability due to the PEG shell and core cross-linking. Moreover, ODN entrapped in the micelles also displayed highly increased stability against nuclease, com‐ pared to that in the micelles without cross-linking. Release of ODN from the dissociated micelles at intracellular GSH concentration suggested the potential for intracellular ODN delivery. It is reported that PEG-peptide (PEG-SS-Cys-Trp-Lys(18): PEG-SS-CWK18) having disulfide cross-linker displayed much enhanced gene transfer efficiency compared to PEGpeptide having non-reducible cross-linker (PEG-VS-CWK18) when complexed with plas‐

For a successful accumulation of the nucleic acid in the target cell, the delivery system requires the attachment of a specific "recognition element". However, since many targeting moieties are rather large, they hinder effective unpacking of the gene vector. Thus, inside cells, it is very important to remove them to facilitate the disassembly of the load. Here, disulfides can be quite useful, as they can be cleaved at the cell surface during cellular entry or in the cytosol [Bauhuber et al., 2009]. The frequency of sulfhydryl occurrence in Antibodies/proteins or other molecules is usually low as compared to other groups like amines or carboxylates. The use of sulfhydryl reactive chemistries thus can restrict modification to only a limited number of sites within a target molecule. N-Succinimidyl 3-(2-pyridyldithio) propionate (SPDP) is one of the most popular heterobi-functional cross-linking agents. The NHS ester end of SPDP reacts with amine groups to form an amide linkage, while the 2-pyridyldithiol group at the other end can react with sulfhydryl residues to form a disulfide linkage. The reagent is also useful in creating sulfhydryls in proteins and other molecules. Once modified with SPDP, a protein can be treated with DTT (or other disulfide reducing agents, to release the pyridine-2-thione leaving group

Iminothiolane (Traut's reagent) can react with primary amines in a ring-opening reac‐ tion that regenerates the free sulfhydryl. An example is the thiolation of antibody us‐ ing Traut's reagent in the preparation of immunoliposomes. It is an excellent thiolation reagent for use in the preparation of immunotoxins. It has also been used to modify and introduce sulfhydryls into oligosaccharides from asparagines linked glycans [Man‐

SiRNA has generated great interest as a research tool and a therapeutic agent because of its ability to efficiently silence specific genes by the mechanism of RNA interference [Vi‐ dugirienė et al., 2007]. However, siRNA cannot penetrate the cellular membrane alone and can be easily degraded by RNase; therefore, effective siRNA carriers are needed for siRNA-based therapies. In this regard, poly ion complexes (PICs) formed from nucleic acids and oppositely charged polycations have been widely studied as a promising nucle‐ ic acids carrier because of the variety of chemical designs of polycations. PICs protect nu‐ cleic acids from enzymatic degradation and show facilitated cellular uptake. Thus, PICs have demonstrated to be useful for plasmid DNA (pDNA) delivery *in vitro* and *in vivo*.

mid DNA [Kim and Kim, 2011].

234 Novel Gene Therapy Approaches

*8.2.3. Disulfides for the attachment of a targeting moiety*

and form the free sulfhydryl [Manjappa et al., 2011].

*8.2.4. Disulfide bonds to enhance the intracellular release of the nucleic acid*

jappa et al., 2011].

The conjugation of nucleic acids including siRNA and antisense oligodeoxynucleotide to polymer such as PEG and hyaluronic acid through a disulfide bond represents a approach to construct GSH-responsive gene delivery systems [Cheng et al., 2011]. Lee *et al*. explored the potential possibility of hyaluronic acid (HA) as a biocompatible, biodegradable, and non-cytotoxic material for delivery of siRNA. Nano-sized HA hydrogels, called HA nano‐ gels, were prepared for target-specific intracellular delivery of siRNA to HA receptor over-expressing cancer cells. HA nanogels crosslinked with disulfide linkages were pre‐ pared by an inverse emulsion method. An aqueous phase containing thiol functionalized HA and siRNA was emulsified in an oil phase under ultrasonication, thus generating self cross-linked HA nanogels encapsulating anti-green fluorescent protein (GFP) siRNA. Re‐ lease profiles of siRNA from HA nanogels were studied in response to various reductive conditions that could cleave the disulfide linkages of HA nanogels to varying extents. The HA/siRNA nanogels were readily taken up by HA receptor positive cells (HCT-116 cells) having HA-specific CD44 receptors on the surface [Lee et al., 2007]. Kam *et al*. conjugated various biological molecules, including oligonucleotides and siRNA, to phospholipid–PEG functionalized single-walled carbon nanotube (SWNT) via cleavable disulfide linkage. Due to the presence of a PEG linker, these SWNT conjugates form highly stable suspensions in aqueous solutions including physiological buffers. The SWNT carriers mediated efficient delivery and release of DNA and siRNA inside cells. Gene silencing experiments using HeLa cells displayed a two-fold higher silencing efficiency compared to lipofectamine at the same siRNA concentration. This is ascribed to a high surface area of SWNT for effi‐ cient siRNA cargo loading, high intracellular transporting ability of SWNT, and high de‐ gree of endosome/lysosome escape owing to the disulfide approach [Kam et al., 2005; Meng et al., 2009].

## **9. Stimuli-sensitive multi-functional nano-particulate nucleic acid delivery systems**

been illustrated in the work by Sawant *et al*. who developed targeted long-circulating PEGy‐ lated liposomes and PEG-phosphatidylethanolamine (PEG-PE)-based micelles possessing several functionalities. Such systems were capable of targeting a specific cell or organ by attaching the monoclonal antibody to their surface via long PEG spacer groups. Second, these liposomes and micelles were additionally modified with TATp moieties attached to the surface of the nanocarrier by using TATp-short PEG-PE derivatives. PEG-PE used for liposome surface modification or for micelle preparation was made degradable by inserting the pH-sensitive hydrazone bond between PEG and PE (PEG-Hz-PE). Under normal pH values, TATp functions on the surface of nanocarriers were ''shielded'' by long protecting PEG chains (pH-degradable PEG 2000-PE or PEG 5000-PE) or by long para- nitrophenyl PEG-PE (pNP-PEG-PE) moieties used to attach antibodies to the nanocarrier (non-pH degradable PEG3400-PE or PEG5000-PE). Following prolonged circulation and uptake into the tumor mass mediated by both the enhanced permeability and retention (EPR) effect and the active targeting agent on the surface of the carrier, the pH responsive bonds are cleaved in the acidic environment of the tumor, thereby releasing the high molecular weight PEG strands from the carrier and exposing the Tat-peptide for enhanced intracellular uptake and intracellular target localization, specifically

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Another example of a triggerable system was created by the synthesis of a liposomal carrier that was comprised of a membrane-permeable ligand and a reductively detachable PEG coating. The surface cover consisted of PEG coupled to DOPE via a thiolytically cleavable linker, while the ligand, an ariginine octamer, was immobilized onto cholesteryl hemisuccinate (CHEMS). These conjugates were mixed with dipalmitoylphosphatidylcholin (DPPC) and unmodified DOPE to form liposomes. DOPE/mPEG–DTP–DSPE liposomes were stable in plasma whereas in the presence of a reducing agent the PEG coating was effectively detached off the liposomal surface, leading to vesicle destabilization and fusion as well as complete release of the entrapped contents. Upon activation of the trigger, the arginine octamer would be exposed, causing cellular uptake of the liposome. Once in the cytosol, the remaining disulfide bonds would be cleaved, ultimately resulting in the destabilization the whole carrier

Nanoparticles have emerged as potential drug delivery carriers to tissues throughout the body. Yet passing the BBB is particularly difficult. The proper design of such engineered 'nanocar‐ riers' becomes very important in transversing the impermeable membranes to facilitate drug delivery. At the same time, it is also required to retain the drug stability and ensure that early degradation of drugs from the nanocarriers does not take place. Therefore, for drugs to be successfully delivered to their target, many factors such as its size, biocompatibility, target specific affinity, avoidance of reticuloendothelial systems, stability in blood, or ability to facilitate controlled drug release need to be considered during manufacture of the NPs. As for nanocarriers to serve as good candidates for drug delivery across the BBB can be summarized as following [Bhaskar et al., 2010]: a) Particle diameter less than 100 nanometers; b) Non-toxic,

located to the target tumor cells [Sawant et al., 2006; Torchilin, 2008].

**10. Nanoparticles mediated brain delivery systems**

[Fig. 2B; Bauhuber et al., 2009].

As mentioned in the previous sections, there are some conflicting demands for overcoming different extra- and intracellular barriers met by nucleic acid-loaded nano-particles during delivery. The delivery systems, which response to only one stimulus and have less function‐ ality, may not be efficient enough to achieve a satisfactory therapeutic effect *in vivo*. Natural gene carriers, such as viruses, have developed sophisticated mechanisms and modular biopolymer designs to overcome these barriers. The development of virus-mimicking, multifunctional gene delivery systems with features that mimic virus modular components and which transfect specific cell lines with high efficacy is considered to be a practical strategy in the future, in particular for intravenous administration. Ideal polymer-based, nucleic acidloaded nanoparticles for potential *in vivo* applications should have several components that function at the appropriate stages during the delivery. The hierarchical nature of the synthetic carriers allows the incorporation of membrane-disrupting peptides, nucleic acid binding components, a protective coat layer, and an outer targeting ligand all in a single nanoparticle, but with functionality such that each is utilized in a specific sequence during the gene delivery process [Du et al., 2010].

The ligands on the particle surface can be used to recognize a specific cell/tissue, and facilitate cellular uptake through receptor-mediated endocytosis. A reversibly removable hydrophilic pole, such as PEG chains, provides stealth protection of the nanoparticles during their circulation in the blood as well as travelling through the ECM, and may reduce toxicity. Moieties that are responsive to different stimulus must be combined to result in an appropriate set of various trigger mechanisms at different time points. Such moieties might be cleaved in acidic environments, show good buffering capacity, be redox active or enzymatically cleava‐ ble, the pole can be removed, which might enhance the cellular uptake and/or endosomal escape. In the inner part of the nanoparticles, nucleic acids can be temporally loaded through electrostatic interaction, covalent conjugation, or physical encapsulation, which will protect the nucleic acid against enzymatic degradation. The inner part should be stable enough until delivery to the correct site (cytoplasm and/or nucleus) where it is disassembled to release the naked nucleic acid upon some kind of stimulus. In addition, it is preferred that the inner part contains endosomolytic components that help the endosomal escape. If cell penetrating peptide (CPP) is incorporated onto the surface of the inner part, cellular uptake may be further improved [Du et al., 2010]. Many experimental results emphasize that the main obstruction of CPPs is the absence of specific cellular delivery [Vivès et al., 2008]. Ideally, the design of a smart delivery system should be built in such a way that during the first phase of delivery, a non-specific cell-penetrating function is shielded by the function of cell-recognition motif which favors the concentration of the drug at the targeted cell type. Upon accumulating in the target, protecting polymer (or specific ligand) attached to the surface of the smart nanocarrier via the stimuli-sensitive bond should detach under the action of local conditions and expose the previously hidden second function (CPP) allowing for the subsequent delivery of the carrier and its cargo inside cells [Torchilin, 2008]. Such engineering of smart nanodevices has been illustrated in the work by Sawant *et al*. who developed targeted long-circulating PEGy‐ lated liposomes and PEG-phosphatidylethanolamine (PEG-PE)-based micelles possessing several functionalities. Such systems were capable of targeting a specific cell or organ by attaching the monoclonal antibody to their surface via long PEG spacer groups. Second, these liposomes and micelles were additionally modified with TATp moieties attached to the surface of the nanocarrier by using TATp-short PEG-PE derivatives. PEG-PE used for liposome surface modification or for micelle preparation was made degradable by inserting the pH-sensitive hydrazone bond between PEG and PE (PEG-Hz-PE). Under normal pH values, TATp functions on the surface of nanocarriers were ''shielded'' by long protecting PEG chains (pH-degradable PEG 2000-PE or PEG 5000-PE) or by long para- nitrophenyl PEG-PE (pNP-PEG-PE) moieties used to attach antibodies to the nanocarrier (non-pH degradable PEG3400-PE or PEG5000-PE). Following prolonged circulation and uptake into the tumor mass mediated by both the enhanced permeability and retention (EPR) effect and the active targeting agent on the surface of the carrier, the pH responsive bonds are cleaved in the acidic environment of the tumor, thereby releasing the high molecular weight PEG strands from the carrier and exposing the Tat-peptide for enhanced intracellular uptake and intracellular target localization, specifically located to the target tumor cells [Sawant et al., 2006; Torchilin, 2008].

Another example of a triggerable system was created by the synthesis of a liposomal carrier that was comprised of a membrane-permeable ligand and a reductively detachable PEG coating. The surface cover consisted of PEG coupled to DOPE via a thiolytically cleavable linker, while the ligand, an ariginine octamer, was immobilized onto cholesteryl hemisuccinate (CHEMS). These conjugates were mixed with dipalmitoylphosphatidylcholin (DPPC) and unmodified DOPE to form liposomes. DOPE/mPEG–DTP–DSPE liposomes were stable in plasma whereas in the presence of a reducing agent the PEG coating was effectively detached off the liposomal surface, leading to vesicle destabilization and fusion as well as complete release of the entrapped contents. Upon activation of the trigger, the arginine octamer would be exposed, causing cellular uptake of the liposome. Once in the cytosol, the remaining disulfide bonds would be cleaved, ultimately resulting in the destabilization the whole carrier [Fig. 2B; Bauhuber et al., 2009].

## **10. Nanoparticles mediated brain delivery systems**

**9. Stimuli-sensitive multi-functional nano-particulate nucleic acid delivery**

As mentioned in the previous sections, there are some conflicting demands for overcoming different extra- and intracellular barriers met by nucleic acid-loaded nano-particles during delivery. The delivery systems, which response to only one stimulus and have less function‐ ality, may not be efficient enough to achieve a satisfactory therapeutic effect *in vivo*. Natural gene carriers, such as viruses, have developed sophisticated mechanisms and modular biopolymer designs to overcome these barriers. The development of virus-mimicking, multifunctional gene delivery systems with features that mimic virus modular components and which transfect specific cell lines with high efficacy is considered to be a practical strategy in the future, in particular for intravenous administration. Ideal polymer-based, nucleic acidloaded nanoparticles for potential *in vivo* applications should have several components that function at the appropriate stages during the delivery. The hierarchical nature of the synthetic carriers allows the incorporation of membrane-disrupting peptides, nucleic acid binding components, a protective coat layer, and an outer targeting ligand all in a single nanoparticle, but with functionality such that each is utilized in a specific sequence during the gene delivery

The ligands on the particle surface can be used to recognize a specific cell/tissue, and facilitate cellular uptake through receptor-mediated endocytosis. A reversibly removable hydrophilic pole, such as PEG chains, provides stealth protection of the nanoparticles during their circulation in the blood as well as travelling through the ECM, and may reduce toxicity. Moieties that are responsive to different stimulus must be combined to result in an appropriate set of various trigger mechanisms at different time points. Such moieties might be cleaved in acidic environments, show good buffering capacity, be redox active or enzymatically cleava‐ ble, the pole can be removed, which might enhance the cellular uptake and/or endosomal escape. In the inner part of the nanoparticles, nucleic acids can be temporally loaded through electrostatic interaction, covalent conjugation, or physical encapsulation, which will protect the nucleic acid against enzymatic degradation. The inner part should be stable enough until delivery to the correct site (cytoplasm and/or nucleus) where it is disassembled to release the naked nucleic acid upon some kind of stimulus. In addition, it is preferred that the inner part contains endosomolytic components that help the endosomal escape. If cell penetrating peptide (CPP) is incorporated onto the surface of the inner part, cellular uptake may be further improved [Du et al., 2010]. Many experimental results emphasize that the main obstruction of CPPs is the absence of specific cellular delivery [Vivès et al., 2008]. Ideally, the design of a smart delivery system should be built in such a way that during the first phase of delivery, a non-specific cell-penetrating function is shielded by the function of cell-recognition motif which favors the concentration of the drug at the targeted cell type. Upon accumulating in the target, protecting polymer (or specific ligand) attached to the surface of the smart nanocarrier via the stimuli-sensitive bond should detach under the action of local conditions and expose the previously hidden second function (CPP) allowing for the subsequent delivery of the carrier and its cargo inside cells [Torchilin, 2008]. Such engineering of smart nanodevices has

**systems**

236 Novel Gene Therapy Approaches

process [Du et al., 2010].

Nanoparticles have emerged as potential drug delivery carriers to tissues throughout the body. Yet passing the BBB is particularly difficult. The proper design of such engineered 'nanocar‐ riers' becomes very important in transversing the impermeable membranes to facilitate drug delivery. At the same time, it is also required to retain the drug stability and ensure that early degradation of drugs from the nanocarriers does not take place. Therefore, for drugs to be successfully delivered to their target, many factors such as its size, biocompatibility, target specific affinity, avoidance of reticuloendothelial systems, stability in blood, or ability to facilitate controlled drug release need to be considered during manufacture of the NPs. As for nanocarriers to serve as good candidates for drug delivery across the BBB can be summarized as following [Bhaskar et al., 2010]: a) Particle diameter less than 100 nanometers; b) Non-toxic, biodegradable and biocompatible; c) Stable in blood (i.e., no opsonisation by proteins); d) BBBtargeted (i.e., use of cell surface, ligands, and receptor mediated endocytosis); e) No activation of neutrophils, non-inflammatory; f) No platelet aggregation; h) Avoidance of the reticuloen‐ dothelial systems; i) Prolonged circulation time; j) Scalable and cost effective with regard to manufacturing process; m) Agreeable to small molecules, peptides, proteins or nucleic acids; n) Controlled drug release or modulation of drug release profiles [Bhaskar et al., 2010].

characterized by a significant lack of efficiency accompanied by a high level of toxicity making them mostly inadequate for *in vivo* applications [Veldhoen et al., 2008; Bolhassani et al., 2011].

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Peptides acting as shuttles for a controlled cellular delivery of nucleic acids represent a new concept to bypass the problem of poor bio-availability and clinical efficacy of such macromo‐ lecules [Veldhoen et al., 2008]. The idea of using peptides as carriers goes back some 20 years when two groups discovered by chance that the HIV-1 transactivating protein Tat is taken up by mammalian cells [Frankel and Pabo, 1998; Green and Loewenstein, 1988]. Just a few years later, the Antennapedia homeodomain of *Drosophila melanogaster* was shown to act similarly [Joliot et al., 1991]. Then, it could be shown that peptides derived from Tat and Antennapedia as well as other proteins are capable of transporting macromolecular cargo molecules into cells [Fawell et al., 1994; Schwarze et al., 1999]. Based on such promising results, a rapidly expanding field focusing on the so-called cell-penetrating peptides (CPPs), also referred to as protein

Up to now numerous CPPs have been described. According to their origin, they can be grouped into three classes. The first group contains CPPs originating from naturally occurring proteins "protein derived CPPs", the second consists of chimeric CPPs composed of different protein domains and the third class includes so-called "model CPPs" which were developed according to structure-fuction relationships without any homology to natural sequences. All known CPPs are basic amino acids causing a net positive charge at physiological pH [Langel, 2002].

At present, a peptide is considered a CPP, if it shows the ability to cross a biological membrane. A cargo can be bound to the CPP covalently or non-covalently. Covalent attachment can be achieved either by expression as a fusion construct or by chemical coupling [Zatsepin et al., 2005]. In some cases, cargo and carrier bind each other non-covalently through ionic interac‐ tions. Depending on the nature of both binding partners assembly of nanoparticles may occur

The studies have shown that the cargo-CPP complexes are taken up by directly penetrating the cell membrane or by an endocytotic pathway. However, the precise mechanism of internalization remains elusive and strongly depends on the properties of both CPP and cargo as well as the transfection conditions and the cell lines used [Maiolo et al., 2005; De Coupade et al., 2005; El-Andaloussi et al., 2007; Bolhassani, 2011]. Recent studies indicate that the uptake mechanism of CPPs can be influenced by the attachment of cargo. For example, Richard *et al*. [Richard et al., 2003; Richard et al., 2005] reported a co-localization of Tat 48-59 with markers of clathrin-mediated endocytosis, whereas Fittipaldi *et al*. [Fittipaldi et al., 2003] found a caveolae/ lipid raft-dependent process for a Tat-GFP fusion protein and Wadia *et al*. [Wadia et al., 2004] described a macropinocytotic uptake pathway for a fusion construct of Tat peptide with Cre

However, further information about the exact mechanism of uptake of such delivery systems is expected in the near future. Furthermore, it has been shown that even minor changes of the physical state of a CPP (e.g., exchange of certain amino acids) can alter translocation properties significantly. This particularly holds true for the attachment of large cargo molecules [Veldhoen et al., 2008]. Thus, it might not be possible to generalize results obtained with a CPP,

transduction domains (PTD) began to develop [Veldhoen et al., 2008].

[Veldhoen et al., 2008].

recombinase.

One of the most important challenges in nano-based diagnostics and drug delivery is the functionalization of nanoparticles. At first, the combination of effective conjugation strategies is needed to develop, in a highly controlled way, specific biomolecules to the surface of nanoparticles. Some of the most prominent candidate biomolecules are cell penetrating peptides such as SynB vectors, penetratin and Tat that facilitate enhanced intracellular delivery, fluorescent dyes (rhodamine, alexa, Cy5.5), tumoral markers for brain and gene therapeutic agents for genetic therapy such as siRNA [Bhaskar et al., 2010].

Functionalization itself requires a profound knowledge of the target organ and its transport mechanisms. The BBB has several transport molecules that can potentially increase the efficiency and kinetics of nanocarriers towards brains such as, growth factors (e.g. epidermal growth factor, vascular endothelial growth factor, basic fibroblast growth factor, insulin-like growth factors (IGF-I and -II), biotin binding proteins (avidin, streptavidin, or neutravidin), insulin, albumin, leptin, lactoferrin, iron binding protein p97 (melanotransferrin), transferrin and Angiopep-2. Some agents play a pivotal role in enhancing the permeability of nanoprobes through BBB. Moreover, by altering the surface of polymeric nanoparticles on coating them with different hydrophilic surfactants, such as polysorbate 80 (Tween® 80) or other polysor‐ bates with 20 polyoxyethylene units, biocompatible coatings of non-viral gene delivery systems, e.g. by PEG attachment for siRNA delivery show significant advantage in brain targeting [Bhaskar et al., 2010].

## **11. Cell penetrating peptides as efficient delivery systems**

Generally, the nucleic acid delivery techniques comprise various physical and chemical methods, viral and non-viral vector systems, and uptake of naked nucleic acids [Veldhoen et al., 2008; Bolhassani et al., 2011]. All of them have certain advantages and dis-advantages and might only be appropriate if particular requirements are performed. For instance, physical and chemical methods like microinjection, electroporation or particle bombardment as well as calcium phosphate co-precipitation are highly efficient but rather harmful for the target cells and lack the potential to be applicable *in vivo*. There is general agreement that viral vector systems are the most efficient vehicles to deliver nucleic acids into cells [Veldhoen et al., 2008]. However, despite substantial efforts over the last 15 years, up to now research has failed to develop suitable and especially safe viral systems. As a result of the difficulties encountered with these viral vectors (e.g., mutagenesis and immune responses), much attention was paid to the development of safer non-viral delivery systems. Currently, liposomes and cationic polymers are used as a standard tool to transfect cells *in vitro*. These approaches are yet characterized by a significant lack of efficiency accompanied by a high level of toxicity making them mostly inadequate for *in vivo* applications [Veldhoen et al., 2008; Bolhassani et al., 2011].

biodegradable and biocompatible; c) Stable in blood (i.e., no opsonisation by proteins); d) BBBtargeted (i.e., use of cell surface, ligands, and receptor mediated endocytosis); e) No activation of neutrophils, non-inflammatory; f) No platelet aggregation; h) Avoidance of the reticuloen‐ dothelial systems; i) Prolonged circulation time; j) Scalable and cost effective with regard to manufacturing process; m) Agreeable to small molecules, peptides, proteins or nucleic acids; n) Controlled drug release or modulation of drug release profiles [Bhaskar et al., 2010].

One of the most important challenges in nano-based diagnostics and drug delivery is the functionalization of nanoparticles. At first, the combination of effective conjugation strategies is needed to develop, in a highly controlled way, specific biomolecules to the surface of nanoparticles. Some of the most prominent candidate biomolecules are cell penetrating peptides such as SynB vectors, penetratin and Tat that facilitate enhanced intracellular delivery, fluorescent dyes (rhodamine, alexa, Cy5.5), tumoral markers for brain and gene

Functionalization itself requires a profound knowledge of the target organ and its transport mechanisms. The BBB has several transport molecules that can potentially increase the efficiency and kinetics of nanocarriers towards brains such as, growth factors (e.g. epidermal growth factor, vascular endothelial growth factor, basic fibroblast growth factor, insulin-like growth factors (IGF-I and -II), biotin binding proteins (avidin, streptavidin, or neutravidin), insulin, albumin, leptin, lactoferrin, iron binding protein p97 (melanotransferrin), transferrin and Angiopep-2. Some agents play a pivotal role in enhancing the permeability of nanoprobes through BBB. Moreover, by altering the surface of polymeric nanoparticles on coating them with different hydrophilic surfactants, such as polysorbate 80 (Tween® 80) or other polysor‐ bates with 20 polyoxyethylene units, biocompatible coatings of non-viral gene delivery systems, e.g. by PEG attachment for siRNA delivery show significant advantage in brain

Generally, the nucleic acid delivery techniques comprise various physical and chemical methods, viral and non-viral vector systems, and uptake of naked nucleic acids [Veldhoen et al., 2008; Bolhassani et al., 2011]. All of them have certain advantages and dis-advantages and might only be appropriate if particular requirements are performed. For instance, physical and chemical methods like microinjection, electroporation or particle bombardment as well as calcium phosphate co-precipitation are highly efficient but rather harmful for the target cells and lack the potential to be applicable *in vivo*. There is general agreement that viral vector systems are the most efficient vehicles to deliver nucleic acids into cells [Veldhoen et al., 2008]. However, despite substantial efforts over the last 15 years, up to now research has failed to develop suitable and especially safe viral systems. As a result of the difficulties encountered with these viral vectors (e.g., mutagenesis and immune responses), much attention was paid to the development of safer non-viral delivery systems. Currently, liposomes and cationic polymers are used as a standard tool to transfect cells *in vitro*. These approaches are yet

therapeutic agents for genetic therapy such as siRNA [Bhaskar et al., 2010].

**11. Cell penetrating peptides as efficient delivery systems**

targeting [Bhaskar et al., 2010].

238 Novel Gene Therapy Approaches

Peptides acting as shuttles for a controlled cellular delivery of nucleic acids represent a new concept to bypass the problem of poor bio-availability and clinical efficacy of such macromo‐ lecules [Veldhoen et al., 2008]. The idea of using peptides as carriers goes back some 20 years when two groups discovered by chance that the HIV-1 transactivating protein Tat is taken up by mammalian cells [Frankel and Pabo, 1998; Green and Loewenstein, 1988]. Just a few years later, the Antennapedia homeodomain of *Drosophila melanogaster* was shown to act similarly [Joliot et al., 1991]. Then, it could be shown that peptides derived from Tat and Antennapedia as well as other proteins are capable of transporting macromolecular cargo molecules into cells [Fawell et al., 1994; Schwarze et al., 1999]. Based on such promising results, a rapidly expanding field focusing on the so-called cell-penetrating peptides (CPPs), also referred to as protein transduction domains (PTD) began to develop [Veldhoen et al., 2008].

Up to now numerous CPPs have been described. According to their origin, they can be grouped into three classes. The first group contains CPPs originating from naturally occurring proteins "protein derived CPPs", the second consists of chimeric CPPs composed of different protein domains and the third class includes so-called "model CPPs" which were developed according to structure-fuction relationships without any homology to natural sequences. All known CPPs are basic amino acids causing a net positive charge at physiological pH [Langel, 2002].

At present, a peptide is considered a CPP, if it shows the ability to cross a biological membrane. A cargo can be bound to the CPP covalently or non-covalently. Covalent attachment can be achieved either by expression as a fusion construct or by chemical coupling [Zatsepin et al., 2005]. In some cases, cargo and carrier bind each other non-covalently through ionic interac‐ tions. Depending on the nature of both binding partners assembly of nanoparticles may occur [Veldhoen et al., 2008].

The studies have shown that the cargo-CPP complexes are taken up by directly penetrating the cell membrane or by an endocytotic pathway. However, the precise mechanism of internalization remains elusive and strongly depends on the properties of both CPP and cargo as well as the transfection conditions and the cell lines used [Maiolo et al., 2005; De Coupade et al., 2005; El-Andaloussi et al., 2007; Bolhassani, 2011]. Recent studies indicate that the uptake mechanism of CPPs can be influenced by the attachment of cargo. For example, Richard *et al*. [Richard et al., 2003; Richard et al., 2005] reported a co-localization of Tat 48-59 with markers of clathrin-mediated endocytosis, whereas Fittipaldi *et al*. [Fittipaldi et al., 2003] found a caveolae/ lipid raft-dependent process for a Tat-GFP fusion protein and Wadia *et al*. [Wadia et al., 2004] described a macropinocytotic uptake pathway for a fusion construct of Tat peptide with Cre recombinase.

However, further information about the exact mechanism of uptake of such delivery systems is expected in the near future. Furthermore, it has been shown that even minor changes of the physical state of a CPP (e.g., exchange of certain amino acids) can alter translocation properties significantly. This particularly holds true for the attachment of large cargo molecules [Veldhoen et al., 2008]. Thus, it might not be possible to generalize results obtained with a CPP, and it might be necessary to characterize each carrier/cargo complex individually. If CPPs are proposed to be used for therapeutic purposes in the future, it is essential to focus on the attachment of functional cargos and analyze their biological effects inside the cell. Data from our lab clearly show that uptake and biological activity of a functional cargo is everything but the same. Therefore, a quantitative comparison of cargo taken up and functionally active cargo is an essential requirement in order to improve therapeutic efficacy. Indeed, only looking for efficient internalization is not sufficient [Veldhoen et al., 2008].

which could be used for gene delivery into mammalian cells. Despite reasonably high transfection efficiency *in vitro*, low gene expression levels were detected in the liver of mice injected intravenously with DNA-Tat complexes, a fact that was attributed to inactivation of the complexes in the bloodstream due to interactions with serum albumin. Interestingly, an endocytosis-dependent mechanism was proposed for the uptake of the DNA-Tat complexes, similar to what was proposed for internalization of complexes of plasmid DNA with other polycationic carriers. A different study, by Tung *et al.*, compared the efficiency of a series of Tat peptides, containing 1–8 Tat moieties. Although, all compounds complexed with plasmid DNA, it was demonstrated that at least eight Tat peptide moieties are required in order to achieve efficient gene delivery. Sandgren *et al.* also studied the cellular uptake of complexes of plasmid DNA and the HIV-Tat derived peptide. According to this study, the Tat peptide stimulated cellular uptake of DNA in a time-, concentration- and temperature-dependent manner, while accumulating in large, acidic, cytoplasmic vesicles, followed by transfer of the cargo into the nuclear compartment and subsequent disappearance from the endolysosomal vesicles. Aiming at increasing the efficiency of the Tat peptide to deliver plasmid DNA, Lo *et al.* made several modifications to the Tat peptide, through the use of histidine and cysteine residues to enhance endosomal escape and complex stability [Trabulo et al., 2010]. Up to 7,000 fold improvement in gene transfection efficiency was observed for the Tat peptide covalently fused with 10 histidine residues (Tat-10H) over the original Tat peptide, and incorporation of two cysteine residues into this peptide resulted in an even higher efficacy (C-5H-Tat-5H-C). The association of CPPs with other non-viral delivery vectors has also been extensively investigated, aiming at exploring the possibility to combine efficient delivery, packaging and

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A combination of a peptide nucleic acids (PNA) with the SV40 core NLS, performed by Branden *et al.*, originated a bifunctional peptide that improved the efficacy of plasmid transfection up to 8-fold when associated with the transfection agent polyethyleneimine (PEI) [Branden et al., 1999]. Several other studies also combined PEI with CPPs. Kleemann *et al.* covalently coupled the Tat peptide to 25 kDa PEI through a heterobi-functional PEG spacer resulting in a Tat-PEG-PEI conjugate. Improved DNA reporter gene complexation and protection were observed for small (approximately 90 nm) polyplexes as well as low toxicity and significantly enhanced transfection efficiency *in vivo* [Kleemann et al., 2005]. Rudolph *et al.* demonstrated that oligomers of the Tat peptide were able to condense plasmid DNA to nanosized particles and protect DNA from nuclease degradation [Rudolph et al., 2003]. Most importantly, when DNA was pre-condensed with Tat peptides and PEI, Superfect or Lipo‐ fectAMINE were added to the mixture, transfection efficiency was enhanced up to 390-fold compared with the standard vectors. Similar studies by Kilk *et al.*, demonstrated that the poor transfection abilities exhibited by TP10 was significantly enhanced in the presence of PEI, increasing several fold compared to PEI alone, particularly at low PEI concentrations, therefore allowing the use of reduced PEI concentration [Kilk et al., 2005]. Using fluorescently labeled liposomes and cargos, Torchilin *et al.* demonstrated that large drug carriers, such as 200 nm liposomes, could be delivered into cells by attaching Tat peptide to the liposome surface. Next, the same group described the formation of non-covalent complexes of Tat, liposomes and DNA that were able to efficiently transfect cells both *in vitro* and *in vivo,* while being less toxic than

targeting moieties within the same system [Trabulo et al., 2010].

The successful clinical application of nucleic acid-based therapeutic strategies has been limited by the poor delivery efficiency achieved by existing vectors. The development of alternative delivery systems for improved biological activity is, therefore, obligatory. Since two decades ago that the Tat protein, and derived peptides, can translocate across biological membranes, cell-penetrating peptides have been considered one of the most promising tools to improve non-invasive cellular delivery of therapeutic molecules. Despite extensive research on the use of CPPs for this purpose, the exact mechanisms underlying their cellular uptake and that of peptide conjugates remain controversial [Trabulo et al., 2010].

There are many examples of CPP-mediated delivery of plasmid DNA into cultured cells and also *in vivo* involving the use of a non-covalent approach [Bolhassani, 2011]. While some approaches involve single component peptide vectors, the major focus has been on the association of CPPs with other non-viral gene delivery methods, such as liposomes, polye‐ thyleneimine (PEI) or nanoparticles. In 1999, Morris and coworkers demonstrated that MPG could be used as a powerful tool for the delivery of nucleic acids. It was shown that MPG is not cytotoxic, insensitive to serum and able to efficiently deliver plasmid DNA into several different cell lines [Morris et al., 1999]. Further studies demonstrated that cell entry of the MPG/ DNA particles is independent of the endosomal pathway and that the NLS of MPG is involved in both electrostatic interactions with DNA and nuclear targeting. Furthermore, it was shown that a mutation affecting the NLS of MPG prevents nuclear delivery of DNA. In an alternative study, Rittner *et al.* described the novel basic amphiphilic peptides, ppTG1 and ppTG20 (20 amino acids), and evaluated their efficiencies *in vitro* and *in vivo* as single-component gene transfer vectors. It was demonstrated that both the ppTG1 and ppTG20 peptides are able to bind nucleic acids and destabilize membranes, in a liposome leakage assay. Complexes of plasmid DNA with ppTG1 originated high levels of gene expression in cell culture experiments and, most importantly, complexes of plasmid DNA with ppTG1 or ppTG20 led to significant gene expression *in vivo* [Rittner et al., 2002]. Peptide modification has also been explored as a means to enhance gene delivery. In particular, stearic acid modification of different membranepermeable arginine-rich peptides, such as HIV-1 Tat (48-60), HIV-1 Rev (34-50), flock house virus (FHV) coat (35-49), (RxR)4 and oligoarginines of 4-16 residues was shown to substantially increase their transfection efficiency. The mechanisms by which stearic acid modification improves plasmid DNA delivery by CPPs have been shown to involve increased efficiency of endosomal escape or enhanced cellular association, as well as higher nuclear delivery. The extensively studied Tat peptide has also been exploited for plasmid DNA delivery by different research groups, with paradoxical results [Trabulo et al., 2010, Bolhassani, 2011]. A study by Ignatovich *et al.*, demonstrated that Tat peptide is able to form complexes with plasmid DNA, which could be used for gene delivery into mammalian cells. Despite reasonably high transfection efficiency *in vitro*, low gene expression levels were detected in the liver of mice injected intravenously with DNA-Tat complexes, a fact that was attributed to inactivation of the complexes in the bloodstream due to interactions with serum albumin. Interestingly, an endocytosis-dependent mechanism was proposed for the uptake of the DNA-Tat complexes, similar to what was proposed for internalization of complexes of plasmid DNA with other polycationic carriers. A different study, by Tung *et al.*, compared the efficiency of a series of Tat peptides, containing 1–8 Tat moieties. Although, all compounds complexed with plasmid DNA, it was demonstrated that at least eight Tat peptide moieties are required in order to achieve efficient gene delivery. Sandgren *et al.* also studied the cellular uptake of complexes of plasmid DNA and the HIV-Tat derived peptide. According to this study, the Tat peptide stimulated cellular uptake of DNA in a time-, concentration- and temperature-dependent manner, while accumulating in large, acidic, cytoplasmic vesicles, followed by transfer of the cargo into the nuclear compartment and subsequent disappearance from the endolysosomal vesicles. Aiming at increasing the efficiency of the Tat peptide to deliver plasmid DNA, Lo *et al.* made several modifications to the Tat peptide, through the use of histidine and cysteine residues to enhance endosomal escape and complex stability [Trabulo et al., 2010]. Up to 7,000 fold improvement in gene transfection efficiency was observed for the Tat peptide covalently fused with 10 histidine residues (Tat-10H) over the original Tat peptide, and incorporation of two cysteine residues into this peptide resulted in an even higher efficacy (C-5H-Tat-5H-C). The association of CPPs with other non-viral delivery vectors has also been extensively investigated, aiming at exploring the possibility to combine efficient delivery, packaging and targeting moieties within the same system [Trabulo et al., 2010].

and it might be necessary to characterize each carrier/cargo complex individually. If CPPs are proposed to be used for therapeutic purposes in the future, it is essential to focus on the attachment of functional cargos and analyze their biological effects inside the cell. Data from our lab clearly show that uptake and biological activity of a functional cargo is everything but the same. Therefore, a quantitative comparison of cargo taken up and functionally active cargo is an essential requirement in order to improve therapeutic efficacy. Indeed, only looking for

The successful clinical application of nucleic acid-based therapeutic strategies has been limited by the poor delivery efficiency achieved by existing vectors. The development of alternative delivery systems for improved biological activity is, therefore, obligatory. Since two decades ago that the Tat protein, and derived peptides, can translocate across biological membranes, cell-penetrating peptides have been considered one of the most promising tools to improve non-invasive cellular delivery of therapeutic molecules. Despite extensive research on the use of CPPs for this purpose, the exact mechanisms underlying their cellular uptake and that of

There are many examples of CPP-mediated delivery of plasmid DNA into cultured cells and also *in vivo* involving the use of a non-covalent approach [Bolhassani, 2011]. While some approaches involve single component peptide vectors, the major focus has been on the association of CPPs with other non-viral gene delivery methods, such as liposomes, polye‐ thyleneimine (PEI) or nanoparticles. In 1999, Morris and coworkers demonstrated that MPG could be used as a powerful tool for the delivery of nucleic acids. It was shown that MPG is not cytotoxic, insensitive to serum and able to efficiently deliver plasmid DNA into several different cell lines [Morris et al., 1999]. Further studies demonstrated that cell entry of the MPG/ DNA particles is independent of the endosomal pathway and that the NLS of MPG is involved in both electrostatic interactions with DNA and nuclear targeting. Furthermore, it was shown that a mutation affecting the NLS of MPG prevents nuclear delivery of DNA. In an alternative study, Rittner *et al.* described the novel basic amphiphilic peptides, ppTG1 and ppTG20 (20 amino acids), and evaluated their efficiencies *in vitro* and *in vivo* as single-component gene transfer vectors. It was demonstrated that both the ppTG1 and ppTG20 peptides are able to bind nucleic acids and destabilize membranes, in a liposome leakage assay. Complexes of plasmid DNA with ppTG1 originated high levels of gene expression in cell culture experiments and, most importantly, complexes of plasmid DNA with ppTG1 or ppTG20 led to significant gene expression *in vivo* [Rittner et al., 2002]. Peptide modification has also been explored as a means to enhance gene delivery. In particular, stearic acid modification of different membranepermeable arginine-rich peptides, such as HIV-1 Tat (48-60), HIV-1 Rev (34-50), flock house virus (FHV) coat (35-49), (RxR)4 and oligoarginines of 4-16 residues was shown to substantially increase their transfection efficiency. The mechanisms by which stearic acid modification improves plasmid DNA delivery by CPPs have been shown to involve increased efficiency of endosomal escape or enhanced cellular association, as well as higher nuclear delivery. The extensively studied Tat peptide has also been exploited for plasmid DNA delivery by different research groups, with paradoxical results [Trabulo et al., 2010, Bolhassani, 2011]. A study by Ignatovich *et al.*, demonstrated that Tat peptide is able to form complexes with plasmid DNA,

efficient internalization is not sufficient [Veldhoen et al., 2008].

240 Novel Gene Therapy Approaches

peptide conjugates remain controversial [Trabulo et al., 2010].

A combination of a peptide nucleic acids (PNA) with the SV40 core NLS, performed by Branden *et al.*, originated a bifunctional peptide that improved the efficacy of plasmid transfection up to 8-fold when associated with the transfection agent polyethyleneimine (PEI) [Branden et al., 1999]. Several other studies also combined PEI with CPPs. Kleemann *et al.* covalently coupled the Tat peptide to 25 kDa PEI through a heterobi-functional PEG spacer resulting in a Tat-PEG-PEI conjugate. Improved DNA reporter gene complexation and protection were observed for small (approximately 90 nm) polyplexes as well as low toxicity and significantly enhanced transfection efficiency *in vivo* [Kleemann et al., 2005]. Rudolph *et al.* demonstrated that oligomers of the Tat peptide were able to condense plasmid DNA to nanosized particles and protect DNA from nuclease degradation [Rudolph et al., 2003]. Most importantly, when DNA was pre-condensed with Tat peptides and PEI, Superfect or Lipo‐ fectAMINE were added to the mixture, transfection efficiency was enhanced up to 390-fold compared with the standard vectors. Similar studies by Kilk *et al.*, demonstrated that the poor transfection abilities exhibited by TP10 was significantly enhanced in the presence of PEI, increasing several fold compared to PEI alone, particularly at low PEI concentrations, therefore allowing the use of reduced PEI concentration [Kilk et al., 2005]. Using fluorescently labeled liposomes and cargos, Torchilin *et al.* demonstrated that large drug carriers, such as 200 nm liposomes, could be delivered into cells by attaching Tat peptide to the liposome surface. Next, the same group described the formation of non-covalent complexes of Tat, liposomes and DNA that were able to efficiently transfect cells both *in vitro* and *in vivo,* while being less toxic than other commonly used transfection reagents. The internalization of this system was claimed to depend on a direct cytoplasmic delivery imparted by the Tat peptide [Torchilin et al., 2001]. A study by Hyndman *et al.* showed that mixing the Tat with liposomes containing DOTAP or Lipofectin and DNA, resulted in complexes that significantly enhance transfection *in vitro* with a marked reduction in the amount of liposomes required, despite the lack of any covalent linkage of the peptide to liposomes. In this study, the use of endosomolytic agents and results from experiments performed at low temperature suggested that the endocytotic pathway was involved in the internalization of the complexes. Another report demonstrated that the increase in gene transfer of Tat-modified lipoplexes is dependent on the amount of cationic lipid in the lipoplexes and on the way, Tat was coupled to the lipoplexes. Moreover, it was shown that the cellular uptake of both Tat-modified and un-modified lipoplexes was very fast and, in contrast to previous publications, temperature-dependent [Hyndman et al., 2004]. A concept called "Programmed Packaging" was proposed by Kogure *et al.*, who developed a multi-functional envelope-type nano device (MEND), consisting of a condensed DNA core and a surrounding lipid envelope. This packaging method involves three steps: (1) DNA condensation with a polycation, (2) lipid film hydration for the electrostatic binding of the condensed DNA and (3) sonication to package the condensed DNA with lipids. MEND, having octa-arginine on the envelope for enhancing cellular uptake, showed a 1000-fold higher transfection activity than a DNA/poly-L-lysine/lipid complex prepared in similar conditions [Kogure et al., 2004]. Another study, by Khalil *et al.*, also described the high-efficiency delivery of nucleic acids to eukaryotic cells using MEND particles containing polycation-condensed nucleic acids encapsulated in an R8-DOPE lipid envelope. MEND particles were shown to be non-cytotoxic and achieved transfection efficiencies as high as *adenovirus* [Khalil et al., 2010]. In this case, the high efficiency of MEND particles was ascribed, at least in part, to R8 which was claimed to promote cellular uptake by macropinocytosis, improving intracellular traf‐ ficking towards more efficient gene expression. Along the same lines, work of the same research group demonstrated that gene expression of condensed plasmid DNA encapsulated in R8-modified nanoparticles was more than one order of magnitude higher than that of K8 modified nanoparticles, and two orders of magnitude higher than gene expression using unmodified nanoparticles. Differences in gene expression achieved with R8- and K8-modified liposomes could not be attributed to differences in cellular uptake, since both kinds of complexes were taken up primarily *via* macropinocytosis at comparable efficiencies. More‐ over, it was described that modification of nanoparticles with a high density of R8 allows their escape from endocytotic vesicles *via* membrane fusion at both acidic and neutral pH, and that the guanidinium groups of arginine residues, and not only their positive charge, are important for efficient endosomal escape [Trabulo et al., 2010]. Recently, MacKay *et al.* described gene transfer using PEGylated bio-responsive nano-lipid particles (NLPs) containing plasmid DNA. In this study, the Tat peptide was attached either directly to a phospholipid (Tatp-lipid) or via a 2 kDa PEG (Tatp-PEG-lipid); incorporation of 0.3 mol% Tatp-PEG into pH-sensitive NLPs improved transfection 100,000-fold compared to NLPs. Although, Tatp-PEG-lipid could dramatically increase gene expression *in vitro*, when tested in brain and in implanted tumors, a restriction of NLP distribution to the vicinity of the infusion catheter reduced the absolute level of gene transfer [MacKay et al., 2008].

Over the last years, a research group focused on the S413-PV cell-penetrating peptide gener‐ ated from the combination of 13-amino acid cell penetrating sequence derived from the Dermaseptin S4 peptide with the SV40 large T antigen nuclear localization signal [Trabulo et al., 2010]. In these studies, complexes obtained through electrostatic association of the S413- PV cell penetrating peptide with plasmid DNA are able to very efficiently mediate transfection, particularly at high peptide/DNA charge ratios (5/1 and higher). Importantly, complexes prepared with the S413-PV or reverse NLS peptides mediate transfection at significantly higher efficiencies than those containing the scrambled version of the peptide, demonstrating the importance of the cell-penetrating sequence derived from the Dermaseptin S4 peptide (amino acids 1-13) to the transfection process. Additionally, we demonstrated that ternary complexes, resulting from association of cationic liposomes to peptide/DNA complexes, are significantly more efficient in mediating transfection than the corresponding peptide/DNA or cationic liposome/DNA complexes [Trabulo et al., 2010]. In agreement with what has been described for oligonucleotides, CPPs seem to be very efficient to mediate the uptake of plasmid DNA, as well as lipoplexes and polyplexes containing DNA, surpassing the cell membrane barrier. However, the challenge of overcoming the entrapment of complexes inside endosomes has not been solved as easily as initially predicted, even taking advantage of the capacity of direct translocation to the cytoplasm of some CPPs. Nevertheless, several of the studies described above present promising strategies to overcome this limitation, such as chemical modification of the peptide backbone or coupling of CPPs to other classes of delivery vectors. Overall, accumulated evidence suggests that CPPs used in combination with other delivery systems are more likely to be effective for gene therapy purposes than CPPs alone [Trabulo et al., 2010].

Challenges in Advancing the Field of Cancer Gene Therapy: An Overview of the Multi-Functional Nanocarriers

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243

As antibiotic resistance increases worldwide, there is an increasing pressure to develop novel classes of antimicrobial compounds to fight infectious disease. Peptide therapeutic represents a novel class of therapeutic agents. Some of them, such as cationic antimicrobial peptides (CAMPs) and peptidoglycan recognition proteins (PGRPs), have been identified from studies of innate immune effector mechanisms, while others are completely novel compounds generated in biological systems. Currently, only selected cationic antimicrobial peptides have been licensed, and for topical applications. However, research using new approaches to identify novel antimicrobial peptide therapeutics, and new approaches to delivery and improving stability, will result in an increased range of peptide therapeutics available in the clinic for broader applications. A potentially rich source of peptide therapeutics that is being investigated by researchers is the innate immune response, the effectors of which are produced

by eukaryotes to defend themselves against microbial attack [Oyston et al., 2009].

Human cancer is one of the most important causes of death in the western countries. In advanced stages of the disease, the therapeutic opportunities are still limited due to the difficulty to target specifically only cancer cells sparing healthy ones. Cancer cells have on their surface antigens that are expressed at higher levels than their normal counterparts. Often these antigens (also called tumor-associated antigens) have receptor activity and bind to specific

**12. Bioactive peptides**

Over the last years, a research group focused on the S413-PV cell-penetrating peptide gener‐ ated from the combination of 13-amino acid cell penetrating sequence derived from the Dermaseptin S4 peptide with the SV40 large T antigen nuclear localization signal [Trabulo et al., 2010]. In these studies, complexes obtained through electrostatic association of the S413- PV cell penetrating peptide with plasmid DNA are able to very efficiently mediate transfection, particularly at high peptide/DNA charge ratios (5/1 and higher). Importantly, complexes prepared with the S413-PV or reverse NLS peptides mediate transfection at significantly higher efficiencies than those containing the scrambled version of the peptide, demonstrating the importance of the cell-penetrating sequence derived from the Dermaseptin S4 peptide (amino acids 1-13) to the transfection process. Additionally, we demonstrated that ternary complexes, resulting from association of cationic liposomes to peptide/DNA complexes, are significantly more efficient in mediating transfection than the corresponding peptide/DNA or cationic liposome/DNA complexes [Trabulo et al., 2010]. In agreement with what has been described for oligonucleotides, CPPs seem to be very efficient to mediate the uptake of plasmid DNA, as well as lipoplexes and polyplexes containing DNA, surpassing the cell membrane barrier. However, the challenge of overcoming the entrapment of complexes inside endosomes has not been solved as easily as initially predicted, even taking advantage of the capacity of direct translocation to the cytoplasm of some CPPs. Nevertheless, several of the studies described above present promising strategies to overcome this limitation, such as chemical modification of the peptide backbone or coupling of CPPs to other classes of delivery vectors. Overall, accumulated evidence suggests that CPPs used in combination with other delivery systems are more likely to be effective for gene therapy purposes than CPPs alone [Trabulo et al., 2010].

## **12. Bioactive peptides**

other commonly used transfection reagents. The internalization of this system was claimed to depend on a direct cytoplasmic delivery imparted by the Tat peptide [Torchilin et al., 2001]. A study by Hyndman *et al.* showed that mixing the Tat with liposomes containing DOTAP or Lipofectin and DNA, resulted in complexes that significantly enhance transfection *in vitro* with a marked reduction in the amount of liposomes required, despite the lack of any covalent linkage of the peptide to liposomes. In this study, the use of endosomolytic agents and results from experiments performed at low temperature suggested that the endocytotic pathway was involved in the internalization of the complexes. Another report demonstrated that the increase in gene transfer of Tat-modified lipoplexes is dependent on the amount of cationic lipid in the lipoplexes and on the way, Tat was coupled to the lipoplexes. Moreover, it was shown that the cellular uptake of both Tat-modified and un-modified lipoplexes was very fast and, in contrast to previous publications, temperature-dependent [Hyndman et al., 2004]. A concept called "Programmed Packaging" was proposed by Kogure *et al.*, who developed a multi-functional envelope-type nano device (MEND), consisting of a condensed DNA core and a surrounding lipid envelope. This packaging method involves three steps: (1) DNA condensation with a polycation, (2) lipid film hydration for the electrostatic binding of the condensed DNA and (3) sonication to package the condensed DNA with lipids. MEND, having octa-arginine on the envelope for enhancing cellular uptake, showed a 1000-fold higher transfection activity than a DNA/poly-L-lysine/lipid complex prepared in similar conditions [Kogure et al., 2004]. Another study, by Khalil *et al.*, also described the high-efficiency delivery of nucleic acids to eukaryotic cells using MEND particles containing polycation-condensed nucleic acids encapsulated in an R8-DOPE lipid envelope. MEND particles were shown to be non-cytotoxic and achieved transfection efficiencies as high as *adenovirus* [Khalil et al., 2010]. In this case, the high efficiency of MEND particles was ascribed, at least in part, to R8 which was claimed to promote cellular uptake by macropinocytosis, improving intracellular traf‐ ficking towards more efficient gene expression. Along the same lines, work of the same research group demonstrated that gene expression of condensed plasmid DNA encapsulated in R8-modified nanoparticles was more than one order of magnitude higher than that of K8 modified nanoparticles, and two orders of magnitude higher than gene expression using unmodified nanoparticles. Differences in gene expression achieved with R8- and K8-modified liposomes could not be attributed to differences in cellular uptake, since both kinds of complexes were taken up primarily *via* macropinocytosis at comparable efficiencies. More‐ over, it was described that modification of nanoparticles with a high density of R8 allows their escape from endocytotic vesicles *via* membrane fusion at both acidic and neutral pH, and that the guanidinium groups of arginine residues, and not only their positive charge, are important for efficient endosomal escape [Trabulo et al., 2010]. Recently, MacKay *et al.* described gene transfer using PEGylated bio-responsive nano-lipid particles (NLPs) containing plasmid DNA. In this study, the Tat peptide was attached either directly to a phospholipid (Tatp-lipid) or via a 2 kDa PEG (Tatp-PEG-lipid); incorporation of 0.3 mol% Tatp-PEG into pH-sensitive NLPs improved transfection 100,000-fold compared to NLPs. Although, Tatp-PEG-lipid could dramatically increase gene expression *in vitro*, when tested in brain and in implanted tumors, a restriction of NLP distribution to the vicinity of the infusion catheter reduced the absolute

242 Novel Gene Therapy Approaches

level of gene transfer [MacKay et al., 2008].

As antibiotic resistance increases worldwide, there is an increasing pressure to develop novel classes of antimicrobial compounds to fight infectious disease. Peptide therapeutic represents a novel class of therapeutic agents. Some of them, such as cationic antimicrobial peptides (CAMPs) and peptidoglycan recognition proteins (PGRPs), have been identified from studies of innate immune effector mechanisms, while others are completely novel compounds generated in biological systems. Currently, only selected cationic antimicrobial peptides have been licensed, and for topical applications. However, research using new approaches to identify novel antimicrobial peptide therapeutics, and new approaches to delivery and improving stability, will result in an increased range of peptide therapeutics available in the clinic for broader applications. A potentially rich source of peptide therapeutics that is being investigated by researchers is the innate immune response, the effectors of which are produced by eukaryotes to defend themselves against microbial attack [Oyston et al., 2009].

Human cancer is one of the most important causes of death in the western countries. In advanced stages of the disease, the therapeutic opportunities are still limited due to the difficulty to target specifically only cancer cells sparing healthy ones. Cancer cells have on their surface antigens that are expressed at higher levels than their normal counterparts. Often these antigens (also called tumor-associated antigens) have receptor activity and bind to specific proteins or peptides. The latter can be used for the specific delivery of anticancer drugs to cancer cells through retargeting strategies and/or for the direct modulation of cancer cell proliferation and survival interacting with cell-surface-specific receptors. These bioactive peptides can be raised against either tumor cells themselves or to the tumor microenvironment cell components (tumor vessels, tumor-associated macrophages, and fibroblasts) [Fields et al., 2009]. However, the feasibility of pharmacological application of peptides depends on absorption and bioavailability in intact forms in target tissues.

so far been limited by a lack of safe and effective gene delivery systems [Nguyen et al., 2008]. We attempted to mention some barriers and solutions (e.g., viral/non-viral methods) for DNA

Challenges in Advancing the Field of Cancer Gene Therapy: An Overview of the Multi-Functional Nanocarriers

http://dx.doi.org/10.5772/54862

245

The main recombinant viral vectors used for gene delivery are *adenovirus*, *adeno-associated virus* (AAVs), *retrovirus* and *lentivirus*. The advantages of adenovirus are infection of a wide range of human cell types, ability to infect non-dividing cells and lower risk of insertional mutagen‐ esis. However, *adenovirus* expression is short lived and *adenoviruses* can cause a severe, even lethal, inflammatory response due to prior immune exposure. AAV, which depends on *adenovirus* or another virus for replication, has also been used for gene delivery with the advantages of predictable chromosomal insertion and no known pathological consequence of infection [Nguyen et al., 2008]. The main advantage of *retroviruses*, their ability to integrate into the host genome for long-term expression, is also their main disadvantage as this integration can cause mutagenesis and potentially cancer. *Retroviruses* are also further limited by their inability to infect non-dividing cells. *Lentiviruses*, which can transfect a broad spectrum of cell types, are the most efficient method to transfect DCs *in vitro* and *in vivo*. Yang *et al*. recently reported very high levels of immune activation and therapeutic tumor rejection following immunization with a lentiviral vector engineered to target DCs by the cell surface receptor DC-SIGN [Yang et al., 2008]. In particular, Merck & Co. has advanced the use of viral DNA vaccines for HIV vaccination. While there have been some successes in using viral gene therapy and many clinical trials are currently ongoing there are currently no approved protocols. Problems with viral delivery systems include immunologic priming to the vector itself, oncogenicity due to insertional mutagenesis, difficult manufacturing and limited DNA cargo capacity [Nguyen et al., 2008]. Clinical trials have highlighted some of these safety risks as viral gene delivery has resulted in both cancer and deaths. Recently, Merck & Co. stopped its Phase III HIV *adenovirus* vaccine prompting renewed questions about the utility of viral vectors. The safety challenges and limitations of viral vectors have resulted in increased interest in non-viral approaches to gene delivery using non-viral materials. In general, the nonviral methods of DNA vaccination utilized in clinical trials, recently reviewed by Lu *et al*., rely on physical methods. Injection of naked DNA plasmids has found limited success in humans particularly when injected intramuscularly, even though in smaller animal models naked DNA vaccination produces robust humoral and cell-mediated responses [Lu et al., 2008]. However, the rapid degradation/clearance [half-life of under 5 min if injected intravenously (IV)] of unprotected nucleic acids, poor induction of humoral immune responses in DNA vaccination in larger animals and requirement for large doses has hindered progress into clinical trials. Clinically, relevant physical methods that have been employed include electroporation, ballistics (gene gun), ultrasound and magnetofection. Encapsulation or complexation of DNA with a biomaterial can significantly enhance DNA stability, cellular uptake of DNA and final protein expression. Materials shown to possess potential for the delivery of genes include inorganic nanoparticles and surfaces that bind to or encapsulate DNA [Lu et al., 2008; Nguyen et al., 2008]. Cationic biomolecules including lipids, polysaccharides, polymers, and dendrim‐ ers can also electrostatically complex anionic DNA to facilitate transfection. Unless specifically

vaccines individually.

**13.1. Viral and non-viral methods for gene delivery**

Moreover, their correct bio-distribution is sometimes hindered by biopharmaceutical obsta‐ cles, that is, protection by circulating protease-mediated degradation or specific accumula‐ tion in tumor tissues. Chemical modification of peptide backbone can increase the stability of peptides in biological fluids. Moreover, the use of delivery systems, and in particular the use of nanotechnologies, not only protects peptides from enzymatic degradation but also improves the delivery of the bioactive peptide in the target tissue. Moreover, peptide conju‐ gation on the surface of nano-vectors can be useful for selective delivery of conventional chemotherapeutic agents in tumor tissues. The requirements for an effective and safe der‐ matological therapeutic or active ingredient are included as following:


Collectively, these are not easily achieved criteria. For a new technology paradigm to emerge, these criteria not only have to be met but be applicable across the wide range of product-acceptable bioactivities. Peptides have significant advantages over many other tech‐ nologies in addressing these criteria primarily based upon their chemistry. Peptides consist of chains of amino acids which can be modified in many ways to increase receptor binding, increase specificity, decrease toxicity, and increase skin penetration, stability, and solubility. In this way, the field of bioactive peptides for dermatological applications has changed sig‐ nificantly in recent years. From modest beginnings of a single peptide capable of stimulating collagen, technological advances have created newer peptides capable of targeting most as‐ pects of dermal health. These advances include neutralizing toxins, stimulating fibroblast scaffolding, reducing inflammation and other desirable effects [Fields et al., 2009].

## **13. Challenges in gene delivery for DNA vaccines**

The goal of DNA vaccination is transfection of an antigen presenting cells (APC) or a bystander cell to produce antigens in an immuno-stimulatory setting. The field of genetic vaccines has so far been limited by a lack of safe and effective gene delivery systems [Nguyen et al., 2008]. We attempted to mention some barriers and solutions (e.g., viral/non-viral methods) for DNA vaccines individually.

#### **13.1. Viral and non-viral methods for gene delivery**

proteins or peptides. The latter can be used for the specific delivery of anticancer drugs to cancer cells through retargeting strategies and/or for the direct modulation of cancer cell proliferation and survival interacting with cell-surface-specific receptors. These bioactive peptides can be raised against either tumor cells themselves or to the tumor microenvironment cell components (tumor vessels, tumor-associated macrophages, and fibroblasts) [Fields et al., 2009]. However, the feasibility of pharmacological application of peptides depends on

Moreover, their correct bio-distribution is sometimes hindered by biopharmaceutical obsta‐ cles, that is, protection by circulating protease-mediated degradation or specific accumula‐ tion in tumor tissues. Chemical modification of peptide backbone can increase the stability of peptides in biological fluids. Moreover, the use of delivery systems, and in particular the use of nanotechnologies, not only protects peptides from enzymatic degradation but also improves the delivery of the bioactive peptide in the target tissue. Moreover, peptide conju‐ gation on the surface of nano-vectors can be useful for selective delivery of conventional chemotherapeutic agents in tumor tissues. The requirements for an effective and safe der‐

**1.** The molecule exhibits a proven specific beneficial bioactivity that would lead to a rational

**2.** The bioactivity does not have a negative consequence either theoretically or experimen‐

**3.** The molecule does not exhibit toxicity such as cytotoxicity, inflammation, immunogenic‐

**5.** The molecule can be formulated in such a way as to be stable, compatible with other

Collectively, these are not easily achieved criteria. For a new technology paradigm to emerge, these criteria not only have to be met but be applicable across the wide range of product-acceptable bioactivities. Peptides have significant advantages over many other tech‐ nologies in addressing these criteria primarily based upon their chemistry. Peptides consist of chains of amino acids which can be modified in many ways to increase receptor binding, increase specificity, decrease toxicity, and increase skin penetration, stability, and solubility. In this way, the field of bioactive peptides for dermatological applications has changed sig‐ nificantly in recent years. From modest beginnings of a single peptide capable of stimulating collagen, technological advances have created newer peptides capable of targeting most as‐ pects of dermal health. These advances include neutralizing toxins, stimulating fibroblast

**4.** The molecule is capable of reaching its desired target intact and in its active form.

components, and be delivered effectively to the skin [Fields et al., 2009].

scaffolding, reducing inflammation and other desirable effects [Fields et al., 2009].

The goal of DNA vaccination is transfection of an antigen presenting cells (APC) or a bystander cell to produce antigens in an immuno-stimulatory setting. The field of genetic vaccines has

**13. Challenges in gene delivery for DNA vaccines**

absorption and bioavailability in intact forms in target tissues.

matological therapeutic or active ingredient are included as following:

demonstrable effect.

244 Novel Gene Therapy Approaches

ity, or mutagenicity.

tally due to its mechanism of action.

The main recombinant viral vectors used for gene delivery are *adenovirus*, *adeno-associated virus* (AAVs), *retrovirus* and *lentivirus*. The advantages of adenovirus are infection of a wide range of human cell types, ability to infect non-dividing cells and lower risk of insertional mutagen‐ esis. However, *adenovirus* expression is short lived and *adenoviruses* can cause a severe, even lethal, inflammatory response due to prior immune exposure. AAV, which depends on *adenovirus* or another virus for replication, has also been used for gene delivery with the advantages of predictable chromosomal insertion and no known pathological consequence of infection [Nguyen et al., 2008]. The main advantage of *retroviruses*, their ability to integrate into the host genome for long-term expression, is also their main disadvantage as this integration can cause mutagenesis and potentially cancer. *Retroviruses* are also further limited by their inability to infect non-dividing cells. *Lentiviruses*, which can transfect a broad spectrum of cell types, are the most efficient method to transfect DCs *in vitro* and *in vivo*. Yang *et al*. recently reported very high levels of immune activation and therapeutic tumor rejection following immunization with a lentiviral vector engineered to target DCs by the cell surface receptor DC-SIGN [Yang et al., 2008]. In particular, Merck & Co. has advanced the use of viral DNA vaccines for HIV vaccination. While there have been some successes in using viral gene therapy and many clinical trials are currently ongoing there are currently no approved protocols. Problems with viral delivery systems include immunologic priming to the vector itself, oncogenicity due to insertional mutagenesis, difficult manufacturing and limited DNA cargo capacity [Nguyen et al., 2008]. Clinical trials have highlighted some of these safety risks as viral gene delivery has resulted in both cancer and deaths. Recently, Merck & Co. stopped its Phase III HIV *adenovirus* vaccine prompting renewed questions about the utility of viral vectors. The safety challenges and limitations of viral vectors have resulted in increased interest in non-viral approaches to gene delivery using non-viral materials. In general, the nonviral methods of DNA vaccination utilized in clinical trials, recently reviewed by Lu *et al*., rely on physical methods. Injection of naked DNA plasmids has found limited success in humans particularly when injected intramuscularly, even though in smaller animal models naked DNA vaccination produces robust humoral and cell-mediated responses [Lu et al., 2008]. However, the rapid degradation/clearance [half-life of under 5 min if injected intravenously (IV)] of unprotected nucleic acids, poor induction of humoral immune responses in DNA vaccination in larger animals and requirement for large doses has hindered progress into clinical trials. Clinically, relevant physical methods that have been employed include electroporation, ballistics (gene gun), ultrasound and magnetofection. Encapsulation or complexation of DNA with a biomaterial can significantly enhance DNA stability, cellular uptake of DNA and final protein expression. Materials shown to possess potential for the delivery of genes include inorganic nanoparticles and surfaces that bind to or encapsulate DNA [Lu et al., 2008; Nguyen et al., 2008]. Cationic biomolecules including lipids, polysaccharides, polymers, and dendrim‐ ers can also electrostatically complex anionic DNA to facilitate transfection. Unless specifically designed to do so, DNA delivered non-virally has low potential for genomic integration [Nguyen et al., 2008]. Non-viral delivery systems for gene therapy are generally cheaper to manufacture, easily scalable from laboratory to GMP-scale production and are typically more robust for long-term storage compared to their viral counterparts. Despite achieving greater efficacy than naked DNA administration (IM, IV or otherwise), physical methods for gene delivery are often limited due to local tissue damage and insufficient gene expression. Research into non-viral gene delivery has been ongoing since the 1970s, and as understanding of the mechanisms of gene delivery has grown, the design of synthetic biomaterials has become more advanced. However, while there have been advances, non-viral methods of gene delivery generally still have lower efficacy than viruses [Nguyen et al., 2008].

brane. However, electrostatic interactions can also rapidly lead to aggregation of these VNPs with serum proteins; as VNP-protein aggregate size increases they can be eliminated from cir‐ culation by the phagocytic MPs of the RES, deposit non-specifically in microvascular beds or crash out of solution, which may cause acute toxicity [Nguyen et al., 2008]. In addition to con‐ taining high concentrations of negatively charged proteins, plasma also has a significant ionic strength. Interactions between serum proteins, blood and interstitial fluid solutes and polyca‐ tionic carrier materials can lead to competitive binding, destabilization of the VNP and subse‐ quent premature release of the nucleic acid payload. Interaction with complement proteins, C3 and C4 in particular, can activate the innate immune responses resulting in acute inflammation and lead to severe acute toxicity or death. Mucosal surfaces and serum also contain DNase and RNase enzymes that specifically degrade nucleic acids. Condensed VNPs prevent the degrada‐ tion of the nucleic acid payload by steric inhibition of these DNases/RNases. The addition of PEG and other hydrophilic polymers can be used to prevent aggregation with serum proteins and subsequent rapid clearance [Nguyen et al., 2008]. This simple functionality can sharply in‐ crease the serum half-life of a particle and prevent acute toxic events due to non-specific interac‐ tions, but also results in lower transfection efficiency and reduced cellular targeting. Toxicity at the cellular level and/or due to interactions with the immune system, liver, kidneys, or other complex organ systems can be a concern with non-viral gene delivery. For example, PEI has been shown to be an effective transfection agent but has also been reported to be toxic in animal models. Polycations such as PEI and cationic lipids such as 1, 2-dioleoyl-3-trimethylammoni‐ um-propane (DOTAP) also tend to activate complement and the reticuloendothelial system (RES) aggregate with serum proteins, and can aggregate with red blood cells as well. Some tox‐ icity issues both *in vitro* and *in vivo* can be addressed by chemical modification of PEI [Nguyen et al., 2008]. For increased safety, biodegradable gene delivery systems have also been developed, including degradable cross-linked PEI [Kim et al., 2005], poly (ortho-esters) (POE) [Heller et al.,

Challenges in Advancing the Field of Cancer Gene Therapy: An Overview of the Multi-Functional Nanocarriers

http://dx.doi.org/10.5772/54862

247

2000] and poly (b-amino esters) (PBAE) [Anderson et al., 2003].

**13.3. Particle uptake by APCs and targeting gene delivery for DNA vaccines**

Particle uptake by phagocytosis (particles > 500 nm), macropinocytosis, and receptor-mediated endocytosis are particularly important routes of entry into APCs. Delivery systems can be designed to exploit these ways. Cell-specific targeting can significantly enhance transfection efficiency and the desired therapeutic outcome. The direct conjugation of targeting moieties such as receptor ligands, peptides, sugars, aptamers and antibodies can increase cell and tissue specificity and transfection efficiency. Additionally, targeting can be based upon size-specific signals to avoid off-target affects. A variety of strategies exist for targeting APCs. First, APCs express Fc-receptors, which bind to the constant region of antibodies to facilitate uptake of antibody-coated foreign bodies. APCs also express complement receptors that help clear complement-opsonized particles [Nguyen et al., 2008]. Lectin-binding receptors, such as the mannose receptor and scavenger receptors that recognize apoptotic bodies, certain bacterial components and other non-self motifs are PRRs commonly found on APCs that can enhance particle uptake and may trigger innate immune activation. Second, unlike most other cell types, immature DCs constitutively sample their extracellular fluid environment nonspecifically through macropinocytosis to maintain immune surveillance and vigilance for

#### **13.2. Barriers to gene delivery for DNA vaccines**

There are many potential blocks that must be overcome for successful DNA delivery, a process broadly defined as transfection. Plasmid DNA must first be packaged into particles. Require‐ ments for gene delivery include protection of plasmid DNA from degradation, localization to the tissue and cell types of interest avoiding off-target distribution, minimal inactivation by in‐ teraction with serum proteins, low clearance from the blood or interstitial space and efficient transport through the extracellular matrix to the surface of target cells. Next, the DNA-contain‐ ing particles must associate with cells and become internalized into them by cellular uptake processes [Nguyen et al., 2008]. Following uptake, DNA-containing particles must escape the endosomal/ phagosomal compartment into the cytoplasm and release their DNA cargo. DNA must finally translocate into the nucleus to be transcribed into mRNA and subsequently trans‐ lated into protein antigen. Viruses have evolved to accomplish these steps and provide a frame‐ work for the design of synthetic delivery particles. Despite efficient uptake of particles of a variety of sizes, *in vitro* and *in vivo* transfection of DCs is still notoriously difficult to achieve. APCs are specialized not only for uptake of antigen but also rapid and efficient antigen process‐ ing. As a key role of APCs is to internalize and process pathogens for immune activation, APCs may have greater protection against foreign (viral) DNA entry into the nucleus, which may be a barrier to DNA vaccination. Whereas *in vitro* investigation of transfection efficiency in cell cul‐ ture can be used to identify promising materials for transfection, there exist multiple extracellu‐ lar barriers to effective DNA vaccination *in vivo*. DCs reside in the blood, in the skin (Langerhans cells), other mucosal barriers, and in lymph nodes [Nguyen et al., 2008]. macrophages (MPs) al‐ so exist in lymph nodes, as circulating precursors in the blood (monocytes) that differentiate as they enter inflamed tissue cites, and as specialized MPs lining the spleen and liver (Kupfer cells) forming the phagocytic part of the reticuloendothelial system (RES). Access to these APCs is therefore determined not only by route of injection, but also by ability of a particle to drain into lymphatic systems or activate inflammatory signals to recruit APCs. For example, Reddy *et al*. have illustrated size-based targeting of lymph-node resident DCs by accessing lymphatic ves‐ sels with 25 nm particles; lymphatic drainage and DC uptake was significantly reduced with 100 nm particles [Reddy et al., 2007]. Many cationic delivery materials, both polymeric and lip‐ id, form vector-nucleic acid particle (VNP) complexes by electrostatic interactions with the neg‐ ative charges on the phosphate groups of the DNA backbone. A net positive surface charge can facilitate transfection by interacting with the negatively charged glycoproteins at the cell mem‐ designed to do so, DNA delivered non-virally has low potential for genomic integration [Nguyen et al., 2008]. Non-viral delivery systems for gene therapy are generally cheaper to manufacture, easily scalable from laboratory to GMP-scale production and are typically more robust for long-term storage compared to their viral counterparts. Despite achieving greater efficacy than naked DNA administration (IM, IV or otherwise), physical methods for gene delivery are often limited due to local tissue damage and insufficient gene expression. Research into non-viral gene delivery has been ongoing since the 1970s, and as understanding of the mechanisms of gene delivery has grown, the design of synthetic biomaterials has become more advanced. However, while there have been advances, non-viral methods of gene delivery

There are many potential blocks that must be overcome for successful DNA delivery, a process broadly defined as transfection. Plasmid DNA must first be packaged into particles. Require‐ ments for gene delivery include protection of plasmid DNA from degradation, localization to the tissue and cell types of interest avoiding off-target distribution, minimal inactivation by in‐ teraction with serum proteins, low clearance from the blood or interstitial space and efficient transport through the extracellular matrix to the surface of target cells. Next, the DNA-contain‐ ing particles must associate with cells and become internalized into them by cellular uptake processes [Nguyen et al., 2008]. Following uptake, DNA-containing particles must escape the endosomal/ phagosomal compartment into the cytoplasm and release their DNA cargo. DNA must finally translocate into the nucleus to be transcribed into mRNA and subsequently trans‐ lated into protein antigen. Viruses have evolved to accomplish these steps and provide a frame‐ work for the design of synthetic delivery particles. Despite efficient uptake of particles of a variety of sizes, *in vitro* and *in vivo* transfection of DCs is still notoriously difficult to achieve. APCs are specialized not only for uptake of antigen but also rapid and efficient antigen process‐ ing. As a key role of APCs is to internalize and process pathogens for immune activation, APCs may have greater protection against foreign (viral) DNA entry into the nucleus, which may be a barrier to DNA vaccination. Whereas *in vitro* investigation of transfection efficiency in cell cul‐ ture can be used to identify promising materials for transfection, there exist multiple extracellu‐ lar barriers to effective DNA vaccination *in vivo*. DCs reside in the blood, in the skin (Langerhans cells), other mucosal barriers, and in lymph nodes [Nguyen et al., 2008]. macrophages (MPs) al‐ so exist in lymph nodes, as circulating precursors in the blood (monocytes) that differentiate as they enter inflamed tissue cites, and as specialized MPs lining the spleen and liver (Kupfer cells) forming the phagocytic part of the reticuloendothelial system (RES). Access to these APCs is therefore determined not only by route of injection, but also by ability of a particle to drain into lymphatic systems or activate inflammatory signals to recruit APCs. For example, Reddy *et al*. have illustrated size-based targeting of lymph-node resident DCs by accessing lymphatic ves‐ sels with 25 nm particles; lymphatic drainage and DC uptake was significantly reduced with 100 nm particles [Reddy et al., 2007]. Many cationic delivery materials, both polymeric and lip‐ id, form vector-nucleic acid particle (VNP) complexes by electrostatic interactions with the neg‐ ative charges on the phosphate groups of the DNA backbone. A net positive surface charge can facilitate transfection by interacting with the negatively charged glycoproteins at the cell mem‐

generally still have lower efficacy than viruses [Nguyen et al., 2008].

**13.2. Barriers to gene delivery for DNA vaccines**

246 Novel Gene Therapy Approaches

brane. However, electrostatic interactions can also rapidly lead to aggregation of these VNPs with serum proteins; as VNP-protein aggregate size increases they can be eliminated from cir‐ culation by the phagocytic MPs of the RES, deposit non-specifically in microvascular beds or crash out of solution, which may cause acute toxicity [Nguyen et al., 2008]. In addition to con‐ taining high concentrations of negatively charged proteins, plasma also has a significant ionic strength. Interactions between serum proteins, blood and interstitial fluid solutes and polyca‐ tionic carrier materials can lead to competitive binding, destabilization of the VNP and subse‐ quent premature release of the nucleic acid payload. Interaction with complement proteins, C3 and C4 in particular, can activate the innate immune responses resulting in acute inflammation and lead to severe acute toxicity or death. Mucosal surfaces and serum also contain DNase and RNase enzymes that specifically degrade nucleic acids. Condensed VNPs prevent the degrada‐ tion of the nucleic acid payload by steric inhibition of these DNases/RNases. The addition of PEG and other hydrophilic polymers can be used to prevent aggregation with serum proteins and subsequent rapid clearance [Nguyen et al., 2008]. This simple functionality can sharply in‐ crease the serum half-life of a particle and prevent acute toxic events due to non-specific interac‐ tions, but also results in lower transfection efficiency and reduced cellular targeting. Toxicity at the cellular level and/or due to interactions with the immune system, liver, kidneys, or other complex organ systems can be a concern with non-viral gene delivery. For example, PEI has been shown to be an effective transfection agent but has also been reported to be toxic in animal models. Polycations such as PEI and cationic lipids such as 1, 2-dioleoyl-3-trimethylammoni‐ um-propane (DOTAP) also tend to activate complement and the reticuloendothelial system (RES) aggregate with serum proteins, and can aggregate with red blood cells as well. Some tox‐ icity issues both *in vitro* and *in vivo* can be addressed by chemical modification of PEI [Nguyen et al., 2008]. For increased safety, biodegradable gene delivery systems have also been developed, including degradable cross-linked PEI [Kim et al., 2005], poly (ortho-esters) (POE) [Heller et al., 2000] and poly (b-amino esters) (PBAE) [Anderson et al., 2003].

#### **13.3. Particle uptake by APCs and targeting gene delivery for DNA vaccines**

Particle uptake by phagocytosis (particles > 500 nm), macropinocytosis, and receptor-mediated endocytosis are particularly important routes of entry into APCs. Delivery systems can be designed to exploit these ways. Cell-specific targeting can significantly enhance transfection efficiency and the desired therapeutic outcome. The direct conjugation of targeting moieties such as receptor ligands, peptides, sugars, aptamers and antibodies can increase cell and tissue specificity and transfection efficiency. Additionally, targeting can be based upon size-specific signals to avoid off-target affects. A variety of strategies exist for targeting APCs. First, APCs express Fc-receptors, which bind to the constant region of antibodies to facilitate uptake of antibody-coated foreign bodies. APCs also express complement receptors that help clear complement-opsonized particles [Nguyen et al., 2008]. Lectin-binding receptors, such as the mannose receptor and scavenger receptors that recognize apoptotic bodies, certain bacterial components and other non-self motifs are PRRs commonly found on APCs that can enhance particle uptake and may trigger innate immune activation. Second, unlike most other cell types, immature DCs constitutively sample their extracellular fluid environment nonspecifically through macropinocytosis to maintain immune surveillance and vigilance for foreign particles, as well as to present endogenous proteins for maintenance of self-tolerance. This constant sampling may explain DC-targeting by particle size. Particle size may also influence the specific route of entry, as reviewed recently by Xiang *et al*. Particle surface characteristics also play a role in uptake, as cationic particles more readily associate with the negatively charged glycoproteins on the cell surface and promote non-specific uptake, and other surface characteristics can activate opsonization [Xiang et al., 2006]. Champion *et al.* have clarified the role of particle shape in influencing phagocytosis by MPs, as well as provided simple methods for creating materials with complex shapes and sizes to take advantage of particle physical properties. While spherical micro- and nano-particles are efficiently phago‐ cytosed by lung alveolar MPs, phagocytosis can be inhibited by contact with odd geometries due to an inability to form the necessary actin structures. In general, size plays a more significant role with particle association with the cell surface than with internalization. These studies suggest a role for particle surface nano and micro-structure in the design of APCtargeted DNA vaccine delivery systems [Champion et al., 2007].

despite decades of study, safe and efficient gene delivery remains a major blockage in human medicine. The current progresses of gene therapy are further focused on synthesized nanoparticle technologies such as polymers (PLGA, chitosan and PEI), lipids and peptides. It is necessary to understand the effects of particle size, surface characteristics and material interactions. Research on cell penetrating peptides (CPPs) as gene/ drug delivery systems has clarified their capacity to promote the efficient internalization of therapeutic biomolecules. Despite differences in size, charge and/or structure between different bioactive molecules, it is clear that CPP-based systems appear to be very versatile and efficient delivery is achievable following proper adjustment of the carrier to the transported biomolecule. Because the development of drug, oligonucleotide or gene delivery systems is aimed at a clinical applica‐ tion, the design of these novel delivery vectors should consider other important issues including safety, bio-distribution, ease of manufacturing, scale-up, reproducibility and analytical and physical characterization. The advance of CPP technology depends on the development of strategies that facilitate endosomal escape and that confer cell specificity to these systems. A careful investigation of the mechanisms of internalization of CPP-cargo complexes or conjugates will greatly help the improvement of this powerful technology. However, a viable non-viral gene vector for systemic delivery depends on its capacity to bypass a series of physiological barriers and its efficiency in carrying nucleic acids to a targeted site within a cell. The concept of a multifunctional delivery system helps to solve the problems associated with various barriers. The availability of delivery devices directed towards each individual barrier, provides a basis for this direction, although the complexity in the devel‐ opment of multifunctional non-viral vectors is much more than a merely combination of various devices into a single system. In order to achieve the optimal gene delivery, the researchers have focused largely on the evolution of "intelligent" bio-responsive materials, as well as on the advances in formulation technologies. In this process, a number of strategies have emerged including the balances between gene packing and controlled release, and optimal control between long-circulation and intracellular trafficking that promotes safer and

Challenges in Advancing the Field of Cancer Gene Therapy: An Overview of the Multi-Functional Nanocarriers

http://dx.doi.org/10.5772/54862

249

more efficient delivery of gene/drug in a systemic context.

and Tayebeh Saleh2

\*Address all correspondence to: azam\_bolhassani@yahoo.com

1 Molecular Immunology and Vaccine Research Lab., Pasteur Institute of Iran, Tehran, Iran

2 Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares Uni‐

\*Address all correspondence to: A\_bolhasani@pasteur.ac.ir

**Author details**

Azam Bolhassani1

versity, Tehran, Iran

#### **13.4. Biomaterials for DNA delivery: Non-polymeric biomaterials for gene delivery**

Many materials have been developed for gene delivery. Early chemical methods of increasing the efficacy of gene delivery focused on co-precipitation of the DNA with salts such as calcium phosphate. More recently, inorganic materials have also been combined with polymers to form hybrid gene delivery nanoparticles. For example, textured surfaces and silica nano-particles have been shown to be effective for gene transfer *in vitro* and organically modified silica nanoparticles have been shown to deliver genes *in vivo*. Gold nano-particles have also been com‐ bined with PEI for hybrid gene delivery systems. Cationic lipids have been the non-viral gene delivery vectors of choice for clinical application since Felgner first introduced their use in 1987. The cationic lipid molecule consists of a hydrophilic positively charged head group, a linker that may impart some functionality such as pH sensitivity and a hydrophobic long chain tail [Felgner et al., 1987]. A prototypical cationic lipid is DOTAP; it is the most widely used lipid for gene delivery. For *in vivo* delivery, nucleic acids are usually encapsulated into liposomes: vesi‐ cles with lipid bilayer membranes that exist as large uni-lamellar vesicles (LUVs) or multi-la‐ mellar vesicles (MLVs). Liposomes generally consist of a single cationic lipid or a mixture of cationic lipids that facilitate nucleic acid binding and transfection, cholesterol or diolelphopha‐ tidylethanolamine (DOPE) to impart some rigidity or stability to the complex, and PEG to shield particles from aggregation, serum components, or other non-specific interactions. Lipids have been used extensively in gene therapy and are the main non-viral delivery vectors used in clini‐ cal trials. Unfortunately, some lipoplexes are toxic, interact nonspecifically with serum proteins and cells, aggregate quickly, activate the complement system, or have low *in vivo* efficacy. One promising approach that may address these problems is to increase the chemical diversity of lipid-like materials through combinatorial synthesis approaches [Nguyen et al., 2008].

#### **14. Conclusion**

Gene therapy possesses great potential for combating a variety of diseases. Initial results are promising and some technologies have advanced to clinical trials. Yet challenges remain, and despite decades of study, safe and efficient gene delivery remains a major blockage in human medicine. The current progresses of gene therapy are further focused on synthesized nanoparticle technologies such as polymers (PLGA, chitosan and PEI), lipids and peptides. It is necessary to understand the effects of particle size, surface characteristics and material interactions. Research on cell penetrating peptides (CPPs) as gene/ drug delivery systems has clarified their capacity to promote the efficient internalization of therapeutic biomolecules. Despite differences in size, charge and/or structure between different bioactive molecules, it is clear that CPP-based systems appear to be very versatile and efficient delivery is achievable following proper adjustment of the carrier to the transported biomolecule. Because the development of drug, oligonucleotide or gene delivery systems is aimed at a clinical applica‐ tion, the design of these novel delivery vectors should consider other important issues including safety, bio-distribution, ease of manufacturing, scale-up, reproducibility and analytical and physical characterization. The advance of CPP technology depends on the development of strategies that facilitate endosomal escape and that confer cell specificity to these systems. A careful investigation of the mechanisms of internalization of CPP-cargo complexes or conjugates will greatly help the improvement of this powerful technology. However, a viable non-viral gene vector for systemic delivery depends on its capacity to bypass a series of physiological barriers and its efficiency in carrying nucleic acids to a targeted site within a cell. The concept of a multifunctional delivery system helps to solve the problems associated with various barriers. The availability of delivery devices directed towards each individual barrier, provides a basis for this direction, although the complexity in the devel‐ opment of multifunctional non-viral vectors is much more than a merely combination of various devices into a single system. In order to achieve the optimal gene delivery, the researchers have focused largely on the evolution of "intelligent" bio-responsive materials, as well as on the advances in formulation technologies. In this process, a number of strategies have emerged including the balances between gene packing and controlled release, and optimal control between long-circulation and intracellular trafficking that promotes safer and more efficient delivery of gene/drug in a systemic context.

## **Author details**

foreign particles, as well as to present endogenous proteins for maintenance of self-tolerance. This constant sampling may explain DC-targeting by particle size. Particle size may also influence the specific route of entry, as reviewed recently by Xiang *et al*. Particle surface characteristics also play a role in uptake, as cationic particles more readily associate with the negatively charged glycoproteins on the cell surface and promote non-specific uptake, and other surface characteristics can activate opsonization [Xiang et al., 2006]. Champion *et al.* have clarified the role of particle shape in influencing phagocytosis by MPs, as well as provided simple methods for creating materials with complex shapes and sizes to take advantage of particle physical properties. While spherical micro- and nano-particles are efficiently phago‐ cytosed by lung alveolar MPs, phagocytosis can be inhibited by contact with odd geometries due to an inability to form the necessary actin structures. In general, size plays a more significant role with particle association with the cell surface than with internalization. These studies suggest a role for particle surface nano and micro-structure in the design of APC-

targeted DNA vaccine delivery systems [Champion et al., 2007].

**13.4. Biomaterials for DNA delivery: Non-polymeric biomaterials for gene delivery**

lipid-like materials through combinatorial synthesis approaches [Nguyen et al., 2008].

Gene therapy possesses great potential for combating a variety of diseases. Initial results are promising and some technologies have advanced to clinical trials. Yet challenges remain, and

**14. Conclusion**

248 Novel Gene Therapy Approaches

Many materials have been developed for gene delivery. Early chemical methods of increasing the efficacy of gene delivery focused on co-precipitation of the DNA with salts such as calcium phosphate. More recently, inorganic materials have also been combined with polymers to form hybrid gene delivery nanoparticles. For example, textured surfaces and silica nano-particles have been shown to be effective for gene transfer *in vitro* and organically modified silica nanoparticles have been shown to deliver genes *in vivo*. Gold nano-particles have also been com‐ bined with PEI for hybrid gene delivery systems. Cationic lipids have been the non-viral gene delivery vectors of choice for clinical application since Felgner first introduced their use in 1987. The cationic lipid molecule consists of a hydrophilic positively charged head group, a linker that may impart some functionality such as pH sensitivity and a hydrophobic long chain tail [Felgner et al., 1987]. A prototypical cationic lipid is DOTAP; it is the most widely used lipid for gene delivery. For *in vivo* delivery, nucleic acids are usually encapsulated into liposomes: vesi‐ cles with lipid bilayer membranes that exist as large uni-lamellar vesicles (LUVs) or multi-la‐ mellar vesicles (MLVs). Liposomes generally consist of a single cationic lipid or a mixture of cationic lipids that facilitate nucleic acid binding and transfection, cholesterol or diolelphopha‐ tidylethanolamine (DOPE) to impart some rigidity or stability to the complex, and PEG to shield particles from aggregation, serum components, or other non-specific interactions. Lipids have been used extensively in gene therapy and are the main non-viral delivery vectors used in clini‐ cal trials. Unfortunately, some lipoplexes are toxic, interact nonspecifically with serum proteins and cells, aggregate quickly, activate the complement system, or have low *in vivo* efficacy. One promising approach that may address these problems is to increase the chemical diversity of

Azam Bolhassani1 and Tayebeh Saleh2

\*Address all correspondence to: azam\_bolhassani@yahoo.com

\*Address all correspondence to: A\_bolhasani@pasteur.ac.ir

1 Molecular Immunology and Vaccine Research Lab., Pasteur Institute of Iran, Tehran, Iran

2 Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares Uni‐ versity, Tehran, Iran

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Challenges in Advancing the Field of Cancer Gene Therapy: An Overview of the Multi-Functional Nanocarriers

http://dx.doi.org/10.5772/54862

251

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[114] Trabulo, S, Cardoso, A. L, & Mano, M. Pedroso de Lima, MC. ((2010). Cell-penetrating peptides-mechanisms of cellular uptake and generation of delivery systems. Pharma‐

[115] Tung, C. H, Mueller, S, & Weissleder, R. (2002). Novel branching membrane translo‐ cational peptide as gene delivery vector. Bioorg. Med. Chem., , 10, 3609-3614.

[116] Undercover genes slip into the brain(2003). New Scientist, http://www.newscient‐

[117] Van Vlerken, L. E, Vyas, T. K, & Amiji, M. M. (2007). Poly (ethylene glycol)-modified nanocarriers for tumor-targeted and intracellular delivery. Pharmaceutical research, ,

[118] Veldhoen, S, Laufer, S. D, & Restle, T. (2008). Recent developments in peptide-based

[119] Varkouhi, A. K, Scholte, M, Storm, G, & Haisma, H. J. (2011). Endosomal escape pathways for delivery of biologicals. Journal of Controlled Release, , 151, 220-228.

[120] Vercauteren, D, Rejman, J, Martens, T. F, Demeester, J, De Smedt, S. C, & Braeckmans, K. (2012). On the cellular processing of non-viral nanomedicines for nucleic acid delivery: Mechanisms and methods. Journal of Controlled Release, , 161(2), 566-581.

[121] Vidugiriene, J, Goueli, S, & Sužiedelis, K. (2007). RNA interference: from a research tool

[122] Vivès, E, Schmidt, J, & Pèlegrin, A. (2008). Cell-penetrating and cell-targeting peptides in drug delivery. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer, , 1786,

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nucleic acid delivery. Int. J. Mol. Sci., , 9, 1276-1320.

for intracellular drug and gene delivery. Peptide Science, , 90, 604-610.


**Chapter 11**

**Cancer Gene Therapy: Targeted Genomedicines**

To date, traditional chemotherapy alone or in combination with immunotherapy and ionizing radiation modalities have been used to obliterate dividing aberrant cells in various tumors,

Given the fact that malignant cells proliferate more rapidly than normal cells, damage to the cancer cells is anticipated to be markedly greater than normal cells. However, cancer cells generate chemoresistance mechanisms within the tumor microenvironment, while undesired toxicity may occur in the normal cells. For example, in colorectal cancer (CRC), there exist well-described sequences of mutational events that evince the shift of normal colon epithelium to premalignant adenoma and malignant adenocarcinoma. These events are 1) loss of the function of the adenomatous polyposis coli (APC) gene (encoding a protein involved in cell adhesion and transcription) in up to 85% of all cases of CRC, 2) mutation of KRAS (a GTP-ase that controls cell proliferation) in 50–60% of all cases of CRC, and 3) downregulated expression of the cell-adhesion transmembrane glycoprotein E-cadherin in almost 50–60% of all cases of CRC. Mutations in the mismatch-repair genes MLH1 and MSH2 contribute to genetic instability. Besides, there exist a number of genes alterations leading cells toward remodeling that include: 1) SMAD4 involved in the transforming growth factor signal transduction suppressing epithelial-cell growth, 2) INK4A involved in the retinoblas‐ toma tumor-suppressor pathway, and 3) TP53 alterations increasing the resistance of cancer cells to apoptosis [1]. Similar molecular/cellular alterations occur in various solid tumors, highlighting the intricacy of biological events leading to initiation and progression of malignancies. Therefore, necessity for development and advancement of more effective modalities targeting such genetic changes is perceptible to achieve successful cancer treatment and cure. After decades of disappointment, targeted therapy of cancer has been advanced by integration of immunotherapy as well as gene and cell therapy. As proof-ofconcept, recent clinical trials (e.g., anti-CTLA4 antibody, ipilimumab) have shown signifi‐

> © 2013 Omidi 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,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

while morbid statistics of cancer therapy show limited clinical successes.

Yadollah Omidi, Jaleh Barar and George Coukos

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54739

**1. Introduction**

## **Cancer Gene Therapy: Targeted Genomedicines**

Yadollah Omidi, Jaleh Barar and George Coukos

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54739

## **1. Introduction**

To date, traditional chemotherapy alone or in combination with immunotherapy and ionizing radiation modalities have been used to obliterate dividing aberrant cells in various tumors, while morbid statistics of cancer therapy show limited clinical successes.

Given the fact that malignant cells proliferate more rapidly than normal cells, damage to the cancer cells is anticipated to be markedly greater than normal cells. However, cancer cells generate chemoresistance mechanisms within the tumor microenvironment, while undesired toxicity may occur in the normal cells. For example, in colorectal cancer (CRC), there exist well-described sequences of mutational events that evince the shift of normal colon epithelium to premalignant adenoma and malignant adenocarcinoma. These events are 1) loss of the function of the adenomatous polyposis coli (APC) gene (encoding a protein involved in cell adhesion and transcription) in up to 85% of all cases of CRC, 2) mutation of KRAS (a GTP-ase that controls cell proliferation) in 50–60% of all cases of CRC, and 3) downregulated expression of the cell-adhesion transmembrane glycoprotein E-cadherin in almost 50–60% of all cases of CRC. Mutations in the mismatch-repair genes MLH1 and MSH2 contribute to genetic instability. Besides, there exist a number of genes alterations leading cells toward remodeling that include: 1) SMAD4 involved in the transforming growth factor signal transduction suppressing epithelial-cell growth, 2) INK4A involved in the retinoblas‐ toma tumor-suppressor pathway, and 3) TP53 alterations increasing the resistance of cancer cells to apoptosis [1]. Similar molecular/cellular alterations occur in various solid tumors, highlighting the intricacy of biological events leading to initiation and progression of malignancies. Therefore, necessity for development and advancement of more effective modalities targeting such genetic changes is perceptible to achieve successful cancer treatment and cure. After decades of disappointment, targeted therapy of cancer has been advanced by integration of immunotherapy as well as gene and cell therapy. As proof-ofconcept, recent clinical trials (e.g., anti-CTLA4 antibody, ipilimumab) have shown signifi‐

© 2013 Omidi 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. © 2013 The Author(s). Licensee InTech. This chapter is 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.

cant increase in survival for patients with metastatic melanoma, for which conventional therapies have failed [2].

lation and histone acetylation/deacetylation) have directed scientists to devise genomedicines to fix the genomic defects. It should be evoked that, unlike treatment strategies for genetic defects that need permanent expression of the corrected genes, cancer gene therapy is based on temporary and locally limited stimulation/suppression effects on desired gene(s). Further, malignant cells display specific gene markers that are different in nature or magnitude compared to the normal cells. These characteristics of cancer cells are deemed to provide a robust platform for specific targeted gene therapy that provides major advantages over current

Cancer Gene Therapy: Targeted Genomedicines

http://dx.doi.org/10.5772/54739

263

Up until now, some domains of cancer gene therapy have been devoted greater attention, including: a) suppression of cancer cells by introducing genes into tumor cells to lead cells toward apoptosis (e.g., herpes simplex virus thymidine kinase, cytosine deaminase); b) inhibition of growth of cancer cells; c) enhancement of cancer cells chemosensitivity (p53, Bax); d) specific stimulation of the host's immune response against the cancer cells (tumor antigen, DNA vaccines, cytokine genes) by introducing the relevant genes into tumor cells or dendritic cells. Although use of genomedicines (e.g., antisense RNA, siRNA, ribozymes, DNAzyme and aptamers) have shown positive outcomes, their combination with other cancer therapy modalities including chemotherapy and immunotherapy can open other avenues for cancer

In addition, immune gene therapies (e.g., targeted DNA vaccine) exploit the lymphocytes and dendritic cells potentials, activating the immune system defense mechanisms against cancer cells. DNA vaccines possess intrinsic ability to activate multiple pathways of innate immunity, that also provide a unique opportunity to guide defined antigens, accompanied by specific activator molecules, through a patient's compromised immune system [11]. Further, suicide gene therapy tackles to deliver genes to the cancer cells, upon which cancer cells convert nontoxic prodrugs into active chemotherapeuties. In this approach, cancerous cells containing suicide genes are solely targeted through a systemic administration of prodrug. The suicide gene therapy is deemed to provide maximal inhibitory effects in cancer cells, but minimal toxic effects in normal cells [12]. Other than these strategies, antisense oligodeoxynucleotides (AS-ODNs) as a new class of molecularly targeted agents are in transitional trajectory from the laboratory into the clinic. A number of very important transcriptomic elements (e.g., VEGF, Ang-1, MDM2, protein kinase C-a, c-myb, integrin subunit b3, PKA-I, H-ras, bcl-2, c-raf, R1/R2 subunits of ribonucleotide reductase) have

In contrast to AS-ODNs technology, the mechanism of silencing an endogenous gene through a homologous double-stranded RNA (dsRNA), which is termed post-transcriptional gene silencing (PTGS) or RNA interference (RNAi), is a natural mechanism by which mammalian cells can regulate expansion of genes. Accordingly, short interfering RNA (siRNA) can be used for gene silencing. It is currently the fastest growing sector for target validation and therapeutic [14]. Further, considering cancer cells scape from immune system within the tumor microen‐ vironment, immune targeted gene therapy appears to provide an effective tactic for activation of the immune systems in such intricate microenvironment, whereby targeted gene therapy

of angiogenesis and lymphangiogenesis bestow robust treatment possibilities [15].

chemotherapy and immunotherapy modalities [6, 7].

therapy [8-10].

successfully targeted by AS-ODNs [13].

Targeted therapy of cancer using mAbs has provided great outcomes [3], while cancer gene therapy has not been as productive as immunotherapy from translational stand point. Efficient gene transfer strategy, as a fundamental step, continues to be the major determining factor for clinical successes of the gene therapy. In fact, there exist some hurdles that make gene therapy a formidable task. There are problems with delivery of sufficient copies of a gene (e.g., short interfering RNA (siRNA), antisense) to all tumor cells, whose biology appear to be very complex and ideally all the cancer-related genes must be controlled. Another barrier is the lack of proper gene delivery systems (GDSs) since the nonspecificity of GDSs makes gene therapy strategy somewhat uncertain. Overall, the current gene therapy approaches are capable of introducing genes into cells in vivo without discrimination within target and non-target cells. However, such unselective approach can impact both normal and aberrant cells. Incorporation of a homing device (e.g., monoclonal antibodies (mAb), antibody (Ab) fragments, or target specific aptamers) with an appropriate delivery system may result in cell-specific targeting and greater clinical outcomes [4].

The main gist of this chapter is to concisely provide information upon the specific gene therapy strategies and gene targets. We will discuss impacts of oncogenes, tumor suppressor genes and apoptosis-inducing genes on cancer gene therapy strategies as well as methods that specifically reactivate pathways that render the mutated cells susceptible to antitumor agents and immunotherapy. We will also remark on the cancer therapy opportunities through exploiting targeted nanogenomedicines.

## **2. Trajectory of gene therapy**

Of many cancer therapy endeavors, cancer gene therapy has granted great hopes even though it is in its developmental trajectory. So far, more than 65% of the gene therapy trials have been devoted to the cancer diseases using various vectors (retrovirus (20%), advenovirus (18%), adeno-associated virusade (5%), lipofection (6%)) and naked/plasmid DNA (18.5%). Despite conducting more than 1186 cancer gene therapy trials (out of 1843), 45 have reached to phase III and only 1 is in phase IV [5]. At this stage, there exist 9 clinical trials of gene therapy that have been conditionally approved [4]. Most of these trials are conducted as adjuvant therapies, which clearly highlight needs for more effective gene therapy systems.

The foremost basis of gene therapy is to fix the genomic defects; nonetheless the gene therapy concept is going to be revolutionized by illumination of epigenomics and targeted genome‐ dicines. In tumor development, the origination of cancer is an intricate biological process, in which molecular changes at genomic/epigenomic levels play a central role. These molecular alterations can equip cancerous cells with unique molecular biostructures that play crucial roles in survival, progression and invasion of cancer cells. Such genomic/epigenomic altera‐ tions (e.g., changes in gene expression, mutations, gene deletion, DNA methylation/demethy‐ lation and histone acetylation/deacetylation) have directed scientists to devise genomedicines to fix the genomic defects. It should be evoked that, unlike treatment strategies for genetic defects that need permanent expression of the corrected genes, cancer gene therapy is based on temporary and locally limited stimulation/suppression effects on desired gene(s). Further, malignant cells display specific gene markers that are different in nature or magnitude compared to the normal cells. These characteristics of cancer cells are deemed to provide a robust platform for specific targeted gene therapy that provides major advantages over current chemotherapy and immunotherapy modalities [6, 7].

cant increase in survival for patients with metastatic melanoma, for which conventional

Targeted therapy of cancer using mAbs has provided great outcomes [3], while cancer gene therapy has not been as productive as immunotherapy from translational stand point. Efficient gene transfer strategy, as a fundamental step, continues to be the major determining factor for clinical successes of the gene therapy. In fact, there exist some hurdles that make gene therapy a formidable task. There are problems with delivery of sufficient copies of a gene (e.g., short interfering RNA (siRNA), antisense) to all tumor cells, whose biology appear to be very complex and ideally all the cancer-related genes must be controlled. Another barrier is the lack of proper gene delivery systems (GDSs) since the nonspecificity of GDSs makes gene therapy strategy somewhat uncertain. Overall, the current gene therapy approaches are capable of introducing genes into cells in vivo without discrimination within target and non-target cells. However, such unselective approach can impact both normal and aberrant cells. Incorporation of a homing device (e.g., monoclonal antibodies (mAb), antibody (Ab) fragments, or target specific aptamers) with an appropriate delivery system may result in cell-specific targeting

The main gist of this chapter is to concisely provide information upon the specific gene therapy strategies and gene targets. We will discuss impacts of oncogenes, tumor suppressor genes and apoptosis-inducing genes on cancer gene therapy strategies as well as methods that specifically reactivate pathways that render the mutated cells susceptible to antitumor agents and immunotherapy. We will also remark on the cancer therapy opportunities through

Of many cancer therapy endeavors, cancer gene therapy has granted great hopes even though it is in its developmental trajectory. So far, more than 65% of the gene therapy trials have been devoted to the cancer diseases using various vectors (retrovirus (20%), advenovirus (18%), adeno-associated virusade (5%), lipofection (6%)) and naked/plasmid DNA (18.5%). Despite conducting more than 1186 cancer gene therapy trials (out of 1843), 45 have reached to phase III and only 1 is in phase IV [5]. At this stage, there exist 9 clinical trials of gene therapy that have been conditionally approved [4]. Most of these trials are conducted as adjuvant therapies,

The foremost basis of gene therapy is to fix the genomic defects; nonetheless the gene therapy concept is going to be revolutionized by illumination of epigenomics and targeted genome‐ dicines. In tumor development, the origination of cancer is an intricate biological process, in which molecular changes at genomic/epigenomic levels play a central role. These molecular alterations can equip cancerous cells with unique molecular biostructures that play crucial roles in survival, progression and invasion of cancer cells. Such genomic/epigenomic altera‐ tions (e.g., changes in gene expression, mutations, gene deletion, DNA methylation/demethy‐

which clearly highlight needs for more effective gene therapy systems.

therapies have failed [2].

262 Novel Gene Therapy Approaches

and greater clinical outcomes [4].

exploiting targeted nanogenomedicines.

**2. Trajectory of gene therapy**

Up until now, some domains of cancer gene therapy have been devoted greater attention, including: a) suppression of cancer cells by introducing genes into tumor cells to lead cells toward apoptosis (e.g., herpes simplex virus thymidine kinase, cytosine deaminase); b) inhibition of growth of cancer cells; c) enhancement of cancer cells chemosensitivity (p53, Bax); d) specific stimulation of the host's immune response against the cancer cells (tumor antigen, DNA vaccines, cytokine genes) by introducing the relevant genes into tumor cells or dendritic cells. Although use of genomedicines (e.g., antisense RNA, siRNA, ribozymes, DNAzyme and aptamers) have shown positive outcomes, their combination with other cancer therapy modalities including chemotherapy and immunotherapy can open other avenues for cancer therapy [8-10].

In addition, immune gene therapies (e.g., targeted DNA vaccine) exploit the lymphocytes and dendritic cells potentials, activating the immune system defense mechanisms against cancer cells. DNA vaccines possess intrinsic ability to activate multiple pathways of innate immunity, that also provide a unique opportunity to guide defined antigens, accompanied by specific activator molecules, through a patient's compromised immune system [11]. Further, suicide gene therapy tackles to deliver genes to the cancer cells, upon which cancer cells convert nontoxic prodrugs into active chemotherapeuties. In this approach, cancerous cells containing suicide genes are solely targeted through a systemic administration of prodrug. The suicide gene therapy is deemed to provide maximal inhibitory effects in cancer cells, but minimal toxic effects in normal cells [12]. Other than these strategies, antisense oligodeoxynucleotides (AS-ODNs) as a new class of molecularly targeted agents are in transitional trajectory from the laboratory into the clinic. A number of very important transcriptomic elements (e.g., VEGF, Ang-1, MDM2, protein kinase C-a, c-myb, integrin subunit b3, PKA-I, H-ras, bcl-2, c-raf, R1/R2 subunits of ribonucleotide reductase) have successfully targeted by AS-ODNs [13].

In contrast to AS-ODNs technology, the mechanism of silencing an endogenous gene through a homologous double-stranded RNA (dsRNA), which is termed post-transcriptional gene silencing (PTGS) or RNA interference (RNAi), is a natural mechanism by which mammalian cells can regulate expansion of genes. Accordingly, short interfering RNA (siRNA) can be used for gene silencing. It is currently the fastest growing sector for target validation and therapeutic [14]. Further, considering cancer cells scape from immune system within the tumor microen‐ vironment, immune targeted gene therapy appears to provide an effective tactic for activation of the immune systems in such intricate microenvironment, whereby targeted gene therapy of angiogenesis and lymphangiogenesis bestow robust treatment possibilities [15].

## **3. Gene silencing as gene therapy modality**

It is clear that the tumorigenesis results from clusters of several genetic and/or epigenetic events. Therefore, identification of the involved genes provides new targets toward effective treatment of malignancies. Of various gene therapy approaches, it is deemed that the silencing of cancer-causing genes can control the biological consequences at its genetic root and thereby cure the disease. Hence, development of agents capable of gene silencing is now considered as a rational strategy for cancer therapy, which can be accomplished by genomedicines. We will review gene silencing technologies in the following sections.

delivered by a recombinant AAV (rAAV). It was found that such genomedicines can inhibit cell proliferation, induce apoptosis, reduce cell migration, and restrain in vivo proliferation of the cervical cancer CaSki cells [24]. Table 1 shows selected oncogenes targeted by AS-ODNs.

**Oncogene Application Ref**

BCL-2 BCL-2 AS-ODN inhibits sensitize small cell lung cancer cells (in vitro and in vivo) to radiation [29] Phase I/II study of G3139 (Bcl-2 AS-ODN) combined with doxorubicin and docetaxel in

Induction of apoptosis and increased chemosensitivity in human prostate cancer cells by

c-fos AS-ODN control prostaglandin E2-induced upregulation of vascular endothelial

c-MYC c-myc AS-ODN sensitize human colorectal cancer cells to chemotherapeutic drugs [36] inhibition of c-MYC by antisense phosphorodiamidate morpholino oligomer in prostate

The effectiveness of the AS-ODNs have so far been shown in both target cells/tissue as in vitro models and animal in vivo models in particular for cancer therapy [13]. The mechanisms of action of the AS-ODNs seem to vary in different systems. In the case of hybridization and intramolecular and/or intermolecular interactions, their degradation pattern appears to be different. Many investigations have shown the anti-oncogenic impacts of AS-ODNs through targeting specific oncogenes. We have used AS-ODNs specific to EGFR and showed substantial inhibition of EGFR in A431 cells [25] as well as A549 lung cancer cells [26] using non-viral vectors as delivery system. The inhibitory impacts of AS-ODNs have been assessed through alterations in growth rate, morphology and molecular analysis. Various oncogenes have been

The siRNA (also called as short interfering RNA or silencing RNA) is double-stranded RNA (dsRNA) molecules of 20-25 nucleotides. The siRNA gene-silencing mechanism is induced by

c-RAF-1 Phase I study of the c-raf-1 AS-ODN (ISIS 5132) combined with carboplatin and paclitaxel in

c-FOS Tissue-targeted antisense c-fos retroviral vector inhibits established breast cancer

patients with advanced non-small cell lung cancer

growth factor in human liver cancer cells

cancer murine models and humans

c-erbB-2 AS-ODN Inhibit serum-induced cell spreading of ovarian cancer cells [27] Inhibitory effects of c-erbB-2 AS-ODN in uterine endometrial cancer Ishikawa cells [28]

Phase I study of the c-raf-1 AS-ODN (ISIS 5132) in patients with advanced cancer [33]

[30]

265

Cancer Gene Therapy: Targeted Genomedicines

http://dx.doi.org/10.5772/54739

[31]

[32]

[34]

[35]

[37]

HER2 (c-erbB-2)

breast cancer

Bcl-2 AS-ODN

xenografts in nude mice

**Table 1.** Selected oncogenes targeted by AS-ODNs.

targeted by AS-ODNs.

**3.2. Small interfering RNA**

#### **3.1. Antisense oligodeoxynucleotides for suppression of mRNA**

During the last decade, we have witnessed emergence of synthetic AS-ODNs. They are primarily designed to attach selectively to the target transcriptomes and to disrupt the expression of a target gene. It should be evoked that the overall utility of AS-ODN as thera‐ peutic agent is dependent upon 1) the expression level of the target mRNA, 2) the optimal design of As-ODN, 3) the specificity of the AS-ODN to the target mRNA and 4) the availability of safe and highly efficient delivery systems. For example, we have shown that most of cationic polymers (CPs) and lipids used as GDSs to shuttle AS-ODNs specific to the epidermal growth factor receptor (EGFR) can also induce intrinsic cytotoxicity and toxicogenomics [16-22]. Previous studies have demonstrated that the viral vectors (e.g., adenovirus, adeno-associated virus (AAV), Epstein-Barr virus (EBV), herpes simplex virus type-1 (HSV-1), retrovirus, lentivirus, poxvirus, baculovirus) vectors can function as efficient vehicles for AS-ODN delivery. Of these, AAV vectors can be constructed to express short, distinct transcripts, a property that is useful for RNA - mediated inhibition of gene expression and successful delivery of the As-ODNs [23, 24]. Fig. 1 and Table 1 respectively represent the mechanism of action of AS-ODNs and their applications.

**Figure 1.** Mechanism of action of AS-ODN. A) Inhibition of proteins by small molecule drugs after translation. B) Sup‐ pression of mRNA by AS-ODN before translation in the presence of RNase H. This figure was adapted with permission from reference [4].

It is known that the oncogene E7 from high-risk human papillomavirus (HPV) strains has the potential to immortalize epithelial cells and increase cellular transformation in culture. Hence, to prevent the cervical cancer growth, the HPV16 E7 was inhibited by AS-ODN that was delivered by a recombinant AAV (rAAV). It was found that such genomedicines can inhibit cell proliferation, induce apoptosis, reduce cell migration, and restrain in vivo proliferation of the cervical cancer CaSki cells [24]. Table 1 shows selected oncogenes targeted by AS-ODNs.


**Table 1.** Selected oncogenes targeted by AS-ODNs.

**3. Gene silencing as gene therapy modality**

264 Novel Gene Therapy Approaches

will review gene silencing technologies in the following sections.

**3.1. Antisense oligodeoxynucleotides for suppression of mRNA**

action of AS-ODNs and their applications.

from reference [4].

It is clear that the tumorigenesis results from clusters of several genetic and/or epigenetic events. Therefore, identification of the involved genes provides new targets toward effective treatment of malignancies. Of various gene therapy approaches, it is deemed that the silencing of cancer-causing genes can control the biological consequences at its genetic root and thereby cure the disease. Hence, development of agents capable of gene silencing is now considered as a rational strategy for cancer therapy, which can be accomplished by genomedicines. We

During the last decade, we have witnessed emergence of synthetic AS-ODNs. They are primarily designed to attach selectively to the target transcriptomes and to disrupt the expression of a target gene. It should be evoked that the overall utility of AS-ODN as thera‐ peutic agent is dependent upon 1) the expression level of the target mRNA, 2) the optimal design of As-ODN, 3) the specificity of the AS-ODN to the target mRNA and 4) the availability of safe and highly efficient delivery systems. For example, we have shown that most of cationic polymers (CPs) and lipids used as GDSs to shuttle AS-ODNs specific to the epidermal growth factor receptor (EGFR) can also induce intrinsic cytotoxicity and toxicogenomics [16-22]. Previous studies have demonstrated that the viral vectors (e.g., adenovirus, adeno-associated virus (AAV), Epstein-Barr virus (EBV), herpes simplex virus type-1 (HSV-1), retrovirus, lentivirus, poxvirus, baculovirus) vectors can function as efficient vehicles for AS-ODN delivery. Of these, AAV vectors can be constructed to express short, distinct transcripts, a property that is useful for RNA - mediated inhibition of gene expression and successful delivery of the As-ODNs [23, 24]. Fig. 1 and Table 1 respectively represent the mechanism of

**Figure 1.** Mechanism of action of AS-ODN. A) Inhibition of proteins by small molecule drugs after translation. B) Sup‐ pression of mRNA by AS-ODN before translation in the presence of RNase H. This figure was adapted with permission

It is known that the oncogene E7 from high-risk human papillomavirus (HPV) strains has the potential to immortalize epithelial cells and increase cellular transformation in culture. Hence, to prevent the cervical cancer growth, the HPV16 E7 was inhibited by AS-ODN that was The effectiveness of the AS-ODNs have so far been shown in both target cells/tissue as in vitro models and animal in vivo models in particular for cancer therapy [13]. The mechanisms of action of the AS-ODNs seem to vary in different systems. In the case of hybridization and intramolecular and/or intermolecular interactions, their degradation pattern appears to be different. Many investigations have shown the anti-oncogenic impacts of AS-ODNs through targeting specific oncogenes. We have used AS-ODNs specific to EGFR and showed substantial inhibition of EGFR in A431 cells [25] as well as A549 lung cancer cells [26] using non-viral vectors as delivery system. The inhibitory impacts of AS-ODNs have been assessed through alterations in growth rate, morphology and molecular analysis. Various oncogenes have been targeted by AS-ODNs.

#### **3.2. Small interfering RNA**

The siRNA (also called as short interfering RNA or silencing RNA) is double-stranded RNA (dsRNA) molecules of 20-25 nucleotides. The siRNA gene-silencing mechanism is induced by dsRNA and it is largely sequence-specific. RNA interference (RNAi) approach appears to be an extremely powerful tool for silencing gene expression in vitro [38]. Accordingly, huge researchers have been conducted to expand this technology toward in vivo applications [39]. Fig. 2 represents mechanism of siRNA in controlling the expression of a target mRNA.

Some chemotherapy agents are substrate of the efflux transporters (e.g., P-glycoprotein (P-gp), multidrug resistance proteins (MRPs)) that are often overexpressed on cancer cells developing resistance, while no safe inhibitor of P-gp is available. Simultaneous delivery of P-gp targeted siRNA and paclitaxel as poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) decorated with biotin has been shown to overcome tumor drug resistance in both in vitro and in vivo [42].

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267

After being discovered in early 1980s, ribozymes as a class of RNA showing catalytic activity to cleave RNA molecules in a sequence specific manner have been used for cancer therapy. They have been shown to perform excellent catalytic reactions with great precision, which can be encoded and transcribed from DNA. It was a decade later that DNAzymes have entered the scene of nucleic acid-mediated catalysis [45]. They are special class of nucleic acid chains, which usually consist of both double and single stranded regions that fold into a specific threedimensional structure performing catalytic functions. Various ribozyme formats (e.g., hammerhead, hairpin, axhead, group I intron, and RNAse P) can be used as trans-acting catalysts. Of these, the hammerhead and hairpin ribozymes seem to be the most commonly used. For example, the efficacy of an anti-KRAS hammerhead ribozyme targeting GUUmutated codon 12 of the KRAS gene was evaluated in a cell-free system and also in cultured pancreatic carcinoma cells [46]. Fig. 3 schematically exemplifies a morphology and cleavage

**Figure 3.** Schematic representation of morphology and cleavage mechanism of Ribozyme (A) and DNAzyme (B). This

Tsuchida *et al.* showed that, in the cell-free system, the anti-KRAS ribozyme specifically cleaved KRAS RNA with GUU-mutation at codon 12. In the cell culture system, they showed that the anti-KRAS ribozyme significantly reduced KRAS mRNA level (GUU-mutated codon 12) in Capan-1 pancreatic carcinoma cells. Further, it has been proposed that trans-splicing ribozyme capable of specifically reprograming the human telomerase reverse transcriptase (hTERT) RNA can be harnessed as a useful tool for tumor-targeted gene therapy. Thus, a transcriptional targeting with the RNA replacement approach was implemented to target liver cancer cells

**3.3. Ribozymes and DNAzyme**

mechanisms of Ribozyme (A) and DNAzyme (B).

figure was adapted with permission from reference [4].

**Figure 2.** Cleavage and degradation of mRNA expression by siRNA. Short interfering RNAs (siRNAs) basically consist of two 21-25 single-stranded RNAs forming double stand RNA with overhangs at 3′ end. The antisense strand of the siR‐ NA bound to RNA-induced silencing complex (RISC) can cleave the target mRNA. This figure was adapted with permis‐ sion from reference [4].

Basically, investigation on RNAi has highlighted two distinct methodologies for gene silencing as: 1) cytoplasmic delivery of siRNA to target cells to imitate an endogenous RNAi mechanism and 2) nuclear delivery of gene expression cassettes expressing a short hairpin RNA (shRNA) that mimic the micro interfering RNA (miRNA) active intermediate of a different endogenous RNAi mechanism [40]. Both these approaches need safe delivery of gene materials into the target sites.

In fact, RNAi that was discovered initially in plants has been applied for various types of cancer as well as other diseases. Besides, RNAi technology seems to be the right tool for delineation of the functions and interactions of the thousands of human genes in high-throughput systems, which can also be harnessed in target validation technology. It is deemed that delivery of siRNAs as nanoformulations may resolve the inefficient delivery problem [41-43]. For example, a micelleplex system based on an amphiphilic and cationic triblock copolymer has been developed for delivery of siRNA specific to the acid ceramidase (AC) gene. In aqueous solution, the triblock copolymer (consisting of monomethoxy poly(ethylene glycol), poly(ep‐ silon-caprolactone) and poly(2-aminoethyl ethylene phosphate)) can self assembles into positively charged (48 mV) micellar nanoparticles (MNPs) with an average diameter of 60 nm. Once exposed to siRNA, it can result in micelleplex that was shown to effectively internalize into the BT474 breast cancer cells and induce significant gene knockdown. Systemic delivery of micelleplex targeting AC gene was shown to significantly inhibit the tumor growth in a BT474 xenograft murine model without activation of the innate immune response [44].

Some chemotherapy agents are substrate of the efflux transporters (e.g., P-glycoprotein (P-gp), multidrug resistance proteins (MRPs)) that are often overexpressed on cancer cells developing resistance, while no safe inhibitor of P-gp is available. Simultaneous delivery of P-gp targeted siRNA and paclitaxel as poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) decorated with biotin has been shown to overcome tumor drug resistance in both in vitro and in vivo [42].

#### **3.3. Ribozymes and DNAzyme**

dsRNA and it is largely sequence-specific. RNA interference (RNAi) approach appears to be an extremely powerful tool for silencing gene expression in vitro [38]. Accordingly, huge researchers have been conducted to expand this technology toward in vivo applications [39]. Fig. 2 represents mechanism of siRNA in controlling the expression of a target mRNA.

**Figure 2.** Cleavage and degradation of mRNA expression by siRNA. Short interfering RNAs (siRNAs) basically consist of two 21-25 single-stranded RNAs forming double stand RNA with overhangs at 3′ end. The antisense strand of the siR‐ NA bound to RNA-induced silencing complex (RISC) can cleave the target mRNA. This figure was adapted with permis‐

Basically, investigation on RNAi has highlighted two distinct methodologies for gene silencing as: 1) cytoplasmic delivery of siRNA to target cells to imitate an endogenous RNAi mechanism and 2) nuclear delivery of gene expression cassettes expressing a short hairpin RNA (shRNA) that mimic the micro interfering RNA (miRNA) active intermediate of a different endogenous RNAi mechanism [40]. Both these approaches need safe delivery of gene materials into the

In fact, RNAi that was discovered initially in plants has been applied for various types of cancer as well as other diseases. Besides, RNAi technology seems to be the right tool for delineation of the functions and interactions of the thousands of human genes in high-throughput systems, which can also be harnessed in target validation technology. It is deemed that delivery of siRNAs as nanoformulations may resolve the inefficient delivery problem [41-43]. For example, a micelleplex system based on an amphiphilic and cationic triblock copolymer has been developed for delivery of siRNA specific to the acid ceramidase (AC) gene. In aqueous solution, the triblock copolymer (consisting of monomethoxy poly(ethylene glycol), poly(ep‐ silon-caprolactone) and poly(2-aminoethyl ethylene phosphate)) can self assembles into positively charged (48 mV) micellar nanoparticles (MNPs) with an average diameter of 60 nm. Once exposed to siRNA, it can result in micelleplex that was shown to effectively internalize into the BT474 breast cancer cells and induce significant gene knockdown. Systemic delivery of micelleplex targeting AC gene was shown to significantly inhibit the tumor growth in a BT474 xenograft murine model without activation of the innate immune response [44].

sion from reference [4].

266 Novel Gene Therapy Approaches

target sites.

After being discovered in early 1980s, ribozymes as a class of RNA showing catalytic activity to cleave RNA molecules in a sequence specific manner have been used for cancer therapy. They have been shown to perform excellent catalytic reactions with great precision, which can be encoded and transcribed from DNA. It was a decade later that DNAzymes have entered the scene of nucleic acid-mediated catalysis [45]. They are special class of nucleic acid chains, which usually consist of both double and single stranded regions that fold into a specific threedimensional structure performing catalytic functions. Various ribozyme formats (e.g., hammerhead, hairpin, axhead, group I intron, and RNAse P) can be used as trans-acting catalysts. Of these, the hammerhead and hairpin ribozymes seem to be the most commonly used. For example, the efficacy of an anti-KRAS hammerhead ribozyme targeting GUUmutated codon 12 of the KRAS gene was evaluated in a cell-free system and also in cultured pancreatic carcinoma cells [46]. Fig. 3 schematically exemplifies a morphology and cleavage mechanisms of Ribozyme (A) and DNAzyme (B).

**Figure 3.** Schematic representation of morphology and cleavage mechanism of Ribozyme (A) and DNAzyme (B). This figure was adapted with permission from reference [4].

Tsuchida *et al.* showed that, in the cell-free system, the anti-KRAS ribozyme specifically cleaved KRAS RNA with GUU-mutation at codon 12. In the cell culture system, they showed that the anti-KRAS ribozyme significantly reduced KRAS mRNA level (GUU-mutated codon 12) in Capan-1 pancreatic carcinoma cells. Further, it has been proposed that trans-splicing ribozyme capable of specifically reprograming the human telomerase reverse transcriptase (hTERT) RNA can be harnessed as a useful tool for tumor-targeted gene therapy. Thus, a transcriptional targeting with the RNA replacement approach was implemented to target liver cancer cells through combining a liver-selective promoter with an hTERT-mediated cancer-specific ribozyme [47]. To this end, Song *et al.* validated it in vivo by constructing an adenovirus encoding the hTERT-targeting trans-splicing ribozyme under the control of a liver-selective phosphoenolpyruvate carboxy kinase promoter. They found that intratumoral injection of this virus produced selective and efficient regression of tumors in mice [47].

Malignant brain tumors (high-grade glioma), pancreatic cancer and malignant melanoma are among the most aggressive tumors known. Despites these facts, necessary translational steps are needed to be fulfilled for their clinical applications. For example, Antisense Pharma has recently taken an AS-ODN medication (Trabedersen or AP 12009) in several clinical trials. Trabedersen is a DNA-oligonucleotide that inhibits the synthesis of the cytokine transforming growth factor beta 2 (TGF-ß2) through specific binding to mRNA of TGF-ß2 that is overex‐ pressed in many highly aggressive tumors suppressing the immune system activity [51-53].

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269

Cancerous cells of different types of tumors often display expression of aberrant genes such as: 1) mutated genes (e.g., mutated P53, RAS, BCR-ABL), 2) unique genes resultant from viral oncogenes (e.g., HPV E6 or E7), 3) overexpressed cancer specific genes (e.g., Her2, TGF-ß2,

These aberrant genes could be recognized by the host immune system, resulting in elimination of the cancerous cells expressing such oncogenes. However, cancer cells can circumvent from the anticancer activity of immune system within the permissive tumor microenvironment. Accordingly, the basis of the tumor antigen-specific vaccines is boosting the immune system harnessing these aberrant antigens. Nevertheless, success of this approach depends on identification and appropriate use of tumor specific genes [54-56]. In fact, vaccination against tumors may provide a selective destruction of malignant cells by the host's immune system, which can be applied as integrated system containing target gene(s) in recombinant vectors. Of viral vectors use in cancer vaccination, the recombinant AAV vectors appear to grant better clinical responses because of their low intrinsic immunogenicity, hence they have been employed to generate immune responses against specific antigens. For example, the safety of cytotoxic T lymphocytes (CTLs) infusion by transfected dendritic cells (DCs) with rAAV carrying carcinoembryonic antigen (CEA) cDNA was investigated in advanced cancer patients. For example, a total of 27 cancer patients with tumor tissue and/or sera-elevated level of CEA were treated with the rAAV-DC immunovaccine, which was well-tolerated showing

As the most potent antigen-presenting cells, DCs originate from the bone marrow and play a key role in the generation of immune responses. Further, peptide-based vaccination in cancer patients using DCs have resulted in promising outcomes. For example, to control the relapse and succumb to progressive disease in patients with advanced ovarian cancer, an immuno‐ therapy approach was applied using CDs loaded with Her2/neu, hTERT, and PADRE peptides in a randomized open-label phase I/II trial. Despite showing modest immune responses, these peptide-loaded DC vaccination showed promising survival rate [58]. Fig. 4 represents the radar

It appears that we need to develop much more rational consolidative strategies for treatment of solidtumorinadvancedstages since the appliedstrategies exploitingDCs (e.g.,peptidepulsing with tumor antigens, transfection with DNA/RNA and transduction with tumor antigens encoding viral vectors) have not substantially generated antitumor immune responses.

**4.1. Tumor antigen–specific vaccines and DNA vaccines**

carcinoembryonic antigen, mucin).

no severe side effects in patients [57].

pattern of DNA vaccines in each phase of clinical trials.

It should be emphasized that the catalytic ribozyme core is basically attached to the specific regions of the target transcript through flanking antisense sequences. They have been designed to effectively cleave the targets transcript resulting in suppressed gene expression. For inhibition of gene expression, it is deemed that ribozymes are more effective than AS-ODNs because they cleave the target transcripts catalytically.

The DNAzyme (the so called deoxyribozyme) molecules consist of the 10-23 nucleotides, which bind to mRNA in a highly sequence-specific manner and cleave the RNA independent from RNase with the relatively stable chemistries used in oligodeoxynucleotide-based antisense reagents. The major obstacle in the further development of these technologies is a phenomenon that requires substantial development efforts invested in drugs of various classes, the uphill battle to affect cellular delivery in a targeted manner. This challenge is being met with a multidisciplinary approach with the hope that a greater understanding of each step of this process will enhance DNAzyme pharmacodynamics [45].

Owing to DNA backbone, DNAzymes have the advantage of being highly stable and costeffective in compassion with RNAzymes and proteins. DNAzymes, similar to aptamers, can be isolated through a combinatorial in vitro selection process. Hence, they can be literally manipulated to meet the requirements and applied for engineering of targeted genoceuti‐ cals. Such characteristics make them excellent choice for dynamic control of nanomaterials assembly [48].

## **4. Target antigens and oncogenes**

Tumor epithelial and endothelial cells as well as tumor associated cells represent unique marker molecules that can be harnessed for targeted therapy of cancer. For example, tumor vasculature varies significantly from its normal counterpart, representing unique cancer marker molecules. This has been emphasized through recent technologies including: immu‐ nohistochemistry laser-capture microdissection (immuno-LCM), genome-wide high-through‐ put screening, and proteomics. It is deemed that the vast array of vascular bed-specific markers may provide an exceptional platform for discovery of new therapeutics that target tumor microvasculature in various malignancies [49]. It is the same for tumor epithelial cells and tumor associated cells (TACs). Regarding the epithelial cells, EGFRs are the most studied cancer marker molecules (CMMs), whose upregulation in cancer cells was shown to be substantially down regulated with gene based medicines such as siRNA and AS-ODN. Likewise, vascular EGF and EGF-receptors have been shown to be upregulated in tumor endothelial cells and they can also be suppressed by genomedicines [50].

Malignant brain tumors (high-grade glioma), pancreatic cancer and malignant melanoma are among the most aggressive tumors known. Despites these facts, necessary translational steps are needed to be fulfilled for their clinical applications. For example, Antisense Pharma has recently taken an AS-ODN medication (Trabedersen or AP 12009) in several clinical trials. Trabedersen is a DNA-oligonucleotide that inhibits the synthesis of the cytokine transforming growth factor beta 2 (TGF-ß2) through specific binding to mRNA of TGF-ß2 that is overex‐ pressed in many highly aggressive tumors suppressing the immune system activity [51-53].

#### **4.1. Tumor antigen–specific vaccines and DNA vaccines**

through combining a liver-selective promoter with an hTERT-mediated cancer-specific ribozyme [47]. To this end, Song *et al.* validated it in vivo by constructing an adenovirus encoding the hTERT-targeting trans-splicing ribozyme under the control of a liver-selective phosphoenolpyruvate carboxy kinase promoter. They found that intratumoral injection of this

It should be emphasized that the catalytic ribozyme core is basically attached to the specific regions of the target transcript through flanking antisense sequences. They have been designed to effectively cleave the targets transcript resulting in suppressed gene expression. For inhibition of gene expression, it is deemed that ribozymes are more effective than AS-ODNs

The DNAzyme (the so called deoxyribozyme) molecules consist of the 10-23 nucleotides, which bind to mRNA in a highly sequence-specific manner and cleave the RNA independent from RNase with the relatively stable chemistries used in oligodeoxynucleotide-based antisense reagents. The major obstacle in the further development of these technologies is a phenomenon that requires substantial development efforts invested in drugs of various classes, the uphill battle to affect cellular delivery in a targeted manner. This challenge is being met with a multidisciplinary approach with the hope that a greater understanding of each step

Owing to DNA backbone, DNAzymes have the advantage of being highly stable and costeffective in compassion with RNAzymes and proteins. DNAzymes, similar to aptamers, can be isolated through a combinatorial in vitro selection process. Hence, they can be literally manipulated to meet the requirements and applied for engineering of targeted genoceuti‐ cals. Such characteristics make them excellent choice for dynamic control of nanomaterials

Tumor epithelial and endothelial cells as well as tumor associated cells represent unique marker molecules that can be harnessed for targeted therapy of cancer. For example, tumor vasculature varies significantly from its normal counterpart, representing unique cancer marker molecules. This has been emphasized through recent technologies including: immu‐ nohistochemistry laser-capture microdissection (immuno-LCM), genome-wide high-through‐ put screening, and proteomics. It is deemed that the vast array of vascular bed-specific markers may provide an exceptional platform for discovery of new therapeutics that target tumor microvasculature in various malignancies [49]. It is the same for tumor epithelial cells and tumor associated cells (TACs). Regarding the epithelial cells, EGFRs are the most studied cancer marker molecules (CMMs), whose upregulation in cancer cells was shown to be substantially down regulated with gene based medicines such as siRNA and AS-ODN. Likewise, vascular EGF and EGF-receptors have been shown to be upregulated in tumor

endothelial cells and they can also be suppressed by genomedicines [50].

virus produced selective and efficient regression of tumors in mice [47].

because they cleave the target transcripts catalytically.

of this process will enhance DNAzyme pharmacodynamics [45].

assembly [48].

268 Novel Gene Therapy Approaches

**4. Target antigens and oncogenes**

Cancerous cells of different types of tumors often display expression of aberrant genes such as: 1) mutated genes (e.g., mutated P53, RAS, BCR-ABL), 2) unique genes resultant from viral oncogenes (e.g., HPV E6 or E7), 3) overexpressed cancer specific genes (e.g., Her2, TGF-ß2, carcinoembryonic antigen, mucin).

These aberrant genes could be recognized by the host immune system, resulting in elimination of the cancerous cells expressing such oncogenes. However, cancer cells can circumvent from the anticancer activity of immune system within the permissive tumor microenvironment. Accordingly, the basis of the tumor antigen-specific vaccines is boosting the immune system harnessing these aberrant antigens. Nevertheless, success of this approach depends on identification and appropriate use of tumor specific genes [54-56]. In fact, vaccination against tumors may provide a selective destruction of malignant cells by the host's immune system, which can be applied as integrated system containing target gene(s) in recombinant vectors. Of viral vectors use in cancer vaccination, the recombinant AAV vectors appear to grant better clinical responses because of their low intrinsic immunogenicity, hence they have been employed to generate immune responses against specific antigens. For example, the safety of cytotoxic T lymphocytes (CTLs) infusion by transfected dendritic cells (DCs) with rAAV carrying carcinoembryonic antigen (CEA) cDNA was investigated in advanced cancer patients. For example, a total of 27 cancer patients with tumor tissue and/or sera-elevated level of CEA were treated with the rAAV-DC immunovaccine, which was well-tolerated showing no severe side effects in patients [57].

As the most potent antigen-presenting cells, DCs originate from the bone marrow and play a key role in the generation of immune responses. Further, peptide-based vaccination in cancer patients using DCs have resulted in promising outcomes. For example, to control the relapse and succumb to progressive disease in patients with advanced ovarian cancer, an immuno‐ therapy approach was applied using CDs loaded with Her2/neu, hTERT, and PADRE peptides in a randomized open-label phase I/II trial. Despite showing modest immune responses, these peptide-loaded DC vaccination showed promising survival rate [58]. Fig. 4 represents the radar pattern of DNA vaccines in each phase of clinical trials.

It appears that we need to develop much more rational consolidative strategies for treatment of solidtumorinadvancedstages since the appliedstrategies exploitingDCs (e.g.,peptidepulsing with tumor antigens, transfection with DNA/RNA and transduction with tumor antigens encoding viral vectors) have not substantially generated antitumor immune responses.

PA2024 consisting of the antigen prostatic acid phosphatase (PAP) and an immune signaling factor granulocyte-macrophage colony stimulating factor (GM-CSF) that helps the APCs to

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271

It should be highlighted that the initiation of cancer is an intricate multi-cause process involving sequential activation of oncogenes and inactivation of tumor suppressor genes (TSGs) and apoptosis inducing genes (AIGs). Such genetic changes, subsequently, yield concomitant phenotypic alterations in the tumor cells resulting in cancer cells survival and progression. Thus, in addition to oncogenes, the tumor suppressor genes must be targeted by

The pivotal roles of the TSGs and AIGs should be considered for cancer gene therapy, while little devotion has given to their biological impacts. The defects/mutated forms of these genes should be corrected through transfecting the normal forms which can be fulfilled through targeted systems; for more details on TSCs and AIGs, reader is directed to see reference [4].

Having harnessed suicide genes, a prodrug can be converted to a toxic metabolite. In fact, suicide gene therapy (SGT) is a unique approach that allows selective targeting through

Using a designated prodrug, which can be activated only in aberrant cells producing the metabolizing enzyme, cancer cells can be specifically targeted by a nontoxic prodrug that metabolized into toxic metabolites. The herpes simplex virus thymidine kinase (HSV-TK) gene is the prototype gene, which can be transferred into tumor cells either by viral vectors or

Suicide gene therapy using gene-directed enzyme/prodrug therapy (GEPT) was shown to improve the therapeutic efficacy of conventional cancer radiotherapy and chemotherapy without side-effects. Of the SGTs, the HSV- TK system gene therapy can sensitizes cells to the cytotoxic effects of designated drugs such as ganciclovir (GCV) and acyclovir (ACV). The HSV-TK-based SGT approach has resulted in promising outcomes in phase I/II study of glioblas‐ toma, showing that brain injections of M11 retroviral vector-producing cells for glioblastoma HSV-1 TK gene therapy were well tolerated and associated with significant therapeutic responses [65]. Similar clinical outcomes have been reported for the treatment of melanoma [66]. In this study, although patients showed disease progression on long-term follow-up, retrovirus vector "M11"-mediated HSV-1 TK gene therapy was well tolerated over a wide dose range. Despite limited tumor response possibly due to poor gene transfer efficiency, necrosis following GCV administration in transduced tumors may indicate a potential for treatment efficacy. The HSV-TK based SGT has been reported as an effective system for treating exper‐

In an interesting study, Aoi et al. capitalized on a physical method using ultrasound (US) and nano/microbubbles (NBs/MBs) to deliver exogenous genomedicines noninvasively into the

mature, and 3) infusion of the activated blood product [62].

**4.3. Suicide gene therapy: A targeted genomedicine modality**

**4.2. Tumor suppressor and apoptosis–inducing genes**

a designated genomedicine [63].

negative selection of malignant cells.

imental human pancreatic cancer [67].

nonviral methods [64].

**Figure 4.** Selected DNA vaccines in clinical trials. Only 4 trials have reached to the phase IV trial, in which 3 of them are targeting human papillomavirus (HPV) and 1 targeting HPV and hepatitis B [59].

Ideally, for effective vaccination of any type of malignant disease, administered vaccine should activate both innate immunity and specific immune effector responses. Basically, the success of the vaccine therapy using immuno-stimulating genes depends on several parameters such as appropriateness of vector, suitable transgene design, inclusion/deletion of specific sequen‐ ces, and optimization of necessary elements to induce secretion of the transgene product from the transduced cells. It also largely relies on the safe and efficient delivery of DNA into target cells. Development of the most current clinical trials has been based upon cytotoxic agents, immunotherapy and vaccination, while the mechanistic function of the DNA vaccines is different from these medicaments. They have several advantages over conventional vaccina‐ tion modalities, including no risk of infection, antigen presentation by both MHC class I and class II molecules, polarizing T-cell helpers toward TH1/TH2 phenotypes, ease of preparation and cost-effectiveness [11, 60].

To date, over 730 DNA vaccines clinical trials have been undertaken. Of these, 156 are challenging different types of cancers [59]. A plasmid DNA encoding human tyrosinase (huTyr) has been approved by the US Department of Agriculture to treat canine melanoma [61]. The results supported the safety and efficacy of the huTyr DNA vaccine in dogs as adjunctive treatment for oral malignant melanoma. To date, no DNA vaccine has been approved by the U.S. Food and Drug Administration (FDA) for human, there exist more than 150 trials for different types of cancers. DNA-based vaccines have the advantage over con‐ ventional vaccines because they are able to induce both cell-mediated and humoral immunity, and to provide long-term responses with lower (in ng range) and fewer doses in a safer manner in comparison with conventional live vaccines. Further, they are cost-effective because of easier manufacturing process [56].

In 2010, sipuleucel-T (PROvenge®, Dendreon, USA) was approved by the FDA for treatment of asymptomatic/minimally symptomatic metastatic hormone-refractory prostate cancer (HRPC). PROvenge® is the first personalized medicine, which is a cellular immunotherapy agent and its administration demands 3 steps, as follow: 1) extraction of patient's antigenpresenting cells (APCs) through a leukapheresis procedure, 2) incubation with a fusion protein PA2024 consisting of the antigen prostatic acid phosphatase (PAP) and an immune signaling factor granulocyte-macrophage colony stimulating factor (GM-CSF) that helps the APCs to mature, and 3) infusion of the activated blood product [62].

#### **4.2. Tumor suppressor and apoptosis–inducing genes**

It should be highlighted that the initiation of cancer is an intricate multi-cause process involving sequential activation of oncogenes and inactivation of tumor suppressor genes (TSGs) and apoptosis inducing genes (AIGs). Such genetic changes, subsequently, yield concomitant phenotypic alterations in the tumor cells resulting in cancer cells survival and progression. Thus, in addition to oncogenes, the tumor suppressor genes must be targeted by a designated genomedicine [63].

The pivotal roles of the TSGs and AIGs should be considered for cancer gene therapy, while little devotion has given to their biological impacts. The defects/mutated forms of these genes should be corrected through transfecting the normal forms which can be fulfilled through targeted systems; for more details on TSCs and AIGs, reader is directed to see reference [4].

#### **4.3. Suicide gene therapy: A targeted genomedicine modality**

Ideally, for effective vaccination of any type of malignant disease, administered vaccine should activate both innate immunity and specific immune effector responses. Basically, the success of the vaccine therapy using immuno-stimulating genes depends on several parameters such as appropriateness of vector, suitable transgene design, inclusion/deletion of specific sequen‐ ces, and optimization of necessary elements to induce secretion of the transgene product from the transduced cells. It also largely relies on the safe and efficient delivery of DNA into target cells. Development of the most current clinical trials has been based upon cytotoxic agents, immunotherapy and vaccination, while the mechanistic function of the DNA vaccines is different from these medicaments. They have several advantages over conventional vaccina‐ tion modalities, including no risk of infection, antigen presentation by both MHC class I and class II molecules, polarizing T-cell helpers toward TH1/TH2 phenotypes, ease of preparation

**Phase III**

**Figure 4.** Selected DNA vaccines in clinical trials. Only 4 trials have reached to the phase IV trial, in which 3 of them are

**Phase IV**

targeting human papillomavirus (HPV) and 1 targeting HPV and hepatitis B [59].

**Phase II**

To date, over 730 DNA vaccines clinical trials have been undertaken. Of these, 156 are challenging different types of cancers [59]. A plasmid DNA encoding human tyrosinase (huTyr) has been approved by the US Department of Agriculture to treat canine melanoma [61]. The results supported the safety and efficacy of the huTyr DNA vaccine in dogs as adjunctive treatment for oral malignant melanoma. To date, no DNA vaccine has been approved by the U.S. Food and Drug Administration (FDA) for human, there exist more than 150 trials for different types of cancers. DNA-based vaccines have the advantage over con‐ ventional vaccines because they are able to induce both cell-mediated and humoral immunity, and to provide long-term responses with lower (in ng range) and fewer doses in a safer manner in comparison with conventional live vaccines. Further, they are cost-effective because of easier

In 2010, sipuleucel-T (PROvenge®, Dendreon, USA) was approved by the FDA for treatment of asymptomatic/minimally symptomatic metastatic hormone-refractory prostate cancer (HRPC). PROvenge® is the first personalized medicine, which is a cellular immunotherapy agent and its administration demands 3 steps, as follow: 1) extraction of patient's antigenpresenting cells (APCs) through a leukapheresis procedure, 2) incubation with a fusion protein

and cost-effectiveness [11, 60].

270 Novel Gene Therapy Approaches

manufacturing process [56].

Having harnessed suicide genes, a prodrug can be converted to a toxic metabolite. In fact, suicide gene therapy (SGT) is a unique approach that allows selective targeting through negative selection of malignant cells.

Using a designated prodrug, which can be activated only in aberrant cells producing the metabolizing enzyme, cancer cells can be specifically targeted by a nontoxic prodrug that metabolized into toxic metabolites. The herpes simplex virus thymidine kinase (HSV-TK) gene is the prototype gene, which can be transferred into tumor cells either by viral vectors or nonviral methods [64].

Suicide gene therapy using gene-directed enzyme/prodrug therapy (GEPT) was shown to improve the therapeutic efficacy of conventional cancer radiotherapy and chemotherapy without side-effects. Of the SGTs, the HSV- TK system gene therapy can sensitizes cells to the cytotoxic effects of designated drugs such as ganciclovir (GCV) and acyclovir (ACV). The HSV-TK-based SGT approach has resulted in promising outcomes in phase I/II study of glioblas‐ toma, showing that brain injections of M11 retroviral vector-producing cells for glioblastoma HSV-1 TK gene therapy were well tolerated and associated with significant therapeutic responses [65]. Similar clinical outcomes have been reported for the treatment of melanoma [66]. In this study, although patients showed disease progression on long-term follow-up, retrovirus vector "M11"-mediated HSV-1 TK gene therapy was well tolerated over a wide dose range. Despite limited tumor response possibly due to poor gene transfer efficiency, necrosis following GCV administration in transduced tumors may indicate a potential for treatment efficacy. The HSV-TK based SGT has been reported as an effective system for treating exper‐ imental human pancreatic cancer [67].

In an interesting study, Aoi et al. capitalized on a physical method using ultrasound (US) and nano/microbubbles (NBs/MBs) to deliver exogenous genomedicines noninvasively into the target cancer cells. They successfully harnessed a low-intensity pulsed ultrasound (1 MHz; 1.3 W/cm2 ) and NBs/MBs to transduce the HSV-TK system using an in vitro model. They showed thatadditionofGCVtothetransducedcellscanleadtoHSV-TK/GCV-dependentapoptosis [68].

Other paradigms of this approach are cytosine deaminase/5-fluorocytosine (CD/5-FC) and carboxyl esterase/irinotecan (CE/CPT-11). Further, genetically engineered stem cells (GES‐ TECs) have also been applied for GEPT [69]. While chemotherapy of brain tumors is often disrupted by the brain blood barrier (BBB) [70], GESTECs (consisting of neural stem cells (NSCs) expressing cytosine deaminase (CD) gene) have been employed as a novel cell therapy modality. The GESTECs were injected to xenograft mouse model of lung cancer metastasis to the brain produced through implanting the 549 lung cancer cells in the right hemisphere of the mouse brain. Two days after the injection of GESTECs, 5-fluorocytosine (5-FC) was administered via intraperitoneal injection. Histological analysis of extracted brain confirmed the therapeutic efficacy of GESTECs that converted the 5-FC into 5-fluorouracil resulting in the decreased density and aggressiveness of lung cancer cells [71].

Likewise, in a study, the GESTECs expressing either cytosine deaminase (CD) or carboxyl esterase (CE) showed profound inhibition of ovarian cancer cells SKOV-3 by converting prodrug 5-FC into 5-FU [72]. Table 2 represents the clinical trials for suicide gene therapy of cancer.

## **5. Immunogene therapy approaches for cancer**

Cancer immunotherapy as an effective alternative treatment modality to chemotherapy arose from the notion that the immune system play a central role in prevention of the development/ progression of tumors, which is also called as immunosurveillance [73]. Perhaps the most compelling evidence for such tumor immunosurveillance is immune system activity in paraneoplastic diseases that are neurological disorders resultant from an anti-tumor immune response [74]. For progression, invasion and metastasis, a solid tumor must develop several critical abilities, including: 1) movement and migration potential, 2) capacity for degradation of extracellular matrix (ECM), 3) survival ability inside and outside of the tumor microenvir‐ onment escaping from immune system activity, and 4) propensity and quality of generation and progression in the new environment [75]. In fact, migrating malignant cells have capabil‐ ities to escape from immune system [76], invade and initiate a new life, perhaps through its pleiotrophic abilities activating a number of unique transcription factors, transporters and enzymes. Nevertheless, various solid tumors show some extend of immune system escape capabilities within the tumor microenvironment [76], where the anti-tumor immunity induced by T cells requires several mechanisms, including: a) recognition of an antigen by T cells receptors, b) co-stimulation by appropriate accessory molecules, and c) initiation of an inflammatory signal (the so called danger signal).

Inherently, based upon innate and adaptive responses of immune system, immunotherapy modalities are performed as "passive therapy" (using antibodies (Abs)/cytokines), "adaptive therapy" (in the form of the graft vs leukemia (GVL) reaction associated with the graft vs host (GVH) reaction) or "active therapy" by stimulating the immune system [77]. Basically, autologous antigen-specific T cells can be expanded ex vivo and then re-infused into patients to boost T cells-based immune system activities. DCs, which play a central role in immune

**Clinical trial US Trial ID Malignancy Intervention Phase Status**

NCT01086735 Hematological

NCT00844623 Carcinoma,

NCT00710892 Lymphoblastic

NCT01494103 Various types of

NCT00423124 Hematological

**Table 2.** Clinical trials for suicide gene therapy of cancer. Ad5-yCD/mutTKSR39rep-ADP: Replication-competent adenovirus; Ad5: Adenovirus; yCD: Yeast cytosine deaminase; ADP: Adenovirus death protein; Td: terminated; Rg: recruiting; Cd: completed; IMRT: Intensity-modulated radiation therapy; TK99UN: An adenoviral vector containing herpes simplex virus's thymidine Kinase; GCV: Ganciclovir; AP1903: a lipid permeable, synthetic organic compound used exclusively in conjunction with a chemical inducers of dimerization (CID) therapy; HSV-TK: herpes simplex virus's

thymidine Kinase. Data were adapted with permission from reference [4].

leukemia; lymphoma

leukemia; non-Hodgkin's lymphoma

malignancies

malignancy

hepatocellular

NCT00964756 Ovarian cancer Genetic: Ad5.SSTR/

NCT00583492 Prostate cancer Biological: Ad5-yCD/

NCT00415454 Pancreatic cancer Genetic: Ad5-yCD/

mutTKSR39rep-ADP;

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http://dx.doi.org/10.5772/54739

mutTKSR39rep-ADP

Biological: donor lymphocyte infusion

TK.RGD; Drug: GCV

Allodepleted T Cells

Biological:

Biological: Allodepleted T cells transduced with caspase 9; Drug: AP1903

Genetic: TK99UN I Cd

Genetic: HSV-TK I/II Active

II/III Rg

273

I Td

I/II Rg

I Rg

I Active

I Rg

Radiation

Randomized trial of suicide gene therapy and prostate cancer

Combining suicide gene therapy with chemoradiotherapy in the treatment of non-metastatic pancreatic adenocarcinoma

Suicide gene therapy for donor lymphocytes infusion after allogeneic hematopoietic stem cell transplantation (ILD-TK01)

TK-based suicide gene therapy for hepatocellular carcinoma

An infectivity enhanced suicide gene expressing adenovirus for ovarian cancer in patients with recurrent ovarian and other selected gynecologic cancers

CASPALLO: Allo-depleted T cells transduced with inducible caspase

Administration of donor T cells with the caspase-9 suicide gene

Infusion of donor lymphocytes transduced with the suicide gene

HSV-TK in patients with haematological malignancies

9 suicide gene


target cancer cells. They successfully harnessed a low-intensity pulsed ultrasound (1 MHz; 1.3

Likewise, in a study, the GESTECs expressing either cytosine deaminase (CD) or carboxyl esterase (CE) showed profound inhibition of ovarian cancer cells SKOV-3 by converting prodrug 5-FC into 5-FU [72]. Table 2 represents the clinical trials for suicide gene therapy of

Cancer immunotherapy as an effective alternative treatment modality to chemotherapy arose from the notion that the immune system play a central role in prevention of the development/ progression of tumors, which is also called as immunosurveillance [73]. Perhaps the most compelling evidence for such tumor immunosurveillance is immune system activity in paraneoplastic diseases that are neurological disorders resultant from an anti-tumor immune response [74]. For progression, invasion and metastasis, a solid tumor must develop several critical abilities, including: 1) movement and migration potential, 2) capacity for degradation of extracellular matrix (ECM), 3) survival ability inside and outside of the tumor microenvir‐ onment escaping from immune system activity, and 4) propensity and quality of generation and progression in the new environment [75]. In fact, migrating malignant cells have capabil‐ ities to escape from immune system [76], invade and initiate a new life, perhaps through its pleiotrophic abilities activating a number of unique transcription factors, transporters and enzymes. Nevertheless, various solid tumors show some extend of immune system escape capabilities within the tumor microenvironment [76], where the anti-tumor immunity induced by T cells requires several mechanisms, including: a) recognition of an antigen by T cells receptors, b) co-stimulation by appropriate accessory molecules, and c) initiation of an

Inherently, based upon innate and adaptive responses of immune system, immunotherapy modalities are performed as "passive therapy" (using antibodies (Abs)/cytokines), "adaptive therapy" (in the form of the graft vs leukemia (GVL) reaction associated with the graft vs host

the decreased density and aggressiveness of lung cancer cells [71].

**5. Immunogene therapy approaches for cancer**

inflammatory signal (the so called danger signal).

) and NBs/MBs to transduce the HSV-TK system using an in vitro model. They showed thatadditionofGCVtothetransducedcellscanleadtoHSV-TK/GCV-dependentapoptosis [68]. Other paradigms of this approach are cytosine deaminase/5-fluorocytosine (CD/5-FC) and carboxyl esterase/irinotecan (CE/CPT-11). Further, genetically engineered stem cells (GES‐ TECs) have also been applied for GEPT [69]. While chemotherapy of brain tumors is often disrupted by the brain blood barrier (BBB) [70], GESTECs (consisting of neural stem cells (NSCs) expressing cytosine deaminase (CD) gene) have been employed as a novel cell therapy modality. The GESTECs were injected to xenograft mouse model of lung cancer metastasis to the brain produced through implanting the 549 lung cancer cells in the right hemisphere of the mouse brain. Two days after the injection of GESTECs, 5-fluorocytosine (5-FC) was administered via intraperitoneal injection. Histological analysis of extracted brain confirmed the therapeutic efficacy of GESTECs that converted the 5-FC into 5-fluorouracil resulting in

W/cm2

272 Novel Gene Therapy Approaches

cancer.

**Table 2.** Clinical trials for suicide gene therapy of cancer. Ad5-yCD/mutTKSR39rep-ADP: Replication-competent adenovirus; Ad5: Adenovirus; yCD: Yeast cytosine deaminase; ADP: Adenovirus death protein; Td: terminated; Rg: recruiting; Cd: completed; IMRT: Intensity-modulated radiation therapy; TK99UN: An adenoviral vector containing herpes simplex virus's thymidine Kinase; GCV: Ganciclovir; AP1903: a lipid permeable, synthetic organic compound used exclusively in conjunction with a chemical inducers of dimerization (CID) therapy; HSV-TK: herpes simplex virus's thymidine Kinase. Data were adapted with permission from reference [4].

(GVH) reaction) or "active therapy" by stimulating the immune system [77]. Basically, autologous antigen-specific T cells can be expanded ex vivo and then re-infused into patients to boost T cells-based immune system activities. DCs, which play a central role in immune system activities due to their ability to control both immune tolerance and immunity, have been extensively used as a cell-based immunotherapy modality [74]. While tumor cells themselves are poor antigen-presenting cells (APCs), DCs are potent APCs. Fundamentally, the aim of DCs based immunotherapy is to elicit tumor-specific effector T cells (CD4+ T cells, CD8+ T cells and B cells) that can effectively reduce the tumor mass and can also induce immunological memory to control tumor relapse [74]. The first step of DCs-based vaccination is to provide DCs with tumor-specific antigens, which can be performed through ex vivo cultivation of the patients-derived DCs with an adjuvant for DC maturation and the tumorspecific antigen. The processed DCs can be then injected back into the patient. Alternatively, DCs can be induced to take up the tumor-specific antigen in vivo [74]. This approach has been harnessed as vaccination modality in various cancers. For example, phase I/II randomized trial of DCs-based vaccination with or without cyclophosphamide have recently been conducted for consolidation therapy of advanced ovarian cancer in first or second remission [58]. It was shown that the peptide-loaded DC vaccination induced modest immune responses, while the survival rate was promising [58].

Administration of endogenous inhibitors of angiogenesis are associated with some hindrances (e.g., high dose requirements and some instability of the corresponding recombinant proteins), hence gene therapy of angiogenesis may be an effective approach to battle malignancies. It should be noted that tumors secrete a number of "angiogenesis" factors, whose encoding genes

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Selected angiogenesis factors include: vascular endothelial growth factor (VEGF), thrombo‐ spondin-1 (THBS1), endostatin, tumstatin, arresten, canstatin, vastatin, restin, angiostatin, 16 kD human prolactin fragment (16K hPRL), platelet factor-4 (PF4), interferon-inducible protein-10 (IIP10), angiopoietins, interleukin-12 (IL-12), interleukin-18 (IL-18), interferons (IFNs), endothelial-monocyte activating polypeptide-II (EMAP-II), tissue Inhibitors of metalloproteinases (TIMPs), tumor necrosis factor-α (TNF-α, transforming growth factor (TGF), pleiotropin, fibroblast growth factor (FGF), placental growth factor (PGF), and platelet derived endothelial cell growth factor (PD-ECGF) [79]. Of these, VEGF is the most studied target. It carries out multifaceted functions in tumor development, in which several isoforms impose distinct biologic functions and clinical implications. Several strategies have been carried out to control VEGF with some successes. Coukos' group has successfully used DNA vector-based RNA interference (RNAi) by inserting RNAi sequences targeting murine VEGF isoforms in downstream of an RNA polymerase III (Pol III) promoter, which may have potential applications in isoform-specific "knock-down" of VEGF. They compared two Pol III promoters, U6 and H1, in their efficiency forsiRNA expression. Large molecular weight VEGF isoforms were specifically reduced in vitro in the presence of isoform-specific RNAi constructs. Additionally, H1 promoter may be superior to U6 promoter when used for vector-based RNAi of VEGF isoforms. They proposed this novel strategy as an effective tool to investigate the functionalities of various VEGF isoforms and also concluded that, to develop such novel RNAi strategy as a practical research tool and feasible cancer therapy approach, identifying the most efficient targeting sequence and developing an efficient delivery system are vital steps [80]. The effect of INF-β gene therapy on the growth of human prostate cancer was determined in nude mice bearing PC3MM2 cells. It was found that the intralesional delivery of adenoviaral vector encoding murine IFN-β was able to suppress the growth of tumor in a dose-dependent manner, perhaps through induction of INF-β and inducible nitric oxide synthase (iNOS) as well as reduction of basic FGF and TGF-β1 resulting in inhibition of angiogenesis [81].

In a study, rAAV vectors were constructed to express endostatin (rAAV-endostatin) or the antiangiogenic domain of thrombospondin-1 3TSR (rAAV-3TSR) and applied to a mouse angiogenesis model. The rAAV-mediated gene delivery resulted in inhibition of VEGFinduced angiogenesis, in which pretreatment of mice with i.m. or intrasplenic injection of

The rAAV vectors carrying IL-12 and angiostatin-like molecule (K1-3) were administered to a subcutaneous hepatoma model in mice (Hepa129 cells in C3H mice). It was found that injection of rAAV-K1-3 or rAAV-IL-12 into tumor nodules resulted in a significant dose-dependent reduction in tumor growth, while the survival rate was significantly improved in the IL-12 treated mice, but not in the K1-3 treated mice. Combined therapy of these genomedicines, however, did not further improve antitumor efficacy compared with the monotherapy [83].

rAAV-endostatin or rAAV-3TSR significantly inhibited tumor growth [82].

Table 3 represents selected examples for angiogenic gene therapy trials.

can be targeted.

In a study, Coukos' group has investigated the mechanism underlying cooperation between oncolytic HSV and host effector immune mechanisms in a syngeneic murine model of ovarian carcinoma. They showed that therapeutic administration of HSV-1716 (a replication-restricted mutant) can result in significant reduction of tumor growth and improved survival rate. Intratumoral injection of HSV-1716 elicited expression of some key elements (IFN-γ, MIG, and IP-10) and significant increase in the number of tumor-associated natural killer (NK) and CD8+ T cells. Ascites from HSV-1716-treated animals efficiently induced in vitro migration of NK and CD8+ T cells that was dependent upon the presence of MIG and IP-10, in which monocytes and DCs appeared to be responsible for the production of MIG and IP-10 [78]. This study clearly indicate that, in ovarian carcinoma, monocyte-derived DCs produced large amounts of IFNgamma and upregulated MIG and IP-10 expression upon HSV-1716 infection, which may favor antitumor immune response upon oncolytic therapy.

Thus far, CDs-based vaccination has been harnessed in over 150 clinical trials [59]. Neverthe‐ less, the overall results obtained from the human clinical trials capitalizing on DCs have shown promising clinical outcomes resulting in significant induction of clinically meaningful antitumor immunity even with no apparent side effects or toxicities. This modality is a perfect paradigm for personalized medicines.

## **6. Anti–angiogenesis gene therapy**

Several critical steps are involved during angiogenesis, including: proliferation of the endo‐ thelial cells (ECs), migration of the ECs, degradation of the basement membrane, and formation of the new lumen organization. Such biological event is controlled by proangiogenesis and antiangiogenesis factors liberated by various cells (activated ECs, monocytes, smooth muscle cells, pericytes and platelets) into the blood circulation [79]. Tumors need angiogenesis for survival and growth, thus inhibition of angiogenesis can be an effective strategy for cancer therapy.

Administration of endogenous inhibitors of angiogenesis are associated with some hindrances (e.g., high dose requirements and some instability of the corresponding recombinant proteins), hence gene therapy of angiogenesis may be an effective approach to battle malignancies. It should be noted that tumors secrete a number of "angiogenesis" factors, whose encoding genes can be targeted.

system activities due to their ability to control both immune tolerance and immunity, have been extensively used as a cell-based immunotherapy modality [74]. While tumor cells themselves are poor antigen-presenting cells (APCs), DCs are potent APCs. Fundamentally,

In a study, Coukos' group has investigated the mechanism underlying cooperation between oncolytic HSV and host effector immune mechanisms in a syngeneic murine model of ovarian carcinoma. They showed that therapeutic administration of HSV-1716 (a replication-restricted mutant) can result in significant reduction of tumor growth and improved survival rate. Intratumoral injection of HSV-1716 elicited expression of some key elements (IFN-γ, MIG, and IP-10) and significant increase in the number of tumor-associated natural killer (NK) and CD8+ T cells. Ascites from HSV-1716-treated animals efficiently induced in vitro migration of NK and CD8+ T cells that was dependent upon the presence of MIG and IP-10, in which monocytes and DCs appeared to be responsible for the production of MIG and IP-10 [78]. This study clearly indicate that, in ovarian carcinoma, monocyte-derived DCs produced large amounts of IFNgamma and upregulated MIG and IP-10 expression upon HSV-1716 infection, which may favor

Thus far, CDs-based vaccination has been harnessed in over 150 clinical trials [59]. Neverthe‐ less, the overall results obtained from the human clinical trials capitalizing on DCs have shown promising clinical outcomes resulting in significant induction of clinically meaningful antitumor immunity even with no apparent side effects or toxicities. This modality is a perfect

Several critical steps are involved during angiogenesis, including: proliferation of the endo‐ thelial cells (ECs), migration of the ECs, degradation of the basement membrane, and formation of the new lumen organization. Such biological event is controlled by proangiogenesis and antiangiogenesis factors liberated by various cells (activated ECs, monocytes, smooth muscle cells, pericytes and platelets) into the blood circulation [79]. Tumors need angiogenesis for survival and growth, thus inhibition of angiogenesis can be an effective strategy for cancer

 T cells and B cells) that can effectively reduce the tumor mass and can also induce immunological memory to control tumor relapse [74]. The first step of DCs-based vaccination is to provide DCs with tumor-specific antigens, which can be performed through ex vivo cultivation of the patients-derived DCs with an adjuvant for DC maturation and the tumorspecific antigen. The processed DCs can be then injected back into the patient. Alternatively, DCs can be induced to take up the tumor-specific antigen in vivo [74]. This approach has been harnessed as vaccination modality in various cancers. For example, phase I/II randomized trial of DCs-based vaccination with or without cyclophosphamide have recently been conducted for consolidation therapy of advanced ovarian cancer in first or second remission [58]. It was shown that the peptide-loaded DC vaccination induced modest immune responses, while the

T cells,

the aim of DCs based immunotherapy is to elicit tumor-specific effector T cells (CD4+

CD8+

274 Novel Gene Therapy Approaches

survival rate was promising [58].

antitumor immune response upon oncolytic therapy.

paradigm for personalized medicines.

therapy.

**6. Anti–angiogenesis gene therapy**

Selected angiogenesis factors include: vascular endothelial growth factor (VEGF), thrombo‐ spondin-1 (THBS1), endostatin, tumstatin, arresten, canstatin, vastatin, restin, angiostatin, 16 kD human prolactin fragment (16K hPRL), platelet factor-4 (PF4), interferon-inducible protein-10 (IIP10), angiopoietins, interleukin-12 (IL-12), interleukin-18 (IL-18), interferons (IFNs), endothelial-monocyte activating polypeptide-II (EMAP-II), tissue Inhibitors of metalloproteinases (TIMPs), tumor necrosis factor-α (TNF-α, transforming growth factor (TGF), pleiotropin, fibroblast growth factor (FGF), placental growth factor (PGF), and platelet derived endothelial cell growth factor (PD-ECGF) [79]. Of these, VEGF is the most studied target. It carries out multifaceted functions in tumor development, in which several isoforms impose distinct biologic functions and clinical implications. Several strategies have been carried out to control VEGF with some successes. Coukos' group has successfully used DNA vector-based RNA interference (RNAi) by inserting RNAi sequences targeting murine VEGF isoforms in downstream of an RNA polymerase III (Pol III) promoter, which may have potential applications in isoform-specific "knock-down" of VEGF. They compared two Pol III promoters, U6 and H1, in their efficiency forsiRNA expression. Large molecular weight VEGF isoforms were specifically reduced in vitro in the presence of isoform-specific RNAi constructs. Additionally, H1 promoter may be superior to U6 promoter when used for vector-based RNAi of VEGF isoforms. They proposed this novel strategy as an effective tool to investigate the functionalities of various VEGF isoforms and also concluded that, to develop such novel RNAi strategy as a practical research tool and feasible cancer therapy approach, identifying the most efficient targeting sequence and developing an efficient delivery system are vital steps [80].

The effect of INF-β gene therapy on the growth of human prostate cancer was determined in nude mice bearing PC3MM2 cells. It was found that the intralesional delivery of adenoviaral vector encoding murine IFN-β was able to suppress the growth of tumor in a dose-dependent manner, perhaps through induction of INF-β and inducible nitric oxide synthase (iNOS) as well as reduction of basic FGF and TGF-β1 resulting in inhibition of angiogenesis [81].

In a study, rAAV vectors were constructed to express endostatin (rAAV-endostatin) or the antiangiogenic domain of thrombospondin-1 3TSR (rAAV-3TSR) and applied to a mouse angiogenesis model. The rAAV-mediated gene delivery resulted in inhibition of VEGFinduced angiogenesis, in which pretreatment of mice with i.m. or intrasplenic injection of rAAV-endostatin or rAAV-3TSR significantly inhibited tumor growth [82].

The rAAV vectors carrying IL-12 and angiostatin-like molecule (K1-3) were administered to a subcutaneous hepatoma model in mice (Hepa129 cells in C3H mice). It was found that injection of rAAV-K1-3 or rAAV-IL-12 into tumor nodules resulted in a significant dose-dependent reduction in tumor growth, while the survival rate was significantly improved in the IL-12 treated mice, but not in the K1-3 treated mice. Combined therapy of these genomedicines, however, did not further improve antitumor efficacy compared with the monotherapy [83]. Table 3 represents selected examples for angiogenic gene therapy trials.


cationic lipids (CLs) and polymers may induce inadvertent intrinsic gene expression, masking/ stimulating some undesired gene activities [17, 19-21, 85, 86]. Fig. 5 represents schematic

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**Figure 5.** Schematic structures of advanced nanogenomedicines. A) Bioconjugations of genes with polymeric back‐ bone and grafted homing and imaging moieties. B) Polymeric and liposomal gene containing nanoformulations. This

Lipids and polymers, based upon their end groups, can be conjugated with different moieties such as imaging devices (fluorescent dyes, quantum dots) and homing devices (antibody, peptide, aptamer). Post-formulation conjugation of NPs are basically performed through chemical grafting using homobifunctional crosslinkers (*e.g.*, N-hydroxysuccinimide (NHS) esters, immidoesters, sulfhydryl-reactive crosslinkers, hydrazides) or heterobifunctional crosslinkers (*e.g.*, sulfhydryl-reactive and photoreactive crosslinkers like N-succinimidyl-3-(2-

figure was adapted with permission from reference [4].

**7.1. Bioconjugation and PEGylation**

structure of the advanced nanoformulations for genomedicines.

**Table 3.** Selected paradigms for angiogenic gene therapy trials. RTVP-1: related to testes-specific, vespid, and pathogenesis protein; Cd: completed; NA: not available; E10A: an adenovirus carrying human endostatin gene [84]; Td: terminated; Rg: recruiting. Data were adapted with permission from reference [4].

## **7. Targeted nanogenomedicines: Nanotechnology and gene therapy integration**

Integration of nanotechnology with gene therapy has resulted in production of advanced nanoscaled genomedicines that can be armed with homing devices to deliver the gene-based cargos to the target sites through both passive and active targeting mechanisms. It should be high‐ lighted that the lipids or polymers used for formulation should be positively charged to be able to condense the negatively charged nucleic acids. However, we have shown that both cationic lipids (CLs) and polymers may induce inadvertent intrinsic gene expression, masking/ stimulating some undesired gene activities [17, 19-21, 85, 86]. Fig. 5 represents schematic structure of the advanced nanoformulations for genomedicines.

**Figure 5.** Schematic structures of advanced nanogenomedicines. A) Bioconjugations of genes with polymeric back‐ bone and grafted homing and imaging moieties. B) Polymeric and liposomal gene containing nanoformulations. This figure was adapted with permission from reference [4].

#### **7.1. Bioconjugation and PEGylation**

**Clinical trial US Trial ID Malignancy Intervention Phase Status**

NCT00634595 Head and neck

NCT00262327 Advanced solid

NCT00004070 Head and neck

NCT00028652 Metastatic

NCT01604031 Chronic

**Table 3.** Selected paradigms for angiogenic gene therapy trials. RTVP-1: related to testes-specific, vespid, and pathogenesis protein; Cd: completed; NA: not available; E10A: an adenovirus carrying human endostatin gene [84];

**7. Targeted nanogenomedicines: Nanotechnology and gene therapy**

Integration of nanotechnology with gene therapy has resulted in production of advanced nanoscaled genomedicines that can be armed with homing devices to deliver the gene-based cargos to the target sites through both passive and active targeting mechanisms. It should be high‐ lighted that the lipids or polymers used for formulation should be positively charged to be able to condense the negatively charged nucleic acids. However, we have shown that both

Td: terminated; Rg: recruiting. Data were adapted with permission from reference [4].

tumor

cancer

cancers

NCT01440816 Skin cancers Biological:

lymphocytic leukemia

NCT00403221 Prostate Cancer Genetic: RTVP-1

squamous carcinoma; Nasopharyngeal carcinoma

Gene

Paclitaxel

Drug:

gene

Biological:

Biological:

Antangiogenesis; Genetic: endostatin

interleukin-12 gene

interleukin-12 gene

interleukin-12 gene; electroporationmediated plasmid DNA vaccine therapy

Biological: B-CLL Vaccine; Drug: Lenalidomide

Drug: E10A, Cisplatin,

I Cd

II NA

I NA

I/II NA

I Td

II Rg

I/II NA

Phase I - Pre-Radical Prostatectomy RTVP-1 Gene Therapy for Prostate

276 Novel Gene Therapy Approaches

Trial of E10A in Head and Neck

Safety and Efficacy of Adenoviral Endostatin in the Treatment of Advanced Solid Tumor

Gene Therapy in Treating Patients With Unresectable, Recurrent, or Refractory Head and Neck Cancer

Interleukin-12 Gene Therapy in Treating Patients With Skin

Interleukin-12 Gene and in Vivo Electroporation-Mediated Plasmid DNA Vaccine Therapy in Treating Patients With Merkel Cell Cancer

Treatment of B-CLL With Autologous IL2 and CD40 Ligand-Expressing Tumor Cells +

Lenalidomide

**integration**

Metastases

Cancer

Cancer

Lipids and polymers, based upon their end groups, can be conjugated with different moieties such as imaging devices (fluorescent dyes, quantum dots) and homing devices (antibody, peptide, aptamer). Post-formulation conjugation of NPs are basically performed through chemical grafting using homobifunctional crosslinkers (*e.g.*, N-hydroxysuccinimide (NHS) esters, immidoesters, sulfhydryl-reactive crosslinkers, hydrazides) or heterobifunctional crosslinkers (*e.g.*, sulfhydryl-reactive and photoreactive crosslinkers like N-succinimidyl-3-(2pyridyldithio)propionate (SPDP), LC-SPDP, and Sulfo-LC-SPDP ) [87]. Decoration with homing devices can arm them to target cancer cells and deliver the gene-based cargo directly to the tumor microenvironment and thereby cancer cells, but not normal cells/tissues. Anti‐ bodies can be modified via amine groups using 2-iminothiolane (Traut's reagent) and conju‐ gated to NPs. They can also be activated with, N-succinimidyl S-acetylthioacetate (SATA) or SPDP, in which the active NHS ester end of SATA or SPDP can react with amino groups in proteins and other molecules to form a stable amide linkage. Further, conjugation of the NPs with PEG (the so called PEGylation) can favor the pharmacokinetics of these NPs prolonging the circulation period that grant a proper time frame for NPs' accumulation in the tumor microenvironment. Although attaching poly ethylene glycol (PEG) (i.e., PEGylation) is the most effective method to reduce protein immunogenicity and to avoid the RES system clearance, several other polymers have successfully been implemented as alternative to PEG, including poloxamer, polyvinyl alcohol, poly(amino acid)s, and polysaccharide. However, PEG is still the most widely used polymer to engineer stealth NPs [88]. For nanoliposomes, PEG-lipid (such as PEG-DSPE) is usually inserted into liposomes to form a hydrated layer on the liposome surface.

from μm to nm, respectively. Sonication, homogenization and extrusion are the methods used for preparation of liposomes. In practice, based on end-point aims, different compositions of lipids can be used to engineer the intended liposomes. Mainly, lipophilic compounds (*e.g.*, phosphatidylcholine (PC), cholesterol (Chol), designated amounts of functionalized lipids and lipophilic drugs) are dissolved in a solvent (*e.g.*, chloroform or 3:1 ratio of chloroform: methanol). To form lipid film, the solvent is then evaporated using a rotary evaporator at 40-60 °C. The lipid film is rehydrated with stirring (~250 rpm) for 1 hr in the presence of surfactant such as Tween 20/Tween 80 under pulsed sonication (20 s ON, 10 s OFF intervals to avoid over-heating) for 10 min. As an alternative approach, the mixture can be homogenized by a high-speed homogenizer at 16,000 rpm for 5 min. To make uniform nanoliposomes, they can be extruded through polycarbonate filters with a designated pore size of (*e.g.*, 200 nm or 100 nm) for several times. The nanoliposomes can be then lyophilized for future use, reader is directed to see [95]. Mixture of polycationic lipids with plasmid DNAs can form self-assembled liposomal structures. To this end, several lipids have been exploited [96, 97], including: mixture of dioleoyl phosphatidylethanolamine (DOPE); 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA); N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,Ndimethy- lammonium trifluoroacetate (DOSPA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); dioctadecylamidoglycyl carboxyspermine (DOGS); N-(2,3-dioley‐ loxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); N-(1,2-dimyristyloxyprop-3 yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE); dioleyl-N,Ndimethylammonium chloride (DODAC); dipalmitoylphosphatidylcholine (DPPC); and 3β- (N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol). However, most of these

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CLs are able to yield relatively high transfection efficiency in vitro and in vivo, and accordingly they have been progressed toward clinical trials. Precise advantages of this approach are 1) the simplicity of the DNA/liposome formulation as lipoplex, 2) the stability of the formulation and gene components protection, and 3) the robustness and applicability of the method for delivery to different types of solid tumors. For example, using cationic lipids, 16K hPRL was formulated as cationic liposomes and administered subcutaneously to a B16F10 mouse melanoma model. The results revealed that administration of the liposomal formulation of 16K hPRL gene can effectively maintain antiangiogenic activities in mice [98]. Table 4

In another study, to monitor breast cancer processes, a unique liposomal formulation was applied as quantitative bioluminescence imaging (BLI) method. A breast cancer model was created by injection of 4T1 cells carrying a reporter system encoding a double fusion reporter gene consisting of firefly luciferase (Fluc) and green fluorescent protein (GFP) into BALB/c mice. Nanoliposomes loaded with a triple fusion gene containing HSV-TK and renilla luciferase (Rluc) and red fluorescent protein (RFP) were administered, and subsequently mice were treated with GCV. This approach resulted in monitoring of the tumor growth by BLI, while the treatment delivery of nanoliposomes was efficiently tracked by Rluc imaging [99]. Further, to avoid rapid clearance by RES after an intravenous injection, stealth PEGylated liposomes have resulted increased serum half-life and greater EPR. These stealth nanolipo‐

cationic lipids induce nonspecific gene expressions [17, 20, 85].

represents selected gene therapy trials using liposomal formulations.

#### **7.2. Bioimpacts of nanogenomedicines**

Typically, tumor microvasculature display discontinuous fenestrated morphology character‐ istics with gaps and pores between endothelial cells, in which the pore sizes are at a range of 100 nm to 1000 nm [89]. For instance, subcutaneously grown tumors were reported to have profound fenestration, showing pore sizes at a range of 200 nm to 1200 nm [90]. Most tissues present tight junctions between cells with intercellular openings smaller than 2 nm and around 6 nm in post-capillary venules, and tissues with discontinuous fenestrated endothelium such as kidney glomerulus and sinusoidal endothelium of liver have larger junctions with pore sizes of 40-60 nm and 70-150 nm, respectively [91]. As a result, NPs with size ranging 150-250 nm can substantially extravasate showing significant enhanced permeation and retention (EPR) effects within the tumor microenvironment [92]. Since long circulation of NPs in blood is a pivotal requirement for their successful *in vivo* applications, they are basically grafted with PEG that provide greater hydrophilicity and longer circulation in blood resulting in greater accumulation within the tumor microenvironment [93]. The naked gene based medicines such as AS-ODN and siRNA can be simply degraded and destroyed by the nuclease enzymes within blood, thereby not being taken up by the target cells and even giving a rise to undesired harmful immune reactions. Thus, nano-scaled protected gene medicines will provide desired canonical outcomes. Recently, it was shown that the siRNA protected by cyclodextrincontaining polymers (the basis of the RONDEL platform) can literally get to the proposed target site and impose the intended impacts [94].

#### **7.3. Liposomal NPs**

Nanoliposomes are basically formulated using solvent evaporation method to make lipid film that is then subjected to hydration. In the presence of surfactant, the hydrated lipids form multilamellar vesicles (MLVs) and unilamellar vesicles (ULVs) with a diverse size, ranging from μm to nm, respectively. Sonication, homogenization and extrusion are the methods used for preparation of liposomes. In practice, based on end-point aims, different compositions of lipids can be used to engineer the intended liposomes. Mainly, lipophilic compounds (*e.g.*, phosphatidylcholine (PC), cholesterol (Chol), designated amounts of functionalized lipids and lipophilic drugs) are dissolved in a solvent (*e.g.*, chloroform or 3:1 ratio of chloroform: methanol). To form lipid film, the solvent is then evaporated using a rotary evaporator at 40-60 °C. The lipid film is rehydrated with stirring (~250 rpm) for 1 hr in the presence of surfactant such as Tween 20/Tween 80 under pulsed sonication (20 s ON, 10 s OFF intervals to avoid over-heating) for 10 min. As an alternative approach, the mixture can be homogenized by a high-speed homogenizer at 16,000 rpm for 5 min. To make uniform nanoliposomes, they can be extruded through polycarbonate filters with a designated pore size of (*e.g.*, 200 nm or 100 nm) for several times. The nanoliposomes can be then lyophilized for future use, reader is directed to see [95]. Mixture of polycationic lipids with plasmid DNAs can form self-assembled liposomal structures. To this end, several lipids have been exploited [96, 97], including: mixture of dioleoyl phosphatidylethanolamine (DOPE); 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA); N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,Ndimethy- lammonium trifluoroacetate (DOSPA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); dioctadecylamidoglycyl carboxyspermine (DOGS); N-(2,3-dioley‐ loxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); N-(1,2-dimyristyloxyprop-3 yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE); dioleyl-N,Ndimethylammonium chloride (DODAC); dipalmitoylphosphatidylcholine (DPPC); and 3β- (N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol). However, most of these cationic lipids induce nonspecific gene expressions [17, 20, 85].

pyridyldithio)propionate (SPDP), LC-SPDP, and Sulfo-LC-SPDP ) [87]. Decoration with homing devices can arm them to target cancer cells and deliver the gene-based cargo directly to the tumor microenvironment and thereby cancer cells, but not normal cells/tissues. Anti‐ bodies can be modified via amine groups using 2-iminothiolane (Traut's reagent) and conju‐ gated to NPs. They can also be activated with, N-succinimidyl S-acetylthioacetate (SATA) or SPDP, in which the active NHS ester end of SATA or SPDP can react with amino groups in proteins and other molecules to form a stable amide linkage. Further, conjugation of the NPs with PEG (the so called PEGylation) can favor the pharmacokinetics of these NPs prolonging the circulation period that grant a proper time frame for NPs' accumulation in the tumor microenvironment. Although attaching poly ethylene glycol (PEG) (i.e., PEGylation) is the most effective method to reduce protein immunogenicity and to avoid the RES system clearance, several other polymers have successfully been implemented as alternative to PEG, including poloxamer, polyvinyl alcohol, poly(amino acid)s, and polysaccharide. However, PEG is still the most widely used polymer to engineer stealth NPs [88]. For nanoliposomes, PEG-lipid (such as PEG-DSPE) is usually inserted into liposomes to form a hydrated layer on

Typically, tumor microvasculature display discontinuous fenestrated morphology character‐ istics with gaps and pores between endothelial cells, in which the pore sizes are at a range of 100 nm to 1000 nm [89]. For instance, subcutaneously grown tumors were reported to have profound fenestration, showing pore sizes at a range of 200 nm to 1200 nm [90]. Most tissues present tight junctions between cells with intercellular openings smaller than 2 nm and around 6 nm in post-capillary venules, and tissues with discontinuous fenestrated endothelium such as kidney glomerulus and sinusoidal endothelium of liver have larger junctions with pore sizes of 40-60 nm and 70-150 nm, respectively [91]. As a result, NPs with size ranging 150-250 nm can substantially extravasate showing significant enhanced permeation and retention (EPR) effects within the tumor microenvironment [92]. Since long circulation of NPs in blood is a pivotal requirement for their successful *in vivo* applications, they are basically grafted with PEG that provide greater hydrophilicity and longer circulation in blood resulting in greater accumulation within the tumor microenvironment [93]. The naked gene based medicines such as AS-ODN and siRNA can be simply degraded and destroyed by the nuclease enzymes within blood, thereby not being taken up by the target cells and even giving a rise to undesired harmful immune reactions. Thus, nano-scaled protected gene medicines will provide desired canonical outcomes. Recently, it was shown that the siRNA protected by cyclodextrincontaining polymers (the basis of the RONDEL platform) can literally get to the proposed

Nanoliposomes are basically formulated using solvent evaporation method to make lipid film that is then subjected to hydration. In the presence of surfactant, the hydrated lipids form multilamellar vesicles (MLVs) and unilamellar vesicles (ULVs) with a diverse size, ranging

the liposome surface.

278 Novel Gene Therapy Approaches

**7.3. Liposomal NPs**

**7.2. Bioimpacts of nanogenomedicines**

target site and impose the intended impacts [94].

CLs are able to yield relatively high transfection efficiency in vitro and in vivo, and accordingly they have been progressed toward clinical trials. Precise advantages of this approach are 1) the simplicity of the DNA/liposome formulation as lipoplex, 2) the stability of the formulation and gene components protection, and 3) the robustness and applicability of the method for delivery to different types of solid tumors. For example, using cationic lipids, 16K hPRL was formulated as cationic liposomes and administered subcutaneously to a B16F10 mouse melanoma model. The results revealed that administration of the liposomal formulation of 16K hPRL gene can effectively maintain antiangiogenic activities in mice [98]. Table 4 represents selected gene therapy trials using liposomal formulations.

In another study, to monitor breast cancer processes, a unique liposomal formulation was applied as quantitative bioluminescence imaging (BLI) method. A breast cancer model was created by injection of 4T1 cells carrying a reporter system encoding a double fusion reporter gene consisting of firefly luciferase (Fluc) and green fluorescent protein (GFP) into BALB/c mice. Nanoliposomes loaded with a triple fusion gene containing HSV-TK and renilla luciferase (Rluc) and red fluorescent protein (RFP) were administered, and subsequently mice were treated with GCV. This approach resulted in monitoring of the tumor growth by BLI, while the treatment delivery of nanoliposomes was efficiently tracked by Rluc imaging [99]. Further, to avoid rapid clearance by RES after an intravenous injection, stealth PEGylated liposomes have resulted increased serum half-life and greater EPR. These stealth nanolipo‐


chemical property of drug and desired emulsion (single or double), solvent evaporation or solvent diffusion methods are mainly recruited [104]. We have developed folate receptor

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281

**Figure 6.** Size and morphology of PLGA nanoparticles. A) Dynamic light scattering (DLC) analysis of PEGylated PLGA NPs encapsulating siRNA. B) Transmission electron microscopy (TEM) micrograph of PLGA NPs. C) Scanning electron microscopy (SEM) micrograph of clusters of PLGA NPs. D) Schematic presentation of antibody armed PEGylated PLGA

Self-assembled micellar nanoformulations are another type of NPs that are widely used as gene delivery nanosystems, in which the positively charged polymers can interact with the negatively charged nucleic acids forming nanomicelles under sonication. Cationic polymers (e.g., linear and branched polyethyleneimine (l-PEI and b-PEI), poly(L-lysine), and polyami‐ doamine (PAMAM) dendrimers) can condense the nucleic acids forming polyplexes as either uni-molecular or multi-molecular complexes. Unfortunately, similar to CLs, cationic polymers can also induce intrinsic toxicogenomics [86] and cytotoxicity [16]. To achieve efficient targetspecific gene transfer, these polymers can covalently be modified by conjugating targeting ligands. The ligand-armed systems can then target the cells that present the specific cellular receptors. Because of their nano-scaled size (100-200 nm in diameter), they can be taken up by cells through receptor-mediated endocytosis. For example, a Trf-modified cyclodextrin polymer-based gene delivery nanosystem has been developed. These Trf-armed PEGylated cyclodextrin show increased stability in biological fluids and active targeting potential via transferrin, retaining high binding affinity toward Trf receptor and profound transfection of the target leukemia cells (K562 cells) through both passive and active targeting [105]. Recently, Hatefi's group engineered a novel multi-domain biopolymer, consisting of: 1) repeating units of arginine and histidine to condense pDNA and lyse endosome membranes, 2) a HER2 affibody as targeting moety, 3) a pH responsive fusogenic peptide to destabilize endosome membranes and enhance endosomolytic activity of histidine residues, and 4) a nuclear localization signal to enhance translocation of pDNA towards the cell nucleus [106]. They showed that pDNA was condensed into biopolymeric Nps and protected from serum endo‐

targeting PEGylated PLGA NPs for delivery of nucleic acids (Fig. 6).

nanoparticle (our unpublished data).

**Table 4.** Gene therapy clinical trials using liposomal formulations. EGFR: epidermal growth factor receptor; NA: not available; Cd: completed; LErafAON: liposomes carrying antisense oligonucleotide against the Raf-1 protein; siRNA: small interfering RNA; EphA2: ephrin type-A receptor 2; C-VISA BikDD: liposome consists of a pancreatic-cancerspecific expression vector "VISA" (VP16-GAL4-WPRE integrated systemic amplifier) and a pancreatic-cancer-specific promoter CCKAR (cholecystokinin type A receptor) (CCKAR-VISA or C-VISA) which drives expression of the gene BikDD, a mutant form of the potent proapoptotic gene Bik (Bcl-2 interacting killer). Data were adapted with permission from reference [4].

somes can be further decorated with homing devices (Ab, ligand) and imaging devices to develop targeted stealth nanoliposome for safe i.v. delivery of gene based medicines [100]. For example, transferrin (Trf) receptor-targeted liposomes (Trf-liposomes) encapsulating anti-BCR-ABL genomedicine (siRNA or AS-ODN) has resulted in significant delivery of cargo genes in chronic myeloid leukemia [101]. Folate receptor-targeted liposomes have also been used as cancer specific vectors [102]. For efficient delivery of siRNA to neuroblastoma (the most common solid tumor in early childhood), Adrian et al. engineered liposomal nanofor‐ mulation (190 to 240 nm) containing siRNA armed with anti-Disialoganglioside (GD2) Ab for selective interaction with neuroblastoma cells [103]. They showed a significant association of liposomes with neuroblastoma cells and effective delivery of siRNA with anti-GD2 Ab-armed liposomes.

#### **7.4. Polymeric gene delivery nanosystems**

To engineer polymeric NPs, various synthetic and biodegradable polymers have so far been exploited. Of these, biodegradable polymers provide better clinical outcomes. Among biodegradable polymers, poly(lactic-co-glycolic acid) (PLGA) as a copolymer approved by FDA is the most widely used polymer. For engineering PLGA NPs, depending on physico‐ chemical property of drug and desired emulsion (single or double), solvent evaporation or solvent diffusion methods are mainly recruited [104]. We have developed folate receptor targeting PEGylated PLGA NPs for delivery of nucleic acids (Fig. 6).

**Figure 6.** Size and morphology of PLGA nanoparticles. A) Dynamic light scattering (DLC) analysis of PEGylated PLGA NPs encapsulating siRNA. B) Transmission electron microscopy (TEM) micrograph of PLGA NPs. C) Scanning electron microscopy (SEM) micrograph of clusters of PLGA NPs. D) Schematic presentation of antibody armed PEGylated PLGA nanoparticle (our unpublished data).

somes can be further decorated with homing devices (Ab, ligand) and imaging devices to develop targeted stealth nanoliposome for safe i.v. delivery of gene based medicines [100]. For example, transferrin (Trf) receptor-targeted liposomes (Trf-liposomes) encapsulating anti-BCR-ABL genomedicine (siRNA or AS-ODN) has resulted in significant delivery of cargo genes in chronic myeloid leukemia [101]. Folate receptor-targeted liposomes have also been used as cancer specific vectors [102]. For efficient delivery of siRNA to neuroblastoma (the most common solid tumor in early childhood), Adrian et al. engineered liposomal nanofor‐ mulation (190 to 240 nm) containing siRNA armed with anti-Disialoganglioside (GD2) Ab for selective interaction with neuroblastoma cells [103]. They showed a significant association of liposomes with neuroblastoma cells and effective delivery of siRNA with anti-GD2 Ab-armed

**Clinical trial US Trial ID Malignancy Intervention Phase Status**

and neck cancer

NCT01455389 Lung cancer DOTAP:Chol-fus1;

Liposomal

antisense

Erlotinib; Dexamethasone

formulation of EGFR

siRNA-EphA2-DOPC

liposomes

BikDD Nanoparticles

LErafAON I Cd

I NA

I/II Active

I NA

I Active

NCT00009841 Advanced head

NCT00024661 Advanced solid

NCT01591356 Advanced solid

NCT00968604 Advanced

tumors

tumors

**Table 4.** Gene therapy clinical trials using liposomal formulations. EGFR: epidermal growth factor receptor; NA: not available; Cd: completed; LErafAON: liposomes carrying antisense oligonucleotide against the Raf-1 protein; siRNA: small interfering RNA; EphA2: ephrin type-A receptor 2; C-VISA BikDD: liposome consists of a pancreatic-cancerspecific expression vector "VISA" (VP16-GAL4-WPRE integrated systemic amplifier) and a pancreatic-cancer-specific promoter CCKAR (cholecystokinin type A receptor) (CCKAR-VISA or C-VISA) which drives expression of the gene BikDD, a mutant form of the potent proapoptotic gene Bik (Bcl-2 interacting killer). Data were adapted with

pancreatic cancer

To engineer polymeric NPs, various synthetic and biodegradable polymers have so far been exploited. Of these, biodegradable polymers provide better clinical outcomes. Among biodegradable polymers, poly(lactic-co-glycolic acid) (PLGA) as a copolymer approved by FDA is the most widely used polymer. For engineering PLGA NPs, depending on physico‐

liposomes.

Gene Therapy in Treating Patients With Advanced Head and Neck

280 Novel Gene Therapy Approaches

FUS1-nanoparticles and Erlotinib in Stage IV Lung Cancer

Study to Determine the Maximum Tolerated Dose of LErafAON in Patients With Advanced Solid

EphA2 Gene Targeting Using Neutral Liposomal Small Interfering RNA Delivery

C-VISA BikDD: Liposome in Advanced Pancreatic Cancer

permission from reference [4].

Cancer

Tumors

**7.4. Polymeric gene delivery nanosystems**

Self-assembled micellar nanoformulations are another type of NPs that are widely used as gene delivery nanosystems, in which the positively charged polymers can interact with the negatively charged nucleic acids forming nanomicelles under sonication. Cationic polymers (e.g., linear and branched polyethyleneimine (l-PEI and b-PEI), poly(L-lysine), and polyami‐ doamine (PAMAM) dendrimers) can condense the nucleic acids forming polyplexes as either uni-molecular or multi-molecular complexes. Unfortunately, similar to CLs, cationic polymers can also induce intrinsic toxicogenomics [86] and cytotoxicity [16]. To achieve efficient targetspecific gene transfer, these polymers can covalently be modified by conjugating targeting ligands. The ligand-armed systems can then target the cells that present the specific cellular receptors. Because of their nano-scaled size (100-200 nm in diameter), they can be taken up by cells through receptor-mediated endocytosis. For example, a Trf-modified cyclodextrin polymer-based gene delivery nanosystem has been developed. These Trf-armed PEGylated cyclodextrin show increased stability in biological fluids and active targeting potential via transferrin, retaining high binding affinity toward Trf receptor and profound transfection of the target leukemia cells (K562 cells) through both passive and active targeting [105]. Recently, Hatefi's group engineered a novel multi-domain biopolymer, consisting of: 1) repeating units of arginine and histidine to condense pDNA and lyse endosome membranes, 2) a HER2 affibody as targeting moety, 3) a pH responsive fusogenic peptide to destabilize endosome membranes and enhance endosomolytic activity of histidine residues, and 4) a nuclear localization signal to enhance translocation of pDNA towards the cell nucleus [106]. They showed that pDNA was condensed into biopolymeric Nps and protected from serum endo‐ nucleases, while targeting HER2 positive cancer cells, and metabolized by endogenous furin enzymes to reduce potential toxicity. Later on, synthesis of a targeted PEI polymer was reported, in which the PEI polymer was conjugated to angiogenic vessel-homing peptide Ala-Pro-Arg-Pro-Gly (APRPG) through PEG spacer [107]. The PEI-PEG-APRPG was shown to effectively condense siRNA into 20-50 nm NPs that can substantially impose inhibitory effects in vitro with profound EPR in vivo targeting tumor vasculature through VEGF.

normal cells in brain using a rat model. It was found that the tumoricidal bystander effect in the HSV-TK gene therapy using MSCs and GCV does not injure normal brain tissues [114]. In another study, umbilical cord blood MSCs were used to deliver the transgenic LIGHT (TNFSF14) to the target tumor cells in vivo. The transfected MSCs with lentiviral vectors carrying LIGHT genes demonstrated a strong suppressive effect on tumor growth, in which pathological sections of the tumor tissues showed significant induction of apoptosis and occurrence of tumor necrosis in tumor cells [115]. The potential of genetically modified MSCs expressing IFN- β was assessed in an immuno-competent mouse model of prostate cancer lung metastasis. Significant reduction in tumor volume in lungs was seen following IFN- β ex‐ pressing MSC therapy, perhaps through induction of apoptosis and increase in the natural kill cell activity [116]. The MSC-based gene therapy is still in its infancy era and need much more investigation prior to its clinical applications even though the MSCs themselves are under

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283

Tissue-specific promoters (TSPs), a powerful tool for decreasing the toxicity of cancer gene therapy to normal tissues, have been used as targeted gene therapy approach. TSPs have been utilized for specific mutation compensation or delivery of prodrug-converting enzymes and also for controlling crucial viral replication regulators and consequent restriction of replication to tumor cells [118]. The safety and contingency of this approach has been shown in some initial clinical trials [119]. Of these, the cytomegalovirus (CMV) immediate-early promoter is often harnessed in gene therapy since it can express target genes at high levels in tumor cells. Lin et al. (2001) examined the effects of the involucrin (INV), keratin 14 (K14) and CMV promoters on the expression of the reporter gene beta-galactosidase. They introduce the plasmid DNA to BALB/c mice using a gene gun, and examined the skin biopsies. They found that the K14 and INV promoter constructs could induce the beta-galactosidase gene expression only in the epidermis, while the CMV promoter was able to elicit gene expression in both the dermis and epidermis [120]. To increase promoter strength while maintaining tissue specific‐ ity, Qiao et al. (2002) constructed a recombinant adenovirus encompassing a binary promoter system with a tumor-specific promoter carcinoembryonic antigen (CEA) driving a transcrip‐ tion transactivator with capability to express a HSV-TK. After successful application in vitro, they employed noninvasive nuclear imaging using a radioiodinated nucleoside (fraluridine (FIAU)) serving as a substrate for HSV-TK in BALB/C mice model. The results indicated the accumulated radioactivity only in the area of CEA-positive tumors after intratumoral injection, in which significantly less spread was observed to the adjacent liver tissue [121]. In another study, a vector with the human minimal tyrosinase promoter and two human enhancer elements (2hE-hTyrP) was compared with different hybrid promoter constructs containing tyrosinase regulatory sequences and the viral simian virus 40 (SV40) promoters. The hybrid SV40-based promoters were effective in vitro, and the in vivo tissue specificity of the 2hEhTyrP vector was demonstrated in subcutaneous xenografted tumors model [122]. Another plausible approach to specifically target tumor cells for gene expression is to harness promoter elements that become activated in chemotherapy-resistant tumor cells [123]. In addition to TSPs, inducible promoters (IPs) can be exploited to minimize target gene expression in normal

clinical trials [117].

**8.3. Tissue–specific promoters and inducible promoters**

## **8. Tissue specific gene therapy**

In addition to targeted nanomedicine gene therapies, tissue specific gene therapy provides a robust targeted approach. We describe some of the cell/tissue specific applications in the following sections.

#### **8.1. Cancer Stem Cells (CSCs)**

Some of the transit-amplifying cells in the cancer population appear to remain immature within the differentiating cells. These classes of cells that act as cancer cells progenitor are called cancer stem cells, which are deemed to be one of the reasons for cancer relapse that are hardly responsive for treatment. The poor prognosis and responsiveness of patients with relapsed aggressive metastatic tumors necessitate the development of more effective tumor-selective therapies towards cells that cause such relapse, while the conventional therapy target the differentiated cancer cells. Therefore, to be maximally effective, gene therapy of cancer should target both the resting stem cells and the proliferating cells of the cancer [108]. To this end, several translational approaches have been undertaken to target CSCs, including use of oncolytic viruses that may offer an effective way to specifically target and eradicate CSCs. Of these, conditionally replicative adenoviruses (CRAd) are considered as promising virotherapy systems [109]. Considering the plasticity of CSCs, apoptosis-inducing strategy can be used to eliminate these cells by harnessing genes such as TRAIL, BCL-2 family and XIAP as targeted therapies [110].

#### **8.2. Mesenchymal Stem Cells (MSCs) as a gene therapy carrier**

To date, as a personalized medicine, cell-based therapy of cancer has been considered as a promising modality. Of these, MSCs seem to hold great potential as targeted-delivery vehicle in cancer gene therapy [111]. Their propagation in culture is simple, also shows contingency toward genetic modification in order to express therapeutic proteins. Above all, MSCs possess inherent tumor-tropic and migratory properties that allow them to serve as robust cell based carrier as targeted drug delivery systems for isolated tumors and metastatic diseases [112]. In a study, the migration ability of MSCs toward prostate cancer cells (in vitro and in vivo) and incorporating into the tumor mass was investigated. The infected cells with HSV-TK gene were shown to maintain their tumor tropism capabilities and significantly inhibited the growth of subcutaneous PC3 prostate cancer xenografts in nude mice in the presence of GCV [113]. Similar strategy was applied to evaluate the impact of the suicide gene therapy by MSCs in normal cells in brain using a rat model. It was found that the tumoricidal bystander effect in the HSV-TK gene therapy using MSCs and GCV does not injure normal brain tissues [114]. In another study, umbilical cord blood MSCs were used to deliver the transgenic LIGHT (TNFSF14) to the target tumor cells in vivo. The transfected MSCs with lentiviral vectors carrying LIGHT genes demonstrated a strong suppressive effect on tumor growth, in which pathological sections of the tumor tissues showed significant induction of apoptosis and occurrence of tumor necrosis in tumor cells [115]. The potential of genetically modified MSCs expressing IFN- β was assessed in an immuno-competent mouse model of prostate cancer lung metastasis. Significant reduction in tumor volume in lungs was seen following IFN- β ex‐ pressing MSC therapy, perhaps through induction of apoptosis and increase in the natural kill cell activity [116]. The MSC-based gene therapy is still in its infancy era and need much more investigation prior to its clinical applications even though the MSCs themselves are under clinical trials [117].

#### **8.3. Tissue–specific promoters and inducible promoters**

nucleases, while targeting HER2 positive cancer cells, and metabolized by endogenous furin enzymes to reduce potential toxicity. Later on, synthesis of a targeted PEI polymer was reported, in which the PEI polymer was conjugated to angiogenic vessel-homing peptide Ala-Pro-Arg-Pro-Gly (APRPG) through PEG spacer [107]. The PEI-PEG-APRPG was shown to effectively condense siRNA into 20-50 nm NPs that can substantially impose inhibitory effects

In addition to targeted nanomedicine gene therapies, tissue specific gene therapy provides a robust targeted approach. We describe some of the cell/tissue specific applications in the

Some of the transit-amplifying cells in the cancer population appear to remain immature within the differentiating cells. These classes of cells that act as cancer cells progenitor are called cancer stem cells, which are deemed to be one of the reasons for cancer relapse that are hardly responsive for treatment. The poor prognosis and responsiveness of patients with relapsed aggressive metastatic tumors necessitate the development of more effective tumor-selective therapies towards cells that cause such relapse, while the conventional therapy target the differentiated cancer cells. Therefore, to be maximally effective, gene therapy of cancer should target both the resting stem cells and the proliferating cells of the cancer [108]. To this end, several translational approaches have been undertaken to target CSCs, including use of oncolytic viruses that may offer an effective way to specifically target and eradicate CSCs. Of these, conditionally replicative adenoviruses (CRAd) are considered as promising virotherapy systems [109]. Considering the plasticity of CSCs, apoptosis-inducing strategy can be used to eliminate these cells by harnessing genes such as TRAIL, BCL-2 family and XIAP as targeted

To date, as a personalized medicine, cell-based therapy of cancer has been considered as a promising modality. Of these, MSCs seem to hold great potential as targeted-delivery vehicle in cancer gene therapy [111]. Their propagation in culture is simple, also shows contingency toward genetic modification in order to express therapeutic proteins. Above all, MSCs possess inherent tumor-tropic and migratory properties that allow them to serve as robust cell based carrier as targeted drug delivery systems for isolated tumors and metastatic diseases [112]. In a study, the migration ability of MSCs toward prostate cancer cells (in vitro and in vivo) and incorporating into the tumor mass was investigated. The infected cells with HSV-TK gene were shown to maintain their tumor tropism capabilities and significantly inhibited the growth of subcutaneous PC3 prostate cancer xenografts in nude mice in the presence of GCV [113]. Similar strategy was applied to evaluate the impact of the suicide gene therapy by MSCs in

**8.2. Mesenchymal Stem Cells (MSCs) as a gene therapy carrier**

in vitro with profound EPR in vivo targeting tumor vasculature through VEGF.

**8. Tissue specific gene therapy**

following sections.

282 Novel Gene Therapy Approaches

therapies [110].

**8.1. Cancer Stem Cells (CSCs)**

Tissue-specific promoters (TSPs), a powerful tool for decreasing the toxicity of cancer gene therapy to normal tissues, have been used as targeted gene therapy approach. TSPs have been utilized for specific mutation compensation or delivery of prodrug-converting enzymes and also for controlling crucial viral replication regulators and consequent restriction of replication to tumor cells [118]. The safety and contingency of this approach has been shown in some initial clinical trials [119]. Of these, the cytomegalovirus (CMV) immediate-early promoter is often harnessed in gene therapy since it can express target genes at high levels in tumor cells. Lin et al. (2001) examined the effects of the involucrin (INV), keratin 14 (K14) and CMV promoters on the expression of the reporter gene beta-galactosidase. They introduce the plasmid DNA to BALB/c mice using a gene gun, and examined the skin biopsies. They found that the K14 and INV promoter constructs could induce the beta-galactosidase gene expression only in the epidermis, while the CMV promoter was able to elicit gene expression in both the dermis and epidermis [120]. To increase promoter strength while maintaining tissue specific‐ ity, Qiao et al. (2002) constructed a recombinant adenovirus encompassing a binary promoter system with a tumor-specific promoter carcinoembryonic antigen (CEA) driving a transcrip‐ tion transactivator with capability to express a HSV-TK. After successful application in vitro, they employed noninvasive nuclear imaging using a radioiodinated nucleoside (fraluridine (FIAU)) serving as a substrate for HSV-TK in BALB/C mice model. The results indicated the accumulated radioactivity only in the area of CEA-positive tumors after intratumoral injection, in which significantly less spread was observed to the adjacent liver tissue [121]. In another study, a vector with the human minimal tyrosinase promoter and two human enhancer elements (2hE-hTyrP) was compared with different hybrid promoter constructs containing tyrosinase regulatory sequences and the viral simian virus 40 (SV40) promoters. The hybrid SV40-based promoters were effective in vitro, and the in vivo tissue specificity of the 2hEhTyrP vector was demonstrated in subcutaneous xenografted tumors model [122]. Another plausible approach to specifically target tumor cells for gene expression is to harness promoter elements that become activated in chemotherapy-resistant tumor cells [123]. In addition to TSPs, inducible promoters (IPs) can be exploited to minimize target gene expression in normal cells. Harnessing the IPs, the timing of the gene expression can be modulated and controlled. Of a large number of inducible systems developed, only a few were translated into clinical gene therapy trials, including radiation-inducible genes [124]. Using this cancer gene therapy modality, promoters of radiation-inducible genes are exploited to drive transcription of transgenes in response to radiation, resulting in increased responsiveness of cancer cells to radiotherapy. These constructs, delivered by adenoviral vectors, can activate a transgene encoding a cytotoxic protein in tumor cells, in which the tumoricidal effects can be then localized temporally and spatially by X-rays. Perhaps, TNFerade (GenVec, Inc) is the best paradigm, which is an adenoviral vector containing radiation-inducible elements of the early growth response-1 promoter upstream of a cDNA encoding human TNF-α [125]. It has been translated into several clinical applications, e.g., as the first-line treatment of locally advanced pancreatic cancer in combination with 5-FU and radiotherapy [126]. However, it has not been approved.

## **9. Translational hurdles**

Over the last couple of decades, various gene-based medicines have been developed in vitro with great potential to be translated for in vivo uses in clinic. It has been evinced that the gene therapy approach by virtue carries a certain degree of risk, thus the design and development of such modality need to meet the entire scientific and regulatory requirements. Some of the risks are procedural hazards (e.g. for parenteral medicaments), while some others happen to be specific to the genomedicine per se (e.g., immunologic reaction of viral vectors or nonspe‐ cific impacts of the delivered genes). All scientific, ethical, legal and social implications of this novel modality to genetic disease are involved for its successful translation. Fig. 7 schematically epitomizes the complexity of the steps for development and translation of gene-medicine for immunogene therapy of ovarian cancer.

**10. Final remarks**

cine.

associated cells, stromal cells and CSCs.

Cancer gene therapy continues to grow even though clinical applications of this approach demand further investigations. Trajectory of gene therapy shows great impacts of genomedi‐ cines (i.e., As-ODNs, siRNA, Ribozymes, DNAzyme) both cell based and animal models, while tumor antigen-specific vaccines and DNA vaccines appear to be the most promising modali‐ ties. While suicide gene therapy, immunogene therapy and angiogenic gene therapy continue to become a mature modality, integration of nanotechnology toward development of multi‐ functional nanoparticles appear to provide a resilient, yet versatile, platform for targeted cancer gene therapy as "nano-genoceuticals". Rise of MSCs based cancer gene therapies may also open a new chapter as "cyto-genoceuticals". More than 65% of the gene therapy trials have been devoted to the cancer diseases; however, less than 3% of these trials have been progressed to the phase II/III stages and only few to the phase IV stage. The first approved cancer vaccination (Sipuleucel-T) has resulted in great clinical corollaries. Still many tumor suppressor and apoptosis-inducing genes can be evaluated for clinical applications. Attribut‐ able to intricate nature of malignant diseases, to achieve more effective gene therapy against cancer, genomedicines need to be advanced to be able to holistically target the most cancer causing genes. It is also essential to target both the tumor cells and other cancer associated players of the tumor microenvironment including: tumor microvasculature and tumor

**Figure 7.** Schematic representation of translational approach for immunogene therapy of ovarian cancer. A) Design of the immunogene therapy based on a) different strategies of gene therapy and b) different gene delivery systems. B) Anatomical and cellular complexity of the ovarian cancer cells that need to be specifically targeted using advanced gene delivery systems and cancer marker molecules (CMMs). C) Various stages for translation of immunogenetic medi‐

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In immunogene therapy, for example, stimulation of the cellular immune system to recognize and obliterate the cancer cells with genes encoding a variety of cytokines (e.g., interleukin-2 (IL-2), granulocyte - macrophage colony-stimulating factor (GM-CSF), co-stimulatory mole‐ cules such as CD80 and CD86) can 1) increase the immunogenicity of the transfected autolo‐ gous tumour cells, 2) increase the likelihood of generating a tumor-specific cytotoxic Tlymphocyte (CTL) response. In the ex vivo transfection of antigen-presenting cells (APCs), the cells are transfected with a gene encoding a tumor-specific antigen (e.g., carcinoembryonic antigen, CEA) that is presented by major histocompatibility complex (MHC) class I molecules to antigen-specific CTLs via the T-cell receptor (TCR). Stimulated CTLs can find and eradicate the residual CEA-expressing tumor cells, in which GM-CSF can increase the activation of APCs and their migration into the tumor microenvironment [1].

Further, some degree of knowledge of industrial drug development is critical for innovation in this new sector, while healthcare systems and industries need to undertake more translatable approaches to fasten the in terms of cancer gene therapy.

**Figure 7.** Schematic representation of translational approach for immunogene therapy of ovarian cancer. A) Design of the immunogene therapy based on a) different strategies of gene therapy and b) different gene delivery systems. B) Anatomical and cellular complexity of the ovarian cancer cells that need to be specifically targeted using advanced gene delivery systems and cancer marker molecules (CMMs). C) Various stages for translation of immunogenetic medi‐ cine.

## **10. Final remarks**

cells. Harnessing the IPs, the timing of the gene expression can be modulated and controlled. Of a large number of inducible systems developed, only a few were translated into clinical gene therapy trials, including radiation-inducible genes [124]. Using this cancer gene therapy modality, promoters of radiation-inducible genes are exploited to drive transcription of transgenes in response to radiation, resulting in increased responsiveness of cancer cells to radiotherapy. These constructs, delivered by adenoviral vectors, can activate a transgene encoding a cytotoxic protein in tumor cells, in which the tumoricidal effects can be then localized temporally and spatially by X-rays. Perhaps, TNFerade (GenVec, Inc) is the best paradigm, which is an adenoviral vector containing radiation-inducible elements of the early growth response-1 promoter upstream of a cDNA encoding human TNF-α [125]. It has been translated into several clinical applications, e.g., as the first-line treatment of locally advanced pancreatic cancer in combination with 5-FU and radiotherapy [126]. However, it has not been

Over the last couple of decades, various gene-based medicines have been developed in vitro with great potential to be translated for in vivo uses in clinic. It has been evinced that the gene therapy approach by virtue carries a certain degree of risk, thus the design and development of such modality need to meet the entire scientific and regulatory requirements. Some of the risks are procedural hazards (e.g. for parenteral medicaments), while some others happen to be specific to the genomedicine per se (e.g., immunologic reaction of viral vectors or nonspe‐ cific impacts of the delivered genes). All scientific, ethical, legal and social implications of this novel modality to genetic disease are involved for its successful translation. Fig. 7 schematically epitomizes the complexity of the steps for development and translation of gene-medicine for

In immunogene therapy, for example, stimulation of the cellular immune system to recognize and obliterate the cancer cells with genes encoding a variety of cytokines (e.g., interleukin-2 (IL-2), granulocyte - macrophage colony-stimulating factor (GM-CSF), co-stimulatory mole‐ cules such as CD80 and CD86) can 1) increase the immunogenicity of the transfected autolo‐ gous tumour cells, 2) increase the likelihood of generating a tumor-specific cytotoxic Tlymphocyte (CTL) response. In the ex vivo transfection of antigen-presenting cells (APCs), the cells are transfected with a gene encoding a tumor-specific antigen (e.g., carcinoembryonic antigen, CEA) that is presented by major histocompatibility complex (MHC) class I molecules to antigen-specific CTLs via the T-cell receptor (TCR). Stimulated CTLs can find and eradicate the residual CEA-expressing tumor cells, in which GM-CSF can increase the activation of APCs

Further, some degree of knowledge of industrial drug development is critical for innovation in this new sector, while healthcare systems and industries need to undertake more translatable

approved.

284 Novel Gene Therapy Approaches

**9. Translational hurdles**

immunogene therapy of ovarian cancer.

and their migration into the tumor microenvironment [1].

approaches to fasten the in terms of cancer gene therapy.

Cancer gene therapy continues to grow even though clinical applications of this approach demand further investigations. Trajectory of gene therapy shows great impacts of genomedi‐ cines (i.e., As-ODNs, siRNA, Ribozymes, DNAzyme) both cell based and animal models, while tumor antigen-specific vaccines and DNA vaccines appear to be the most promising modali‐ ties. While suicide gene therapy, immunogene therapy and angiogenic gene therapy continue to become a mature modality, integration of nanotechnology toward development of multi‐ functional nanoparticles appear to provide a resilient, yet versatile, platform for targeted cancer gene therapy as "nano-genoceuticals". Rise of MSCs based cancer gene therapies may also open a new chapter as "cyto-genoceuticals". More than 65% of the gene therapy trials have been devoted to the cancer diseases; however, less than 3% of these trials have been progressed to the phase II/III stages and only few to the phase IV stage. The first approved cancer vaccination (Sipuleucel-T) has resulted in great clinical corollaries. Still many tumor suppressor and apoptosis-inducing genes can be evaluated for clinical applications. Attribut‐ able to intricate nature of malignant diseases, to achieve more effective gene therapy against cancer, genomedicines need to be advanced to be able to holistically target the most cancer causing genes. It is also essential to target both the tumor cells and other cancer associated players of the tumor microenvironment including: tumor microvasculature and tumor associated cells, stromal cells and CSCs.

## **Author details**

Yadollah Omidi1,2, Jaleh Barar1,2 and George Coukos1,3

1 Ovarian Cancer Research Center, Perelman School of Medicine, University of Pennsylva‐ nia, Philadelphia, PA, USA

feron-beta gene therapy and 5-fluorouracil significantly reduces growth of metastatic human breast cancer in SCID mouse lungs. Cancer Invest. (2008). , 26(7), 662-70.

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287

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3 Ludwig Center for Cancer Research, University of Lausanne, Lausanne, Switzerland

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**Chapter 12**

**Identification and Validation of Targets for Cancer**

The link between immune responses and cancer is evident from findings such as, a com‐ promised immune system resulting in an increased tumour incidence [1] and cancer pa‐ tient sera evidencing recognition of autologous cancer antigens [2]. The identification of tumour associated antigens (TAAs) plays a central role in our understanding of how can‐ cer cells can inhibit the immune system and how we can overcome this tumour immune suppression to break tolerance and achieve cancer destruction [3]. Antibodies reacting with TAAs on the surface of cancer cells provoke an extremely effective immune re‐ sponse [4] which can be exquisitely specific to the tumour cells present in the body. However not many surface proteins are present on tumour cells and limited otherwise in

TAAs are most often proteins which have acquired mutations or have elevated expression levels which are expressed at the sub-cellular level. The ideal immunotherapy targets should also play a role in tumour progression [5]. For example p53 [6] is one of the most desirable targets for immunotherapy – targeting p53 can kill both the evolving tumour cell population and any cancer "stem" cell which harbours this as an early tumourigenesis aberration and supports further tumour growth. In addition, a number of tumour antigens have been shown

In this chapter, we will examine how tumour antigens are identified and characterised to demonstrate their potential as immunotherapy targets and examine their role as biomarkers

and reproduction in any medium, provided the original work is properly cited.

© 2013 Khan 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,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

for treatment response and patient survival, and targets for personalised therapies.

**Immunotherapy: From the Bench-to-Bedside**

Ghazala Khan, Suzanne E. Brooks, Frances Denniss,

Dagmar Sigurdardottir and Barbara-ann Guinn

Additional information is available at the end of the chapter

expression to healthy non-essential tissues.

to be useful biomarkers for cancer diagnosis [7] and survival [8].

http://dx.doi.org/10.5772/54698

**1. Introduction**

## **Identification and Validation of Targets for Cancer Immunotherapy: From the Bench-to-Bedside**

Ghazala Khan, Suzanne E. Brooks, Frances Denniss, Dagmar Sigurdardottir and Barbara-ann Guinn

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54698

## **1. Introduction**

The link between immune responses and cancer is evident from findings such as, a com‐ promised immune system resulting in an increased tumour incidence [1] and cancer pa‐ tient sera evidencing recognition of autologous cancer antigens [2]. The identification of tumour associated antigens (TAAs) plays a central role in our understanding of how can‐ cer cells can inhibit the immune system and how we can overcome this tumour immune suppression to break tolerance and achieve cancer destruction [3]. Antibodies reacting with TAAs on the surface of cancer cells provoke an extremely effective immune re‐ sponse [4] which can be exquisitely specific to the tumour cells present in the body. However not many surface proteins are present on tumour cells and limited otherwise in expression to healthy non-essential tissues.

TAAs are most often proteins which have acquired mutations or have elevated expression levels which are expressed at the sub-cellular level. The ideal immunotherapy targets should also play a role in tumour progression [5]. For example p53 [6] is one of the most desirable targets for immunotherapy – targeting p53 can kill both the evolving tumour cell population and any cancer "stem" cell which harbours this as an early tumourigenesis aberration and supports further tumour growth. In addition, a number of tumour antigens have been shown to be useful biomarkers for cancer diagnosis [7] and survival [8].

In this chapter, we will examine how tumour antigens are identified and characterised to demonstrate their potential as immunotherapy targets and examine their role as biomarkers for treatment response and patient survival, and targets for personalised therapies.

© 2013 Khan 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. © 2013 The Author(s). Licensee InTech. This chapter is 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. Which tumour antigens?**

Many of the tumour antigens identified by serological identification of antigens by recombi‐ nant expression cloning (SEREX) can be classified into one or more categories which are: cancer-testis, mutational, differentiation, amplified/overexpressed, splice variant and viral antigens [9]. Cancer-testis (CT) antigens [10] have been found to be highly expressed in tu‐ mours but not in normal tissues with the exception of immunologically protected sites (those tissues which lack major histocompatibility complex (MHC) class I and therefore do not present self-antigens). CT antigens make attractive targets as due to their limited expres‐ sion, they should therefore induce specific anti-tumour immune responses and less toxicity to healthy tissue [11]. However some debate remains as to the definition of a CT antigen [12] with some suggestion of differential levels of expression and others suggesting no expres‐ sion in normal tissues expect those in immunologically protected sites (such as ovary, pla‐ centa and testis). TAAs that are frequently found in tumours and provide excellent targets for immunotherapy include Wilms tumour 1 [13] and PRAME [14].

**3.3. Serological identification of antigens by recombinant expression cloning (SEREX)**

ry screening, prior to cDNA sequencing of phagemid inserts [31].

preparative gel and indentified using mass spectrometry [33, 34].

**3.4. Serological proteome analysis (SERPA)**

**3.5. CDNA microarrays**

**3.6. Mass Spectroscopy (MS)**

Serological identification of antigens by recombinant expression cloning (SEREX) provided a much needed boost to the area of antigen identification at a time when few cancer antigen identification options existed [2]. SEREX was not limited to immunogenic cancers such as melanoma and has now been used to identify more than 2,000 antigens [22-23] in a large range of different solid [24-26] and haematological malignancies [27-30]. cDNA libraries are created from tumour samples, cell lines or healthy normal donor cells (such as testes). RNA from these cells were reverse transcribed and inserted as cDNA into phage vectors and ex‐ pressed as recombinant proteins on the capsid surface of phage which survived on permis‐ sive E.coli. Expressed proteins were transferred to nitrocellulose membranes and following the removal of excess E.coli waste, phage plaques were immunoscreened using pre-cleared patient sera. Any positive plaques were isolated, eluted and used for secondary confirmato‐

Identification of Targets for Immunotherapy http://dx.doi.org/10.5772/54698 299

Serological proteome analysis (SERPA) was first described by Klade *et al* in 2001 [32]. Proteins were extracted from primary tumours or cell lines, separated concurrently on two 2D gels and transferred to nitrocellulose membranes. A third gel is stained with Coomassie Blue as a prepa‐ rative gel. The membranes are incubated with cancer patient's sera and normal control. The two gels are directly compared and any bright spots on the cancer sera membrane were cut from the

The differential expression of tumour antigens and/or protein biomarkers between cell and dis‐ ease subtypes have been directly compared on cDNA microarrays and has allowed our im‐ proved understanding of lymphomas [35] and aided our development of personalised therapies [36]. Microarray technology is able to distinguish between different subtypes of a par‐ ticular cancer as well as identify the expression of novel antigens [37]. Minimal residual disease is a very important tool in the detection of impending relapse in patients who have had some form of treatment. Markers for minimal residual disease in acute lymphocytic leukaemia were identified by gene profiling [38]. cDNA microarray has been used to identify the frequency of elevated tumour antigen expression in acute myeloid leukaemia [28] and also associations be‐ tween specific cytogenetic abnormalities and relative levels of tumour antigen expression [39]. Micorarray has also been used to elucidate the possible function of tumour antigens such as Synovial Sarcoma X breakpoint 2 Interacting Protein (SSX2IP) in the subversion of cells har‐ bouring cytogenetic abnormalities (t(8;21) associated with mitotic spindle failure and the asso‐ ciation between the elevated expression of some tumour antigens (SSX2IP, RHAMM and SURVIVIN) at disease presentation and patient survival [8] in acute myeloid leukaemia.

Mass Spectroscopy (MS) involves the analysis of peptides eluted from the MHC of antigen presenting cells [40-42] or proteins in serum [43]. This area is reviewed more completely in the

## **3. Identification of tumour antigens**

Strategies are required to help identify potential targets which can be used for cancer immuno‐ therapy. Some of the most commonly used and successful techniques are described as follows:-

#### **3.1. Reverse-Transcription-Polymerase Chain Reaction (RT-PCR) and real-time PCR (RQ-PCR)**

Reverse-transcription-polymerase chain reaction (RT-PCR) and real-time PCR (RQ-PCR) has been used to examine known TAA expression in a range of solid and haematological malig‐ nancies [15-19]. Although this has provided important expression information and a good starting point to identify potential antigenic targets in a range of cancers, these studies are entirely limited to tumour antigens which had already been discovered.

#### **3.2. Representational difference analysis**

Representational difference analysis was developed by Thierry Boon's group and used to discover a number of CT antigens [20, 21] including the MAGE family of antigens, typically from melanoma with one exception, RAGE, from renal cancer. Briefly, total RNA was ex‐ tracted from normal tissue (driver) and a tumour sample (tester) and used to construct dou‐ ble-stranded cDNA. Both cDNA samples were digested with the restriction enzymes DpnII and ligated to adapters which contained primer binding sites. The fragments were amplified by PCR, the adapters removed and new adapters for unrelated primers ligated to the tester. The tester and driver were then mixed and hybridized leading to three combinations of product: driver-driver (no amplification), tester-driver (linear amplification) and tester-test‐ er (exponential amplification). A further two hybridization and amplification steps generate greater variation in the products which are subsequently cloned and sequenced.

#### **3.3. Serological identification of antigens by recombinant expression cloning (SEREX)**

Serological identification of antigens by recombinant expression cloning (SEREX) provided a much needed boost to the area of antigen identification at a time when few cancer antigen identification options existed [2]. SEREX was not limited to immunogenic cancers such as melanoma and has now been used to identify more than 2,000 antigens [22-23] in a large range of different solid [24-26] and haematological malignancies [27-30]. cDNA libraries are created from tumour samples, cell lines or healthy normal donor cells (such as testes). RNA from these cells were reverse transcribed and inserted as cDNA into phage vectors and ex‐ pressed as recombinant proteins on the capsid surface of phage which survived on permis‐ sive E.coli. Expressed proteins were transferred to nitrocellulose membranes and following the removal of excess E.coli waste, phage plaques were immunoscreened using pre-cleared patient sera. Any positive plaques were isolated, eluted and used for secondary confirmato‐ ry screening, prior to cDNA sequencing of phagemid inserts [31].

#### **3.4. Serological proteome analysis (SERPA)**

Serological proteome analysis (SERPA) was first described by Klade *et al* in 2001 [32]. Proteins were extracted from primary tumours or cell lines, separated concurrently on two 2D gels and transferred to nitrocellulose membranes. A third gel is stained with Coomassie Blue as a prepa‐ rative gel. The membranes are incubated with cancer patient's sera and normal control. The two gels are directly compared and any bright spots on the cancer sera membrane were cut from the preparative gel and indentified using mass spectrometry [33, 34].

#### **3.5. CDNA microarrays**

**2. Which tumour antigens?**

298 Novel Gene Therapy Approaches

Many of the tumour antigens identified by serological identification of antigens by recombi‐ nant expression cloning (SEREX) can be classified into one or more categories which are: cancer-testis, mutational, differentiation, amplified/overexpressed, splice variant and viral antigens [9]. Cancer-testis (CT) antigens [10] have been found to be highly expressed in tu‐ mours but not in normal tissues with the exception of immunologically protected sites (those tissues which lack major histocompatibility complex (MHC) class I and therefore do not present self-antigens). CT antigens make attractive targets as due to their limited expres‐ sion, they should therefore induce specific anti-tumour immune responses and less toxicity to healthy tissue [11]. However some debate remains as to the definition of a CT antigen [12] with some suggestion of differential levels of expression and others suggesting no expres‐ sion in normal tissues expect those in immunologically protected sites (such as ovary, pla‐ centa and testis). TAAs that are frequently found in tumours and provide excellent targets

Strategies are required to help identify potential targets which can be used for cancer immuno‐ therapy. Some of the most commonly used and successful techniques are described as follows:-

**3.1. Reverse-Transcription-Polymerase Chain Reaction (RT-PCR) and real-time PCR (RQ-**

Reverse-transcription-polymerase chain reaction (RT-PCR) and real-time PCR (RQ-PCR) has been used to examine known TAA expression in a range of solid and haematological malig‐ nancies [15-19]. Although this has provided important expression information and a good starting point to identify potential antigenic targets in a range of cancers, these studies are

Representational difference analysis was developed by Thierry Boon's group and used to discover a number of CT antigens [20, 21] including the MAGE family of antigens, typically from melanoma with one exception, RAGE, from renal cancer. Briefly, total RNA was ex‐ tracted from normal tissue (driver) and a tumour sample (tester) and used to construct dou‐ ble-stranded cDNA. Both cDNA samples were digested with the restriction enzymes DpnII and ligated to adapters which contained primer binding sites. The fragments were amplified by PCR, the adapters removed and new adapters for unrelated primers ligated to the tester. The tester and driver were then mixed and hybridized leading to three combinations of product: driver-driver (no amplification), tester-driver (linear amplification) and tester-test‐ er (exponential amplification). A further two hybridization and amplification steps generate

greater variation in the products which are subsequently cloned and sequenced.

for immunotherapy include Wilms tumour 1 [13] and PRAME [14].

entirely limited to tumour antigens which had already been discovered.

**3. Identification of tumour antigens**

**3.2. Representational difference analysis**

**PCR)**

The differential expression of tumour antigens and/or protein biomarkers between cell and dis‐ ease subtypes have been directly compared on cDNA microarrays and has allowed our im‐ proved understanding of lymphomas [35] and aided our development of personalised therapies [36]. Microarray technology is able to distinguish between different subtypes of a par‐ ticular cancer as well as identify the expression of novel antigens [37]. Minimal residual disease is a very important tool in the detection of impending relapse in patients who have had some form of treatment. Markers for minimal residual disease in acute lymphocytic leukaemia were identified by gene profiling [38]. cDNA microarray has been used to identify the frequency of elevated tumour antigen expression in acute myeloid leukaemia [28] and also associations be‐ tween specific cytogenetic abnormalities and relative levels of tumour antigen expression [39]. Micorarray has also been used to elucidate the possible function of tumour antigens such as Synovial Sarcoma X breakpoint 2 Interacting Protein (SSX2IP) in the subversion of cells har‐ bouring cytogenetic abnormalities (t(8;21) associated with mitotic spindle failure and the asso‐ ciation between the elevated expression of some tumour antigens (SSX2IP, RHAMM and SURVIVIN) at disease presentation and patient survival [8] in acute myeloid leukaemia.

#### **3.6. Mass Spectroscopy (MS)**

Mass Spectroscopy (MS) involves the analysis of peptides eluted from the MHC of antigen presenting cells [40-42] or proteins in serum [43]. This area is reviewed more completely in the following reviews [44,45]. By using mass spectrometry, it has been demonstrated that as many as 10,000 different peptide species are presented by individual class I MHC alleles [46]. The technique, its strengths and limitations are extensively reviewed [47].

**4.4. Immunoprecipitation**

4,6'-diamino-2-phenylindole (DAPI).

sub-cellular localisation are not possible.

**4.5. Immunocytochemistry/histochemistry**

The protein of interest can be purified by incubating lysed cell extracts with its specific antibody in solution. Once the antibody has bonded with the protein, the resulting complex can be precipitated using agarose or G Sepharose beads which remove the required protein. The complex can be separated using sodium dodecyl sulphate-polyacrylamide gel electro‐ phoresis [55]. The sample can then be used to determine how much protein is present relative to other cells or other treatment conditions but denaturation is often required and details about

Identification of Targets for Immunotherapy http://dx.doi.org/10.5772/54698 301

The antigen of interest can be detected in cells (cytochemistry) or in tissues (histochemistry). The cells or tissue sections are fixed onto slides using a fixative such as paraformaldehyde to immobilise them. They are incubated with the primary antibody and then the secondary which is labelled with a detection molecule. The technique is qualitative informing the user about the sub-cellular localisation of the antigen in tissue and which cell types express it (Figure 1). However quantitation is often lacking and like most methods this requires optimised reagents.

**Figure 1.** Demonstration of the sub-cellular localisation of the tumour antigen SSX2IP in K562 cells using immuno‐ fluoresence microscopy. Cells were air dried for 4-18hours onto glass microscope slides and stored at -20oC wrapped in saranwrap. Cells were defrosted and stained with antigen specific primary and fluorescently labelled secondary an‐ tibodies. Using confocal microscopy we detected SSX2IP expression (observed as a green colour by virtue of anti-SSX2IP-fluorescein isothiocyanate) was detectable on the surface of the K562 cells. Cell nuclei were stained blue using

The advent of multiple tissue arrays from collaborators or commercial sources provides a

screening opportunity once the cancer(s) of interest for the antigen has been defined.

#### **3.7. Protein microarrays**

Protein microarrays involve the immunoscreening of protein arrays (approximately 9,000 full length proteins and functional domains) which may be purchased from companies such as Invitrogen, Functional Genomics or Cambridge Protein Arrays. Antibodies in sera from patients [33,48,49] can be detected using generic secondary antibodies (fluorescently conju‐ gated anti-human IgG) and visualised on microarray scanners.

## **4. Validation of the expression of tumour antigens in tumour cells**

Once TAAs have been identified their expression in tumour cells needs to be confirmed. There are a number of assays which can be used to validate the expression of antigens in tumour cells. Many of the most frequently used rely on an available antibody which has been validated [50,51]. Techniques frequently used include:-

#### **4.1. Reverse Transcription (RT-PCR)/Real-time PCR**

Total RNA is extracted from cells and used to make cDNA using reverse transcriptase. The cDNA product is amplified by PCR and run on an agarose gel to identify the presence of the transcribed gene in the cell [52]. This technique is sensitive and real-time PCR can provide relative quantitation, however both techniques only indicate the presence/level of gene ex‐ pression and not protein translation, which can vary greatly between antigens.

#### **4.2. Enzyme-Linked Immunosorbent Assay (ELISA)**

Enzyme-Linked Immunosorbent Assay (ELISA) is a straightforward procedure which can be used to detect an antigen using an antibody [53]. The antigen is attached to the bottom of a 96 well plate, or bound by a capture antibody on the bottom of a plate (in the case of a sandwich ELISA). The protein of interest is then incubated with a chemically labelled detection anti‐ body. In most experiments the chemical label is an enzyme and a substrate is added which will produce a colour change detectable by a microplate reader. The technique is sensitive and quan‐ titative when used in conjunction with appropriate protein concentration controls but is better fitted to the analysis of protein in urine and blood, rather than in tissues.

#### **4.3. Immunoblotting**

Other systems which use antigen-antibody interactions are techniques such as Western blot. Extracted proteins from tumours or cells are separated by 2-dimensional electrophoretic gels and then blotted onto nitrocellulose membranes. The membranes are incubated with primary and then secondary antibody. The secondary antibody is covalently labelled with an enzyme which reacts with a substrate solution generating colour, which then can be measured [54].

#### **4.4. Immunoprecipitation**

following reviews [44,45]. By using mass spectrometry, it has been demonstrated that as many as 10,000 different peptide species are presented by individual class I MHC alleles [46]. The

Protein microarrays involve the immunoscreening of protein arrays (approximately 9,000 full length proteins and functional domains) which may be purchased from companies such as Invitrogen, Functional Genomics or Cambridge Protein Arrays. Antibodies in sera from patients [33,48,49] can be detected using generic secondary antibodies (fluorescently conju‐

Once TAAs have been identified their expression in tumour cells needs to be confirmed. There are a number of assays which can be used to validate the expression of antigens in tumour cells. Many of the most frequently used rely on an available antibody which has

Total RNA is extracted from cells and used to make cDNA using reverse transcriptase. The cDNA product is amplified by PCR and run on an agarose gel to identify the presence of the transcribed gene in the cell [52]. This technique is sensitive and real-time PCR can provide relative quantitation, however both techniques only indicate the presence/level of gene ex‐

Enzyme-Linked Immunosorbent Assay (ELISA) is a straightforward procedure which can be used to detect an antigen using an antibody [53]. The antigen is attached to the bottom of a 96 well plate, or bound by a capture antibody on the bottom of a plate (in the case of a sandwich ELISA). The protein of interest is then incubated with a chemically labelled detection anti‐ body. In most experiments the chemical label is an enzyme and a substrate is added which will produce a colour change detectable by a microplate reader. The technique is sensitive and quan‐ titative when used in conjunction with appropriate protein concentration controls but is better

Other systems which use antigen-antibody interactions are techniques such as Western blot. Extracted proteins from tumours or cells are separated by 2-dimensional electrophoretic gels and then blotted onto nitrocellulose membranes. The membranes are incubated with primary and then secondary antibody. The secondary antibody is covalently labelled with an enzyme which reacts with a substrate solution generating colour, which then can be measured [54].

pression and not protein translation, which can vary greatly between antigens.

fitted to the analysis of protein in urine and blood, rather than in tissues.

**4. Validation of the expression of tumour antigens in tumour cells**

technique, its strengths and limitations are extensively reviewed [47].

gated anti-human IgG) and visualised on microarray scanners.

been validated [50,51]. Techniques frequently used include:-

**4.1. Reverse Transcription (RT-PCR)/Real-time PCR**

**4.2. Enzyme-Linked Immunosorbent Assay (ELISA)**

**3.7. Protein microarrays**

300 Novel Gene Therapy Approaches

**4.3. Immunoblotting**

The protein of interest can be purified by incubating lysed cell extracts with its specific antibody in solution. Once the antibody has bonded with the protein, the resulting complex can be precipitated using agarose or G Sepharose beads which remove the required protein. The complex can be separated using sodium dodecyl sulphate-polyacrylamide gel electro‐ phoresis [55]. The sample can then be used to determine how much protein is present relative to other cells or other treatment conditions but denaturation is often required and details about sub-cellular localisation are not possible.

#### **4.5. Immunocytochemistry/histochemistry**

The antigen of interest can be detected in cells (cytochemistry) or in tissues (histochemistry). The cells or tissue sections are fixed onto slides using a fixative such as paraformaldehyde to immobilise them. They are incubated with the primary antibody and then the secondary which is labelled with a detection molecule. The technique is qualitative informing the user about the sub-cellular localisation of the antigen in tissue and which cell types express it (Figure 1). However quantitation is often lacking and like most methods this requires optimised reagents.

**Figure 1.** Demonstration of the sub-cellular localisation of the tumour antigen SSX2IP in K562 cells using immuno‐ fluoresence microscopy. Cells were air dried for 4-18hours onto glass microscope slides and stored at -20oC wrapped in saranwrap. Cells were defrosted and stained with antigen specific primary and fluorescently labelled secondary an‐ tibodies. Using confocal microscopy we detected SSX2IP expression (observed as a green colour by virtue of anti-SSX2IP-fluorescein isothiocyanate) was detectable on the surface of the K562 cells. Cell nuclei were stained blue using 4,6'-diamino-2-phenylindole (DAPI).

The advent of multiple tissue arrays from collaborators or commercial sources provides a screening opportunity once the cancer(s) of interest for the antigen has been defined.

#### **4.6. Flow cytometry**

This technique allows the analyses of cells with a variety of parameters such as extracellular or intracellular markers, granularity, size and shape. Cancer cells are labelled with fluorescent antibodies for the required antigen. The cells are passed in a stream and intersected with a laser beam. The intensity of the fluorescence is measured and plotted in the form of dot plots and histograms. This technique is sensitive and informative to allow specific cell types to be "gated" by virtue of size, granularity and detectable protein expression. Machines can measure up to 19 parameters in the most sophisticated machines allowing multiple proteins and cell types to be analysed simultaneously [56]. However the technique requires validated antibodies that have been shown to be appropriate for fluorescence activated cell sorting analysis and enough tumour cells in suspension for analysis.

A2, indicating how long T cells will have to interact with peptide bound HLA-A2 before the

Identification of Targets for Immunotherapy http://dx.doi.org/10.5772/54698 303

There are a number of databases which can be mined to find epitopes which have already been shown to bind to HLA molecules. These have been used to identify established epitopes that

The SYFPEITHI database allows the prediction of MHC class I and II binding ligands for different mammalian species. When a search is carried out using a protein sequence, a prediction is made based on the amino acids in the anchor and auxiliary anchor posi‐ tions and other frequent amino acids which can bind to MHC molecules. A score is then calculated which follows certain rules which are: a numerical value of 10 is given to ami‐ no acids that regularly arise in anchor positions, the value 8 is set for amino acids occur‐ ring in a significant number of ligands, six is for unusual anchors such as auxiliary anchors and less frequent residues of the same set have a value of four. Preferred amino acids have coefficients between 1–4 depending on the signal strength in pool sequencing or the occurrence of individual sequences. Amino acids that are considered as having an adverse effect on binding have a coefficient of –1 to –3 [60]. SYFPEITHI database gets updated regularly and has been used to identify various ligands; p28 peptide as an epit‐ ope for the CT antigen PLAC1 in breast cancer [61], p101-111 is the first CTA-derived peptide which induces CD4(+), CD8(+), and B-cell responses *in vitro* [62], p43-57 epitope stimulates T cells in HCA587-derived tumours [63] and PASD1(1) – PASD1(5) [51].

Bioinformatics and Molecular Analysis Section (BIMAS) develops computational processes to analyse data generated from molecular biology and genetics research; and provides bioinfor‐ matics guidance, support and resources for the collection, management, and display of biological sequence and genomic information for scientists involved in genomics and genetic analysis [64]. Other online software which can be used to identify epitopes includes EpiJen,

There are a number of assays which can be used to determine if T cells are activated in response

**7. Cell based assays —** *In vitro* **demonstration of T cell reactivity**

complex falls apart.

**6.1. The SYFPEITHI**

**6.** *In silico* **identification of epitopes**

may be used in immunotherapy strategies.

**6.2. Bioinformatics and Molecular Analysis Section (BIMAS)**

Rankpep, nHLApred, NetCTL and Multipred [65].

to antigen.

## **5. Identification of HLA-binding epitopes —** *in vitro* **assays**

Immune responses in the body can ensure that any foreign matter is eliminated effectively. Class I and II major histocompatability complexes (MHC) are present on the surface of nucleated cells and present processed peptides from proteins inside the cell to T cells. T cells can destroy infected cells if peptides in the context of "danger" are detected [57]. MHC in humans is known as the human leukocyte antigen (HLA) system. MHC class I HLA molecules are highly polymorphic and generally the best defence against infections.

#### **5.1. MHC Peptide binding assay**

Peptide antigens are stripped from the HLA class I molecules by mild acid treatment, cells are then incubated with a fluorescent reference peptide together with different concentrations of the peptide of interest. The efficiency with which the required peptide competes for binding to the HLA class I molecules is examined by measuring the amount of HLA-bound reference peptide with fluorescence activated cell sorting analysis [58].

#### **5.2. T2** *in vitro* **HLA-A2 binding assay**

T2 *in vitro* HLA-A2 binding assay is more frequently used to determine the strength of peptide binding to the most common HLA molecule in Caucasian populations. The HLA-A2 express‐ ing, TAP-1 deficient human T-cell line T2 is used as an assay of HLA-A2 peptide binding efficiency. T2 cells are washed and resuspended in serum-free RPMI media and plated in 96 well microtitre plates. Human β2-microglobulin and often nonamers (nine amino acids long peptides) are added and the cells are incubated overnight at 37°C/5% CO2. The cells are washed and probed with a HLA-A2-specific monoclonal antibody and appropriate secondary anti‐ body prior to flow cytometry. Only HLA-A2 molecules bound to peptide are stabilised and detectable on the cell surface. Results are reported as a relative mean fluorescence index (MFI), calculated as the MFI of peptide-pulsed T2 cells compared with the MFI of unpulsed T2 cells [59]. Time course assays may be used to indicate how long the peptide remains on the HLA- A2, indicating how long T cells will have to interact with peptide bound HLA-A2 before the complex falls apart.

## **6.** *In silico* **identification of epitopes**

There are a number of databases which can be mined to find epitopes which have already been shown to bind to HLA molecules. These have been used to identify established epitopes that may be used in immunotherapy strategies.

#### **6.1. The SYFPEITHI**

**4.6. Flow cytometry**

302 Novel Gene Therapy Approaches

enough tumour cells in suspension for analysis.

**5.1. MHC Peptide binding assay**

**5.2. T2** *in vitro* **HLA-A2 binding assay**

**5. Identification of HLA-binding epitopes —** *in vitro* **assays**

are highly polymorphic and generally the best defence against infections.

peptide with fluorescence activated cell sorting analysis [58].

This technique allows the analyses of cells with a variety of parameters such as extracellular or intracellular markers, granularity, size and shape. Cancer cells are labelled with fluorescent antibodies for the required antigen. The cells are passed in a stream and intersected with a laser beam. The intensity of the fluorescence is measured and plotted in the form of dot plots and histograms. This technique is sensitive and informative to allow specific cell types to be "gated" by virtue of size, granularity and detectable protein expression. Machines can measure up to 19 parameters in the most sophisticated machines allowing multiple proteins and cell types to be analysed simultaneously [56]. However the technique requires validated antibodies that have been shown to be appropriate for fluorescence activated cell sorting analysis and

Immune responses in the body can ensure that any foreign matter is eliminated effectively. Class I and II major histocompatability complexes (MHC) are present on the surface of nucleated cells and present processed peptides from proteins inside the cell to T cells. T cells can destroy infected cells if peptides in the context of "danger" are detected [57]. MHC in humans is known as the human leukocyte antigen (HLA) system. MHC class I HLA molecules

Peptide antigens are stripped from the HLA class I molecules by mild acid treatment, cells are then incubated with a fluorescent reference peptide together with different concentrations of the peptide of interest. The efficiency with which the required peptide competes for binding to the HLA class I molecules is examined by measuring the amount of HLA-bound reference

T2 *in vitro* HLA-A2 binding assay is more frequently used to determine the strength of peptide binding to the most common HLA molecule in Caucasian populations. The HLA-A2 express‐ ing, TAP-1 deficient human T-cell line T2 is used as an assay of HLA-A2 peptide binding efficiency. T2 cells are washed and resuspended in serum-free RPMI media and plated in 96 well microtitre plates. Human β2-microglobulin and often nonamers (nine amino acids long peptides) are added and the cells are incubated overnight at 37°C/5% CO2. The cells are washed and probed with a HLA-A2-specific monoclonal antibody and appropriate secondary anti‐ body prior to flow cytometry. Only HLA-A2 molecules bound to peptide are stabilised and detectable on the cell surface. Results are reported as a relative mean fluorescence index (MFI), calculated as the MFI of peptide-pulsed T2 cells compared with the MFI of unpulsed T2 cells [59]. Time course assays may be used to indicate how long the peptide remains on the HLA-

The SYFPEITHI database allows the prediction of MHC class I and II binding ligands for different mammalian species. When a search is carried out using a protein sequence, a prediction is made based on the amino acids in the anchor and auxiliary anchor posi‐ tions and other frequent amino acids which can bind to MHC molecules. A score is then calculated which follows certain rules which are: a numerical value of 10 is given to ami‐ no acids that regularly arise in anchor positions, the value 8 is set for amino acids occur‐ ring in a significant number of ligands, six is for unusual anchors such as auxiliary anchors and less frequent residues of the same set have a value of four. Preferred amino acids have coefficients between 1–4 depending on the signal strength in pool sequencing or the occurrence of individual sequences. Amino acids that are considered as having an adverse effect on binding have a coefficient of –1 to –3 [60]. SYFPEITHI database gets updated regularly and has been used to identify various ligands; p28 peptide as an epit‐ ope for the CT antigen PLAC1 in breast cancer [61], p101-111 is the first CTA-derived peptide which induces CD4(+), CD8(+), and B-cell responses *in vitro* [62], p43-57 epitope stimulates T cells in HCA587-derived tumours [63] and PASD1(1) – PASD1(5) [51].

#### **6.2. Bioinformatics and Molecular Analysis Section (BIMAS)**

Bioinformatics and Molecular Analysis Section (BIMAS) develops computational processes to analyse data generated from molecular biology and genetics research; and provides bioinfor‐ matics guidance, support and resources for the collection, management, and display of biological sequence and genomic information for scientists involved in genomics and genetic analysis [64]. Other online software which can be used to identify epitopes includes EpiJen, Rankpep, nHLApred, NetCTL and Multipred [65].

## **7. Cell based assays —** *In vitro* **demonstration of T cell reactivity**

There are a number of assays which can be used to determine if T cells are activated in response to antigen.

### **7.1. Carboxyfluorescein diacetate Succinimidyl Ester (CFSE)**

Carboxyfluorescein diacetate succinimidyl ester (CFSE) is a cytoplasmic dye which is absorbed by all cell types. Once the CFSE labelled cells divide, the dye is shared amongst the daughter cells equally therefore the fluorescence is halved after each round of the cell cycle. This difference in fluorescence can be measured. The more cells proliferate, the greater the decrease in the fluorescent signal. The fluorescence peaks can be measured by flow cytometry [66]. CFSE labelling is increasingly used to measure target tumour cell killing [67], superseding radiation based assays, as well as T cell proliferation in response to tumour cells *in vivo* [68]. CFSE labelling can also be performed *in vivo* where the dye is injected into the host animal's spleen or lymph nodes, however the labelling is not uniform and it is sometimes difficult to obtain individual peaks once lymphocyte cell division has occurred [66].

been generated but won't determine which T cells, if they are indeed T cells, have been activated. pMHCs, often referred to as tetramers, can be used to identify antigen specific T cells. They are produced through the refolding of β2-microglobulin and heavy chains in MHC molecules with the appropriate epitope of interest. The pMHC is then labelled with biotin using BirA enzyme. A streptavidin molecule conjugated to a fluorescent detector binds to four (tetramers) pMHCs or can used to create multimers (for example dimers, pentamers, dex‐ tramers) of these constructed MHC molecules courtesy of the biotin-avidin interaction [73]. T cell populations are added to this mixture and T cells with the specific receptor for the epitope of interest will bind and be measurable by flow cytometry [74]. Shen *et al* [74] have found that cross-reactive T cells i.e. T cells which recognise two different antigens can be identified providing an extra tool in vaccine development. In some cases antigen specific T cells may not bind tetramers due to being undifferentiated and unable to accumulate T cell receptor (TCR) molecules close to the antigen. Another reason could be low affinity between TCR and MHC [75]. Other techniques based on the use of pMHCs include pMHC arrays [76] (Section 7.5), NACS [77] and the combinatorial approach [78,79]. These techniques all provide high through‐ put analysis of multiple T cell populations with a variety of pros and cons to each technique including issues with background, specificity/binding capacity of individual pMHC com‐ plexes, activated induced cell death of pMHC bound T cells, internalisation of pMHCs following T cell binding [80], cost and labour intensity. Sequencing of TCRs (2-3 million every 2-3 days) by companies such as TRON gGmbH (Johannes Gutenberg University Mainz, Germany) and Adaptive Biotechnologies (Seattle, USA) will provide a new way of analysing T cell populations which will be informative with regards to which TCRs are present but not necessarily whether they are present on mature, anergic, activated or functional T cells nor which sub-group of T cells are harbouring them (helper T cells, cytotoxic T cells, Th17 cells or indeed regulatory T cells (Tregs)). This technology allows the first opportunity to examine an extremely large number of TCRs in a very short time and will revolutionise how we examine

Identification of Targets for Immunotherapy http://dx.doi.org/10.5772/54698 305

pMHC or tetramer arrays [76,81] (Figure 2) provides a strategy to determine which spe‐ cific CD8+ T cell populations are present in the peripheral blood of patients. Antigens identified by the techniques described already can be used to help expand the pMHC ar‐ ray for future studies. In addition, the pMHC array provides a means to investigate epit‐ ope spreading and changes in T cell specificities with disease progression. The technique benefits from the low number of purified CD8+ T cells required for each array (0.5-2 x

), which can be purified from 20ml of patient peripheral blood using StemCell CD8+ negative isolation beads providing "untouched" T cells (Bonney, Guinn *et al*, in prepara‐ tion). The purified CD8+ T cells are then lipophyllically dyed with DiD (Molecular Probes), washed and incubated with the pMHC array. The pMHC array has a detection limit of 0.02% matching the sensitivity we can reproducibly achieve with flow cytometry when analysing patient samples. Where sample availability permits, pMHC array data should be validated by flow cytometry [82] using the same pMHC tetramers as in the pMHC array. The pMHC array has the added advantage that it can be used for the ini‐

T cell responses in patients in the future.

**7.5. pMHC arrays**

106

### **7.2. Lymphocyte proliferation assays**

Lymphocyte proliferation assays can be used to determine activation of T cells. Peripheral blood mononuclear cells are isolated and cultured in microtitre plates. The specific antigen is incubated with the cells, which causes the T cells to divide and grow. The MTT colorimetric assay is based upon (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazolium salt which gets cleaved by enzymes in the mitochondria to produce blue formazan. Viable, dividing cells will create more formazan which can be quantified using a plate reader. MTT assay is very convenient, however some considerations need to be assessed to avoid false positives such as cell densities, correct culture medium, filtration of media to remove precip‐ itate, optimisation of MTT concentrations and incubation times [69].

#### **7.3. [3 H]-Thymidine incorporation assay**

[ 3 H]-Thymidine incorporation assay is based on the use of [3 H]-thymidine a radioactive molecule which can be incorporated into DNA during the S-phase of cell division. As new DNA is synthesised, occasional thymidine bases are replaced by [3 H]-thymidine and subsequently the incorporated radioactivity is measured, following washes to remove un‐ incorporated radioactivity, using a Scintillation Counter [70]. As [3 H]-thymidine is radio‐ active an analogue called bromodeoxyuridine (BrdU) was developed to replace it in assays. BrdU integrates into DNA strands and can be measured using immunohisto‐ chemistry and flow cytometry protocols using fluorescent conjugates and can be ob‐ served over a longer duration. BrdU is also used to look at the number of cells in each part of the cell cycle by flow cytometry [71]. However BrdU has been found to be more toxic than [3 H]-thymidine, possibly because it is structurally very different to the original DNA nucleotides. It also adversely affects cell division, the pattern of cell migration, fi‐ nal position of migrating cells and the fate of labelled cells [72].

#### **7.4. Peptide-MHC (pMHCs)**

Peptide-MHC (pMHCs) based assays circumvent issues caused by measuring T cell prolifer‐ ation. T cell proliferation assays can provide information on whether an immune response has been generated but won't determine which T cells, if they are indeed T cells, have been activated. pMHCs, often referred to as tetramers, can be used to identify antigen specific T cells. They are produced through the refolding of β2-microglobulin and heavy chains in MHC molecules with the appropriate epitope of interest. The pMHC is then labelled with biotin using BirA enzyme. A streptavidin molecule conjugated to a fluorescent detector binds to four (tetramers) pMHCs or can used to create multimers (for example dimers, pentamers, dex‐ tramers) of these constructed MHC molecules courtesy of the biotin-avidin interaction [73]. T cell populations are added to this mixture and T cells with the specific receptor for the epitope of interest will bind and be measurable by flow cytometry [74]. Shen *et al* [74] have found that cross-reactive T cells i.e. T cells which recognise two different antigens can be identified providing an extra tool in vaccine development. In some cases antigen specific T cells may not bind tetramers due to being undifferentiated and unable to accumulate T cell receptor (TCR) molecules close to the antigen. Another reason could be low affinity between TCR and MHC [75]. Other techniques based on the use of pMHCs include pMHC arrays [76] (Section 7.5), NACS [77] and the combinatorial approach [78,79]. These techniques all provide high through‐ put analysis of multiple T cell populations with a variety of pros and cons to each technique including issues with background, specificity/binding capacity of individual pMHC com‐ plexes, activated induced cell death of pMHC bound T cells, internalisation of pMHCs following T cell binding [80], cost and labour intensity. Sequencing of TCRs (2-3 million every 2-3 days) by companies such as TRON gGmbH (Johannes Gutenberg University Mainz, Germany) and Adaptive Biotechnologies (Seattle, USA) will provide a new way of analysing T cell populations which will be informative with regards to which TCRs are present but not necessarily whether they are present on mature, anergic, activated or functional T cells nor which sub-group of T cells are harbouring them (helper T cells, cytotoxic T cells, Th17 cells or indeed regulatory T cells (Tregs)). This technology allows the first opportunity to examine an extremely large number of TCRs in a very short time and will revolutionise how we examine T cell responses in patients in the future.

#### **7.5. pMHC arrays**

**7.1. Carboxyfluorescein diacetate Succinimidyl Ester (CFSE)**

individual peaks once lymphocyte cell division has occurred [66].

itate, optimisation of MTT concentrations and incubation times [69].

H]-Thymidine incorporation assay is based on the use of [3

new DNA is synthesised, occasional thymidine bases are replaced by [3

incorporated radioactivity, using a Scintillation Counter [70]. As [3

nal position of migrating cells and the fate of labelled cells [72].

**7.2. Lymphocyte proliferation assays**

304 Novel Gene Therapy Approaches

**H]-Thymidine incorporation assay**

**7.3. [3**

toxic than [3

**7.4. Peptide-MHC (pMHCs)**

[ 3

Carboxyfluorescein diacetate succinimidyl ester (CFSE) is a cytoplasmic dye which is absorbed by all cell types. Once the CFSE labelled cells divide, the dye is shared amongst the daughter cells equally therefore the fluorescence is halved after each round of the cell cycle. This difference in fluorescence can be measured. The more cells proliferate, the greater the decrease in the fluorescent signal. The fluorescence peaks can be measured by flow cytometry [66]. CFSE labelling is increasingly used to measure target tumour cell killing [67], superseding radiation based assays, as well as T cell proliferation in response to tumour cells *in vivo* [68]. CFSE labelling can also be performed *in vivo* where the dye is injected into the host animal's spleen or lymph nodes, however the labelling is not uniform and it is sometimes difficult to obtain

Lymphocyte proliferation assays can be used to determine activation of T cells. Peripheral blood mononuclear cells are isolated and cultured in microtitre plates. The specific antigen is incubated with the cells, which causes the T cells to divide and grow. The MTT colorimetric assay is based upon (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazolium salt which gets cleaved by enzymes in the mitochondria to produce blue formazan. Viable, dividing cells will create more formazan which can be quantified using a plate reader. MTT assay is very convenient, however some considerations need to be assessed to avoid false positives such as cell densities, correct culture medium, filtration of media to remove precip‐

molecule which can be incorporated into DNA during the S-phase of cell division. As

subsequently the incorporated radioactivity is measured, following washes to remove un‐

active an analogue called bromodeoxyuridine (BrdU) was developed to replace it in assays. BrdU integrates into DNA strands and can be measured using immunohisto‐ chemistry and flow cytometry protocols using fluorescent conjugates and can be ob‐ served over a longer duration. BrdU is also used to look at the number of cells in each part of the cell cycle by flow cytometry [71]. However BrdU has been found to be more

DNA nucleotides. It also adversely affects cell division, the pattern of cell migration, fi‐

Peptide-MHC (pMHCs) based assays circumvent issues caused by measuring T cell prolifer‐ ation. T cell proliferation assays can provide information on whether an immune response has

H]-thymidine, possibly because it is structurally very different to the original

H]-thymidine a radioactive

H]-thymidine and

H]-thymidine is radio‐

pMHC or tetramer arrays [76,81] (Figure 2) provides a strategy to determine which spe‐ cific CD8+ T cell populations are present in the peripheral blood of patients. Antigens identified by the techniques described already can be used to help expand the pMHC ar‐ ray for future studies. In addition, the pMHC array provides a means to investigate epit‐ ope spreading and changes in T cell specificities with disease progression. The technique benefits from the low number of purified CD8+ T cells required for each array (0.5-2 x 106 ), which can be purified from 20ml of patient peripheral blood using StemCell CD8+ negative isolation beads providing "untouched" T cells (Bonney, Guinn *et al*, in prepara‐ tion). The purified CD8+ T cells are then lipophyllically dyed with DiD (Molecular Probes), washed and incubated with the pMHC array. The pMHC array has a detection limit of 0.02% matching the sensitivity we can reproducibly achieve with flow cytometry when analysing patient samples. Where sample availability permits, pMHC array data should be validated by flow cytometry [82] using the same pMHC tetramers as in the pMHC array. The pMHC array has the added advantage that it can be used for the ini‐ tial screening of a relatively small number of CD8+ T cells against a large number of pMHCs on the array, and a short-list of T cell populations which are shown to exist on the pMHC array can then be quantitated by flow cytometry (limiting the amount of sam‐ ple required in subsequent studies). The pMHC array can be used to analyse patient samples at a number of disease time-points (presentation, post-treatment (surgery and/or radiotherapy) and with disease progression) to examine how T cell responses to tumour antigens change with treatment, to examine epitope spreading and to correlate changing immune responses with clinical responses.

printed multiplexed AF532-conjugated pMHCs at a concentration of 0.5mg/ml so that 1ng was placed in each spot (shown as coloured ovals). Each spot is 400uM wide with a 700uM inter-spot distance. (B) At least six spots per pMHC are applied in each of two independent sites. Each hydrogel slide can hold up to 1,000 spots of tetramer in total. (C) We applied 0.8- 1.5 x 10<sup>6</sup> lipophillically-dyed CD8<sup>+</sup> T cells/slide (seen in red). pMHCs included a range of HLA restrictions and had already been shown to work in flow cytometry studies,(i) pMHCs are spotted across the gel, (seen as green spots) (ii) even some HLA-A2 positive patients show no reactivity with any pMHCs (iii) while others show the presence of multiple T cell populations which recognise tumour antigens. Based on figure in [144]. **Figure 2.** pMHC arrays for the simultaneous detection of T cell populations in patient peripheral blood. (A) Using a QArray2 printer and HPLF 0.3mm solid tip pins (Genetix) we printed multiplexed AF532-conjugated pMHCs at a con‐ centration of 0.5mg/ml so that 1ng was placed in each spot (shown as coloured ovals). Each spot is 400uM wide with a 700uM inter-spot distance. (B) At least six spots per pMHC are applied in each of two independent sites. Each hydro‐ gel slide can hold up to 1,000 spots of tetramer in total. (C) We applied 0.8-1.5 x 106 lipophillically-dyed CD8+ T cells/ slide (seen in red). pMHCs included a range of HLA restrictions and had already been shown to work in flow cytometry studies,(i) pMHCs are spotted across the gel, (seen as green spots) (ii) even some HLA-A2 positive patients show no reactivity with any pMHCs (iii) while others show the presence of multiple T cell populations which recognise tumour antigens. Based on figure in [144].

**Figure 2.** pMHC arrays for the simultaneous detection of T cell populations in patient peripheral blood. (A) Using a QArray2 printer and HPLF 0.3mm solid tip pins (Genetix) we

39

**7.6. Intracellular cytokine staining assay**

ing 6-16 hours of culture only IFN-γ cytokine was found [84].

cytotoxic T cell response in the presence of longer CD4<sup>+</sup>

sometimes longer proteins can inhibit CD8+

**8. Cell based assays —** *In vivo* **assays**

molecules as described in this section.

**8.1. Immunodeficient mice**

on the constituents of individual protein sequences.

Intracellular cytokine staining assay detects particular cytokines released by immune cells which can provide a useful insight into the responding T cell populations. Such cy‐ tokines could include interferon-gamma (IFNγ), interleukin-2 (IL-2), IL-4 and tumour ne‐ crosis factor-*α*. Cells are plated and incubated with the antigen to stimulate cytokine production. To prevent the cytokine from exiting the cell a transport inhibitor is added e.g. brefeldin A. The cells are then fixed in paraformaldehyde and permeabilized to al‐ low the anti-cytokine antibody to bind. The results are analysed by flow cytometry. The use of intracellular cytokine staining assay to detect the cytokine IFNγ shows high repro‐ ducibility and linearity with little background [83]. Duration of culture prior to antigen stimulation, as well as the cytokine accumulation period, are critical parameters of these methods. In both murine and cattle models, following 2-6 hours in culture, T cells pro‐ duced a mixture of cytokines IFNγ, IL-2 and tumour necrosis factor-α, however follow‐

Using multiple peptides from distinct TAAs to stimulate immune cells may prove the most effective for peptide vaccines. A cocktail of four multiple myeloma antigen peptides were used to stimulate T lymphocytes from HLA-A2 positive people to induce IFNγ production, cell proliferation and cytotoxicity against HLA-A2 positive multiple myeloma patients' cells [85]. Indeed long peptides may offer the advantage of allowing the immune system to choose the epitope(s) it can best process and present from a peptide sequence and induce an effective

There are a number of approaches that can be taken when using mouse models to detect T cell immune responses. Animals can be used in transplantable tumour (xenografts) models or genetically engineered tumour models. In xenograft models human tumour cells are taken and injected into immunodeficient mice so that complex immune respons‐ es involving multiple cell types can be investigated. In contrast, in genetically engineered models, genes known to cause cancer are activated or tumour suppressor genes are "switched off" to allow their effects on tumour growth to be examined. In addition transgenic mice can be used to examine T cell responses to epitopes presented on MHC

Immunodeficient mice such as athymic nude mice, severely compromised immunodefi‐ cient (SCID) mice and non-obese diabetic severe combined immunodeficiency mice will accept xenografts of human cells [88]. Depending upon the number of cells injected, or

helper motifs [86]. Conversely

Identification of Targets for Immunotherapy http://dx.doi.org/10.5772/54698 307

T cells responses [87] but this may vary depending

#### **7.6. Intracellular cytokine staining assay**

tial screening of a relatively small number of CD8+ T cells against a large number of pMHCs on the array, and a short-list of T cell populations which are shown to exist on the pMHC array can then be quantitated by flow cytometry (limiting the amount of sam‐ ple required in subsequent studies). The pMHC array can be used to analyse patient samples at a number of disease time-points (presentation, post-treatment (surgery and/or radiotherapy) and with disease progression) to examine how T cell responses to tumour antigens change with treatment, to examine epitope spreading and to correlate changing

**Figure 2.** pMHC arrays for the simultaneous detection of T cell populations in patient peripheral blood. (A) Using a QArray2 printer and HPLF 0.3mm solid tip pins (Genetix) we printed multiplexed AF532-conjugated pMHCs at a concentration of 0.5mg/ml so that 1ng was placed in each spot (shown as coloured ovals). Each spot is 400uM wide with a 700uM inter-spot distance. (B) At least six spots per pMHC are applied in each of two independent sites. Each hydrogel slide can hold up to 1,000 spots of tetramer in total. (C) We applied 0.8- 1.5 x 10<sup>6</sup> lipophillically-dyed CD8<sup>+</sup> T cells/slide (seen in red). pMHCs included a range of HLA restrictions and had already been shown to work in flow cytometry studies,(i) pMHCs are spotted across the gel, (seen as green spots) (ii) even some HLA-A2 positive patients show no reactivity with any pMHCs (iii) while others show the presence of multiple T cell

**Figure 2.** pMHC arrays for the simultaneous detection of T cell populations in patient peripheral blood. (A) Using a QArray2 printer and HPLF 0.3mm solid tip pins (Genetix) we printed multiplexed AF532-conjugated pMHCs at a con‐ centration of 0.5mg/ml so that 1ng was placed in each spot (shown as coloured ovals). Each spot is 400uM wide with a 700uM inter-spot distance. (B) At least six spots per pMHC are applied in each of two independent sites. Each hydro‐ gel slide can hold up to 1,000 spots of tetramer in total. (C) We applied 0.8-1.5 x 106 lipophillically-dyed CD8+ T cells/ slide (seen in red). pMHCs included a range of HLA restrictions and had already been shown to work in flow cytometry studies,(i) pMHCs are spotted across the gel, (seen as green spots) (ii) even some HLA-A2 positive patients show no reactivity with any pMHCs (iii) while others show the presence of multiple T cell populations which recognise tumour

populations which recognise tumour antigens. Based on figure in [144].

antigens. Based on figure in [144].

immune responses with clinical responses.

306 Novel Gene Therapy Approaches

Intracellular cytokine staining assay detects particular cytokines released by immune cells which can provide a useful insight into the responding T cell populations. Such cy‐ tokines could include interferon-gamma (IFNγ), interleukin-2 (IL-2), IL-4 and tumour ne‐ crosis factor-*α*. Cells are plated and incubated with the antigen to stimulate cytokine production. To prevent the cytokine from exiting the cell a transport inhibitor is added e.g. brefeldin A. The cells are then fixed in paraformaldehyde and permeabilized to al‐ low the anti-cytokine antibody to bind. The results are analysed by flow cytometry. The use of intracellular cytokine staining assay to detect the cytokine IFNγ shows high repro‐ ducibility and linearity with little background [83]. Duration of culture prior to antigen stimulation, as well as the cytokine accumulation period, are critical parameters of these methods. In both murine and cattle models, following 2-6 hours in culture, T cells pro‐ duced a mixture of cytokines IFNγ, IL-2 and tumour necrosis factor-α, however follow‐ ing 6-16 hours of culture only IFN-γ cytokine was found [84].

Using multiple peptides from distinct TAAs to stimulate immune cells may prove the most effective for peptide vaccines. A cocktail of four multiple myeloma antigen peptides were used to stimulate T lymphocytes from HLA-A2 positive people to induce IFNγ production, cell proliferation and cytotoxicity against HLA-A2 positive multiple myeloma patients' cells [85]. Indeed long peptides may offer the advantage of allowing the immune system to choose the epitope(s) it can best process and present from a peptide sequence and induce an effective cytotoxic T cell response in the presence of longer CD4<sup>+</sup> helper motifs [86]. Conversely sometimes longer proteins can inhibit CD8+ T cells responses [87] but this may vary depending on the constituents of individual protein sequences.

### **8. Cell based assays —** *In vivo* **assays**

There are a number of approaches that can be taken when using mouse models to detect T cell immune responses. Animals can be used in transplantable tumour (xenografts) models or genetically engineered tumour models. In xenograft models human tumour cells are taken and injected into immunodeficient mice so that complex immune respons‐ es involving multiple cell types can be investigated. In contrast, in genetically engineered models, genes known to cause cancer are activated or tumour suppressor genes are "switched off" to allow their effects on tumour growth to be examined. In addition transgenic mice can be used to examine T cell responses to epitopes presented on MHC molecules as described in this section.

#### **8.1. Immunodeficient mice**

39

Immunodeficient mice such as athymic nude mice, severely compromised immunodefi‐ cient (SCID) mice and non-obese diabetic severe combined immunodeficiency mice will accept xenografts of human cells [88]. Depending upon the number of cells injected, or the size of the tumour transplanted, the tumour can develop over weeks to months and the response to appropriate therapeutic agents studied *in vivo* [89]. Indeed such models have been used to examine the effectiveness of various immunotherapeutic strategies in‐ cluding whole cell vaccines [90], dendritic cell (DC) vaccines [91], peptide vaccines [92] and DNA vaccines [67,87,93].

monoclonal antibodies (mAb) have become standard treatments for cancer. Ultimately if there is an antibody and it recognises a surface antigen solely on cancer and non-essential cells then this will likely be the most effective way to cause tumour destruction. mAbs are derived by vaccinating an animal with the target antigen and testing to see if the B cells are producing antibodies against it. Then the B cells are extracted from its spleen and infused with myeloma cells to produce hybridomas. Hybridomas divide perpetually and produce the mAb to the

Rosenberg *et al* [107] showed that only 2.6% of immunotherapy clinical trials had worked and therefore an overhaul was needed in the practice of immunotherapy. Subse‐ quently the same group showed that adoptive T cell therapy could be very promising with cell numbers being returned to the patient [108] and their status – activated but not mature [109] and cell numbers being the main issues. It is also likely that the best strat‐ egy may include a combination of conventional and immunotherapy techniques [110] or even a combination of immunotherapy techniques as demonstrated in increasing num‐

DCs are antigen presenting cells therefore they have received some attention for possible use in cancer immunotherapy. DCs pulsed by peptide and injected into the skin showed a response rate of 28%. This percentage increased to 35.7% when immature DCs are injected straight into

Tumour-infiltrating T cells (TIL) therapy has been used in stage IV melanoma patients. TILs are obtained from the blood, lymph nodes or from a tumour tissue biopsy. TILs are isolated, activated and expanded using IL-2 *in vitro*. The patient undergoes lympho-depleting chemo‐

When a tumour antigen is secreted into the circulation in high levels immune tolerance

antigens in the blood, which can induce tolerance through Tregs or negative selection. Tregs are cells which are part of the tolerance system which prevents autoimmunity [115, 116]. Simultaneous Treg depletions (using anti-CD25 antibodies for instance) may aid the effectiveness of immunotherapy in some cancer types where Treg infiltration into the tu‐

There are a number of reviews in this area of research which aim to look into effective immunotherapy strategies for the future. These include cellular immunotherapy [119], whole call vaccines [120], multidrug resistance [121] and DCs [122]. Targeted therapeutic strategies along with ever improving designs in clinical trials pave the way for further

Clinical trials are undertaken after a large amount of data has been obtained on the antigen of interest in the lab. This data is required to ensure treatment safety and efficacy as far as is

, a subset of DCs, are able to capture tumour

Identification of Targets for Immunotherapy http://dx.doi.org/10.5772/54698 309

Sirpα<sup>+</sup>

the tumour and even higher to 40% for advanced pancreatic cancer [113].

therapy prior to the T cells being injected back in to the blood [114].

antigen in large amounts [106].

bers of mouse models [111] and clinical trials [112].

can be induced in the thymus. CD8α−

mour is rife [117,118].

success [123].

**10. Clinical trials**

#### **8.2. Genetically modified mice**

Genetically modified mice may have genes which are overexpressed or deleted and the effects of these genes on tumour development can be studied. Examples include p53 null and heterogenous mice [94,95] demonstrated that these genes can act as oncogenes and lead to tumour development. Possible therapies for these oncogenes/tumour suppressor genes are tested for their response in an *in vivo*, full organism context [89] and examples include Ad-p53, AAV-HGFK1 [96].

HLA-A2 transgenic mice have genes inserted into the DNA so they will express the HLA molecules known in mice as H-2. In order to prevent the presentation of murine H-2-restricted cytotoxic T lymphocyte (CTL) epitopes in HLA-A2 (AAD) transgenic mice, HLA-A2.1 transgenic/H-2 class I knockout mice (HHD mice) were created [97]. In HHD mice, the H-2 class I gene is knocked out, and a chimeric HLA-A2.1 monochain (HHD) is produced by linking the C terminal of the human β2-microglobulin (unit of the class 1 MHC) covalently to the Nterminus of the chimeric HLA-A2 heavy chain (which contain the α1&2 domains of HLA-A2.1 and the α3 domain of H-2Db) by means of a peptide bond. This guarantees the sole expression of the HHD molecule on the cell surface, making sure that any identified CTL epitopes are HLA-A2 restricted [98]. HHD mice allow epitopes which are presented on human HLA-A2 to be examined for their ability to induce T cell responses in a variety of studies; for example STEAP, a prostate tumour antigen has been shown to be targeted by anti-tumour T cells [99] and DNA vaccines encoding Wilms tumour antigen 1 induce cytotoxic responses in mice [100] using this model system.

## **9. Modes of immunotherapy**

One of the biggest debates in cancer immunotherapy remains which mode will be the most effective. The National Cancer Institute have suggested that immunotherapy stud‐ ies should focus on a limited number of antigenic targets to maximise the chances of success [101]. However for some cancers effective immunotherapy targets have yet to be discovered (i.e. ovarian cancer, adult acute lymphocytic leukaemia) and better targets may yet be determined.

When T cells were found to be able to identify cancer cells [102] it was thought that T cell therapies would be the most effective with the aim being to stimulate CD8+ /CTL cells to kill tumour cells. This can be achieved by a number of ways such as through the use of DCs [103], peptide vaccines [85], DNA vaccines [104] and natural killer cells [105]. In recent years monoclonal antibodies (mAb) have become standard treatments for cancer. Ultimately if there is an antibody and it recognises a surface antigen solely on cancer and non-essential cells then this will likely be the most effective way to cause tumour destruction. mAbs are derived by vaccinating an animal with the target antigen and testing to see if the B cells are producing antibodies against it. Then the B cells are extracted from its spleen and infused with myeloma cells to produce hybridomas. Hybridomas divide perpetually and produce the mAb to the antigen in large amounts [106].

Rosenberg *et al* [107] showed that only 2.6% of immunotherapy clinical trials had worked and therefore an overhaul was needed in the practice of immunotherapy. Subse‐ quently the same group showed that adoptive T cell therapy could be very promising with cell numbers being returned to the patient [108] and their status – activated but not mature [109] and cell numbers being the main issues. It is also likely that the best strat‐ egy may include a combination of conventional and immunotherapy techniques [110] or even a combination of immunotherapy techniques as demonstrated in increasing num‐ bers of mouse models [111] and clinical trials [112].

DCs are antigen presenting cells therefore they have received some attention for possible use in cancer immunotherapy. DCs pulsed by peptide and injected into the skin showed a response rate of 28%. This percentage increased to 35.7% when immature DCs are injected straight into the tumour and even higher to 40% for advanced pancreatic cancer [113].

Tumour-infiltrating T cells (TIL) therapy has been used in stage IV melanoma patients. TILs are obtained from the blood, lymph nodes or from a tumour tissue biopsy. TILs are isolated, activated and expanded using IL-2 *in vitro*. The patient undergoes lympho-depleting chemo‐ therapy prior to the T cells being injected back in to the blood [114].

When a tumour antigen is secreted into the circulation in high levels immune tolerance can be induced in the thymus. CD8α− Sirpα<sup>+</sup> , a subset of DCs, are able to capture tumour antigens in the blood, which can induce tolerance through Tregs or negative selection. Tregs are cells which are part of the tolerance system which prevents autoimmunity [115, 116]. Simultaneous Treg depletions (using anti-CD25 antibodies for instance) may aid the effectiveness of immunotherapy in some cancer types where Treg infiltration into the tu‐ mour is rife [117,118].

There are a number of reviews in this area of research which aim to look into effective immunotherapy strategies for the future. These include cellular immunotherapy [119], whole call vaccines [120], multidrug resistance [121] and DCs [122]. Targeted therapeutic strategies along with ever improving designs in clinical trials pave the way for further success [123].

## **10. Clinical trials**

the size of the tumour transplanted, the tumour can develop over weeks to months and the response to appropriate therapeutic agents studied *in vivo* [89]. Indeed such models have been used to examine the effectiveness of various immunotherapeutic strategies in‐ cluding whole cell vaccines [90], dendritic cell (DC) vaccines [91], peptide vaccines [92]

Genetically modified mice may have genes which are overexpressed or deleted and the effects of these genes on tumour development can be studied. Examples include p53 null and heterogenous mice [94,95] demonstrated that these genes can act as oncogenes and lead to tumour development. Possible therapies for these oncogenes/tumour suppressor genes are tested for their response in an *in vivo*, full organism context [89] and examples include Ad-p53,

HLA-A2 transgenic mice have genes inserted into the DNA so they will express the HLA molecules known in mice as H-2. In order to prevent the presentation of murine H-2-restricted cytotoxic T lymphocyte (CTL) epitopes in HLA-A2 (AAD) transgenic mice, HLA-A2.1 transgenic/H-2 class I knockout mice (HHD mice) were created [97]. In HHD mice, the H-2 class I gene is knocked out, and a chimeric HLA-A2.1 monochain (HHD) is produced by linking the C terminal of the human β2-microglobulin (unit of the class 1 MHC) covalently to the Nterminus of the chimeric HLA-A2 heavy chain (which contain the α1&2 domains of HLA-A2.1 and the α3 domain of H-2Db) by means of a peptide bond. This guarantees the sole expression of the HHD molecule on the cell surface, making sure that any identified CTL epitopes are HLA-A2 restricted [98]. HHD mice allow epitopes which are presented on human HLA-A2 to be examined for their ability to induce T cell responses in a variety of studies; for example STEAP, a prostate tumour antigen has been shown to be targeted by anti-tumour T cells [99] and DNA vaccines encoding Wilms tumour antigen 1 induce cytotoxic responses in mice [100]

One of the biggest debates in cancer immunotherapy remains which mode will be the most effective. The National Cancer Institute have suggested that immunotherapy stud‐ ies should focus on a limited number of antigenic targets to maximise the chances of success [101]. However for some cancers effective immunotherapy targets have yet to be discovered (i.e. ovarian cancer, adult acute lymphocytic leukaemia) and better targets

When T cells were found to be able to identify cancer cells [102] it was thought that T cell

tumour cells. This can be achieved by a number of ways such as through the use of DCs [103], peptide vaccines [85], DNA vaccines [104] and natural killer cells [105]. In recent years

/CTL cells to kill

therapies would be the most effective with the aim being to stimulate CD8+

and DNA vaccines [67,87,93].

308 Novel Gene Therapy Approaches

**8.2. Genetically modified mice**

AAV-HGFK1 [96].

using this model system.

may yet be determined.

**9. Modes of immunotherapy**

Clinical trials are undertaken after a large amount of data has been obtained on the antigen of interest in the lab. This data is required to ensure treatment safety and efficacy as far as is possible. It remains imperative in most countries that treatments have been tested on live animals prior to first-in-man clinical trials and that favourable results are apparent in order for treatments to be taken into clinical trials. People, often patients, are recruited as compen‐ sated or full volunteers. The drug is given to participants initially to show that it is safe and then that it is effective. Dose escalation is also important so that an effective and safe dosage in humans is used.

**11.1. Enzyme-Linked Immunosorbent Spot (ELISPOT) assay**

through the detection of drug-specific T cells in their blood [132].

revealed the existence of longer-lasting T cell memory responses [133].

focus of other excellent reviews in the field [134-136].

**11.2. Cultured ELISPOT**

**12. Conclusions**

patient quality of life.

Enzyme-Linked Immunosorbent Spot (ELISPOT) assay was developed by Cecil Czer‐ kinskdy in 1983 [131] and shows most similarity to the ELISA technique. It is based on the use of a 96 well plate with a polyvinyl-difluoride membrane to which antigen specif‐ ic monoclonal "capture" antibodies are attached. The cells are grown in media on the capture antibody coated membrane usually for several hours to overnight and secreted protein (often cytokines such as IFNγ) bind to the capture antibody. A second "detec‐ tion" antibody specific to the protein is used. This is often conjugated to an enzyme al‐ lowing a chemical reaction to occur. Black spots form on the membrane wherever protein is present and these can be counted by an ELISPOT reader [131]. ELISPOT as‐ says are one of the most sensitive *ex vivo* detection methods available with low detection thresholds in peripheral blood. ELISPOT is also able to identify patients with allergies

Cultured ELISPOT measures memory T cells. Cells are stimulated and plated on a 24-well plate. After an incubation period half of the cell culture supernatant is removed and replaced with Lymphocult (an IL-2 containing growth factor supplement). Fresh Lymphocult is added again on day 7. On day 9, the cells are incubated overnight. On day 10, around 2.5x104

originally plated cells are plated for a standard ELISPOT assay. Cultured ELISPOT assays

This chapter has focussed predominantly on the identification of epitopes within tumour antigens and their validation as they enter clinical trials. Focus on clinical trials using antibody therapies, DCs, natural killer cells, and adoptive therapy among many other options are the

Cancer immunotherapy is a vital area of research that continues to progress at a pace. Our understanding of the immune response and its potential to recognise and kill tu‐ mour cells with mans guidance offers hope to the patients for whom few other treat‐ ment options exist. Many tumour antigens have been identified, but some cancers still lack antigen targets that are expressed in the majority of the cancer cells by the majority of cancer patients. New techniques to extend antigen discovery will allow the improve‐ ment of immunotherapy strategies while the identification of new biomarkers will assist in the development of personalised therapies. Personalised therapies will decrease the cost (quantitative and qualitative) of treatment on patients who are unlikely to respond to it, allowing patients to avoid unpleasant and harmful side effects while maximising

of the

311

Identification of Targets for Immunotherapy http://dx.doi.org/10.5772/54698

Clinical trials have four phases, very basically as follows: І – evaluation of safety, ІІ – safety and efficacy (with Phase I/II often including dose escalation), ІІІ – efficacy in a large cohort of patients and ІV – post-approval studies. Phase І trials look at the safety and the best dose of the drug to administer. Such trials often involve 13 patients or less, and these patients are often have late stage cancer and are refractory to all other treat‐ ments with little chance of recovery. Phase ІІ trials start to look at the efficacy of the medicine and often involve 20 patients with late stage disease. Phase І and ІІ trials have to be completed successfully in order for testing to proceed to Phase ІІІ. Current "best practise" treatment is compared to the new drug being tested in phase ІІІ trials. Only if the new drug offers an improvement over best practise does the new medicine have a chance of becoming the standard treatment. At this time the drug will need to be li‐ censed and approved by the authoritative body e.g. the US Food and Drug Administra‐ tion, and once licensed, phase ІV trials investigate the long term benefits and unexpected side effects. In some cases the drug may go through one of the phases more than once before moving forward and even then may get rejected [124].

It is very important that trials follow certain rules for the results to be considered legitimate. Phase ІІІ trials need to be randomised i.e. a computer randomly puts people into one of two groups. These can also be double-blind trials so that neither the patient nor the investigator knows which treatment is being given, thus avoiding any bias. In some cases there may not be any treatment to compare a new therapy against, in which case a placebo (inactive treat‐ ment) is given to one group [125]. In the UK the Medicines and Healthcare products Regulatory Agency is responsible for the regulation of medicines and medical devices and equipment used in healthcare, and the investigation of harmful incidents as well as overseeing the use of blood and blood products [126].

Cohen *et al* [127] have created an online website called BreastCancerTrials.org which matches patients to current trials taking place depending on the information they provide. This provides a valuable source for cancer patients who may want initial guidance on which clinical trials may be beneficial to them.

## **11. Assays to demonstrate efficacy of the response**

Assays tend to reflect the immunotherapy strategy employed with the efficacy of antibody therapies being measured by tumour destruction, ertumaxomab destroys tumour cells expressing HER2/neu [128], bispecific antibodies represent a new class of anticancer thera‐ peutics [129] and antibody-targeted delivery of a vaccine can improve tumour cell killing [130].

#### **11.1. Enzyme-Linked Immunosorbent Spot (ELISPOT) assay**

Enzyme-Linked Immunosorbent Spot (ELISPOT) assay was developed by Cecil Czer‐ kinskdy in 1983 [131] and shows most similarity to the ELISA technique. It is based on the use of a 96 well plate with a polyvinyl-difluoride membrane to which antigen specif‐ ic monoclonal "capture" antibodies are attached. The cells are grown in media on the capture antibody coated membrane usually for several hours to overnight and secreted protein (often cytokines such as IFNγ) bind to the capture antibody. A second "detec‐ tion" antibody specific to the protein is used. This is often conjugated to an enzyme al‐ lowing a chemical reaction to occur. Black spots form on the membrane wherever protein is present and these can be counted by an ELISPOT reader [131]. ELISPOT as‐ says are one of the most sensitive *ex vivo* detection methods available with low detection thresholds in peripheral blood. ELISPOT is also able to identify patients with allergies through the detection of drug-specific T cells in their blood [132].

#### **11.2. Cultured ELISPOT**

possible. It remains imperative in most countries that treatments have been tested on live animals prior to first-in-man clinical trials and that favourable results are apparent in order for treatments to be taken into clinical trials. People, often patients, are recruited as compen‐ sated or full volunteers. The drug is given to participants initially to show that it is safe and then that it is effective. Dose escalation is also important so that an effective and safe dosage

Clinical trials have four phases, very basically as follows: І – evaluation of safety, ІІ – safety and efficacy (with Phase I/II often including dose escalation), ІІІ – efficacy in a large cohort of patients and ІV – post-approval studies. Phase І trials look at the safety and the best dose of the drug to administer. Such trials often involve 13 patients or less, and these patients are often have late stage cancer and are refractory to all other treat‐ ments with little chance of recovery. Phase ІІ trials start to look at the efficacy of the medicine and often involve 20 patients with late stage disease. Phase І and ІІ trials have to be completed successfully in order for testing to proceed to Phase ІІІ. Current "best practise" treatment is compared to the new drug being tested in phase ІІІ trials. Only if the new drug offers an improvement over best practise does the new medicine have a chance of becoming the standard treatment. At this time the drug will need to be li‐ censed and approved by the authoritative body e.g. the US Food and Drug Administra‐ tion, and once licensed, phase ІV trials investigate the long term benefits and unexpected side effects. In some cases the drug may go through one of the phases more than once

It is very important that trials follow certain rules for the results to be considered legitimate. Phase ІІІ trials need to be randomised i.e. a computer randomly puts people into one of two groups. These can also be double-blind trials so that neither the patient nor the investigator knows which treatment is being given, thus avoiding any bias. In some cases there may not be any treatment to compare a new therapy against, in which case a placebo (inactive treat‐ ment) is given to one group [125]. In the UK the Medicines and Healthcare products Regulatory Agency is responsible for the regulation of medicines and medical devices and equipment used in healthcare, and the investigation of harmful incidents as well as overseeing the use of blood

Cohen *et al* [127] have created an online website called BreastCancerTrials.org which matches patients to current trials taking place depending on the information they provide. This provides a valuable source for cancer patients who may want initial guidance on which clinical

Assays tend to reflect the immunotherapy strategy employed with the efficacy of antibody therapies being measured by tumour destruction, ertumaxomab destroys tumour cells expressing HER2/neu [128], bispecific antibodies represent a new class of anticancer thera‐ peutics [129] and antibody-targeted delivery of a vaccine can improve tumour cell killing [130].

before moving forward and even then may get rejected [124].

**11. Assays to demonstrate efficacy of the response**

in humans is used.

310 Novel Gene Therapy Approaches

and blood products [126].

trials may be beneficial to them.

Cultured ELISPOT measures memory T cells. Cells are stimulated and plated on a 24-well plate. After an incubation period half of the cell culture supernatant is removed and replaced with Lymphocult (an IL-2 containing growth factor supplement). Fresh Lymphocult is added again on day 7. On day 9, the cells are incubated overnight. On day 10, around 2.5x104 of the originally plated cells are plated for a standard ELISPOT assay. Cultured ELISPOT assays revealed the existence of longer-lasting T cell memory responses [133].

## **12. Conclusions**

This chapter has focussed predominantly on the identification of epitopes within tumour antigens and their validation as they enter clinical trials. Focus on clinical trials using antibody therapies, DCs, natural killer cells, and adoptive therapy among many other options are the focus of other excellent reviews in the field [134-136].

Cancer immunotherapy is a vital area of research that continues to progress at a pace. Our understanding of the immune response and its potential to recognise and kill tu‐ mour cells with mans guidance offers hope to the patients for whom few other treat‐ ment options exist. Many tumour antigens have been identified, but some cancers still lack antigen targets that are expressed in the majority of the cancer cells by the majority of cancer patients. New techniques to extend antigen discovery will allow the improve‐ ment of immunotherapy strategies while the identification of new biomarkers will assist in the development of personalised therapies. Personalised therapies will decrease the cost (quantitative and qualitative) of treatment on patients who are unlikely to respond to it, allowing patients to avoid unpleasant and harmful side effects while maximising patient quality of life.


**Abbreviations**

**Acknowledgements**

Innovations grant.

**Author details**

Barbara-ann Guinn1,2,3\*

Southampton, Southampton, UK

Rayne Institute, London, UK

Ghazala Khan1

Germany

**References**

BIMAS: Bioinformatics and Molecular Analysis Section; BrdU: bromodeoxyuridine; CFSE: Car‐ boxyfluorescein diacetate succinimidyl ester; CTL: Cytotoxic T lymphocyte; CT: cancer-testis; DC: dendritic cell; ELISA: Enzyme-linked immunosorbent assay; ELISPOT: Enzyme-linked im‐ munosorbence assay; HLA: human leukocyte antigen; IFNγ: interferon-gamma; IL: interleukin; mAb: monoclonal antibodies; MFI: mean fluorescence index; MHC: major histocompatibility complex; PCR: polymerise chain reaction; pMHC: peptide and major histocompatibility com‐ plex; SEREX: Serological identification of antigens by recombinant expression cloning; SSX2IP: Synovial Sarcoma X breakpoint 2 Interacting Protein; TAA: tumour associated antigens; TCR: T

SEB was funded by Cancer Research U.K. Research by our group has been supported by Leukaemia and Lymphoma Research, Wessex Cancer Trust and a Wessex Medical Research

, Frances Denniss3

2 Cancer Sciences Division (MP824), Southampton University Hospitals, University of

3 Department of Haematological Medicine, King's College London School of Medicine, The

4 Department of Immunology, Institute for Cell Biology, University of Tübingen, Tübingen,

[1] Penn, C.G. Halgrimson, T.E. Starzl, De novo malignant tumors in organ transplant

, Dagmar Sigurdardottir4

and

Identification of Targets for Immunotherapy http://dx.doi.org/10.5772/54698 313

cell receptor; TIL: Tumour-infiltrating T cells; Tregs: regulatory T cells.

, Suzanne E. Brooks2

\*Address all correspondence to: barbara.guinn@beds.ac.uk

recipients, Transplant. Proc. 1 (1971) 773-778.

1 Division of Science, University of Bedfordshire, Park Square, Luton, UK

**Table 1.** Examples of completed clinical trials showing the cancer antigen targets. Representation of the various modes of immunotherapy employed to date, cancer patients treated and the outcome of the trials.

## **Abbreviations**

**Target antigen Mode of**

312 Novel Gene Therapy Approaches

TG4010 targeting MUC1 &

IL-2

CD22 Monoclonal antibody

LY6K and TTK peptide vaccines in

CTLA-4 Monoclonal antibody

**immunotherapy**

conjugated to calecheamicin

Poxvirus (modified vaccinia virus Ankara) in combination with

combination with CpG-7909

with glycoprotein 100 (gp100) peptide

vaccine

CD20 Monoclonal antibody Relapsed or

anti-idiotype antibody

CA-125 Abagovomab, an

first-line chemotherapy **Patient group Phase of**

Refractory and relapsed acute lymphocytic leukaemia

Advanced nonsmall-cell lung cancer

Metastatic oesophageal squamous cell carcinoma

Previously treated metastatic melanoma

refractory follicular lymphoma

**Table 1.** Examples of completed clinical trials showing the cancer antigen targets. Representation of the various

modes of immunotherapy employed to date, cancer patients treated and the outcome of the trials.

Prostate-specific antigen Poxviral vaccines Prostate cancer Phase 2 The primary end point was

**clinical trial**

**Outcome Reference**

[137]

[138]

[139]

[140]

[141]

[142]

[143]

Phase 2 18% patients had complete

treatment

Phase 2B 6-month progression-free

Phase 1 Vaccination with peptides in

populations

Phase 3 The median overall survival was 10.0 months among patients receiving ipilimumab plus gp100, as compared with 6.4 months among patients receiving gp100 alone

> progression-free survival which was similar in the two groups (treated, controls). However, at 3 years post study, treated patients had a overall survival rate of 30% compared to 17% of controls

Phase 1/2 Treatment caused immediate

(six injections)

Ovarian Cancer Phase 1 Improved CA125-specific

and profound B-cell depletion, and 65% of patients reverted to negative BCL2 status.

cellular cytotoxicity might indicate that longer vaccination (nine injections) would be preferred to short

response, 39% had marrow complete response, 39% had resistant disease, and 4% died within 4 weeks of starting

survival was 43·2% in the TG4010 plus chemotherapy group, and 35·1% in the chemotherapy alone group

combination with CpG-7909 increased and activated pDC populations and NK cell

BIMAS: Bioinformatics and Molecular Analysis Section; BrdU: bromodeoxyuridine; CFSE: Car‐ boxyfluorescein diacetate succinimidyl ester; CTL: Cytotoxic T lymphocyte; CT: cancer-testis; DC: dendritic cell; ELISA: Enzyme-linked immunosorbent assay; ELISPOT: Enzyme-linked im‐ munosorbence assay; HLA: human leukocyte antigen; IFNγ: interferon-gamma; IL: interleukin; mAb: monoclonal antibodies; MFI: mean fluorescence index; MHC: major histocompatibility complex; PCR: polymerise chain reaction; pMHC: peptide and major histocompatibility com‐ plex; SEREX: Serological identification of antigens by recombinant expression cloning; SSX2IP: Synovial Sarcoma X breakpoint 2 Interacting Protein; TAA: tumour associated antigens; TCR: T cell receptor; TIL: Tumour-infiltrating T cells; Tregs: regulatory T cells.

## **Acknowledgements**

SEB was funded by Cancer Research U.K. Research by our group has been supported by Leukaemia and Lymphoma Research, Wessex Cancer Trust and a Wessex Medical Research Innovations grant.

## **Author details**

Ghazala Khan1 , Suzanne E. Brooks2 , Frances Denniss3 , Dagmar Sigurdardottir4 and Barbara-ann Guinn1,2,3\*

\*Address all correspondence to: barbara.guinn@beds.ac.uk

1 Division of Science, University of Bedfordshire, Park Square, Luton, UK

2 Cancer Sciences Division (MP824), Southampton University Hospitals, University of Southampton, Southampton, UK

3 Department of Haematological Medicine, King's College London School of Medicine, The Rayne Institute, London, UK

4 Department of Immunology, Institute for Cell Biology, University of Tübingen, Tübingen, Germany

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324 Novel Gene Therapy Approaches


**Chapter 13**

**Targeting Intercellular Communication in**

Cells dedicate a considerable amount of energy and regulatory mechanisms to ensure cell-cell communication, for this biological process is an important aspect of their machinery of survival, behavior and fate within their immediate environment. For cells, communicating is vital not only because they are part of organs and tissues of which they contribute to main‐ taining the integrity and proper function [1-5], but also because many of their functions need to be coordinated, quantitatively fine-tuned and/or limited in space and time. Furthermore, cells make use of communication to minimize the energetic and signaling burden, whereas a single minimal signal could be amplified and propagated, as is for instance the case of gap junction-mediated transfer of pro-apoptotic signals [6-8]. Many types of intercellular commu‐ nication have been studied, among which direct cell-cell interactions could be distinguished from cellular interactions via released growth factors and cytokines. Their studies have revealed a significant potential for use in cancer therapy. The importance of cell-cell commu‐ nication is particularly well revealed when defects in this process result in serious diseases, as

exemplified by mutations identified in many gap and tight junction proteins [9, 10].

The diversity of the types of intercellular communications (i.e. gap junctions (GJ), tight junctions (TJ), adherens junctions (AJ) and desmosomes), implicates a diversity of signaling pathways and biological functions at stake. It further emphasizes the need for cells to com‐ municate in different ways and for different purposes: transfer of small molecules, reciprocal signaling, establishment of barriers and polarity, control of paracellular permeability and transmission of cytoskeleton-generated forces. All of these processes have been implicated in cancer development as reviewed previously for GJs [11-13], TJs [14, 15] and desmosomes [16]. In this chapter we will present an overview of how various types of direct cell-cell communi‐ cation and different groups of intercellular-dependent protein interactions have been used in

and reproduction in any medium, provided the original work is properly cited.

© 2013 Amessou and Kandouz; 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.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Mohamed Amessou and Mustapha Kandouz

Additional information is available at the end of the chapter

**Cancer Gene Therapy**

http://dx.doi.org/10.5772/54860

**1. Introduction**

## **Targeting Intercellular Communication in Cancer Gene Therapy**

Mohamed Amessou and Mustapha Kandouz

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54860

## **1. Introduction**

Cells dedicate a considerable amount of energy and regulatory mechanisms to ensure cell-cell communication, for this biological process is an important aspect of their machinery of survival, behavior and fate within their immediate environment. For cells, communicating is vital not only because they are part of organs and tissues of which they contribute to main‐ taining the integrity and proper function [1-5], but also because many of their functions need to be coordinated, quantitatively fine-tuned and/or limited in space and time. Furthermore, cells make use of communication to minimize the energetic and signaling burden, whereas a single minimal signal could be amplified and propagated, as is for instance the case of gap junction-mediated transfer of pro-apoptotic signals [6-8]. Many types of intercellular commu‐ nication have been studied, among which direct cell-cell interactions could be distinguished from cellular interactions via released growth factors and cytokines. Their studies have revealed a significant potential for use in cancer therapy. The importance of cell-cell commu‐ nication is particularly well revealed when defects in this process result in serious diseases, as exemplified by mutations identified in many gap and tight junction proteins [9, 10].

The diversity of the types of intercellular communications (i.e. gap junctions (GJ), tight junctions (TJ), adherens junctions (AJ) and desmosomes), implicates a diversity of signaling pathways and biological functions at stake. It further emphasizes the need for cells to com‐ municate in different ways and for different purposes: transfer of small molecules, reciprocal signaling, establishment of barriers and polarity, control of paracellular permeability and transmission of cytoskeleton-generated forces. All of these processes have been implicated in cancer development as reviewed previously for GJs [11-13], TJs [14, 15] and desmosomes [16].

In this chapter we will present an overview of how various types of direct cell-cell communi‐ cation and different groups of intercellular-dependent protein interactions have been used in

© 2013 Amessou and Kandouz; 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. © 2013 The Author(s). Licensee InTech. This chapter is 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.

strategies of gene therapy of cancer. Important concepts and paradigms as well as successful approaches, limitations and possibilities for the future will be discussed.

Originally tried using retroviral vectors, the same approach adapted to adenoviral vectors was later introduced and used successfully [42-44]. These and subsequent studies, all have in common the use of an efficient delivery system, mostly adenoviral, modified to improve the transduction efficiency or selectivity, in combination with an enzyme/prodrug system, most often the HSVtk/GCV, to achieve cancer cells' cytotoxicity. Virus-free delivery has also been attempted using liposomes for instance, with more or less good efficacy [45-47], but

Targeting Intercellular Communication in Cancer Gene Therapy

http://dx.doi.org/10.5772/54860

329

**Figure 1. The enzyme/prodrug system and the bystander effect.** Delivery via viral or non viral vectors of DNA se‐ quences expressing an enzyme, here the herpes simplex virus thymidine kinase gene (TK) in the presence of the pro‐ drug inactive substrate, here ganciclovir (GCV), results in the synthesis of the active metabolite, here GCV triphosphate (GCV-tp), which kills not only the target cell, but the neighboring bystander cell as well. This 'bystander effect' is medi‐ ated by a direct transfer of cytotoxic signals through gap junctions (GJ)-mediated intercellular communication.

most of the studies have used viral delivery.

## **2. Intercellular communication & gene therapy: The enzyme/prodrug strategy**

Cancer gene therapy has since its beginnings faced a major hurdle, the inefficiency of the methods of gene delivery to target cells (i.e. transfection and infection). While attempts have later been made to identify promising alternatives, a key development was the discovery that gap junctions could provide an efficient method that, without directly reaching every cell, could transfer the cytotoxic signal originating from a limited number of target cells to their bystander neighboring cells, thus amplifying the therapeutic effect. This process has subse‐ quently been called "bystander effect" (BE) [17]. Triggering apoptotic death process in target cells results in the transfer of the pro-apoptotic signaling molecules to other cells with which they interact via gap junction intercellular communications (GJICs), and ultimately in the demise of both cells. The BE thus plays an important role in the efficiency of cancer therapy [18]. It also impacts the therapeutic cytotoxic side effects: since high doses of drugs are not required to kill tumor cells, normal tissues may not be reached by the treatment.

## **3. Use of the bystander effect in the enzyme/prodrug cancer gene therapy**

Gene therapy soon became the major therapeutic application of the BE in the so-called "suicide gene therapy" involving the use of Enzyme/Prodrug cytotoxic systems, whereby target cells express an enzyme that converts a prodrug into the cytotoxic active drug, which is then transferred via gap junctions to the interacting cells [19]. The general mechanism is that the active molecules are therefore transmitted to neighboring cells via GJIC and trigger their death [20]. GJIC and connexins are essential for the BE-based enzyme/prodrug therapy [21-26] (Figure 1). Different enzymes/prodrugs have been assayed among which cytosine deaminase (CD)/5-fluorocytosine (5-FC), carboxylesterase/Camptothecin, and Herpes Simplex Virusthymidine kinase (HSV/tk)/Ganciclovir (GCV) are prominent [27]. The CD/5-FC combination is based on the conversion of the nontoxic prodrug 5-FC by bacterial or yeast enzyme cytosine deaminase into active 5-fluorouracil (5-FU) [28]. Similarly, GCV, a nontoxic purine analogue, is phosphorylated by the enzyme HSVtk and by endogenous kinases to GCV-triphosphate, which kills cells by inhibiting DNA synthesis [29] [30]. The carboxylesterase activates the prodrug irinotecan,7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11) to the active metabolite SN-38. Another combination including the uracil phosphor‐ ibosyltransferase (UPRT) of E. coli and 5-fluorouracil (5-FU), has also been used in BE-based gene therapy, along with other less known systems. UPRT is an enzyme that catalyzes the synthesis of UMP from uracil and 5-phosphoribosyl-alpha-1-diphosphate [31].

The therapeutic potential of the HSVtk and nucleosides' combination has been assayed as early as the 70's and later extended to many types of cancers both *in vitro* and *in vivo* [32-41]. Originally tried using retroviral vectors, the same approach adapted to adenoviral vectors was later introduced and used successfully [42-44]. These and subsequent studies, all have in common the use of an efficient delivery system, mostly adenoviral, modified to improve the transduction efficiency or selectivity, in combination with an enzyme/prodrug system, most often the HSVtk/GCV, to achieve cancer cells' cytotoxicity. Virus-free delivery has also been attempted using liposomes for instance, with more or less good efficacy [45-47], but most of the studies have used viral delivery.

strategies of gene therapy of cancer. Important concepts and paradigms as well as successful

Cancer gene therapy has since its beginnings faced a major hurdle, the inefficiency of the methods of gene delivery to target cells (i.e. transfection and infection). While attempts have later been made to identify promising alternatives, a key development was the discovery that gap junctions could provide an efficient method that, without directly reaching every cell, could transfer the cytotoxic signal originating from a limited number of target cells to their bystander neighboring cells, thus amplifying the therapeutic effect. This process has subse‐ quently been called "bystander effect" (BE) [17]. Triggering apoptotic death process in target cells results in the transfer of the pro-apoptotic signaling molecules to other cells with which they interact via gap junction intercellular communications (GJICs), and ultimately in the demise of both cells. The BE thus plays an important role in the efficiency of cancer therapy [18]. It also impacts the therapeutic cytotoxic side effects: since high doses of drugs are not

**2. Intercellular communication & gene therapy: The enzyme/prodrug**

required to kill tumor cells, normal tissues may not be reached by the treatment.

synthesis of UMP from uracil and 5-phosphoribosyl-alpha-1-diphosphate [31].

The therapeutic potential of the HSVtk and nucleosides' combination has been assayed as early as the 70's and later extended to many types of cancers both *in vitro* and *in vivo* [32-41].

**3. Use of the bystander effect in the enzyme/prodrug cancer gene therapy**

Gene therapy soon became the major therapeutic application of the BE in the so-called "suicide gene therapy" involving the use of Enzyme/Prodrug cytotoxic systems, whereby target cells express an enzyme that converts a prodrug into the cytotoxic active drug, which is then transferred via gap junctions to the interacting cells [19]. The general mechanism is that the active molecules are therefore transmitted to neighboring cells via GJIC and trigger their death [20]. GJIC and connexins are essential for the BE-based enzyme/prodrug therapy [21-26] (Figure 1). Different enzymes/prodrugs have been assayed among which cytosine deaminase (CD)/5-fluorocytosine (5-FC), carboxylesterase/Camptothecin, and Herpes Simplex Virusthymidine kinase (HSV/tk)/Ganciclovir (GCV) are prominent [27]. The CD/5-FC combination is based on the conversion of the nontoxic prodrug 5-FC by bacterial or yeast enzyme cytosine deaminase into active 5-fluorouracil (5-FU) [28]. Similarly, GCV, a nontoxic purine analogue, is phosphorylated by the enzyme HSVtk and by endogenous kinases to GCV-triphosphate, which kills cells by inhibiting DNA synthesis [29] [30]. The carboxylesterase activates the prodrug irinotecan,7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11) to the active metabolite SN-38. Another combination including the uracil phosphor‐ ibosyltransferase (UPRT) of E. coli and 5-fluorouracil (5-FU), has also been used in BE-based gene therapy, along with other less known systems. UPRT is an enzyme that catalyzes the

approaches, limitations and possibilities for the future will be discussed.

**strategy**

328 Novel Gene Therapy Approaches

**Figure 1. The enzyme/prodrug system and the bystander effect.** Delivery via viral or non viral vectors of DNA se‐ quences expressing an enzyme, here the herpes simplex virus thymidine kinase gene (TK) in the presence of the pro‐ drug inactive substrate, here ganciclovir (GCV), results in the synthesis of the active metabolite, here GCV triphosphate (GCV-tp), which kills not only the target cell, but the neighboring bystander cell as well. This 'bystander effect' is medi‐ ated by a direct transfer of cytotoxic signals through gap junctions (GJ)-mediated intercellular communication.

#### **3.1. Combination of oncolytic viruses and enzyme-prodrug gene therapy**

**3.2. Combined use of the enzyme/prodrug cancer gene therapy and gap junction**

**3.3. Applications of the enzyme/prodrug gene targeting of stem cells**

Although since the beginning of the use of the enzyme/prodrug approach, it was found that the BE involves effects that do not depend on direct cell-cell interaction and are rather related to diffusible molecules released extracellularly and possibly to immune-related effects [48-51], the role of gap junctions-mediated intercellular communication (GJIC) and connexins was deemed essential [25, 26, 52-54] [55]. In light of the observed loss of connexins' expression in many cancers, the efficiency of the enzyme/prodrug approach could be limited by the ability of tumor cells to undergo GJICs between gene-transduced and bystander non-transduced cells. The levels of connexins and GJIC could modulate the impact of the bystander effect of the prodrug/enzyme systems, as shown for HSVtk/GCV *in vitro* and *in vivo* [56, 57]. This was suggested to be a reason behind the limited efficacy of the viral HSVtk/GCV delivery in many reports [58-60]. Nevertheless, many attempts have been made to bypass this limitation by restoring connexins' expression and the ability to undergo GJIC. This could be achieved either by the direct delivery of Cx-encoding vectors [61-64] or by pharmacological induction of Cx expression. The later approach involved for instance treating with DNA demethylating agents [65], histone deacetylases' inhibitors (HDACi) [66-68], ATP-sensitive potassium (KATP) channels' inhibitors [69], treatment with all-trans retinoic acid [70] or cyclic-AMP [71-73].

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Cellular vectors, including stem cells, have been used for effective gene delivery in cancer therapy. Stem and progenitor cells have been acknowledged as important for both normal and cancer homeostasis. In particular, according to the cancer stem cells' theory, tumors contain a very small sub-population of self-renewing and highly proliferating cells called cancer stem cells (CSCs), which are responsible for the tumorigenic activity [89]. Mesenchymal stem cells (MSCs), which have a strong tropism for tumor cells, are another type of stem cells of impor‐ tance in cancer understanding and therapeutic targeting [90]. The use of allogeneic and hence escaping immune vigilance mesenchymal stromal cells (MSCs), sometimes called mesenchy‐ mal stem cells, as Trojan horses to deliver the enzyme/prodrug within the tumor mass is a relatively new development in gene therapy. MSCs are used as carriers of the enzyme via viral transduction, which subsequently activates the prodrug and kills not only the MSCs but their neighboring cancer cells (Figure 2). This strategy has been tested in many cancers, as illustrated

It has been shown that MSCs localize primarily to the perivascular environment in many organs and, when implanted or injected into animals, they show a tropism for primary tumors and metastases, and specifically for the perivascular niches within tumors [91, 92]. Based on this preferential migration, MSCs have been used as a vehicle in gene therapy strategies [93, 94]. The cytosine deaminase prodrug system has been partnered with the human MSCs and the combination increased the bystander effect and selective cytotoxicity on target tumor cells *in vitro* and *in vivo* [95-97]. Similarly, human neural stem cells (NSCs) have been successfully used to therapeutically target brain cancers. In fact, both MSCs and NSCs show high tropism for brain cancers and have been combined with the prodrug system to target brainstem

**communication restoration**

by the following examples.

Viruses are preferred vehicles for the transfer and delivery of engineered genes into host cells in gene therapy approaches. Recently, they have emerged as not only delivery vec‐ tors, but as *bona fide* therapeutic agents [74-77] (Figure 2). Oncolytic replication-compe‐ tent viruses infect, replicate in and kill tumor cells. Examples abound of attempts to combine gene therapy and oncolytic virotherapy. Furthermore, the enzyme/prodrug sys‐ tems have been used to improve the anti-tumor efficacy of oncolytic viruses. Early stud‐ ies addressing the use of HSV vectors as oncolytic agents, showed that HSV-mediated oncolysis is enhanced by ganciclovir treatment through bystander effect [78]. A recombi‐ nant HSV (M012) was constructed to express the bacterial CD gene and was shown to enhance the prodrug-mediated anti-tumor effects after intracranial delivery in murine neuroblastoma and human glioma cells [79]. An oncolytic adenovirus modified to bear the human telomerase promoter (hTERT), was used to deliver the gene for the prodrugactivating enzyme carboxypeptidase G2 (CPG2) to tumors. The CPG2 metabolizes the prodrug ZD2767P into a cytotoxic drug and this strategy was shown to be effective in colorectal carcinomas via bystander effects and induction of apoptosis [80]. A recombi‐ nant Vesicular stomatitis virus (rVSV) encoding the CD/UPRT fusion gene was delivered intratumorally in the presence of the systemically administered 5-FU and significantly re‐ duced growth of lymphoma and breast cancer cells *in vivo*. This effect involved three mechanisms: a strong bystander effect, the viral oncolytic activity as well as the activa‐ tion of the immune system against the tumor [81]. Recombinant vesicular stomatitis vi‐ rus (VSV) made to express CD/UPRT was delivered to breast cancer cells in combination with 5-fluorocytosine (5FC) [82]. An oncolytic adenovirus Ad5/3-Delta24FCU1 expressing the fusion suicide gene FCU1, which encodes a bifunctional fusion protein that metabo‐ lizes 5-FC, was found to exert significant anti-tumor activity *in vitro* and *in vivo* in a murine model of head and neck squamous cell carcinoma [83]. ONYX-015 (*dl*1520), a conditionally replicating adenovirus (CRAd) made of an E1B-55k-deleted oncolytic ade‐ novirus and which has anti-tumor effects [84], has been combined with the CD/5-FC sys‐ tem and the enzyme/prodrug system involving *E. coli* nitroreductase (NTR) which can reduce nitro(hetero)aromatic compounds to hydroxylamines and amines, and both com‐ binations showed enhanced efficacy *in vitro* and *in vivo* [85, 86]. Similarly, an oncolytic measles virus (MV) armed with the prodrug convertase, purine nucleoside phosphory‐ lase (PNP) and the prodrug 6-methylpurine-2'-deoxyriboside (MeP-dR), was tested in a model of murine colon adenocarcinoma cells in syngeneic C57BL/6 mice and shown to have anti-tumorigenic effects after systemic delivery [87]. In spite of this available litera‐ ture, many questions remain open. The factors defining the efficacy of this combinatorial therapy are not clearly identified and the strategy might not have any advantage in cer‐ tain contexts. For instance, an oncolytic adenovirus, selective for the Rb/p16 pathway, kil‐ led ovarian cancer cells effectively by Tk/GCV-driven BE. However, while GCV improved the adenoviruses' antitumor efficacy over the replication-deficient virus coun‐ terpart, it did not further enhance its efficacy *in vivo*, suggesting that the prodrug strat‐ egy may not add antitumor activity to highly potent oncolysis [88].

#### **3.2. Combined use of the enzyme/prodrug cancer gene therapy and gap junction communication restoration**

**3.1. Combination of oncolytic viruses and enzyme-prodrug gene therapy**

330 Novel Gene Therapy Approaches

egy may not add antitumor activity to highly potent oncolysis [88].

Viruses are preferred vehicles for the transfer and delivery of engineered genes into host cells in gene therapy approaches. Recently, they have emerged as not only delivery vec‐ tors, but as *bona fide* therapeutic agents [74-77] (Figure 2). Oncolytic replication-compe‐ tent viruses infect, replicate in and kill tumor cells. Examples abound of attempts to combine gene therapy and oncolytic virotherapy. Furthermore, the enzyme/prodrug sys‐ tems have been used to improve the anti-tumor efficacy of oncolytic viruses. Early stud‐ ies addressing the use of HSV vectors as oncolytic agents, showed that HSV-mediated oncolysis is enhanced by ganciclovir treatment through bystander effect [78]. A recombi‐ nant HSV (M012) was constructed to express the bacterial CD gene and was shown to enhance the prodrug-mediated anti-tumor effects after intracranial delivery in murine neuroblastoma and human glioma cells [79]. An oncolytic adenovirus modified to bear the human telomerase promoter (hTERT), was used to deliver the gene for the prodrugactivating enzyme carboxypeptidase G2 (CPG2) to tumors. The CPG2 metabolizes the prodrug ZD2767P into a cytotoxic drug and this strategy was shown to be effective in colorectal carcinomas via bystander effects and induction of apoptosis [80]. A recombi‐ nant Vesicular stomatitis virus (rVSV) encoding the CD/UPRT fusion gene was delivered intratumorally in the presence of the systemically administered 5-FU and significantly re‐ duced growth of lymphoma and breast cancer cells *in vivo*. This effect involved three mechanisms: a strong bystander effect, the viral oncolytic activity as well as the activa‐ tion of the immune system against the tumor [81]. Recombinant vesicular stomatitis vi‐ rus (VSV) made to express CD/UPRT was delivered to breast cancer cells in combination with 5-fluorocytosine (5FC) [82]. An oncolytic adenovirus Ad5/3-Delta24FCU1 expressing the fusion suicide gene FCU1, which encodes a bifunctional fusion protein that metabo‐ lizes 5-FC, was found to exert significant anti-tumor activity *in vitro* and *in vivo* in a murine model of head and neck squamous cell carcinoma [83]. ONYX-015 (*dl*1520), a conditionally replicating adenovirus (CRAd) made of an E1B-55k-deleted oncolytic ade‐ novirus and which has anti-tumor effects [84], has been combined with the CD/5-FC sys‐ tem and the enzyme/prodrug system involving *E. coli* nitroreductase (NTR) which can reduce nitro(hetero)aromatic compounds to hydroxylamines and amines, and both com‐ binations showed enhanced efficacy *in vitro* and *in vivo* [85, 86]. Similarly, an oncolytic measles virus (MV) armed with the prodrug convertase, purine nucleoside phosphory‐ lase (PNP) and the prodrug 6-methylpurine-2'-deoxyriboside (MeP-dR), was tested in a model of murine colon adenocarcinoma cells in syngeneic C57BL/6 mice and shown to have anti-tumorigenic effects after systemic delivery [87]. In spite of this available litera‐ ture, many questions remain open. The factors defining the efficacy of this combinatorial therapy are not clearly identified and the strategy might not have any advantage in cer‐ tain contexts. For instance, an oncolytic adenovirus, selective for the Rb/p16 pathway, kil‐ led ovarian cancer cells effectively by Tk/GCV-driven BE. However, while GCV improved the adenoviruses' antitumor efficacy over the replication-deficient virus coun‐ terpart, it did not further enhance its efficacy *in vivo*, suggesting that the prodrug strat‐

Although since the beginning of the use of the enzyme/prodrug approach, it was found that the BE involves effects that do not depend on direct cell-cell interaction and are rather related to diffusible molecules released extracellularly and possibly to immune-related effects [48-51], the role of gap junctions-mediated intercellular communication (GJIC) and connexins was deemed essential [25, 26, 52-54] [55]. In light of the observed loss of connexins' expression in many cancers, the efficiency of the enzyme/prodrug approach could be limited by the ability of tumor cells to undergo GJICs between gene-transduced and bystander non-transduced cells. The levels of connexins and GJIC could modulate the impact of the bystander effect of the prodrug/enzyme systems, as shown for HSVtk/GCV *in vitro* and *in vivo* [56, 57]. This was suggested to be a reason behind the limited efficacy of the viral HSVtk/GCV delivery in many reports [58-60]. Nevertheless, many attempts have been made to bypass this limitation by restoring connexins' expression and the ability to undergo GJIC. This could be achieved either by the direct delivery of Cx-encoding vectors [61-64] or by pharmacological induction of Cx expression. The later approach involved for instance treating with DNA demethylating agents [65], histone deacetylases' inhibitors (HDACi) [66-68], ATP-sensitive potassium (KATP) channels' inhibitors [69], treatment with all-trans retinoic acid [70] or cyclic-AMP [71-73].

#### **3.3. Applications of the enzyme/prodrug gene targeting of stem cells**

Cellular vectors, including stem cells, have been used for effective gene delivery in cancer therapy. Stem and progenitor cells have been acknowledged as important for both normal and cancer homeostasis. In particular, according to the cancer stem cells' theory, tumors contain a very small sub-population of self-renewing and highly proliferating cells called cancer stem cells (CSCs), which are responsible for the tumorigenic activity [89]. Mesenchymal stem cells (MSCs), which have a strong tropism for tumor cells, are another type of stem cells of impor‐ tance in cancer understanding and therapeutic targeting [90]. The use of allogeneic and hence escaping immune vigilance mesenchymal stromal cells (MSCs), sometimes called mesenchy‐ mal stem cells, as Trojan horses to deliver the enzyme/prodrug within the tumor mass is a relatively new development in gene therapy. MSCs are used as carriers of the enzyme via viral transduction, which subsequently activates the prodrug and kills not only the MSCs but their neighboring cancer cells (Figure 2). This strategy has been tested in many cancers, as illustrated by the following examples.

It has been shown that MSCs localize primarily to the perivascular environment in many organs and, when implanted or injected into animals, they show a tropism for primary tumors and metastases, and specifically for the perivascular niches within tumors [91, 92]. Based on this preferential migration, MSCs have been used as a vehicle in gene therapy strategies [93, 94]. The cytosine deaminase prodrug system has been partnered with the human MSCs and the combination increased the bystander effect and selective cytotoxicity on target tumor cells *in vitro* and *in vivo* [95-97]. Similarly, human neural stem cells (NSCs) have been successfully used to therapeutically target brain cancers. In fact, both MSCs and NSCs show high tropism for brain cancers and have been combined with the prodrug system to target brainstem gliomas, a form of childhood central nervous system tumors with poor prognosis or medul‐ loblastomas [98-101], and even in disseminated brain metastases of non-neuronal origins such as melanoma and breast cancer [102-104]. The success of this approach now warrants clinical trials such as the one recently started to study the feasibility of intracerebral administration of NSCs in combination with oral 5-FC in patients with recurrent high-grade gliomas [105].

Based on the tropism shown by neural stem cells (NSCs) for glioma cells, the herpes simplex virus-thymidine kinase (HSVtk)/GCV system has also been used in targeting gliomas [106-108]. However, for practical reasons related to the availability of cells, the use of MSCs might be more relevant clinically than the use of NSCs [109]. The system has also been test‐ ed for AT-MSCs [110] and bone marrow-derived tumor-infiltrating cells (BM-TICs) target‐ ing of gliomas [111]. It was also proven to have a strong anti-tumor growth in

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333

As discussed earlier, a major limitation to the efficacy of the therapeutic use of GJIC is the deficiency in the bystander effect due to low expression levels of connexins. Expectedly, this is also a challenge when using the prodrug/stem cells combined therapy. This can be bypassed by restoring connexin levels. For instance, GSCs showed more reduced GJIC and connexin levels than differentiated glioma cells [113]. Valproic acid (VPA) was able to upregulate Cx43 and Cx26 and to enhance the bystander effect of suicide gene therapy by human bone marrow MSCs expressing HSV-TK (MSCs-TK) [114]. In another study, the use of Bone marrow-derived stem cells (BMSCs) in combination with the (HSV-TK)/GCV suicide gene therapy of gliomas

The MSC/Prodrug and Oncovirus/Prodrug strategies are often combined. For instance, MSCs transduced with an adenoviral vector modified to express integrin-binding motifs (Ad5lucRGD) for better transduction efficiency, and expressing thymidine kinase were able not only to kill ovarian cancer cells via bystander effect, but also support replication of

MSCs can also act through an anti-angiogenic mechanism. They have been shown to target endothelial cells and inhibit capillary growth, establish Cx43-based GJIC with the target ECs, and to increase the production of reactive oxygen species (ROS). This effect culminates in the

Curiously, unlike gap junctions, the number of studies delivering tight and adherens junctions or desmosomal proteins for cytotoxic gene therapy is limited. The adenoviral delivery of TK and E-cadherin genes improved TK/GCV cytotoxicity and antitumoral activity in pancreatic

Nevertheless, other cell-cell adhesion proteins, either or not with known links to these junctions, have been targeted in the enzyme/prodrug approach, as illustrated by the following examples. Carcinoembryonic antigen (CEA), a glycoprotein involved in cell-cell adhesion as well as cell-extracellular substrate adhesion, is a particularly prolific case. The expression of CEA in cancer cells with the exclusion of adult normal cells has been used in multiple ways to provide specificity to the Enzyme/Prodrug system. This directed enzyme/prodrug therapy, involves the generation of a recombinant plasmid, containing CEA promoter to specifically drive the expression of the enzyme/prodrug systems in CEA-expressing cancer cells [119-121]. The E. coli purine nucleoside phosphorylase (ePNP) under the control of CEA promoter sequences greatly improved the antitumor efficacy of the ePNP/MePdR killing system in

induction of apoptosis, thus inhibiting tumor growth in a model of melanomas [117].

**3.4. The enzyme/prodrug approach in non-gap junctional communications**

was improved by Cx43 overexpression *in vitro* and *in vivo* [115].

adenoviruses which could result in further sustaining the effect [116].

medulloblastomas [112].

cancer cells [118].

**Figure 2. Different approaches of intercellular communication-based gene therapy.** Tumor cells (TC) are targeted with oncolytic viruses which, in addition to their proper cytotoxic effects [1], could be combined with the bystander effect ensured by the enzyme/prodrug system, here for example the TK/GCV pair [3]. TCs are made sensitive to the bystander effect cytotoxic effects by inducing connexin (Cx) expression and the formation of gap junction intercellular communication. This is achieved by either 4) viral vectors, or 5) pharmacological inducers. Cellular delivery of the viral vectors for the enzyme/prodrug system could also be achieved using mesenchymal stem cells (MSCs) and other types of stem/progenitor cells [6].

Based on the tropism shown by neural stem cells (NSCs) for glioma cells, the herpes simplex virus-thymidine kinase (HSVtk)/GCV system has also been used in targeting gliomas [106-108]. However, for practical reasons related to the availability of cells, the use of MSCs might be more relevant clinically than the use of NSCs [109]. The system has also been test‐ ed for AT-MSCs [110] and bone marrow-derived tumor-infiltrating cells (BM-TICs) target‐ ing of gliomas [111]. It was also proven to have a strong anti-tumor growth in medulloblastomas [112].

gliomas, a form of childhood central nervous system tumors with poor prognosis or medul‐ loblastomas [98-101], and even in disseminated brain metastases of non-neuronal origins such as melanoma and breast cancer [102-104]. The success of this approach now warrants clinical trials such as the one recently started to study the feasibility of intracerebral administration of NSCs in combination with oral 5-FC in patients with recurrent high-grade gliomas [105].

**Figure 2. Different approaches of intercellular communication-based gene therapy.** Tumor cells (TC) are targeted with oncolytic viruses which, in addition to their proper cytotoxic effects [1], could be combined with the bystander effect ensured by the enzyme/prodrug system, here for example the TK/GCV pair [3]. TCs are made sensitive to the bystander effect cytotoxic effects by inducing connexin (Cx) expression and the formation of gap junction intercellular communication. This is achieved by either 4) viral vectors, or 5) pharmacological inducers. Cellular delivery of the viral vectors for the enzyme/prodrug system could also be achieved using mesenchymal stem cells (MSCs) and other types

of stem/progenitor cells [6].

332 Novel Gene Therapy Approaches

As discussed earlier, a major limitation to the efficacy of the therapeutic use of GJIC is the deficiency in the bystander effect due to low expression levels of connexins. Expectedly, this is also a challenge when using the prodrug/stem cells combined therapy. This can be bypassed by restoring connexin levels. For instance, GSCs showed more reduced GJIC and connexin levels than differentiated glioma cells [113]. Valproic acid (VPA) was able to upregulate Cx43 and Cx26 and to enhance the bystander effect of suicide gene therapy by human bone marrow MSCs expressing HSV-TK (MSCs-TK) [114]. In another study, the use of Bone marrow-derived stem cells (BMSCs) in combination with the (HSV-TK)/GCV suicide gene therapy of gliomas was improved by Cx43 overexpression *in vitro* and *in vivo* [115].

The MSC/Prodrug and Oncovirus/Prodrug strategies are often combined. For instance, MSCs transduced with an adenoviral vector modified to express integrin-binding motifs (Ad5lucRGD) for better transduction efficiency, and expressing thymidine kinase were able not only to kill ovarian cancer cells via bystander effect, but also support replication of adenoviruses which could result in further sustaining the effect [116].

MSCs can also act through an anti-angiogenic mechanism. They have been shown to target endothelial cells and inhibit capillary growth, establish Cx43-based GJIC with the target ECs, and to increase the production of reactive oxygen species (ROS). This effect culminates in the induction of apoptosis, thus inhibiting tumor growth in a model of melanomas [117].

### **3.4. The enzyme/prodrug approach in non-gap junctional communications**

Curiously, unlike gap junctions, the number of studies delivering tight and adherens junctions or desmosomal proteins for cytotoxic gene therapy is limited. The adenoviral delivery of TK and E-cadherin genes improved TK/GCV cytotoxicity and antitumoral activity in pancreatic cancer cells [118].

Nevertheless, other cell-cell adhesion proteins, either or not with known links to these junctions, have been targeted in the enzyme/prodrug approach, as illustrated by the following examples. Carcinoembryonic antigen (CEA), a glycoprotein involved in cell-cell adhesion as well as cell-extracellular substrate adhesion, is a particularly prolific case. The expression of CEA in cancer cells with the exclusion of adult normal cells has been used in multiple ways to provide specificity to the Enzyme/Prodrug system. This directed enzyme/prodrug therapy, involves the generation of a recombinant plasmid, containing CEA promoter to specifically drive the expression of the enzyme/prodrug systems in CEA-expressing cancer cells [119-121]. The E. coli purine nucleoside phosphorylase (ePNP) under the control of CEA promoter sequences greatly improved the antitumor efficacy of the ePNP/MePdR killing system in pancreatic cancer cells [122]. The use of the double system including TK/GCV and CD/5-FC, in CEA-positive lung cancer cells, resulted in enhanced cytotoxicity [123]. A CEA promoterregulated oncolytic adenovirus vector driving the Hsp70 gene expression in CEA-positive pancreatic cancer cells was also active *in vitro* and *in vivo* [124]. Similar results were obtained by targeting suicide gene CD expression to colon cancer cells [125]. An E1A, E1B doublerestricted oncolytic adenovirus, AxdAdB-3, improved the therapeutic efficacy of the HSVtk/GCV system in gallbladder cancers when directed by the CEA promoter [126]. A modification of the approach done earlier, involved the addition of four tandem-linked NFkappaB DNA-binding sites (kappaB4) and a kappaB4 enhancer upstream of the CEA promoter, thus sensitizing colon cancer cells to the thymidine phosphorylase (TP)/ 5-fluorouracil (5-FU) or 5'-deoxy-5-fluorouradine (5'-DFUR) combinations [127]. A different way of targeted delivery of adenoviral vectors involved the generation of a bispecific adapter protein (sCAR-MFE), consisting of a fusion of the ectodomain of the coxsackie/adenovirus receptor (sCAR) with a single-chain anti-CEA antibody (MFE-23) [128]. A specific CEA RNA-targeting ribozyme was developed and used for selective delivery of HSVtk/GCV cytotoxic activity, into CEA-expressing cancer cells [129].

**4.1. GJIC-independent effects**

of their subcellular localization.

suppressor activities *in vivo* [154].

**4.2. Desmosomes, adherens and tight junctions in gene therapy**

The key players in the BE are connexins, the building blocks of gap junctional intercellular communication (GJIC) [23, 134, 135]. Even though the effectiveness of restoring Connexins' and GJIC's levels has traditionally been associated with the bystander effect in gene therapy, it has become clear that many functions of connexins, could be dissociated from both GJIC and the bystander effects [136-138] [139] [140] [141]. In this case, delivery of Cxs-encoding vectors could be used as a gene therapy approach, regardless of the use of enzyme/prodrug systems. However, future use of such application requires a better understanding of the non GJICrelated functions of these proteins, including their interacting partners and the mechanisms

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Adherens junctions and their related desmosomes, as well as tight junctions are essential types of cell-cell adhesion in both normal homeostasis and tumor progression [142-148]. Claudins are key tight junction proteins whose expression is deregulated in many cancers [146, 149]. Claudins CLDN3 and CLDN4 function as receptors for the Clostridium perfringens entero‐ toxin (CPE) produced by the bacterial Clostridium type A strain, resulting in cell death. A gene therapy application based on CPE gene transfer-mediated cytoxicity has been achieved but, as expected, was limited to CLDN3- and CLDN4-overexpressing tumors [150]. SiRNAmediated silencing of the expression of Epithelial Cell Adhesion Molecule (EpCAM or CD326), a cell-surface protein involved in tight junctions and metastasis in colon, breast and other epithelial carcinomas, was effective in decreasing the growth of breast cancer cells [151]. The same approach was used with an antibody against the carcinoembryonic antigen (CEA) in gastric cancer [152]. In fact, CEA has been extensively targeted in gene therapy approaches in different ways. A recombinant form of the oncolytic measles virus Edmonston strain (MV-Edm) changed to express CEA, demonstrated high cytotoxicity towards hepatocellular carcinoma cells *in vitro* and *in vivo* after either Intratumoral or intravenous delivery [153]. The cell adhesion molecule CECAM1, or carcinoembryonic antigen-related cell adhesion molecule 1, has served in an adenoviral gene therapy targeting prostate cancer cells and showed tumor

It is noteworthy that even when targeting these cell-cell communications could not be di‐ rectly performed or if it fails to affect tumor growth, there is no doubt about their impact on gene therapy applications. Cell-cell communications could indeed constitute a source of impediment to gene therapy, by constituting physical barriers to tumor targeting with oncolytic viruses *in vivo* [155] [156, 157]. This is particularly important in tissues such as the lung, intestine and reproductive system which show natural mechanisms of resistance to viral infection and might thus be less amenable to viral gene delivery. In fact, many junction proteins have been shown to be receptors for many viruses. The protein original‐ ly known as coxsackie-adenovirus receptor (hCAR), which was used in adenoviral-based gene therapy for cancer before realizing that it is a component of epithelial tight junctions [158, 159], affects the efficacy of the adenoviral gene therapy approach [160, 161]. Desmo‐ glein-2 (DSG-2), a desmosomal adhesion glycoprotein, is a receptor used by adenoviruses

A high affinity antibody for Neural cell adhesion molecule 2 (NCAM2), a cell-cell adhe‐ sion molecule, which is also capable of cell-extracellular matrix adhesion, was useful in increasing transduction efficiency of a fiber-modified adenoviral vector Adv-FZ33 in prostate and breast cancers, and restoring sensitivity to the UPRT/5-FU system in previ‐ ously resistant cells [130]. An Adenoviral vector incorporating an IgG Fc-binding motif (Z33) from the Staphylococcus protein A (Ad-FZ33) combined with tumor-specific anti-EpCAM (epithelial cell adhesion molecule) antibodies improved the viral transduction and the growth suppression of biliary cancer xenografts in nude mice in response to the UPRT/FU combination in human biliary cancers [131]. A similar approach used the en‐ zyme/prodrug system comprised of the enzyme carboxylesterase (CE) and its substrate the anticancer agent CPT-11 (irinotecan or 7-ethyl-10[4-(1-piperidino)-1-piperidino] car‐ bonyloxycamptothecin). An adenoviral vector Ad.C28-sCE2 containing a fusion gene en‐ coding a secreted form of human liver CE2 targeted to EpCAM was efficient in colon cancer spheroids [132]. As for CEA, the validation of the use of the EpCAM promoter to target the HSVtk/GCV therapy to cancer cells has been performed [133].

## **4. Gene therapy using bystander effect-independent intercellular communications**

The prominence of BE-based gene therapy in the literature should not eclipse the importance of other intercellular communications which do not involve the BE as candidates for gene therapy. These include in addition to a GJIC-independent role of connexins, other types of cellcell junctions as well as other types of protein-protein (ligand-receptor) interactions who depend on cell-cell interactions for their functions. Although to different extents, all these intercellular events have proven very amenable to gene therapy strategies.

#### **4.1. GJIC-independent effects**

pancreatic cancer cells [122]. The use of the double system including TK/GCV and CD/5-FC, in CEA-positive lung cancer cells, resulted in enhanced cytotoxicity [123]. A CEA promoterregulated oncolytic adenovirus vector driving the Hsp70 gene expression in CEA-positive pancreatic cancer cells was also active *in vitro* and *in vivo* [124]. Similar results were obtained by targeting suicide gene CD expression to colon cancer cells [125]. An E1A, E1B doublerestricted oncolytic adenovirus, AxdAdB-3, improved the therapeutic efficacy of the HSVtk/GCV system in gallbladder cancers when directed by the CEA promoter [126]. A modification of the approach done earlier, involved the addition of four tandem-linked NFkappaB DNA-binding sites (kappaB4) and a kappaB4 enhancer upstream of the CEA promoter, thus sensitizing colon cancer cells to the thymidine phosphorylase (TP)/ 5-fluorouracil (5-FU) or 5'-deoxy-5-fluorouradine (5'-DFUR) combinations [127]. A different way of targeted delivery of adenoviral vectors involved the generation of a bispecific adapter protein (sCAR-MFE), consisting of a fusion of the ectodomain of the coxsackie/adenovirus receptor (sCAR) with a single-chain anti-CEA antibody (MFE-23) [128]. A specific CEA RNA-targeting ribozyme was developed and used for selective delivery of HSVtk/GCV cytotoxic activity, into

A high affinity antibody for Neural cell adhesion molecule 2 (NCAM2), a cell-cell adhe‐ sion molecule, which is also capable of cell-extracellular matrix adhesion, was useful in increasing transduction efficiency of a fiber-modified adenoviral vector Adv-FZ33 in prostate and breast cancers, and restoring sensitivity to the UPRT/5-FU system in previ‐ ously resistant cells [130]. An Adenoviral vector incorporating an IgG Fc-binding motif (Z33) from the Staphylococcus protein A (Ad-FZ33) combined with tumor-specific anti-EpCAM (epithelial cell adhesion molecule) antibodies improved the viral transduction and the growth suppression of biliary cancer xenografts in nude mice in response to the UPRT/FU combination in human biliary cancers [131]. A similar approach used the en‐ zyme/prodrug system comprised of the enzyme carboxylesterase (CE) and its substrate the anticancer agent CPT-11 (irinotecan or 7-ethyl-10[4-(1-piperidino)-1-piperidino] car‐ bonyloxycamptothecin). An adenoviral vector Ad.C28-sCE2 containing a fusion gene en‐ coding a secreted form of human liver CE2 targeted to EpCAM was efficient in colon cancer spheroids [132]. As for CEA, the validation of the use of the EpCAM promoter to

target the HSVtk/GCV therapy to cancer cells has been performed [133].

intercellular events have proven very amenable to gene therapy strategies.

**4. Gene therapy using bystander effect-independent intercellular**

The prominence of BE-based gene therapy in the literature should not eclipse the importance of other intercellular communications which do not involve the BE as candidates for gene therapy. These include in addition to a GJIC-independent role of connexins, other types of cellcell junctions as well as other types of protein-protein (ligand-receptor) interactions who depend on cell-cell interactions for their functions. Although to different extents, all these

CEA-expressing cancer cells [129].

334 Novel Gene Therapy Approaches

**communications**

The key players in the BE are connexins, the building blocks of gap junctional intercellular communication (GJIC) [23, 134, 135]. Even though the effectiveness of restoring Connexins' and GJIC's levels has traditionally been associated with the bystander effect in gene therapy, it has become clear that many functions of connexins, could be dissociated from both GJIC and the bystander effects [136-138] [139] [140] [141]. In this case, delivery of Cxs-encoding vectors could be used as a gene therapy approach, regardless of the use of enzyme/prodrug systems. However, future use of such application requires a better understanding of the non GJICrelated functions of these proteins, including their interacting partners and the mechanisms of their subcellular localization.

#### **4.2. Desmosomes, adherens and tight junctions in gene therapy**

Adherens junctions and their related desmosomes, as well as tight junctions are essential types of cell-cell adhesion in both normal homeostasis and tumor progression [142-148]. Claudins are key tight junction proteins whose expression is deregulated in many cancers [146, 149]. Claudins CLDN3 and CLDN4 function as receptors for the Clostridium perfringens entero‐ toxin (CPE) produced by the bacterial Clostridium type A strain, resulting in cell death. A gene therapy application based on CPE gene transfer-mediated cytoxicity has been achieved but, as expected, was limited to CLDN3- and CLDN4-overexpressing tumors [150]. SiRNAmediated silencing of the expression of Epithelial Cell Adhesion Molecule (EpCAM or CD326), a cell-surface protein involved in tight junctions and metastasis in colon, breast and other epithelial carcinomas, was effective in decreasing the growth of breast cancer cells [151]. The same approach was used with an antibody against the carcinoembryonic antigen (CEA) in gastric cancer [152]. In fact, CEA has been extensively targeted in gene therapy approaches in different ways. A recombinant form of the oncolytic measles virus Edmonston strain (MV-Edm) changed to express CEA, demonstrated high cytotoxicity towards hepatocellular carcinoma cells *in vitro* and *in vivo* after either Intratumoral or intravenous delivery [153]. The cell adhesion molecule CECAM1, or carcinoembryonic antigen-related cell adhesion molecule 1, has served in an adenoviral gene therapy targeting prostate cancer cells and showed tumor suppressor activities *in vivo* [154].

It is noteworthy that even when targeting these cell-cell communications could not be di‐ rectly performed or if it fails to affect tumor growth, there is no doubt about their impact on gene therapy applications. Cell-cell communications could indeed constitute a source of impediment to gene therapy, by constituting physical barriers to tumor targeting with oncolytic viruses *in vivo* [155] [156, 157]. This is particularly important in tissues such as the lung, intestine and reproductive system which show natural mechanisms of resistance to viral infection and might thus be less amenable to viral gene delivery. In fact, many junction proteins have been shown to be receptors for many viruses. The protein original‐ ly known as coxsackie-adenovirus receptor (hCAR), which was used in adenoviral-based gene therapy for cancer before realizing that it is a component of epithelial tight junctions [158, 159], affects the efficacy of the adenoviral gene therapy approach [160, 161]. Desmo‐ glein-2 (DSG-2), a desmosomal adhesion glycoprotein, is a receptor used by adenoviruses Ad3, Ad7, Ad11 and Ad14, which subsequently results in epithelial-to-mesenchymal tran‐ sition-like changes and transient opening of intercellular junctions, a finding that could have an impact on the adenoviral gene delivery to normal or cancer cells [162, 163]. Adhe‐ rens junction proteins Nectin-1 and -2 are entry receptors for the herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) [164-166]. Increasing Nectin-1 expression resulted in in‐ creased susceptibility to HSV-1 infection and oncolytic activity and hence enhanced tumor regression *in vivo* [167]. Attenuated HSV-2 viral production in WB rat liver epithelial cells was found to depend on the viral protein co-localization with adherens junction proteins rather than by the status of gap junctions [168]. Taken together, these studies demonstrate the importance of junctional proteins in the infectivity of viruses and suggest that they might impact the efficacy of the viral oncolytic gene therapies. Compounds could thus be identified for example to improve viral gene transfer [169].

**5. Concluding remarks & perspectives**

to render cancer cells sensitive to enzyme/prodrug therapies.

they provide, will constitute a way for the future.

importance.

**Author details**

USA

Mohamed Amessou1

Over the years, it has become clear that various systems of cell-cell communication play critical roles not only in the normal development, architecture, remodeling and function of various tissues and organs, but in the onset of diseases as well. Cells are social entities and need to interact with each other in a way that ensures a favorable response to input from their immediate micro-environment (growth, survival, cytotoxicity) and a flexible adaptation to various roles and stress conditions. They also need to communicate during their death and demise. These communication processes are subject to various regulatory mechanisms which, when going awry, could result in various pathologies. One such instance where cell-cell communication has a particularly dramatic role is cancer progression, metastasis and response to therapeutic interventions. This reliance of cancer cells on cell-cell communication provides a therapeutic opportunity that will be fully exploited only if the mechanisms of its normal and aberrant functions are elucidated. This is for instance obvious when attempting to restore GJIC

Targeting Intercellular Communication in Cancer Gene Therapy

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337

Also, cancer cells share their microenvironment with many other cell types who are not just neutral bystanders. In particular, invasive cancer cells have very unstable intercellular contacts, as they keep migrating, constantly adhering to and detaching from cells on their way and thus changing the nature of their cell-cell communications. This might be a challenging fact when thinking of gene therapy strategies, and in fact any other type of therapy. Thus understanding these dynamics of change during the course of tumor progression is of utmost

As progress continues in developing strategies for a more efficient and selective viral delivery of gene therapeutics, the role of different junctions in the resistance of cancer epithelial cells to viral infections, needs to be balanced by the advantageous use of these proteins to render this approach more cancer-specific. In this respect, the enzyme/prodrug strategies need to be reconsidered in the light of the new findings that involve both gap junctions and other types of intercellular communications in the bystander effect. Examining the links between the

Finally, the impact of protein-protein interactions which are not necessarily engaged in cell junctions but are involved in direct cell-cell interactions, and the therapeutic opportunities

1 Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan,

different types of cell-cell communication will be critical for future applications.

and Mustapha Kandouz1,2

2 Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA

#### **4.3. Intercellular communications-dependent protein-protein interactions**

Many proteins, although not *bona fide* components of cell-cell junctions, are either affect‐ ed by these interactions or are very important in the function of direct cell-cell interac‐ tions, whether junctional or not. Prototypes of these proteins are the ones involved in axon guidance, such as the Eph/Ephrin proteins. The Eph family is the largest family of receptor tyrosine kinases, and includes the A-type Eph (EphA1–10) and B-type Eph (EphB1–6) receptors as well as A-type Ephrin (EphrinA1-6) and B-type Ephrin (Eph‐ rinB1-3) ligands. A particularity of this family is that, with few exceptions, the receptorligand interactions depend on direct cell-cell contacts, as both Ephs and Ephrins are anchored in interacting cellular membranes and in fact their role in cell-cell repulsion/ attraction and cell sorting is one of their main features. Study of Ephs/Ephrins' role in cancer has dramatically boomed in the last decade [170] and attempts are currently un‐ derway to target them in cancer therapy. Targeting of Ephs and Ephrins for gene thera‐ py has been very timid so far. EphA2 is probably one of the most sought after receptors of this family, as its expression is increased in many cancers and it has shown pro-onco‐ genic functions. A human adenoviral type 5 (HAd) vector expressing a secreted fusion protein constituted of the extracellular domain of EphrinA1, an EphA2 ligand, fused to the Fc portion of IgG1, was used to infect mammary epithelial cells and was found to activate and induce the degradation of EphA2, thus showing anti-tumor effects. After in‐ tratumoral inoculation, the HAd-EphrinA1-Fc vector significantly inhibited tumor growth *in vivo* [171, 172]. On the other hand, taking advantage of the high expression levels of EphA2 in cancer cells, an EphA2-binding peptide has been added to an Adenoviral vec‐ tor (Ad) to target pancreatic cancer cells and bypass the limitation of low Ad transduc‐ tion due to low levels of the major Ad receptor called Coxsackie and Ad receptor (CAR) [173]. Recently, EphA2 has been shown to be an essential receptor for the Kaposi's sarco‐ ma–associated herpesvirus, a major oncogenic virus in endothelial cells [174, 175]. Eph‐ rinB2 and EphrinB3, other family members, have also been identified as entry receptors for the Hendra virus and Nipah virus [176-178]. These data suggest that interfering with Ephs and Ephrins could be an interesting strategy in gene therapy applications by im‐ proving the transduction of viral vectors.

## **5. Concluding remarks & perspectives**

Ad3, Ad7, Ad11 and Ad14, which subsequently results in epithelial-to-mesenchymal tran‐ sition-like changes and transient opening of intercellular junctions, a finding that could have an impact on the adenoviral gene delivery to normal or cancer cells [162, 163]. Adhe‐ rens junction proteins Nectin-1 and -2 are entry receptors for the herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) [164-166]. Increasing Nectin-1 expression resulted in in‐ creased susceptibility to HSV-1 infection and oncolytic activity and hence enhanced tumor regression *in vivo* [167]. Attenuated HSV-2 viral production in WB rat liver epithelial cells was found to depend on the viral protein co-localization with adherens junction proteins rather than by the status of gap junctions [168]. Taken together, these studies demonstrate the importance of junctional proteins in the infectivity of viruses and suggest that they might impact the efficacy of the viral oncolytic gene therapies. Compounds could thus be

Many proteins, although not *bona fide* components of cell-cell junctions, are either affect‐ ed by these interactions or are very important in the function of direct cell-cell interac‐ tions, whether junctional or not. Prototypes of these proteins are the ones involved in axon guidance, such as the Eph/Ephrin proteins. The Eph family is the largest family of receptor tyrosine kinases, and includes the A-type Eph (EphA1–10) and B-type Eph (EphB1–6) receptors as well as A-type Ephrin (EphrinA1-6) and B-type Ephrin (Eph‐ rinB1-3) ligands. A particularity of this family is that, with few exceptions, the receptorligand interactions depend on direct cell-cell contacts, as both Ephs and Ephrins are anchored in interacting cellular membranes and in fact their role in cell-cell repulsion/ attraction and cell sorting is one of their main features. Study of Ephs/Ephrins' role in cancer has dramatically boomed in the last decade [170] and attempts are currently un‐ derway to target them in cancer therapy. Targeting of Ephs and Ephrins for gene thera‐ py has been very timid so far. EphA2 is probably one of the most sought after receptors of this family, as its expression is increased in many cancers and it has shown pro-onco‐ genic functions. A human adenoviral type 5 (HAd) vector expressing a secreted fusion protein constituted of the extracellular domain of EphrinA1, an EphA2 ligand, fused to the Fc portion of IgG1, was used to infect mammary epithelial cells and was found to activate and induce the degradation of EphA2, thus showing anti-tumor effects. After in‐ tratumoral inoculation, the HAd-EphrinA1-Fc vector significantly inhibited tumor growth *in vivo* [171, 172]. On the other hand, taking advantage of the high expression levels of EphA2 in cancer cells, an EphA2-binding peptide has been added to an Adenoviral vec‐ tor (Ad) to target pancreatic cancer cells and bypass the limitation of low Ad transduc‐ tion due to low levels of the major Ad receptor called Coxsackie and Ad receptor (CAR) [173]. Recently, EphA2 has been shown to be an essential receptor for the Kaposi's sarco‐ ma–associated herpesvirus, a major oncogenic virus in endothelial cells [174, 175]. Eph‐ rinB2 and EphrinB3, other family members, have also been identified as entry receptors for the Hendra virus and Nipah virus [176-178]. These data suggest that interfering with Ephs and Ephrins could be an interesting strategy in gene therapy applications by im‐

identified for example to improve viral gene transfer [169].

336 Novel Gene Therapy Approaches

proving the transduction of viral vectors.

**4.3. Intercellular communications-dependent protein-protein interactions**

Over the years, it has become clear that various systems of cell-cell communication play critical roles not only in the normal development, architecture, remodeling and function of various tissues and organs, but in the onset of diseases as well. Cells are social entities and need to interact with each other in a way that ensures a favorable response to input from their immediate micro-environment (growth, survival, cytotoxicity) and a flexible adaptation to various roles and stress conditions. They also need to communicate during their death and demise. These communication processes are subject to various regulatory mechanisms which, when going awry, could result in various pathologies. One such instance where cell-cell communication has a particularly dramatic role is cancer progression, metastasis and response to therapeutic interventions. This reliance of cancer cells on cell-cell communication provides a therapeutic opportunity that will be fully exploited only if the mechanisms of its normal and aberrant functions are elucidated. This is for instance obvious when attempting to restore GJIC to render cancer cells sensitive to enzyme/prodrug therapies.

Also, cancer cells share their microenvironment with many other cell types who are not just neutral bystanders. In particular, invasive cancer cells have very unstable intercellular contacts, as they keep migrating, constantly adhering to and detaching from cells on their way and thus changing the nature of their cell-cell communications. This might be a challenging fact when thinking of gene therapy strategies, and in fact any other type of therapy. Thus understanding these dynamics of change during the course of tumor progression is of utmost importance.

As progress continues in developing strategies for a more efficient and selective viral delivery of gene therapeutics, the role of different junctions in the resistance of cancer epithelial cells to viral infections, needs to be balanced by the advantageous use of these proteins to render this approach more cancer-specific. In this respect, the enzyme/prodrug strategies need to be reconsidered in the light of the new findings that involve both gap junctions and other types of intercellular communications in the bystander effect. Examining the links between the different types of cell-cell communication will be critical for future applications.

Finally, the impact of protein-protein interactions which are not necessarily engaged in cell junctions but are involved in direct cell-cell interactions, and the therapeutic opportunities they provide, will constitute a way for the future.

## **Author details**

Mohamed Amessou1 and Mustapha Kandouz1,2

1 Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan, USA

2 Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA

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[166] Warner MS, Geraghty RJ, Martinez WM, Montgomery RI, Whitbeck JC, Xu R, et al. A cell surface protein with herpesvirus entry activity (HveB) confers susceptibility to infection by mutants of herpes simplex virus type 1, herpes simplex virus type 2, and

[167] Yu Z, Li S, Huang YY, Fong Y, Wong RJ. Calcium depletion enhances nectin-1 expres‐ sion and herpes oncolytic therapy of squamous cell carcinoma. Cancer Gene Ther

[168] Miezeiewski B, McShane-Kay K, Woodruff RI, Mbuy GK, Knabb MT. Role of Adherens Junction Proteins in Differential Herpes Simplex Virus Type 2 Infectivity in Commu‐

[169] Sorscher EJ, Harris J, Alexander M, Rottgers A, Hardy K, Ponnazhagan S, et al. Activators of viral gene expression in polarized epithelial monolayers identified by

[170] Kandouz M. The Eph/Ephrin family in cancer metastasis: communication at the service

[171] Noblitt LW, Bangari DS, Shukla S, Knapp DW, Mohammed S, Kinch MS, et al. De‐ creased tumorigenic potential of EphA2-overexpressing breast cancer cells following treatment with adenoviral vectors that express EphrinA1. Cancer Gene Ther

[172] Noblitt LW, Bangari DS, Shukla S, Mohammed S, Mittal SK. Immunocompetent mouse model of breast cancer for preclinical testing of EphA2-targeted therapy. Cancer Gene

nication-Competent and -Deficient Cell Lines. Intervirology 2012.

rapid-throughput drug screening. Gene Ther 2006;13:781-8.

of invasion. Cancer Metastasis Rev 2012;31:353-73.

adenovirus serotypes 3, 7, 11 and 14. Nat Med 2011;17:96-104.

Res 2000;60:5031-6.

350 Novel Gene Therapy Approaches

1998;72:7064-74.

2007;14:738-47.

2004;11:757-66.

Ther 2005;12:46-53.

the vaginal mucosa. J Virol 2004;78:2530-6.

pseudorabies virus. Virology 1998;246:179-89.


**Chapter 14**

**Cancer Gene Therapy with Small Oligonucleotides**

Although enormous advances have been made in medical research, cancer still remains as one of the leading causes of death. The effects of cancer impacts on many lives and patients' families. Also, this insidious disease represents a huge financial and socioeconomic burden

Cancer is a multigenetic, multicellular and multisystemic disease. Recently, the International Agency for Research on Cancer (IARC) announced that 7.6 million deaths were due to can‐ cer and that there is on average 12.7 million new cases per year worldwide [1]. Current

Current conventional treatment options include surgery, chemotherapy and radiotherapy which can be used independently or sometimes, in combination. However, many of these treat‐ ment options are restricted to early stage tumours and even after surgery, there is still a high possibility of the tumour recurrence in these patients. In addition to the conventional treat‐ ments of cancer, there are also a number of relatively new therapies that include targeted cancer therapy, biological or immunotherapy and gene therapy. In contrast to conventional methods,

Cancer has two major forms: haemological cancers which are cancers arising from abnormal blood or bone marrow cells and solid tumours, which are tumours that grow into a solid mass. Traditionally, a solid mass were thought to be all rapidly dividing cells and all thera‐ peutics were designed to stop or reverse cellular proliferation. More recent studies have de‐ termined finer details of the nature of solid tumours and their microenvironment in order to

and reproduction in any medium, provided the original work is properly cited.

© 2013 Sakiragaoglu 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.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

trends indicate that 63% of cancer deaths are from developing countries [2], [3].

these newly developed treatments can be more effective and have fewer side effects.

Onur Sakiragaoglu, David Good and Ming Q. Wei

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54782

to both the family and health care systems.

**2. Solid tumour and its microenvironment**

**1. Introduction**

## **Cancer Gene Therapy with Small Oligonucleotides**

Onur Sakiragaoglu, David Good and Ming Q. Wei

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54782

## **1. Introduction**

Although enormous advances have been made in medical research, cancer still remains as one of the leading causes of death. The effects of cancer impacts on many lives and patients' families. Also, this insidious disease represents a huge financial and socioeconomic burden to both the family and health care systems.

Cancer is a multigenetic, multicellular and multisystemic disease. Recently, the International Agency for Research on Cancer (IARC) announced that 7.6 million deaths were due to can‐ cer and that there is on average 12.7 million new cases per year worldwide [1]. Current trends indicate that 63% of cancer deaths are from developing countries [2], [3].

Current conventional treatment options include surgery, chemotherapy and radiotherapy which can be used independently or sometimes, in combination. However, many of these treat‐ ment options are restricted to early stage tumours and even after surgery, there is still a high possibility of the tumour recurrence in these patients. In addition to the conventional treat‐ ments of cancer, there are also a number of relatively new therapies that include targeted cancer therapy, biological or immunotherapy and gene therapy. In contrast to conventional methods, these newly developed treatments can be more effective and have fewer side effects.

## **2. Solid tumour and its microenvironment**

Cancer has two major forms: haemological cancers which are cancers arising from abnormal blood or bone marrow cells and solid tumours, which are tumours that grow into a solid mass. Traditionally, a solid mass were thought to be all rapidly dividing cells and all thera‐ peutics were designed to stop or reverse cellular proliferation. More recent studies have de‐ termined finer details of the nature of solid tumours and their microenvironment in order to

© 2013 Sakiragaoglu 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. © 2013 The Author(s). Licensee InTech. This chapter is 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.

identify more specific targets for therapeutics and potential avenues for new cancer gene therapy. Hanahan and Weinberg [4] identified six hallmarks common to cancer:

mour-associated stromal cells [12]. There are also the extracellular matrix in solid tu‐ mours which is composed of complex secretions of proteins and proteoglycans produced by both neoplastic and normal stromal cells. This network continuously regulates signal‐ ing between tumour and normal stromal cells [13]. Traditionally, this microenvironment limits or prevents the effectiveness of many traditional as well as new therapies. Clearly, researchers need to develop new therapeutic strategies if we wish to successfully cure this disease. One such approach may be cancer gene therapy; however, research needs to also look at effective delivery of these agents in order to overcome the barriers set by these tu‐

Cancer Gene Therapy with Small Oligonucleotides

http://dx.doi.org/10.5772/54782

355

Targeted cancer therapeutics are chemical agents or monoclonal antibodies that specifical‐ ly inhibit the growth and spread of cancer by interfering with cell proliferation. These strategies interfere with cancer cell division and spread in different ways. Many of these therapies focus on proteins that are involved in cell signaling pathways, which form a complex communication system that governs basic cellular functions and activities. By blocking signaling pathways that make cancer cells grow and divide uncontrollably, tar‐ geted cancer therapies may induce cancer cell death through a process known as apopto‐

Targeted monoclonal antibodies may also be able to modulate immune responses, which raise the possibility that these treatment strategies can be combined with other therapeutic approaches to improve clinical outcomes [20]. Many targeted therapies against tumours af‐ fect pathways that are also crucial for immune development and function. This suggests the possibility that targeted therapies may help to optimize anti-tumour immune responses from immunotherapies. Similarly, immunotherapies may serve to consolidate impressive clinical responses from targeted therapies into long-lasting clinical remissions [21]. Immuno‐ therapy on the other hand, endeavors to stimulate a host immune response that effectuates

Biological therapy (immunotherapy or biotherapy,) is a method that uses a biological agent or the body's immune system, either directly or indirectly, to fight against cancer whereas traditional therapies target the tumour itself. For this reason, biological therapy can be used to lessen the side effects caused by other cancer treatments. For instance, dendritic cell-acti‐ vated cytokine-induced killer cells, used after chemotherapy in patients with advanced nonsmall cell lung cancer, improve immune response associated with up-regulation of

Using sipuleucel T and ipilimumab in phase III clinical trials the principle that immunother‐ apy can also extend cancer patient survival has been validated [23]. Sipuleucel T, which has recently been approved by the US Food and Drug Administration (FDA) for use in metastat‐

mours and their microenvironment.

**3. Targeted cancer therapy**

long-lived tumour destruction.

**3.1. Biological therapy**

sis, thus helping to stop cancer progression [18], [19].

cytokines that are involved in the anti-tumour activity [22].

*Sustaining proliferative signaling:* Within normal tissues, there is a carefully controlled pro‐ duction and release of growth-promoting signals. Cancer cells deregulate these signals and use them to their own advantage and may produce growth factor ligands for their own re‐ ceptors. In addition to this autocrine proliferative stimulation, cancer cells may send signals to stimulate normal cells, within the tumour microenvironment or surrounding tissue, to supply the cancer cells with various growth factors [5], [6].

*Evading growth suppressors:* Cancer cells must evade the actions of tumour suppressor genes, which limit cell growth and proliferation. The two prototypical tumour suppressors encode the retinoblastoma-associated protein (RB) and tumour protein p53 (TP53) which govern the decision of cells to proliferate or to activate senescence (biological aging).

*Resisting cell death:* Programmed cell death by apoptosis plays an important role in cells as a natural barrier to cancer development. The structure of the apoptotic machinery and how cancer cells can avoid these mechanisms has been widely studied since beginning of last decade. Tumour cells evolve many strategies to limit or evade apoptosis. The most common one is the loss of TP53 tumour suppressor function [7].

*Enabling replicative immortality:* Cancer cells must have unlimited replicative potential in or‐ der to generate macroscopic tumours. Multiple studies suggest that telomeres, which are re‐ petitive sequences at the ends of chromosomes, are centrally involved in the seemingly unlimited proliferation [8], [9].

*Inducing angiogenesis:* Like normal cells, tumour cells require the uptake of nutrients and oxygen as well as discharge carbon dioxide and metabolic waste. Since tumour cells grow faster than normal vasculature, tumour cells keep demanding the growth of the surround‐ ing vasculature. The induction of angiogenesis addresses some needs in this vicious cycle, facilitating sustained expansion of neoplastic growth [10].

*Activating invasion and metastasis:* Metastasis is responsible for as much as 90% of cancer-as‐ sociated mortality. In order for a primary tumour to metastasize it must achieve the follow‐ ing steps: *1) Intravasation*: At first, a cancer cell locally invades tissues in close proximity and thereby enters the microvasculature of the circulatory and lymphatic systems. *2) Extravasa‐ tion:* In the microvasculature, a tumour cell maintains itself and begins its movement through the bloodstream to microvessels of distant tissues where it leaves the bloodstream. *3) Colonization:* The migrated tumour cell survives within the microenvironment of its new location and uses the local tissue for cell proliferation and a secondary macroscopic tumour formation [11].

More recent studies showed that, once a solid tumour reaches approximately 2 mm, it contains hypoxic regions as a result of the failure of angiogenesis to keep pace with ab‐ normal tumourous tissue growth. Studies have shown that hypoxia can inhibit tumour cell differentiation and promote maintenance of cancer stem cells. Hypoxia also blocks the differentiation of mesenchymal stem/progenitor cells, which is a potential source of tu‐ mour-associated stromal cells [12]. There are also the extracellular matrix in solid tu‐ mours which is composed of complex secretions of proteins and proteoglycans produced by both neoplastic and normal stromal cells. This network continuously regulates signal‐ ing between tumour and normal stromal cells [13]. Traditionally, this microenvironment limits or prevents the effectiveness of many traditional as well as new therapies. Clearly, researchers need to develop new therapeutic strategies if we wish to successfully cure this disease. One such approach may be cancer gene therapy; however, research needs to also look at effective delivery of these agents in order to overcome the barriers set by these tu‐ mours and their microenvironment.

## **3. Targeted cancer therapy**

identify more specific targets for therapeutics and potential avenues for new cancer gene

*Sustaining proliferative signaling:* Within normal tissues, there is a carefully controlled pro‐ duction and release of growth-promoting signals. Cancer cells deregulate these signals and use them to their own advantage and may produce growth factor ligands for their own re‐ ceptors. In addition to this autocrine proliferative stimulation, cancer cells may send signals to stimulate normal cells, within the tumour microenvironment or surrounding tissue, to

*Evading growth suppressors:* Cancer cells must evade the actions of tumour suppressor genes, which limit cell growth and proliferation. The two prototypical tumour suppressors encode the retinoblastoma-associated protein (RB) and tumour protein p53 (TP53) which govern the

*Resisting cell death:* Programmed cell death by apoptosis plays an important role in cells as a natural barrier to cancer development. The structure of the apoptotic machinery and how cancer cells can avoid these mechanisms has been widely studied since beginning of last decade. Tumour cells evolve many strategies to limit or evade apoptosis. The most common

*Enabling replicative immortality:* Cancer cells must have unlimited replicative potential in or‐ der to generate macroscopic tumours. Multiple studies suggest that telomeres, which are re‐ petitive sequences at the ends of chromosomes, are centrally involved in the seemingly

*Inducing angiogenesis:* Like normal cells, tumour cells require the uptake of nutrients and oxygen as well as discharge carbon dioxide and metabolic waste. Since tumour cells grow faster than normal vasculature, tumour cells keep demanding the growth of the surround‐ ing vasculature. The induction of angiogenesis addresses some needs in this vicious cycle,

*Activating invasion and metastasis:* Metastasis is responsible for as much as 90% of cancer-as‐ sociated mortality. In order for a primary tumour to metastasize it must achieve the follow‐ ing steps: *1) Intravasation*: At first, a cancer cell locally invades tissues in close proximity and thereby enters the microvasculature of the circulatory and lymphatic systems. *2) Extravasa‐ tion:* In the microvasculature, a tumour cell maintains itself and begins its movement through the bloodstream to microvessels of distant tissues where it leaves the bloodstream. *3) Colonization:* The migrated tumour cell survives within the microenvironment of its new location and uses the local tissue for cell proliferation and a secondary macroscopic tumour

More recent studies showed that, once a solid tumour reaches approximately 2 mm, it contains hypoxic regions as a result of the failure of angiogenesis to keep pace with ab‐ normal tumourous tissue growth. Studies have shown that hypoxia can inhibit tumour cell differentiation and promote maintenance of cancer stem cells. Hypoxia also blocks the differentiation of mesenchymal stem/progenitor cells, which is a potential source of tu‐

therapy. Hanahan and Weinberg [4] identified six hallmarks common to cancer:

supply the cancer cells with various growth factors [5], [6].

one is the loss of TP53 tumour suppressor function [7].

facilitating sustained expansion of neoplastic growth [10].

unlimited proliferation [8], [9].

354 Novel Gene Therapy Approaches

formation [11].

decision of cells to proliferate or to activate senescence (biological aging).

Targeted cancer therapeutics are chemical agents or monoclonal antibodies that specifical‐ ly inhibit the growth and spread of cancer by interfering with cell proliferation. These strategies interfere with cancer cell division and spread in different ways. Many of these therapies focus on proteins that are involved in cell signaling pathways, which form a complex communication system that governs basic cellular functions and activities. By blocking signaling pathways that make cancer cells grow and divide uncontrollably, tar‐ geted cancer therapies may induce cancer cell death through a process known as apopto‐ sis, thus helping to stop cancer progression [18], [19].

Targeted monoclonal antibodies may also be able to modulate immune responses, which raise the possibility that these treatment strategies can be combined with other therapeutic approaches to improve clinical outcomes [20]. Many targeted therapies against tumours af‐ fect pathways that are also crucial for immune development and function. This suggests the possibility that targeted therapies may help to optimize anti-tumour immune responses from immunotherapies. Similarly, immunotherapies may serve to consolidate impressive clinical responses from targeted therapies into long-lasting clinical remissions [21]. Immuno‐ therapy on the other hand, endeavors to stimulate a host immune response that effectuates long-lived tumour destruction.

### **3.1. Biological therapy**

Biological therapy (immunotherapy or biotherapy,) is a method that uses a biological agent or the body's immune system, either directly or indirectly, to fight against cancer whereas traditional therapies target the tumour itself. For this reason, biological therapy can be used to lessen the side effects caused by other cancer treatments. For instance, dendritic cell-acti‐ vated cytokine-induced killer cells, used after chemotherapy in patients with advanced nonsmall cell lung cancer, improve immune response associated with up-regulation of cytokines that are involved in the anti-tumour activity [22].

Using sipuleucel T and ipilimumab in phase III clinical trials the principle that immunother‐ apy can also extend cancer patient survival has been validated [23]. Sipuleucel T, which has recently been approved by the US Food and Drug Administration (FDA) for use in metastat‐ ic prostate cancer, aims to stimulate T cells that are specific for prostatic acid phosphatase (PAP), a protein that is overexpressed in prostate carcinoma cells [24]. Although the precise basis of action for sipuleucel T remains under study, treatment with this drug increases sur‐ vival by an average of 4 months with minimal toxicity.

viral vectors for gene delivery. A large barrier for systemic gene therapy is reduced efficacy of transduction. Some of the other obstacles that affect efficacy of cancer gene therapy in‐ clude: 1). identification of key target genes responsible for the disease pathology and pro‐ gression; 2). identification of therapeutic genes that can inhibit disease progression; 3). optimal trans-gene expression for suppressing the target gene; and 4). delivery of therapeu‐ tic product to the target tissue at an efficacious dose [33]. Components of gene therapy for cancer can be replacement of tumour suppressor gene (p53), inhibition of oncogenes with antisense oligonucleotides, ribozymes and short inhibitory RNA, and activation of apoptosis genes [33]- [37]. However, sometimes the inhibition of the target gene and its pathway is not sufficient to inhibit the disease process because the cells have built abundant or alternative

Cancer Gene Therapy with Small Oligonucleotides

http://dx.doi.org/10.5772/54782

357

The efficient transgene expression requires appropriate promoters and enhancers in order to extend the duration of transgene expression in the cell or tissue. There are two types of pro‐ moters: constitutive or inducible. The constitutive promoters can be either viral or tissue specific promoters, such as melanin for melanoma. Inducible promoters can be induced to express transgenes with hormones or small molecules. Location of enhancers is upstream of the promoters and their function is to increase transgene expression 2-100 fold if the amount of gene product is required in very high amounts in the cell. In cancer cells, the duration of

Delivery of therapeutic genes is one of the most difficult issues in gene therapy studies. All viral gene therapy strategies have significant delivery limitations and very narrow ap‐ plications for cells and tissues. The best method for delivering genes will depend on the type of tissue to be targeted [36]. Commonly used vectors are retroviruses, adenoviruses, adeno-associated viruses and herpes simplex viruses. For cancer gene therapy, replication competent viruses such as the Newcastle disease virus offer a promising delivery technol‐

In addition to viral delivery methods there are non-viral technologies that offer several ad‐ vantages including less toxicity, reduced adverse immune responses and ease of producing larger quantities of vectors [39]. Chemically synthesized nanoparticles like DNA/stearyl pol‐ ylysine coated lipids or DNA coated with glycine oligomers (Peptoids) offer new advances for systemic gene therapy delivery. These molecules have been shown to be effective in can‐

In addition, bacterium has been developed for gene delivery purposes in cancer patients. The hypoxia and solid tumour microenvironment provide a living haven for anaerobic bac‐ teria. These so far fall into three classes. 1). Gram-positive lactic acid producing anaerobic bacteria; 2). Gram-negative intracellular, facultative anaerobes; and 3). the Gram-positive strictly anaerobic, saccharolytic/proteolytic Clostridia. Some of these modified bacterial cells, such as *Salmonella* and *Clostridium novyi* are already in phase 1 clinical studies [41].

pathways to compensate [35].

**5. Gene delivery**

ogy for human tumour therapy [38].

cer related angiogenesis [40].

transgene expression can be up to 30 days [33], [38].

Ipilimumab, an antibody, bolsters T cell responses and potentiates tumour destruction by blocking an important inhibitory signal for activated T cells. Ipilimumab, which has recently been approved by the FDA for use in patients with advanced melanoma, enhances overall survival compared with standard care and, most notably, achieves durable benefits (more than 2.5 years) for 15–20% of treated patients [26], [27].

Agents that target interleukins have also been used in cancer therapy. Blocking IL-6 signal‐ ing is a potential therapeutic strategy for cancer characterized by pathological IL-6 overpro‐ duction [27]. Researchers have demonstrated that the recombinant immunotoxin IL6 (T23)- PE38KDEL kills IL6R-overexpressing cancer cells, and causes significant tumour regression [28]. Other studies have shown that using viral and non-viral vectors to overexpress IL-24 in human cancer cells results in inhibition of tumour growth both *in vitro* and *in vivo* [29]. Tar‐ geted therapies and cancer immunotherapies have begun to enter clinical practice recently and when they were used together they may become promising treatments; however, these combinations have not been well studied.

## **4. Gene therapy**

Gene therapy is a relatively new method compared to other conventional treatments. It in‐ volves a therapeutic gene that is selectively delivered to a specific cell or tissue using a vec‐ tor or delivery vehicle. The first successful treatment of a human disease using gene therapy techniques (as an *ex vivo* gene replacement therapy) was for the treatment of X-linked severe combined immunodeficiency (X-SCID). The replacement of the wild type gene in the bone marrow stem cells was stably expressed and conferred selective growth advantage over the defective T cells. Following treatment, eight patients were cured of this disease but unfortu‐ nately 2 patients developed abnormal white blood cell growth due to the oncogenesis ability of the retroviral vector used for gene delivery [30].

Viral vectors are the most widely used vector system for gene therapy. Within Europe and the United States, gene therapy protocols are mostly used for cancer. Cancer gene therapy research has focused mainly on melanoma, prostate cancer, ovarian cancer and leukemia [31]. Some of these protocols for cancer gene therapy include the thymidine kinase gene and the genes for immunomodulatory cytokines such as IL-2 or granulocyte-macrophage colo‐ ny-stimulating factor (GM-CSF) and have been met with varying success [32]. Early clinical trials of gene therapy used *ex vivo* delivery of therapeutic genes to patients with monogenet‐ ic diseases. Such therapeutic genes, ie.: cytokine genes and viral thymidine kinase genes were transduced into autologous cells, normal cells or/and cancer cells. However, delivery of these therapeutic genes had limited efficacy due to their inability to achieve a pharmaco‐ logical dose of the gene at the target tissue. *In vivo* gene therapy protocols have used mostly viral vectors for gene delivery. A large barrier for systemic gene therapy is reduced efficacy of transduction. Some of the other obstacles that affect efficacy of cancer gene therapy in‐ clude: 1). identification of key target genes responsible for the disease pathology and pro‐ gression; 2). identification of therapeutic genes that can inhibit disease progression; 3). optimal trans-gene expression for suppressing the target gene; and 4). delivery of therapeu‐ tic product to the target tissue at an efficacious dose [33]. Components of gene therapy for cancer can be replacement of tumour suppressor gene (p53), inhibition of oncogenes with antisense oligonucleotides, ribozymes and short inhibitory RNA, and activation of apoptosis genes [33]- [37]. However, sometimes the inhibition of the target gene and its pathway is not sufficient to inhibit the disease process because the cells have built abundant or alternative pathways to compensate [35].

The efficient transgene expression requires appropriate promoters and enhancers in order to extend the duration of transgene expression in the cell or tissue. There are two types of pro‐ moters: constitutive or inducible. The constitutive promoters can be either viral or tissue specific promoters, such as melanin for melanoma. Inducible promoters can be induced to express transgenes with hormones or small molecules. Location of enhancers is upstream of the promoters and their function is to increase transgene expression 2-100 fold if the amount of gene product is required in very high amounts in the cell. In cancer cells, the duration of transgene expression can be up to 30 days [33], [38].

## **5. Gene delivery**

ic prostate cancer, aims to stimulate T cells that are specific for prostatic acid phosphatase (PAP), a protein that is overexpressed in prostate carcinoma cells [24]. Although the precise basis of action for sipuleucel T remains under study, treatment with this drug increases sur‐

Ipilimumab, an antibody, bolsters T cell responses and potentiates tumour destruction by blocking an important inhibitory signal for activated T cells. Ipilimumab, which has recently been approved by the FDA for use in patients with advanced melanoma, enhances overall survival compared with standard care and, most notably, achieves durable benefits (more

Agents that target interleukins have also been used in cancer therapy. Blocking IL-6 signal‐ ing is a potential therapeutic strategy for cancer characterized by pathological IL-6 overpro‐ duction [27]. Researchers have demonstrated that the recombinant immunotoxin IL6 (T23)- PE38KDEL kills IL6R-overexpressing cancer cells, and causes significant tumour regression [28]. Other studies have shown that using viral and non-viral vectors to overexpress IL-24 in human cancer cells results in inhibition of tumour growth both *in vitro* and *in vivo* [29]. Tar‐ geted therapies and cancer immunotherapies have begun to enter clinical practice recently and when they were used together they may become promising treatments; however, these

Gene therapy is a relatively new method compared to other conventional treatments. It in‐ volves a therapeutic gene that is selectively delivered to a specific cell or tissue using a vec‐ tor or delivery vehicle. The first successful treatment of a human disease using gene therapy techniques (as an *ex vivo* gene replacement therapy) was for the treatment of X-linked severe combined immunodeficiency (X-SCID). The replacement of the wild type gene in the bone marrow stem cells was stably expressed and conferred selective growth advantage over the defective T cells. Following treatment, eight patients were cured of this disease but unfortu‐ nately 2 patients developed abnormal white blood cell growth due to the oncogenesis ability

Viral vectors are the most widely used vector system for gene therapy. Within Europe and the United States, gene therapy protocols are mostly used for cancer. Cancer gene therapy research has focused mainly on melanoma, prostate cancer, ovarian cancer and leukemia [31]. Some of these protocols for cancer gene therapy include the thymidine kinase gene and the genes for immunomodulatory cytokines such as IL-2 or granulocyte-macrophage colo‐ ny-stimulating factor (GM-CSF) and have been met with varying success [32]. Early clinical trials of gene therapy used *ex vivo* delivery of therapeutic genes to patients with monogenet‐ ic diseases. Such therapeutic genes, ie.: cytokine genes and viral thymidine kinase genes were transduced into autologous cells, normal cells or/and cancer cells. However, delivery of these therapeutic genes had limited efficacy due to their inability to achieve a pharmaco‐ logical dose of the gene at the target tissue. *In vivo* gene therapy protocols have used mostly

vival by an average of 4 months with minimal toxicity.

than 2.5 years) for 15–20% of treated patients [26], [27].

combinations have not been well studied.

of the retroviral vector used for gene delivery [30].

**4. Gene therapy**

356 Novel Gene Therapy Approaches

Delivery of therapeutic genes is one of the most difficult issues in gene therapy studies. All viral gene therapy strategies have significant delivery limitations and very narrow ap‐ plications for cells and tissues. The best method for delivering genes will depend on the type of tissue to be targeted [36]. Commonly used vectors are retroviruses, adenoviruses, adeno-associated viruses and herpes simplex viruses. For cancer gene therapy, replication competent viruses such as the Newcastle disease virus offer a promising delivery technol‐ ogy for human tumour therapy [38].

In addition to viral delivery methods there are non-viral technologies that offer several ad‐ vantages including less toxicity, reduced adverse immune responses and ease of producing larger quantities of vectors [39]. Chemically synthesized nanoparticles like DNA/stearyl pol‐ ylysine coated lipids or DNA coated with glycine oligomers (Peptoids) offer new advances for systemic gene therapy delivery. These molecules have been shown to be effective in can‐ cer related angiogenesis [40].

In addition, bacterium has been developed for gene delivery purposes in cancer patients. The hypoxia and solid tumour microenvironment provide a living haven for anaerobic bac‐ teria. These so far fall into three classes. 1). Gram-positive lactic acid producing anaerobic bacteria; 2). Gram-negative intracellular, facultative anaerobes; and 3). the Gram-positive strictly anaerobic, saccharolytic/proteolytic Clostridia. Some of these modified bacterial cells, such as *Salmonella* and *Clostridium novyi* are already in phase 1 clinical studies [41].

#### **5.1. Bacterial oncolysis**

A surgeon named William B. Coley described for the first time that bacteria could be used as anticancer agents in 1890 [14]. Since then, scientists have been researching, and engineering, microorganisms such as *Clostridium, Bifidobacterium, Salmonella, Mycobacterium*, and *Bacillus* which have the ability to specifically target cancer cells and cause oncolysis. These anaerobic bacteria grow in the hypoxic core of solid tumours, where most traditional and many emerging therapeutics are unsuccessful. Due to their specificity for the tumour microenvir‐ onment, these bacteria are also promising vectors for delivering therapeutic genes to the cancer patients [15].

Moreover, siRNAs are able to act as primers for an RNA-dependent RNA polymerase that synthesizes extra dsRNA, that results in additional siRNA, which reinforces the effect of the

Cancer Gene Therapy with Small Oligonucleotides

http://dx.doi.org/10.5772/54782

359

Short hairpin RNA (shRNA) has been developed for long-term gene silencing [51]- [53]. shRNA is transcribed in the nucleus from short double-stranded DNA sequence with a hair‐ pin loop. After that the shRNA transcript is processed and incorporates with RISC in the cy‐ toplasm in a process that is the same as siRNA. However, there are some differences between siRNA and shRNA. Firstly, less than 1% of duplex siRNA remains in the cells 48 hours after introduction to the cells due to the high rate of degradation and turnover, where‐ as shRNA is constantly synthesized in host cells, leading to more durable gene silencing. Secondly, vector-based shRNA can only be modified by manipulating the expression strat‐ egy because it is firstly synthesized in the nucleus then transported to cytoplasm for further processes. Major component of RISC is the argonaute proteins. Within these protein family only Ago2 shows endonuclease activity to cleave shRNA in order to make it active single

MicroRNA (miRNA) is another group of small non-coding RNAs. miRNAs are important for gene regulation and highly conserved in cells. miRNA is firstly transcribed from precur‐ sors, that are located within intergenic sequences or introns, as a primary transcript (primiRNA) in the nucleus. Secondly, pre-miRNA is processed by an RNase III endonuclease called Drosha and then is exported to the cytoplasm. In the cytoplasm, pre-miRNA is cleaved by Dicer, another RNase III enzyme, to make 20-23 base pair long mature miRNA that consists of both guide and passengers strands with mismatches. Mature miRNA coop‐

One major difference between siRNA, shRNA, and miRNA is that both siRNA and shRNA require a complete match with the target mRNA but miRNA does not. Change in expression of a single miRNA may affect more than hundreds different genes [56]. miRNA takes part in gene regulation in different ways. Firstly miRNA binds to the 3' UTR region of the target mRNA and repress translation [55]. Nevertheless, a number of studies have shown that miRNA can also recognize coding region or the 5' UTR region to inhibit gene expression, although with less efficiency than at the 3' UTR [57], [58]. Other studies have also shown that miRNA can bind to the 5' UTR region of an mRNA and promote protein translation or can bind to DNA and induce gene expression [59], [60]. It has been stated that failure in reg‐ ulation of miRNA can cause a various human diseases, including cancer [61]. Better under‐ standing of mechanism and regulation of miRNA can contributes to develop effective RNAi

erates with RISC to inhibit translation with target mRNA degradation [55].

original siRNA [49], [50].

**6.2. shRNA**

stranded [54].

**6.3. MicroRNA**

therapies of cancer and other diseases.

The hypoxic nature of solid tumours is a haven for bacterial colonization and proliferation. It has been suggested that the anaerobic nature of hypoxic-necrotic regions within tumours provide faster growth of anaerobic and facultative anaerobic bacteria. Necrotic areas may al‐ so provide purines to the further growth of bacteria [16], [17].

## **6. The use of small oligonucleotides for gene silencing**

In 1998, Fire et al [42] discovered a mechanism, which is called RNA interference (RNAi), that moderates the activity of genes by using small single-stranded ribosomal nucleic acids.. These nucleic acids can bind to other molecules and play important roles in cells. It has been shown that small RNAs have the ability to control gene expression and other activities that were assumed to be carried out only by proteins. As a result RNAi has become a promising tool for researchers in the treatment of genetic diseases and cancer.

RNAi applications have a huge potential for use in inhibiting targets. To compare with molecular drugs, RNAi technology promises more specificity and wide range target ca‐ pacity. Small RNAs that used in RNAi technology currently have been grouped into four major classes: small interfering RNAs (siRNA), short hairpin RNA (shRNA), microRNAs (miRNAs), and P-element-induced wimpy testis (PIWI) interacting RNAs (piRNA). In ad‐ dition to these there are also qiRNA and other unknown small RNAs still to be discov‐ ered [43], [44].

#### **6.1. siRNA**

Synthetic siRNA was used in gene silencing firstly as an RNAi technology [45]. In the proc‐ ess, long dsRNA molecules were cut into 19-23 nucleotide RNAs, called siRNAs, which guide for cleaveage of complementary RNAs [46]. siRNA directly incorporates into RNAinduced silencing complex (RISC), where its guide-strand binds to and cleaves the comple‐ mentary mRNA. After the cleaved mRNA is released and degraded, the RISC binds to another mRNA and starts a new cycle of cleavage [47]. siRNA can cleave its target RNA in both the cytoplasm and the nucleus [48].

Moreover, siRNAs are able to act as primers for an RNA-dependent RNA polymerase that synthesizes extra dsRNA, that results in additional siRNA, which reinforces the effect of the original siRNA [49], [50].

#### **6.2. shRNA**

**5.1. Bacterial oncolysis**

358 Novel Gene Therapy Approaches

cancer patients [15].

ered [43], [44].

both the cytoplasm and the nucleus [48].

**6.1. siRNA**

A surgeon named William B. Coley described for the first time that bacteria could be used as anticancer agents in 1890 [14]. Since then, scientists have been researching, and engineering, microorganisms such as *Clostridium, Bifidobacterium, Salmonella, Mycobacterium*, and *Bacillus* which have the ability to specifically target cancer cells and cause oncolysis. These anaerobic bacteria grow in the hypoxic core of solid tumours, where most traditional and many emerging therapeutics are unsuccessful. Due to their specificity for the tumour microenvir‐ onment, these bacteria are also promising vectors for delivering therapeutic genes to the

The hypoxic nature of solid tumours is a haven for bacterial colonization and proliferation. It has been suggested that the anaerobic nature of hypoxic-necrotic regions within tumours provide faster growth of anaerobic and facultative anaerobic bacteria. Necrotic areas may al‐

In 1998, Fire et al [42] discovered a mechanism, which is called RNA interference (RNAi), that moderates the activity of genes by using small single-stranded ribosomal nucleic acids.. These nucleic acids can bind to other molecules and play important roles in cells. It has been shown that small RNAs have the ability to control gene expression and other activities that were assumed to be carried out only by proteins. As a result RNAi has become a promising

RNAi applications have a huge potential for use in inhibiting targets. To compare with molecular drugs, RNAi technology promises more specificity and wide range target ca‐ pacity. Small RNAs that used in RNAi technology currently have been grouped into four major classes: small interfering RNAs (siRNA), short hairpin RNA (shRNA), microRNAs (miRNAs), and P-element-induced wimpy testis (PIWI) interacting RNAs (piRNA). In ad‐ dition to these there are also qiRNA and other unknown small RNAs still to be discov‐

Synthetic siRNA was used in gene silencing firstly as an RNAi technology [45]. In the proc‐ ess, long dsRNA molecules were cut into 19-23 nucleotide RNAs, called siRNAs, which guide for cleaveage of complementary RNAs [46]. siRNA directly incorporates into RNAinduced silencing complex (RISC), where its guide-strand binds to and cleaves the comple‐ mentary mRNA. After the cleaved mRNA is released and degraded, the RISC binds to another mRNA and starts a new cycle of cleavage [47]. siRNA can cleave its target RNA in

so provide purines to the further growth of bacteria [16], [17].

**6. The use of small oligonucleotides for gene silencing**

tool for researchers in the treatment of genetic diseases and cancer.

Short hairpin RNA (shRNA) has been developed for long-term gene silencing [51]- [53]. shRNA is transcribed in the nucleus from short double-stranded DNA sequence with a hair‐ pin loop. After that the shRNA transcript is processed and incorporates with RISC in the cy‐ toplasm in a process that is the same as siRNA. However, there are some differences between siRNA and shRNA. Firstly, less than 1% of duplex siRNA remains in the cells 48 hours after introduction to the cells due to the high rate of degradation and turnover, where‐ as shRNA is constantly synthesized in host cells, leading to more durable gene silencing. Secondly, vector-based shRNA can only be modified by manipulating the expression strat‐ egy because it is firstly synthesized in the nucleus then transported to cytoplasm for further processes. Major component of RISC is the argonaute proteins. Within these protein family only Ago2 shows endonuclease activity to cleave shRNA in order to make it active single stranded [54].

#### **6.3. MicroRNA**

MicroRNA (miRNA) is another group of small non-coding RNAs. miRNAs are important for gene regulation and highly conserved in cells. miRNA is firstly transcribed from precur‐ sors, that are located within intergenic sequences or introns, as a primary transcript (primiRNA) in the nucleus. Secondly, pre-miRNA is processed by an RNase III endonuclease called Drosha and then is exported to the cytoplasm. In the cytoplasm, pre-miRNA is cleaved by Dicer, another RNase III enzyme, to make 20-23 base pair long mature miRNA that consists of both guide and passengers strands with mismatches. Mature miRNA coop‐ erates with RISC to inhibit translation with target mRNA degradation [55].

One major difference between siRNA, shRNA, and miRNA is that both siRNA and shRNA require a complete match with the target mRNA but miRNA does not. Change in expression of a single miRNA may affect more than hundreds different genes [56]. miRNA takes part in gene regulation in different ways. Firstly miRNA binds to the 3' UTR region of the target mRNA and repress translation [55]. Nevertheless, a number of studies have shown that miRNA can also recognize coding region or the 5' UTR region to inhibit gene expression, although with less efficiency than at the 3' UTR [57], [58]. Other studies have also shown that miRNA can bind to the 5' UTR region of an mRNA and promote protein translation or can bind to DNA and induce gene expression [59], [60]. It has been stated that failure in reg‐ ulation of miRNA can cause a various human diseases, including cancer [61]. Better under‐ standing of mechanism and regulation of miRNA can contributes to develop effective RNAi therapies of cancer and other diseases.

### **6.4. piRNA**

P-element-induced wimpy testis (PIWI) interacting RNAs (piRNAs) are small non-coding RNAs which interact PIWI proteins. These proteins are clade of argonaute proteins and are expressed predominantly in the germlines of a variety of organisms such as *Drosophila* and mammals. piRNAs help to maintain silence repetitive elements, the integrity of the genome, and the development of gametes. It has been suggested that both PIWI proteins and piRNAs are required for transposon silencing. In addition, a subset of piRNAs in *Drosophila* has been shown to function in silencing protein-coding genes [62].

Angiogenesis is a charecteristic for neoplasia and tumour metastasis. The vascular endothe‐ lial growth factor (VEGF) pathway is the most important pathway in angiogenesis. siRNA has been used to selectively silence VEGF and VEGF receptors to arrest tumour growth and angiogenesis successfully. Tumour growth was markedly suppressed [70]. Moreover, the siRNA targeting VEGF receptor 2 (VEGFR2) presented a significant inhibition of tumour growth with reduced VEGFR2 expression [71]. miRNAs affect malignant process by either resulting in overexpression or downregulation of a gene product. miRNA has been used as a tumour repressor in tumours with reduced expression of tumour supressor genes or other key genes. For example, miR-26a is highly expressed in normal liver tissues but its expres‐ sion is downregulated in liver tumours. Patients who have low miR-26a expression have de‐ creased overall survival compared with patients who have high miR-26a expression [72]. Further, miR-34c, miR-145, and miR-142-5p also show tumour suppresion properties in sev‐ eral lung cancers. Replacement of downregulated miRNA causes discontinue the growth of

Cancer Gene Therapy with Small Oligonucleotides

http://dx.doi.org/10.5772/54782

361

Due to miRNAs ability to supress tumours, miRNA gene therapy can be used for retriving miRNA gene expression and prevent tumour development. This approach is principally similar to that used for siRNA/shRNA therapeutics except that miRNAs are used to regain miRNA expression. For instance miR-34a is usually lost in human cancers especially lung cancer and prostate cancer. Using a neutral lipid emulsion (NLE), systemic delivery of syn‐ thetic miR-43a causes accumulation of miR-34a in normal lung tissues and lung tumours [74]. Furthermore, miR-34a and miR-16 are tumour suppressors of prostate cancer. miR-34a blocks metastasis of prostate cancer by repressing CD44 while miR-16 uses as target CDK1 and CDK2 genes which involves cell-cycle progression and cell proliferation [75], [76]. An‐ other miRNA subtype miR-22 induces cellular senescence. In a breast cancer xenograft mod‐ el, synthetic miR-22 induced cellular senescence and inhibited tumour growth by

The effective delivery of miRNA for cancer therapy can be achieved with either plasmid or virus. Kota *et al* has shown that miR-26a, of which re-expression in liver cancer cells inhibits cyclin D2 and E2 and induces G1 arrest, was delivered into hepatocellular tumour by using adeno-associated virus where it was sucessful in inhibiting of tumour development [78].

In general, normal cells produce most of the ATP from glucose through oxidative phosphor‐ ylation [79]. On the contrary, many cancer cells produce ATP by conversion of glucose to lactate and show lower oxidative phosphorylation activity. Tumour cells keep high yields of lactic acid and produce ATP by aerobic glycolysis with or without oxygen. This phenomen‐

Accelerated glycolysis provides ATP levels to the fast proliferating tumour cells in a hypoxic environment. Along with increased glutaminolysis, it also supplies metabolic intermediates that are essential for macromolecule biosynthesis and necessary for cell growth and division

**8. Modulating key genes controlling cancer metabolism**

lung cancer cells [73].

intratumoural delivery [77].

on is called "Warburg effect" [80].

piRNA–PIWI complexes are assumed to directly control transposon activity. piRNAs bound to PIWI proteins show homology-dependent target cleavage *in vitro*. Therefore, transposons are probably silenced through post-transcriptional transcript destruction [63].

piRNAs are different from siRNAs and miRNAs in several ways: 1) piRNAs consist of most‐ ly 24–31 nucleotides whereas other non-coding small RNAs are approximately 21 nucleoti‐ des; 2) opposite to several hundred species of miRNAs, piRNAs have 50,000 cloned species; 3) many piRNAs match to the genome in clusters of 20–90 kilobases in a strand-specific manner. In some clusters, one strand is changed abruptly to another strand which suggest that these bidirectional clusters may be transcribed divergently from a central promoter, however, siRNAs and miRNAs are derived from double-stranded and short hairpin RNA precursors, respectively. 4) Some piRNAs may be involved in epigenetic regulation whereas siRNAs and miRNAs generally target mRNAs [64], [65].

## **7. RNAi phenomena and its use in cancer therapy**

Due to their robustness and specificity, siRNA and shRNA have been extensively used to silence cancer-related gene targets. For instance, metastatic pancreatic cancer is one of the most deadly cancers. The overexpression of pancreatic duodenal homebox-1 (PDX-1) in pan‐ creatic adenocarcinoma has been shown to act as an oncogene. A plasmid vector encoding shRNA was used to target PDX-1 expression in a pancreatic animal model. Further examina‐ tion showed that the expression of PDX-1 was significantly reduced compared with that of the control group. As a result, silencing of PDX-1 expression inhibited tumour growth in malignant pancreatic cancer [66], [67].

Another example is human enhancer of zeste homolog 2 (EZH2) or p110-alpha silencing by siRNA with A systemic delivery vector in advanced prostate cancer in which tumour cells frequently metastasize to bones and regional lymph nodes. It has been shown that siRNA targeted to these proteins inhibit tumour metastasis in these cells [68]. Ryo *et al* have also shown that retrovirus-encoded shRNA was used to silence Pin1 expression in a prostate cancer model. Pin1 is a peptidyl-prolyl isomerase which catalyzes the cis/trans isomerization of peptidyl-prolyl peptide bonds [69]. It is highly overexpressed in prostate and breast can‐ cers. Pin1 shRNA significantly inhibited tumour growth and tumour metastasis.

Angiogenesis is a charecteristic for neoplasia and tumour metastasis. The vascular endothe‐ lial growth factor (VEGF) pathway is the most important pathway in angiogenesis. siRNA has been used to selectively silence VEGF and VEGF receptors to arrest tumour growth and angiogenesis successfully. Tumour growth was markedly suppressed [70]. Moreover, the siRNA targeting VEGF receptor 2 (VEGFR2) presented a significant inhibition of tumour growth with reduced VEGFR2 expression [71]. miRNAs affect malignant process by either resulting in overexpression or downregulation of a gene product. miRNA has been used as a tumour repressor in tumours with reduced expression of tumour supressor genes or other key genes. For example, miR-26a is highly expressed in normal liver tissues but its expres‐ sion is downregulated in liver tumours. Patients who have low miR-26a expression have de‐ creased overall survival compared with patients who have high miR-26a expression [72]. Further, miR-34c, miR-145, and miR-142-5p also show tumour suppresion properties in sev‐ eral lung cancers. Replacement of downregulated miRNA causes discontinue the growth of lung cancer cells [73].

**6.4. piRNA**

360 Novel Gene Therapy Approaches

P-element-induced wimpy testis (PIWI) interacting RNAs (piRNAs) are small non-coding RNAs which interact PIWI proteins. These proteins are clade of argonaute proteins and are expressed predominantly in the germlines of a variety of organisms such as *Drosophila* and mammals. piRNAs help to maintain silence repetitive elements, the integrity of the genome, and the development of gametes. It has been suggested that both PIWI proteins and piRNAs are required for transposon silencing. In addition, a subset of piRNAs in *Drosophila* has been

piRNA–PIWI complexes are assumed to directly control transposon activity. piRNAs bound to PIWI proteins show homology-dependent target cleavage *in vitro*. Therefore, transposons

piRNAs are different from siRNAs and miRNAs in several ways: 1) piRNAs consist of most‐ ly 24–31 nucleotides whereas other non-coding small RNAs are approximately 21 nucleoti‐ des; 2) opposite to several hundred species of miRNAs, piRNAs have 50,000 cloned species; 3) many piRNAs match to the genome in clusters of 20–90 kilobases in a strand-specific manner. In some clusters, one strand is changed abruptly to another strand which suggest that these bidirectional clusters may be transcribed divergently from a central promoter, however, siRNAs and miRNAs are derived from double-stranded and short hairpin RNA precursors, respectively. 4) Some piRNAs may be involved in epigenetic regulation whereas

Due to their robustness and specificity, siRNA and shRNA have been extensively used to silence cancer-related gene targets. For instance, metastatic pancreatic cancer is one of the most deadly cancers. The overexpression of pancreatic duodenal homebox-1 (PDX-1) in pan‐ creatic adenocarcinoma has been shown to act as an oncogene. A plasmid vector encoding shRNA was used to target PDX-1 expression in a pancreatic animal model. Further examina‐ tion showed that the expression of PDX-1 was significantly reduced compared with that of the control group. As a result, silencing of PDX-1 expression inhibited tumour growth in

Another example is human enhancer of zeste homolog 2 (EZH2) or p110-alpha silencing by siRNA with A systemic delivery vector in advanced prostate cancer in which tumour cells frequently metastasize to bones and regional lymph nodes. It has been shown that siRNA targeted to these proteins inhibit tumour metastasis in these cells [68]. Ryo *et al* have also shown that retrovirus-encoded shRNA was used to silence Pin1 expression in a prostate cancer model. Pin1 is a peptidyl-prolyl isomerase which catalyzes the cis/trans isomerization of peptidyl-prolyl peptide bonds [69]. It is highly overexpressed in prostate and breast can‐

cers. Pin1 shRNA significantly inhibited tumour growth and tumour metastasis.

are probably silenced through post-transcriptional transcript destruction [63].

shown to function in silencing protein-coding genes [62].

siRNAs and miRNAs generally target mRNAs [64], [65].

malignant pancreatic cancer [66], [67].

**7. RNAi phenomena and its use in cancer therapy**

Due to miRNAs ability to supress tumours, miRNA gene therapy can be used for retriving miRNA gene expression and prevent tumour development. This approach is principally similar to that used for siRNA/shRNA therapeutics except that miRNAs are used to regain miRNA expression. For instance miR-34a is usually lost in human cancers especially lung cancer and prostate cancer. Using a neutral lipid emulsion (NLE), systemic delivery of syn‐ thetic miR-43a causes accumulation of miR-34a in normal lung tissues and lung tumours [74]. Furthermore, miR-34a and miR-16 are tumour suppressors of prostate cancer. miR-34a blocks metastasis of prostate cancer by repressing CD44 while miR-16 uses as target CDK1 and CDK2 genes which involves cell-cycle progression and cell proliferation [75], [76]. An‐ other miRNA subtype miR-22 induces cellular senescence. In a breast cancer xenograft mod‐ el, synthetic miR-22 induced cellular senescence and inhibited tumour growth by intratumoural delivery [77].

The effective delivery of miRNA for cancer therapy can be achieved with either plasmid or virus. Kota *et al* has shown that miR-26a, of which re-expression in liver cancer cells inhibits cyclin D2 and E2 and induces G1 arrest, was delivered into hepatocellular tumour by using adeno-associated virus where it was sucessful in inhibiting of tumour development [78].

## **8. Modulating key genes controlling cancer metabolism**

In general, normal cells produce most of the ATP from glucose through oxidative phosphor‐ ylation [79]. On the contrary, many cancer cells produce ATP by conversion of glucose to lactate and show lower oxidative phosphorylation activity. Tumour cells keep high yields of lactic acid and produce ATP by aerobic glycolysis with or without oxygen. This phenomen‐ on is called "Warburg effect" [80].

Accelerated glycolysis provides ATP levels to the fast proliferating tumour cells in a hypoxic environment. Along with increased glutaminolysis, it also supplies metabolic intermediates that are essential for macromolecule biosynthesis and necessary for cell growth and division [81]. Although the conversion of pyruvate into lactate occurs in normal cells in hypoxic con‐ ditions, tumour cells produce excessive amounts of lactate even when oxygen is not a limit‐ ing factor. It has been stated that this glycolytic phenotype results from the adaptation of premalignant lesions to spasmodic hypoxia [82].

reported in many types of human cancers, including leukemias, lymphomas, and carcino‐ mas [97]. Cimmino *et al* demonstrate that miR-15a and miR-16-1 expression is inversely cor‐ related to BCL2 expression in chronic lymphocytic leukemia (CLL) [98]. Both these miRNAs negatively regulate BCL2 at a posttranscriptional level. BCL2 repression by miR-15a and miR-16-1 induces apoptosis in a leukemic cell line model. As a result, miR-15 and miR-16 are natural BCL2 inhibitors that could be used for therapy of tumours in which BCL-2 overex‐

Cancer Gene Therapy with Small Oligonucleotides

http://dx.doi.org/10.5772/54782

363

Another protein which is cyclooxygenase-2 (COX-2) enzyme has been involved in the tu‐ mourgenesis and in the progression of colorectal cancer (CRC) [99]. The use of developing RNAi-based techniques allowed researchers to better study the molecular and phenotypical loss of function of COX-2 gene by doing experiments based on a strong COX-2 silencing ef‐ fects. Denkert and colleagues [100] tested the effect of an anti-COX-2 siRNA (siCOX-2) on OVCAR-3 cells derived from human ovarian carcinoma. A comparison with the COX-2 in‐ hibitory drug NS-398 has shown that a different effect of siCOX-2 occured due to its highly specific mechanism of action. Even though COX-2 protein levels significantly reduced in both cases, NS-398 treatment induced a G0/G1 cell cycle arrest in OVCAR-3 cells but only after another factor stimulation. This effect was probably due to the action of NS-398 on oth‐

Research performed by Charames *et al* and Kobayashi *et al* demonstrated that an siRNAs can efficiently knockdown COX-2 in HT-29 human colon cancer cells and bovine Cumulus-Granulosa (CG) cells [101], [102]. Based on their results, it is clear that RNAi, compared with non-steroidal anti-inflammatory drug (NSAIDs), are more powerful and selective tools for

RNAi-mediated COX-2 silencing proved to be highly effective using anti-COX-2 shRNAs (shCOX-2). In 2006, Strillaci *et al* have illustrated that an *in vitro* strategy in which COX-2 is stably knockdowned in colon cancer cells (HT- 29) [107]. There are several studies that have implicated failure of miR expression in carcinogenic mechanisms [108], [109]. miR concen‐ trations may be increased or repressed in hepatocellular carcinoma, which suggests that these sequences may act as oncogenes or suppressors of hepatocyte transformation. Recent studies using miRNA microarrays showed that high expression of miR-21 can contribute to growth and spread of human hepatocellular cancer (HCC) by inhibiting phosphatase and tensin homolog (PTEN) tumour suppressor, whereas low levels of miR-122a which target to

One of the cancer-related genes is the multiple drug resistance (MDR1) gene which provides resistance to vinca alkaloids (vinblastine, vincristine), anthracyclins (adriamycin, daunorubi‐ cin), etoposide and paclitaxel. In order to reverse the MDR1 gene-dependent multidrug re‐ sistance (MDR), two siRNA constructs were designed to inhibit MDR1 expression by RNA interference. Some data indicate that this approach may be applicable to cancer patients to change from tumouric P-glycoprotein-dependent MDR phenotype back to a drug-sensitive one [112]. An Eppstein Barr Virus (EBV)-encoded product, latent membrane protein (LMP-1), is considered to be an oncogene playing an essential role in cell transformation and metastasis. EBV-encoded LMP-1 was inhibited by RNAi and selective inhibition of LMP-1

presses.

er cellular targets involved in cell proliferation.

studying *in vitro* COX-2 functioning [103]- [106].

cyclin G1 mRNA result in increased HCC [110], [111].

Down-regulation or completely silencing genes that are related with cancer metabolism may be the key of future methods of cancer treatment. Hexokinase II and pyruvate kinase M2 are some of metabolic genes that have been focused on in siRNA studies. It has been shown that down-regulated hexokinase II by RNA interference resulted in increased apoptosis rate in colon cancer cells [83]. Inhibition of Pyruvate Kinase M2, a metabolic enzyme whose expres‐ sion in cancer cells results in aerobic glycolysis causes substantial tumor regression [84]. An‐ other study have indicated that combined therapy with siRNA and cisplatin drug resulted in enhanced antitumor activity [85].

## **9. Silencing telomerase activity by RNAi**

There are specialized, repeated structures called telomere which protect the ends of all chro‐ mosomes in eukaryotic organisms [86]. Telomeres are essential for chromosome stability. Also, it is suggested that telomeres are responsible for cellular aging since it acts as a mitotic clock [87], [88]. Telomere shortening triggers the senescence check point so-called Hayflick limit in human somatic cells [89]. Escape from this check point is the first step in cellular im‐ mortalization [90].

In most organisms the main mechanism of telomere length maintenance is carried out by te‐ lomerase, a ribonucleoprotein complex [91]. This enzyme elongates the telomeres at the 3' end of the DNA [92]. Although the telomerase complex contains a number of components that provide telomerase activity *in vivo*, the basic components of telomerase enzyme are telo‐ merase reverse transcriptase (TERT) and telomerase RNA [93]. Increase expression of these Proteins results in high telomerase activity and has been demonstrated in 85-90% of all hu‐ man tumours [94].

Currently, attempts are underway for reducing telomerase activity which may provide a po‐ tential avenue for cancer gene therapy. Kosciolek *et al* has shown that telomerase activity in human cancer cells can be inhibited by siRNAs targeting telomerase components [95]. Hu‐ man cancer cell lines were transfected with 21 nucleotide double-stranded RNA homolo‐ gous to either the catalytic subunit of telomerase (hTERT) or to its template RNA (hTR). Both agents reduced telomerase activity in a variety of human cancer cell lines which in‐ cluded both carcinomas and sarcomas.

## **10. Other gene silencing approaches in cancer therapy**

B cell lymphoma 2 (BCL2) is an important gene in eukaryotic cells as its expression causes uncontrolled growth by inhibiting cell death [96]. Overexpression of BCL2 protein has been reported in many types of human cancers, including leukemias, lymphomas, and carcino‐ mas [97]. Cimmino *et al* demonstrate that miR-15a and miR-16-1 expression is inversely cor‐ related to BCL2 expression in chronic lymphocytic leukemia (CLL) [98]. Both these miRNAs negatively regulate BCL2 at a posttranscriptional level. BCL2 repression by miR-15a and miR-16-1 induces apoptosis in a leukemic cell line model. As a result, miR-15 and miR-16 are natural BCL2 inhibitors that could be used for therapy of tumours in which BCL-2 overex‐ presses.

[81]. Although the conversion of pyruvate into lactate occurs in normal cells in hypoxic con‐ ditions, tumour cells produce excessive amounts of lactate even when oxygen is not a limit‐ ing factor. It has been stated that this glycolytic phenotype results from the adaptation of

Down-regulation or completely silencing genes that are related with cancer metabolism may be the key of future methods of cancer treatment. Hexokinase II and pyruvate kinase M2 are some of metabolic genes that have been focused on in siRNA studies. It has been shown that down-regulated hexokinase II by RNA interference resulted in increased apoptosis rate in colon cancer cells [83]. Inhibition of Pyruvate Kinase M2, a metabolic enzyme whose expres‐ sion in cancer cells results in aerobic glycolysis causes substantial tumor regression [84]. An‐ other study have indicated that combined therapy with siRNA and cisplatin drug resulted

There are specialized, repeated structures called telomere which protect the ends of all chro‐ mosomes in eukaryotic organisms [86]. Telomeres are essential for chromosome stability. Also, it is suggested that telomeres are responsible for cellular aging since it acts as a mitotic clock [87], [88]. Telomere shortening triggers the senescence check point so-called Hayflick limit in human somatic cells [89]. Escape from this check point is the first step in cellular im‐

In most organisms the main mechanism of telomere length maintenance is carried out by te‐ lomerase, a ribonucleoprotein complex [91]. This enzyme elongates the telomeres at the 3' end of the DNA [92]. Although the telomerase complex contains a number of components that provide telomerase activity *in vivo*, the basic components of telomerase enzyme are telo‐ merase reverse transcriptase (TERT) and telomerase RNA [93]. Increase expression of these Proteins results in high telomerase activity and has been demonstrated in 85-90% of all hu‐

Currently, attempts are underway for reducing telomerase activity which may provide a po‐ tential avenue for cancer gene therapy. Kosciolek *et al* has shown that telomerase activity in human cancer cells can be inhibited by siRNAs targeting telomerase components [95]. Hu‐ man cancer cell lines were transfected with 21 nucleotide double-stranded RNA homolo‐ gous to either the catalytic subunit of telomerase (hTERT) or to its template RNA (hTR). Both agents reduced telomerase activity in a variety of human cancer cell lines which in‐

B cell lymphoma 2 (BCL2) is an important gene in eukaryotic cells as its expression causes uncontrolled growth by inhibiting cell death [96]. Overexpression of BCL2 protein has been

premalignant lesions to spasmodic hypoxia [82].

**9. Silencing telomerase activity by RNAi**

in enhanced antitumor activity [85].

mortalization [90].

362 Novel Gene Therapy Approaches

man tumours [94].

cluded both carcinomas and sarcomas.

**10. Other gene silencing approaches in cancer therapy**

Another protein which is cyclooxygenase-2 (COX-2) enzyme has been involved in the tu‐ mourgenesis and in the progression of colorectal cancer (CRC) [99]. The use of developing RNAi-based techniques allowed researchers to better study the molecular and phenotypical loss of function of COX-2 gene by doing experiments based on a strong COX-2 silencing ef‐ fects. Denkert and colleagues [100] tested the effect of an anti-COX-2 siRNA (siCOX-2) on OVCAR-3 cells derived from human ovarian carcinoma. A comparison with the COX-2 in‐ hibitory drug NS-398 has shown that a different effect of siCOX-2 occured due to its highly specific mechanism of action. Even though COX-2 protein levels significantly reduced in both cases, NS-398 treatment induced a G0/G1 cell cycle arrest in OVCAR-3 cells but only after another factor stimulation. This effect was probably due to the action of NS-398 on oth‐ er cellular targets involved in cell proliferation.

Research performed by Charames *et al* and Kobayashi *et al* demonstrated that an siRNAs can efficiently knockdown COX-2 in HT-29 human colon cancer cells and bovine Cumulus-Granulosa (CG) cells [101], [102]. Based on their results, it is clear that RNAi, compared with non-steroidal anti-inflammatory drug (NSAIDs), are more powerful and selective tools for studying *in vitro* COX-2 functioning [103]- [106].

RNAi-mediated COX-2 silencing proved to be highly effective using anti-COX-2 shRNAs (shCOX-2). In 2006, Strillaci *et al* have illustrated that an *in vitro* strategy in which COX-2 is stably knockdowned in colon cancer cells (HT- 29) [107]. There are several studies that have implicated failure of miR expression in carcinogenic mechanisms [108], [109]. miR concen‐ trations may be increased or repressed in hepatocellular carcinoma, which suggests that these sequences may act as oncogenes or suppressors of hepatocyte transformation. Recent studies using miRNA microarrays showed that high expression of miR-21 can contribute to growth and spread of human hepatocellular cancer (HCC) by inhibiting phosphatase and tensin homolog (PTEN) tumour suppressor, whereas low levels of miR-122a which target to cyclin G1 mRNA result in increased HCC [110], [111].

One of the cancer-related genes is the multiple drug resistance (MDR1) gene which provides resistance to vinca alkaloids (vinblastine, vincristine), anthracyclins (adriamycin, daunorubi‐ cin), etoposide and paclitaxel. In order to reverse the MDR1 gene-dependent multidrug re‐ sistance (MDR), two siRNA constructs were designed to inhibit MDR1 expression by RNA interference. Some data indicate that this approach may be applicable to cancer patients to change from tumouric P-glycoprotein-dependent MDR phenotype back to a drug-sensitive one [112]. An Eppstein Barr Virus (EBV)-encoded product, latent membrane protein (LMP-1), is considered to be an oncogene playing an essential role in cell transformation and metastasis. EBV-encoded LMP-1 was inhibited by RNAi and selective inhibition of LMP-1 had anti-proliferation effect on Nasopharyngeal carcinoma (NPC) cell. RNAi could be a powerful method in further investigations of LMP-1 [113]. A recombinant adeno-associated virus type 2 vector was used to deliver shRNA targeting EBV-LMP-1 into the EBV-positive human NPC C666-1 cells. Results showed that long-term suppression of EBV-encoded LMP-1 *in vivo* is an effective way for preventing NPC metastasis [114].

Small oligonucleic acids can form complex secondary and tertiary structures. These nucleic acids can bind to other molecules and play an important role in cells. It has been shown that small RNAs have the ability to control gene expression and other activities that previously were assumed to be carried out only by proteins. As a result, small fragments of RNA may be tools for researchers to cure cancer. Small RNAs that are used in RNAi technology cur‐ rently have been grouped into three major classes: small interfering RNAs (siRNA), micro‐

Cancer Gene Therapy with Small Oligonucleotides

http://dx.doi.org/10.5772/54782

365

siRNA and shRNA have been extensively used to silence cancer-related targets. miRNA, as a tumour supressor, can be used in gene therapy for retrieving miRNA gene expression and preventing tumour development. Tumour cells keep high yields of lactic acid and produce ATP by aerobic glycolysis with or without oxygen. Accelerated glycolysis provides ATP lev‐ els to the fast proliferating tumour cells in a hypoxic environment. It is proposed that with RNA interference technologies metabolic genes in cancer cells can be silenced. Therefore tu‐ mour proliferation can be inhibited. Beside that, studies have shown that genes which are related to cancer such as Telomerase, BCL-2, COX-2 can be silenced for preventing cancer.

1 Division of Molecular and Gene Therapies, Griffith Health Institute and School of Medical

[1] International Agency for Research on Cancer (IARC): GLOBOCAN (2008). Cancer in‐

[2] Jemal, A, Bray, F, Center, M. M, Ferlay, J, Ward, E, & Forman, D. Global cancer statis‐

[3] Ferlay, J, Shin, H. R, Bray, F, Forman, D, Mathers, C, & Parkin, D. M. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer. (2010). , 127,

[5] Cheng, N, Chytil, A, Shyr, Y, Joly, A, & Moses, H. L. Transforming growth factorbeta signaling-deficient fibroblasts enhance hepatocyte growth factor signaling in

[4] Hanahan, D, & Weinberg, R. The hallmarks of cancer. Cell. (2000). , 100, 57-70.

2 School of Physiotherapy, Australian Catholic University, Banyo, QLD, Australia

cidence and mortality worldwide. Lyon, France: IARC. 2010.

, David Good1,2 and Ming Q. Wei1\*

RNAs (miRNAs), and PIWI interacting RNAs (piRNA).

\*Address all correspondence to: m.wei@griffith.edu.au

Science, Griffith University, Gold Coast, QLD, Australia

tics. CA Cancer J Clin. (2011). , 61, 69-90.

**Author details**

Onur Sakiragaoglu1

**References**

2893-2917.

One of the most important signaling pathways to control growth and proliferation of our cells is the mitogen-activated protein kinase (MAPK) pathway. Ras, which is an enzyme in this pathway, is turned to an oncogenic form in about 15% of human cancer. Suppression of tumourgenicity was done by virus-mediated RNAi to inhibit specifically the oncogenic al‐ lele of K-ras (K-rasV12) in human tumour cells [115]. Other studies have reported that the use of siRNA can further block the Ras to Map kinase cascade, at either the Raf level or through NADPH oxidase1 (Nox1) [116]- [118].

## **11. Conclusion**

Cancer is widely recognised as one of the largest burdens to health world wide. Some main features of cancers are its ability to sustain proliferative signaling, evade growth suppres‐ sors, resist cell death, enable replicative immortality, induce angiogenesis, and activate inva‐ sion and metastasis. Until now, many methods have been developed for the treatment of cancer. Conventional treatment methods, ie.: surgery, chemotherapy and radiotherapy, are still widely used in the treatment of most cancers. However, these methods result in a high recurrence of cancer in patients. Clearly, there is an urgent need for the development of new therapies. In contrast to conventional methods, targeted gene therapy, immunotherapy, and gene therapy offer promising alternatives that are more effective and produce less side ef‐ fects. Both targeted therapies and cancer immunotherapies have recently been used in clinic and these therapies can be succesful when used together, nevertheless, there are still limita‐ tions with these therapies.

Gene therapy has already begun to show great promise and is expected to be more effective in curing cancer. Targets for cancer gene therapy may include tumour suppressor genes (e.g. p53), oncogenes,and apoptosis genes. The most problematic issue for cancer gene therapy studies is the delivery of the therapheutic gene to the tumour cells. Although viral delivery methods are widely in use, there are non-viral technologies that offer several advantages that include less toxicity, reduced adverse immune responses and easier to producing large amounts of gene products. More recently, bacteria have also been used in cancer treatment. The hypoxic nature of solid tumours provides considerable conditions for growth of bacte‐ ria and bacterial colonisation. Necrotic areas can also supply purines to further facilitate growth of bacteria.

Bacterial delivery of RNA silencing tools combined with benefit of bacterial oncolysis can contribute to the treatment of cancer. Exploiting of small oligonucleic acids which are car‐ ried by spesific bacteria to cancer cells can be an effective way to cut energy supply and lysis of tumor cells.

Small oligonucleic acids can form complex secondary and tertiary structures. These nucleic acids can bind to other molecules and play an important role in cells. It has been shown that small RNAs have the ability to control gene expression and other activities that previously were assumed to be carried out only by proteins. As a result, small fragments of RNA may be tools for researchers to cure cancer. Small RNAs that are used in RNAi technology cur‐ rently have been grouped into three major classes: small interfering RNAs (siRNA), micro‐ RNAs (miRNAs), and PIWI interacting RNAs (piRNA).

siRNA and shRNA have been extensively used to silence cancer-related targets. miRNA, as a tumour supressor, can be used in gene therapy for retrieving miRNA gene expression and preventing tumour development. Tumour cells keep high yields of lactic acid and produce ATP by aerobic glycolysis with or without oxygen. Accelerated glycolysis provides ATP lev‐ els to the fast proliferating tumour cells in a hypoxic environment. It is proposed that with RNA interference technologies metabolic genes in cancer cells can be silenced. Therefore tu‐ mour proliferation can be inhibited. Beside that, studies have shown that genes which are related to cancer such as Telomerase, BCL-2, COX-2 can be silenced for preventing cancer.

## **Author details**

had anti-proliferation effect on Nasopharyngeal carcinoma (NPC) cell. RNAi could be a powerful method in further investigations of LMP-1 [113]. A recombinant adeno-associated virus type 2 vector was used to deliver shRNA targeting EBV-LMP-1 into the EBV-positive human NPC C666-1 cells. Results showed that long-term suppression of EBV-encoded

One of the most important signaling pathways to control growth and proliferation of our cells is the mitogen-activated protein kinase (MAPK) pathway. Ras, which is an enzyme in this pathway, is turned to an oncogenic form in about 15% of human cancer. Suppression of tumourgenicity was done by virus-mediated RNAi to inhibit specifically the oncogenic al‐ lele of K-ras (K-rasV12) in human tumour cells [115]. Other studies have reported that the use of siRNA can further block the Ras to Map kinase cascade, at either the Raf level or

Cancer is widely recognised as one of the largest burdens to health world wide. Some main features of cancers are its ability to sustain proliferative signaling, evade growth suppres‐ sors, resist cell death, enable replicative immortality, induce angiogenesis, and activate inva‐ sion and metastasis. Until now, many methods have been developed for the treatment of cancer. Conventional treatment methods, ie.: surgery, chemotherapy and radiotherapy, are still widely used in the treatment of most cancers. However, these methods result in a high recurrence of cancer in patients. Clearly, there is an urgent need for the development of new therapies. In contrast to conventional methods, targeted gene therapy, immunotherapy, and gene therapy offer promising alternatives that are more effective and produce less side ef‐ fects. Both targeted therapies and cancer immunotherapies have recently been used in clinic and these therapies can be succesful when used together, nevertheless, there are still limita‐

Gene therapy has already begun to show great promise and is expected to be more effective in curing cancer. Targets for cancer gene therapy may include tumour suppressor genes (e.g. p53), oncogenes,and apoptosis genes. The most problematic issue for cancer gene therapy studies is the delivery of the therapheutic gene to the tumour cells. Although viral delivery methods are widely in use, there are non-viral technologies that offer several advantages that include less toxicity, reduced adverse immune responses and easier to producing large amounts of gene products. More recently, bacteria have also been used in cancer treatment. The hypoxic nature of solid tumours provides considerable conditions for growth of bacte‐ ria and bacterial colonisation. Necrotic areas can also supply purines to further facilitate

Bacterial delivery of RNA silencing tools combined with benefit of bacterial oncolysis can contribute to the treatment of cancer. Exploiting of small oligonucleic acids which are car‐ ried by spesific bacteria to cancer cells can be an effective way to cut energy supply and lysis

LMP-1 *in vivo* is an effective way for preventing NPC metastasis [114].

through NADPH oxidase1 (Nox1) [116]- [118].

**11. Conclusion**

364 Novel Gene Therapy Approaches

tions with these therapies.

growth of bacteria.

of tumor cells.

Onur Sakiragaoglu1 , David Good1,2 and Ming Q. Wei1\*

\*Address all correspondence to: m.wei@griffith.edu.au

1 Division of Molecular and Gene Therapies, Griffith Health Institute and School of Medical Science, Griffith University, Gold Coast, QLD, Australia

2 School of Physiotherapy, Australian Catholic University, Banyo, QLD, Australia

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**Chapter 15**

**Poly(amino ester)s-Based Polymeric Gene Carriers in**

Gene therapy is a novel approach that broadly defined as the transfer of genetic material into a cell, tissue, or whole organ, with the goal of curing a disease or at least improving the clinical status of a patient [1]. Gene therapy refers to local or systemic administration of a nucleic acid construct that can prevent, treat and even cure diseases by changing the expression of genes that are responsible for the pathological condition [2]. As a form of molecular medicine, gene therapy hold great promises to provide new treatments for a large number of inherited and acquired diseases, such as cancer. It has also been considered as suitable substitute for conventional protein therapy, since it can overcome inherent problems associated with administration of protein drugs in terms of bioavailability, systemic toxicity and manufactur‐

There are two essential components in current gene therapy: an effective therapeutic genetic agent and the gene delivery system [4, 5]. The most extensively studied approach involves the delivery of plasmid DNA (pDNA) for expressing therapeutic transgenes. Considerable efforts have been made in plasmid design. This includes removal of extraneous CG dinucleotides, incorporation of scaffold/matrix attached region sequences to prolong expression, promoter selection for gene expression, and improving plasmid entry into the nucleus [6]. The recently emerged RNA interference (RNAi) has also become recognized as pivotal cellular regulator of genetic events and a useful tool in elucidating pathways during stages of development, pathogenesis and senescent cell regulation [7]. RNAi encompasses the range of endogenous or synthetic short double or single stranded oligonucleotides, including microRNAs (miR‐ NAs), small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), piwi interacting RNAs (piRNAs) and antisense oligonucleotides (ASOs) [8]. The intracellular delivery of genetic

> © 2013 Kim 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,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**Cancer Gene Therapy**

http://dx.doi.org/10.5772/54740

**1. Introduction**

ing cost [3].

You-Kyoung Kim, Can Zhang, Chong-Su Cho,

Additional information is available at the end of the chapter

Myung-Haing Cho and Hu-Lin Jiang


## **Poly(amino ester)s-Based Polymeric Gene Carriers in Cancer Gene Therapy**

You-Kyoung Kim, Can Zhang, Chong-Su Cho, Myung-Haing Cho and Hu-Lin Jiang

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54740

## **1. Introduction**

[114] Li, G, Li, X. P, Peng, Y, Liu, X, & Li, X. H. Effective inhibition of EB virus encoded latent membrane protein-1 by siRNA in EB virus (+) nasopharyngeal carcinoma cell.

[115] Li, X, Liu, X, Li, C. Y, Ding, Y, Chau, D, Li, G, Kung, H. F, Lin, M. C, & Peng, Y. Re‐ combinant adeno-associated virus mediated RNA interference inhibits metastasis of nasopharyngeal cancer cells in vivo and in vitro by suppression of Epstein-Barr virus

[116] Brummelkamp, T. R, Bernards, R, & Agami, R. Stable suppression of tumourigenicity

[117] Hingorani, S. R, Jacobetz, M. A, Robertson, G. P, Herlyn, M, & Tuveson, D. A. Sup‐ pression of BRAFV599E in human melanoma abrogates transformation. Cancer Res.

[118] Sumimoto, H, Miyagishi, M, Miyoshi, H, Yamagata, S, Shimizu, A, Taira, K, & Kawa‐ kami, Y. Inhibition of growth and invasive ability of melanoma by inactivation of mutated BRAF with lentivirus-mediated RNA interference. Oncogene. (2004). ,

[119] Mitsushita, J, Lambeth, J. D, & Kamata, T. The superoxidegenerating oxidase Nox1 is functionally required for Ras oncogene transformation. Cancer Res. (2004). , 64,

Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. (2005). , 40, 406-410.

by virus-mediated RNA interference. Cancer Cell. (2002). , 2, 243-247.

encoded LMP-1. Int J Oncol. (2006). , 29, 595-603.

(2003). , 63, 5198-5202.

23(36), 6031-9.

374 Novel Gene Therapy Approaches

3580-3585.

Gene therapy is a novel approach that broadly defined as the transfer of genetic material into a cell, tissue, or whole organ, with the goal of curing a disease or at least improving the clinical status of a patient [1]. Gene therapy refers to local or systemic administration of a nucleic acid construct that can prevent, treat and even cure diseases by changing the expression of genes that are responsible for the pathological condition [2]. As a form of molecular medicine, gene therapy hold great promises to provide new treatments for a large number of inherited and acquired diseases, such as cancer. It has also been considered as suitable substitute for conventional protein therapy, since it can overcome inherent problems associated with administration of protein drugs in terms of bioavailability, systemic toxicity and manufactur‐ ing cost [3].

There are two essential components in current gene therapy: an effective therapeutic genetic agent and the gene delivery system [4, 5]. The most extensively studied approach involves the delivery of plasmid DNA (pDNA) for expressing therapeutic transgenes. Considerable efforts have been made in plasmid design. This includes removal of extraneous CG dinucleotides, incorporation of scaffold/matrix attached region sequences to prolong expression, promoter selection for gene expression, and improving plasmid entry into the nucleus [6]. The recently emerged RNA interference (RNAi) has also become recognized as pivotal cellular regulator of genetic events and a useful tool in elucidating pathways during stages of development, pathogenesis and senescent cell regulation [7]. RNAi encompasses the range of endogenous or synthetic short double or single stranded oligonucleotides, including microRNAs (miR‐ NAs), small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), piwi interacting RNAs (piRNAs) and antisense oligonucleotides (ASOs) [8]. The intracellular delivery of genetic

© 2013 Kim 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. © 2013 The Author(s). Licensee InTech. This chapter is 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.

agents for RNAi such as siRNA takes benefits from existing expertise in pDNA transfer, as they share common properties. However, they face distinct challenges due to apparent differences in size, stability of the formed nucleic acid complexes, the location and mechanism of action [9].

thylenimine (PEI), chitosan, dendrimers etc. have now been extensively investigated as polymer-based non-viral vector gene delivery systems [24, 25]. The success of these agents is directly correlated with their ability to overcome issues of low efficiency and inconsistent

Poly(amino ester)s-Based Polymeric Gene Carriers in Cancer Gene Therapy

http://dx.doi.org/10.5772/54740

377

Among polymeric vectors, the wildly used PEI with appropriate molecular weight can electrostatically interact with negatively charged phosphate groups of genetic agents to form particulate polyelectrolyte complexes, which exhibit superior transfection efficiency due to high buffering capacity [26]. However, high molecular weight of PEI shows high cytotoxicity, and when further decreasing the molecular weight, both cellular toxicity and transfection efficiency are decreased [27]. Polyethylene glycol (PEG) was conjugated to PEI to ameliorate its cell cytotoxicity and develop functionality which limit its clinical application [28-32]. However, free PEI is not easily biodegradable in cellular space, which may remain additional safety concerns. Therefore, it calls for the development of functional biocompatible materials with favorable gene transfer efficiency to substitute for PEI [33, 34]. Poly(amino ester)s-based polymers are a promising class of polymeric gene vector due to their biocompatibility [35-37]. Poly(amino ester)s can be synthesized by Michael addition reaction using functional amines including a primary and a secondary amine to diacrylate ester [38]. The ease in synthesis and lack of byproducts make them even more favorable gene vector candidates with biocompati‐

Here, we are focused on recent progress of different strategies of functionalization of synthetic biocompatible poly(amino ester)s and the applications of these. The characterization of physicochemical properties, degradation kinetics, transfection efficiency and toxicity in vitro

Poly(amino ester)s are promising and efficient gene delivery vectors due to their high trans‐ fection efficiency and biocompatibility, which were first synthesized by Langer et al. [44-46].

Varieties of linear poly(amino ester)s are synthesized by Michael addition reaction of small molecular weight monomers and diacrylate monomers. The Langer group initially investi‐ gated the synthesis of poly(β-amino ester)s via the addition of N,N´-dimethylethylenediamine, piperazine, and 4,4´-trimethylenedipiperidine to 1,4-butanediol diacrylate as shown in Fig. 1(A). They reported that addition of secondary amines to diacrylate moieties results into tertiary amines which do not participate in subsequent addition reaction, that otherwise leads

One of the major merit of poly(β-amino ester)s is degradation. Due to the hydrolysis of the ester bonds in the polymer backbones, poly(β-amino ester)s can easily degraded. The degra‐

preparation that have plagued previous non-viral vector delivery systems.

bility and biodegradability properties [38-43].

and in vivo were covered in this chapter.

*2.1.1. Linear poly(amino ester)s*

to polymer branching or cross linking.

**2. Poly(amino ester)s-based gene therapy**

**2.1. Poly(amino ester)s synthesis and degradation kinetics**

Naked genetic therapeutics is vulnerable to enzyme degradation, rapid clearance by renal filtration, poor cellular uptake due to anionic charges of the phosphate backbone, inefficiently escape from endosome into cytosol. Therefore, the development of gene vectors for effectively carrying genes into cells has made a great deal of progress in recent years [5, 10]. Vectors as gene delivery system that have been developed fall into two broad categories: viral and nonviral vectors. Vectors based upon many different viral systems, including retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses (AAV) (Table 1), currently offer the best choice for efficient gene delivery [11, 12]. They are all highly efficient in specific circum‐ stances, but the potential risks of undesired immune response and the risk of insertional mutagenesis following long term viral gene transfer and toxic side reactions have raised concerns [13-15].


**Table 1.** Viral vector delivery systems [12].

Although viral vector has the advantages in terms of gene transfer efficiency, non-viral gene therapy has the advantage over viral vector therapies with its ability to target specific cells, being less immunogenic and non-integrating into the host genome, low production cost, scalability despite most studies showing less sustained gene expression [16-18]. Non-viral vectors have been investigated even more aggressively since the death of a patient in a virusbased gene therapy trial [19] and the occurrence of leukemia following gene therapy of children with X-linked severe combined immune deficiency using a retroviral gene therapy vector [20]. Previous efforts focused primarily on cationic lipid/DNA complexes frequently composed of combinations of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOT‐ MA) [21], dioleoyl phosphatidylethanolamine (DOPE) [22], and dimethylaminoethanecarbamoyl cholesterol (DC-Chol) [23]. These complexes stabilize incorporated DNA against physical and enzymatic damage. On the other hand, numerous agents poly(L-lysine), polye‐ thylenimine (PEI), chitosan, dendrimers etc. have now been extensively investigated as polymer-based non-viral vector gene delivery systems [24, 25]. The success of these agents is directly correlated with their ability to overcome issues of low efficiency and inconsistent preparation that have plagued previous non-viral vector delivery systems.

Among polymeric vectors, the wildly used PEI with appropriate molecular weight can electrostatically interact with negatively charged phosphate groups of genetic agents to form particulate polyelectrolyte complexes, which exhibit superior transfection efficiency due to high buffering capacity [26]. However, high molecular weight of PEI shows high cytotoxicity, and when further decreasing the molecular weight, both cellular toxicity and transfection efficiency are decreased [27]. Polyethylene glycol (PEG) was conjugated to PEI to ameliorate its cell cytotoxicity and develop functionality which limit its clinical application [28-32]. However, free PEI is not easily biodegradable in cellular space, which may remain additional safety concerns. Therefore, it calls for the development of functional biocompatible materials with favorable gene transfer efficiency to substitute for PEI [33, 34]. Poly(amino ester)s-based polymers are a promising class of polymeric gene vector due to their biocompatibility [35-37]. Poly(amino ester)s can be synthesized by Michael addition reaction using functional amines including a primary and a secondary amine to diacrylate ester [38]. The ease in synthesis and lack of byproducts make them even more favorable gene vector candidates with biocompati‐ bility and biodegradability properties [38-43].

Here, we are focused on recent progress of different strategies of functionalization of synthetic biocompatible poly(amino ester)s and the applications of these. The characterization of physicochemical properties, degradation kinetics, transfection efficiency and toxicity in vitro and in vivo were covered in this chapter.

## **2. Poly(amino ester)s-based gene therapy**

Poly(amino ester)s are promising and efficient gene delivery vectors due to their high trans‐ fection efficiency and biocompatibility, which were first synthesized by Langer et al. [44-46].

#### **2.1. Poly(amino ester)s synthesis and degradation kinetics**

#### *2.1.1. Linear poly(amino ester)s*

agents for RNAi such as siRNA takes benefits from existing expertise in pDNA transfer, as they share common properties. However, they face distinct challenges due to apparent differences in size, stability of the formed nucleic acid complexes, the location and mechanism

Naked genetic therapeutics is vulnerable to enzyme degradation, rapid clearance by renal filtration, poor cellular uptake due to anionic charges of the phosphate backbone, inefficiently escape from endosome into cytosol. Therefore, the development of gene vectors for effectively carrying genes into cells has made a great deal of progress in recent years [5, 10]. Vectors as gene delivery system that have been developed fall into two broad categories: viral and nonviral vectors. Vectors based upon many different viral systems, including retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses (AAV) (Table 1), currently offer the best choice for efficient gene delivery [11, 12]. They are all highly efficient in specific circum‐ stances, but the potential risks of undesired immune response and the risk of insertional mutagenesis following long term viral gene transfer and toxic side reactions have raised

**Virus Genome Size Advantages Disadvantages**

cells and tissues

Structurally simple Provoke less of a host-cell

response

Retrovirus ssRNA 7-10 kb Long-term expression Application is limited to replicating cells

Capable of very efficient episomal gene transfer in a wide range of

Easy to grow in large amounts

Although viral vector has the advantages in terms of gene transfer efficiency, non-viral gene therapy has the advantage over viral vector therapies with its ability to target specific cells, being less immunogenic and non-integrating into the host genome, low production cost, scalability despite most studies showing less sustained gene expression [16-18]. Non-viral vectors have been investigated even more aggressively since the death of a patient in a virusbased gene therapy trial [19] and the occurrence of leukemia following gene therapy of children with X-linked severe combined immune deficiency using a retroviral gene therapy vector [20]. Previous efforts focused primarily on cationic lipid/DNA complexes frequently composed of combinations of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOT‐ MA) [21], dioleoyl phosphatidylethanolamine (DOPE) [22], and dimethylaminoethanecarbamoyl cholesterol (DC-Chol) [23]. These complexes stabilize incorporated DNA against physical and enzymatic damage. On the other hand, numerous agents poly(L-lysine), polye‐

Possibility of insertional mutagenesis

The host response to the virus appears to limit the duration of expression and

Extremely difficult to grow in large

the ability to repeat dosing

amounts

of action [9].

376 Novel Gene Therapy Approaches

concerns [13-15].

Adenovirus dsDNA 36 kb

AAV ssDNA 5 kb

**Table 1.** Viral vector delivery systems [12].

Varieties of linear poly(amino ester)s are synthesized by Michael addition reaction of small molecular weight monomers and diacrylate monomers. The Langer group initially investi‐ gated the synthesis of poly(β-amino ester)s via the addition of N,N´-dimethylethylenediamine, piperazine, and 4,4´-trimethylenedipiperidine to 1,4-butanediol diacrylate as shown in Fig. 1(A). They reported that addition of secondary amines to diacrylate moieties results into tertiary amines which do not participate in subsequent addition reaction, that otherwise leads to polymer branching or cross linking.

One of the major merit of poly(β-amino ester)s is degradation. Due to the hydrolysis of the ester bonds in the polymer backbones, poly(β-amino ester)s can easily degraded. The degra‐ dation of poly(β-amino ester)s presents a particularly attractive basis for the development of new polymeric gene carriers for two reasons: firstly, poly(β-amino ester)s degrade into nontoxic byproducts to increase the safety of gene carrier; secondary, degradation of poly(βamino ester)s will increases the transfection efficiency. While complexation of DNA with cationic polymers is required to compact and protect DNA during early events in the trans‐ fection process, DNA and polymer must ultimately decomplex to allow efficient transcription [44]. In view of this requirement, degradable poly(β-amino ester)s could play an important role in "vector unpackaging" events in the cells [44, 47]. As shown in Fig. 1(C), the polymers degraded more slowly at pH 5.1 than at pH 7.4, and at pH 5.1 each polymer having a half-life of approximately 7-8 h. In contrast, polymers 1 and 3 [Fig. 1(B)] were completely degraded in less than 5 h at pH 7.4. These results are consistent with the pH-degradation profiles of other amine-containing polyesters, such as poly(4-hydroxy-L-proline ester), in which pendant amine functionalities are hypothesized to act as intramolecular nucleophilic catalysts and contribute to more rapid degradation at higher pH [44]. The degradation of polymer 2 occurred more slowly at pH 7.4 than at pH 5.1 due to the incomplete solubility of polymer 2 at pH 7.4 and the resulting heterogeneous nature of the degradation mileu [44].

than at pH 5.1 due to the incomplete solubility of polymer 2 at pH 7.4 and the resulting heterogeneous

Fig. 1. The synthesis of poly(β-amino ester) from butanediol diacrylate and piperazine (A) and degradation of polymers 1-3 (B) at 37 °C at pH 5.1 and 7.4 (C). [Source from Ref. [44]].

After that, the same group reported a parallel approach suitable for synthesis of hundreds to thousands of structurally unique poly(amino ester)s and application of these libraries to rapid and high throughput identification of new gene delivery agents and structure-function trends although they did not report the degradation profiles of poly(amino ester)s in this study [38]. The high throughput method indicated that synthesis of poly(β-amino ester)s are easy to controlable. The advantage of combinatorial chemistry and automated highthroughput synthesis is that it has revolutionized modern drug discovery by rapid synthesis and evaluation with greater precision. As shown in Fig. 2, 140 different poly(β-amino ester)s were synthesized from the 7 diacrylate monomers and 20 amine-based monomers as a screening library. Polymerization reactions were conducted simultaneously as an array of individually labeled vials and the reactions were performed in methylene chloride at 45 °C for 5

Fig. 2. Synthesis of poly(β-amino ester)s. Poly(β-amino ester)s were synthesized by the conjugate addition of primary or bis(secondary amines) to diacrylates using methylene chloride solvent (A) and diacrylate (A-G 7 set) and amine (1-20) monomers chosen for the synthesis of an initial screening library (B). [Source from Ref. [38]]. **Figure 2.** Synthesis of poly(β-amino ester)s. Poly(β-amino ester)s were synthesized by the conjugate addition of pri‐ mary or bis(secondary amines) to diacrylates using methylene chloride solvent (A) and diacrylate (A-G 7 set) and

Based high throughput methods, in 2003, Anderson synthesized over 2,350 poly(β-amino ester)s as shown in Fig. 3 [48]. Polymerization reactions were performed in 1.6M DMSO at 56 °C for 5 days. Anderson et al. observed that reaction conditions such as optimum temperature and solvent play an important role during the synthesis of poly(β-amino ester)s. Even though maximizing monomer concentration in reaction is desirable to obtain high molecular weight poly(β-amino ester)s and it

Fig. 3. Synthesis of poly(β-amino ester)s. Poly(β-amino ester)s were synthesized by the conjugate addition of primary or bis(secondary amines) to diacrylates using DMSO solvent (A). Amino

**Figure 3.** Synthesis of poly(β-amino ester)s. Poly(β-amino ester)s were synthesized by the conjugate addition of pri‐ mary or bis(secondary amines) to diacrylates using DMSO solvent (A). Amino (numbers) and diacrylate (letters) mono‐

Park et al. reported the synthesis of linear poly(amino ester)s from three different molecular weights

(numbers) and diacrylate (letters) monomers (B). [Source from Ref. [48]].

of PEG diacrylate and low molecular weight PEI As shown in Fig. 4 [37].

mers (B). [Source from Ref. [48]].

amine (1-20) monomers chosen for the synthesis of an initial screening library (B). [Source from Ref. [38]].

(A)

(A)

Poly(amino ester)s-Based Polymeric Gene Carriers in Cancer Gene Therapy

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379

(B)

(A)

(B)

(B) (C)

nature of the degradation mileu [44].

days.

leads to insoluble gel formation [49].

than at pH 5.1 due to the incomplete solubility of polymer 2 at pH 7.4 and the resulting heterogeneous

Fig. 1. The synthesis of poly(β-amino ester) from butanediol diacrylate and piperazine (A) and degradation of polymers 1-3 (B) at 37 °C at pH 5.1 and 7.4 (C). [Source from Ref. [44]]. After that, the same group reported a parallel approach suitable for synthesis of hundreds to thousands of structurally unique poly(amino ester)s and application of these libraries to rapid and high **Figure 1.** The synthesis of poly(β-amino ester)s from butanediol diacrylate and piperazine (A) and degradation of pol‐ ymer 1-3 at 37 °C at pH 5.1 and 7.4 (B and C). [Source from Ref. [44]].

throughput identification of new gene delivery agents and structure-function trends although they did not report the degradation profiles of poly(amino ester)s in this study [38]. The high throughput method indicated that synthesis of poly(β-amino ester)s are easy to controlable. The advantage of combinatorial chemistry and automated highthroughput synthesis is that it has revolutionized modern drug discovery by rapid synthesis and evaluation with greater precision. As shown in Fig. 2, 140 different poly(β-amino ester)s were synthesized from the 7 diacrylate monomers and 20 amine-based monomers as a screening library. Polymerization reactions were conducted simultaneously as an array of individually labeled vials and the reactions were performed in methylene chloride at 45 °C for 5 days. (A) After that, the same group reported a parallel approach suitable for synthesis of hundreds to thousands of structurally unique poly(amino ester)s and application of these libraries to rapid and high throughput identification of new gene delivery agents and structure-function trends although they did not report the degradation profiles of poly(amino ester)s in this study [38]. The high throughput method indicated that synthesis of poly(β-amino ester)s are easy to controlable. The advantage of combinatorial chemistry and automated high throughput synthesis is that it has revolutionized modern drug discovery by rapid synthesis and evalua‐ tion with greater precision. As shown in Fig. 2, 140 different poly(β-amino ester)s were synthesized from the 7 diacrylate monomers and 20 amine-based monomers as a screening library. Polymerization reactions were conducted simultaneously as an array of individually labeled vials and the reactions were performed in methylene chloride at 45 °C for 5 days.

> Fig. 2. Synthesis of poly(β-amino ester)s. Poly(β-amino ester)s were synthesized by the conjugate addition of primary or bis(secondary amines) to diacrylates using methylene chloride solvent (A) and diacrylate (A-G 7 set) and amine (1-20) monomers chosen for the synthesis of an initial screening

library (B). [Source from Ref. [38]].

(B)

(A)

(B) (C)

than at pH 5.1 due to the incomplete solubility of polymer 2 at pH 7.4 and the resulting heterogeneous

Fig. 1. The synthesis of poly(β-amino ester) from butanediol diacrylate and piperazine (A) and degradation of polymers 1-3 (B) at 37 °C at pH 5.1 and 7.4 (C). [Source from Ref. [44]].

After that, the same group reported a parallel approach suitable for synthesis of hundreds to thousands of structurally unique poly(amino ester)s and application of these libraries to rapid and high throughput identification of new gene delivery agents and structure-function trends although they did not report the degradation profiles of poly(amino ester)s in this study [38]. The high throughput method indicated that synthesis of poly(β-amino ester)s are easy to controlable. The advantage of

monomers as a screening library. Polymerization reactions were conducted simultaneously as an array of individually labeled vials and the reactions were performed in methylene chloride at 45 °C for 5

nature of the degradation mileu [44].

days.

leads to insoluble gel formation [49].

dation of poly(β-amino ester)s presents a particularly attractive basis for the development of new polymeric gene carriers for two reasons: firstly, poly(β-amino ester)s degrade into nontoxic byproducts to increase the safety of gene carrier; secondary, degradation of poly(βamino ester)s will increases the transfection efficiency. While complexation of DNA with cationic polymers is required to compact and protect DNA during early events in the trans‐ fection process, DNA and polymer must ultimately decomplex to allow efficient transcription [44]. In view of this requirement, degradable poly(β-amino ester)s could play an important role in "vector unpackaging" events in the cells [44, 47]. As shown in Fig. 1(C), the polymers degraded more slowly at pH 5.1 than at pH 7.4, and at pH 5.1 each polymer having a half-life of approximately 7-8 h. In contrast, polymers 1 and 3 [Fig. 1(B)] were completely degraded in less than 5 h at pH 7.4. These results are consistent with the pH-degradation profiles of other amine-containing polyesters, such as poly(4-hydroxy-L-proline ester), in which pendant amine functionalities are hypothesized to act as intramolecular nucleophilic catalysts and contribute to more rapid degradation at higher pH [44]. The degradation of polymer 2 occurred more slowly at pH 7.4 than at pH 5.1 due to the incomplete solubility of polymer 2 at pH 7.4

than at pH 5.1 due to the incomplete solubility of polymer 2 at pH 7.4 and the resulting heterogeneous

Fig. 1. The synthesis of poly(β-amino ester) from butanediol diacrylate and piperazine (A) and

After that, the same group reported a parallel approach suitable for synthesis of hundreds to thousands of structurally unique poly(amino ester)s and application of these libraries to rapid and high throughput identification of new gene delivery agents and structure-function trends although they did not report the degradation profiles of poly(amino ester)s in this study [38]. The high throughput method indicated that synthesis of poly(β-amino ester)s are easy to controlable. The advantage of combinatorial chemistry and automated highthroughput synthesis is that it has revolutionized modern drug discovery by rapid synthesis and evaluation with greater precision. As shown in Fig. 2, 140 different poly(β-amino ester)s were synthesized from the 7 diacrylate monomers and 20 amine-based monomers as a screening library. Polymerization reactions were conducted simultaneously as an array of individually labeled vials and the reactions were performed in methylene chloride at 45 °C for 5

After that, the same group reported a parallel approach suitable for synthesis of hundreds to thousands of structurally unique poly(amino ester)s and application of these libraries to rapid and high throughput identification of new gene delivery agents and structure-function trends although they did not report the degradation profiles of poly(amino ester)s in this study [38]. The high throughput method indicated that synthesis of poly(β-amino ester)s are easy to controlable. The advantage of combinatorial chemistry and automated high throughput synthesis is that it has revolutionized modern drug discovery by rapid synthesis and evalua‐ tion with greater precision. As shown in Fig. 2, 140 different poly(β-amino ester)s were synthesized from the 7 diacrylate monomers and 20 amine-based monomers as a screening library. Polymerization reactions were conducted simultaneously as an array of individually labeled vials and the reactions were performed in methylene chloride at 45 °C for 5 days.

**Figure 1.** The synthesis of poly(β-amino ester)s from butanediol diacrylate and piperazine (A) and degradation of pol‐

Fig. 2. Synthesis of poly(β-amino ester)s. Poly(β-amino ester)s were synthesized by the conjugate addition of primary or bis(secondary amines) to diacrylates using methylene chloride solvent (A) and diacrylate (A-G 7 set) and amine (1-20) monomers chosen for the synthesis of an initial screening

degradation of polymers 1-3 (B) at 37 °C at pH 5.1 and 7.4 (C). [Source from Ref. [44]].

(A)

(A)

(B)

(B) (C)

and the resulting heterogeneous nature of the degradation mileu [44].

nature of the degradation mileu [44].

378 Novel Gene Therapy Approaches

ymer 1-3 at 37 °C at pH 5.1 and 7.4 (B and C). [Source from Ref. [44]].

days.

library (B). [Source from Ref. [38]].

addition of primary or bis(secondary amines) to diacrylates using methylene chloride solvent (A) and diacrylate (A-G 7 set) and amine (1-20) monomers chosen for the synthesis of an initial screening library (B). [Source from Ref. [38]]. **Figure 2.** Synthesis of poly(β-amino ester)s. Poly(β-amino ester)s were synthesized by the conjugate addition of pri‐ mary or bis(secondary amines) to diacrylates using methylene chloride solvent (A) and diacrylate (A-G 7 set) and amine (1-20) monomers chosen for the synthesis of an initial screening library (B). [Source from Ref. [38]]. Based high throughput methods, in 2003, Anderson synthesized over 2,350 poly(β-amino ester)s as shown in Fig. 3 [48]. Polymerization reactions were performed in 1.6M DMSO at 56 °C for 5 days. Anderson et al. observed that reaction conditions such as optimum temperature and solvent play an important role during the synthesis of poly(β-amino ester)s. Even though maximizing monomer

concentration in reaction is desirable to obtain high molecular weight poly(β-amino ester)s and it

Fig. 2. Synthesis of poly(β-amino ester)s. Poly(β-amino ester)s were synthesized by the conjugate

addition of primary or bis(secondary amines) to diacrylates using DMSO solvent (A). Amino (numbers) and diacrylate (letters) monomers (B). [Source from Ref. [48]]. Park et al. reported the synthesis of linear poly(amino ester)s from three different molecular weights **Figure 3.** Synthesis of poly(β-amino ester)s. Poly(β-amino ester)s were synthesized by the conjugate addition of pri‐ mary or bis(secondary amines) to diacrylates using DMSO solvent (A). Amino (numbers) and diacrylate (letters) mono‐ mers (B). [Source from Ref. [48]].

of PEG diacrylate and low molecular weight PEI As shown in Fig. 4 [37].

Fig. 3. Synthesis of poly(β-amino ester)s. Poly(β-amino ester)s were synthesized by the conjugate

Based high throughput methods, in 2003, Anderson synthesized over 2,350 poly(β-amino es‐ ter)s as shown in Fig. 3 [48]. Polymerization reactions were performed in 1.6M DMSO at 56 °C for 5 days. Anderson et al. observed that reaction conditions such as optimum tempera‐ ture and solvent play an important role during the synthesis of poly(β-amino ester)s. Even though maximizing monomer concentration in reaction is desirable to obtain high molecular weight poly(β-amino ester)s and it leads to insoluble gel formation [49].

(A) (B)

Poly(amino ester)s-Based Polymeric Gene Carriers in Cancer Gene Therapy

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381

(A) (B)

(A) (B)

(A) (B)

Fig. 4. Proposed reaction scheme for copolymer formation (A) and degradation of copolymers (B).

It was found that molecular weights of poly(amino ester)s were maintained relatively constant at about 4kDa during the degradation aftger 72 h regardless of molecular weight of PEG diacylate. However, half life was observed depending on molecular weight of PEG diacrylate. Poly(β-amino ester)s composed of PEG diacylate (Mn: 575) showed an half life of 8 h while those of 25 h for poly(β-amino ester)s with PEG diacylate (Mn: 700). This rapid degradation in case of linear poly(βamino ester)s is plausible as even few cleavages may reduce chain length rapidly with quick drop in

Liu et al. synthesized the branched poly(amino ester)s by the polymerization of 1-(2 aminoethyl)piperazine (AEPZ) with 1,4-butanediol diacrylate (BDA), which was carried out by adding BDA dropwise to an equimolar solution of AEPZ in chloroform at 45 °C as shown in Fig. 5 [50]. After the polymerization was performed for around 72 h, a water-soluble polymer, BDA-AEPZ, was obtained by precipitating the solution into acetone containing HCl. The molecular weight of

Fig. 5. Possible routes of the Michael addition polymerization of trifunctional amine monomers and diacrylates (A) and the structure of poly(amino ester) obtained and enlarged 13C-NMR (INVGATE) spectrum of methylene carbons attached to the hydrochloride salts of amines in BDA-AEPZ (B).

**Figure 5.** Possible routes of the Michael addition polymerization of trifunctional amine monomers and diacrylates (A) and the structure of poly(amino ester)s obtained and enlarged 13C-NMR (INVGATE) spectrum of methylene carbons

The polymerization of AEPZ with diacylate monomers was reported but branched poly(amino ester)s with primary, secondary and tertiary amines were supposed to be formed based on un-solidified experimental conditions, suggesting that secondary amines are more reactive than primary ones in case of trifunctional amines [50]. Wu et al. also synthesized protonated hyperbranched poly(amino ester)s and characterized as gene delivery carriers as shown in Fig. 6(A) [51]. It was found that all

The polymerization of AEPZ with diacylate monomers was reported but branched poly(amino ester)s with primary, secondary and tertiary amines were supposed to be formed based on unsolidified experimental conditions, suggesting that secondary amines are more reactive than primary ones in case of trifunctional amines [50]. Wu et al. also synthesized protonated hyperbranched poly(amino ester)s and characterized as gene delivery carriers as shown in Fig. 6(A) [51]. It was found that all these hyperbranched poly(amino ester)s degraded in a controlled manner within 50 days and it was speculated that this phenomenon may be due to the lesser water accessibility of the ester groups in hyperbranched sturctures [Fig. 6(B)]. these hyperbranched poly(amino ester)s degraded in a controlled manner within 50 days and it was speculated that this phenomenon may be due to the lesser water accessibility of the ester groups in

> Fig. 6. Structure of linear and hyperbranched poly(amino ester)s obtained via Michael addition polymerization of trifunctional amines with diacrylates and triacrylates (A) and comparison of the hydrolysis profiles of protonated hyperbranched poly(BDA2-AEPZ1)-MPZ, hyperbranched poly(TMPTA1-AEPZ2), and linear poly(BDA-AEPZ) in aqueous solutions (B). [Source from Ref.

**Figure 6.** Structure of linear and hyperbranched poly(amino ester)s obtained via Michael addition polymerization of trifunctional amines with diacrylates and triacrylates (A) and comparison of the hydrolysis profiles of protonated hy‐ perbranched poly(BDA2-AEPZ1)-MPZ, hyperbranched poly(TMPTA1-AEPZ2), and linear poly(BDA-AEPZ) in aqueous

> Cho's group also reported the synthesis of branch poly(amino ester)s by Michael addition, based on hydrophobic polycaprolactone diacrylate and low molecular weight PEI [Fig. 7(A)] [40]. It was simply an indication of application of ester linkage which supports the easy degradation leaving nontox building blocks, thereby increased transfection efficiency and reduced cytotoxicity. The branched poly(amino ester)s showed controlled degradation with the half life of 4-4.5 days as shown

Cho's group also reported the synthesis of branch poly(amino ester)s by Michael addition, based on hydrophobic polycaprolactone diacrylate and low molecular weight PEI [Fig. 7(A)] [40]. It was simply an indication of application of ester linkage which supports the easy degradation leaving nontoxic building blocks, thereby increased transfection efficiency and reduced cytotoxicity. The branched poly(amino ester)s showed controlled degradation with

Fig. 7. The synthetic scheme of PEA by Michael addition (A) and degradation of PEAs (PCL/PEI-1.2

Same group also reported another degradable branched poly(amino ester)s based on poloxamer diacrylate and low molecular weight PEI [52]. These hyperbranched poly(amino ester)s can be easily synthesized by Michael type addition reaction between poloxamer diacrylate and low molecular weight PEI [Fig. 8(A)] and the hyperbranched poly(amino ester)s showed slow degradation at physiological conditions which was greatly dependent on hydrophilicity of poloxamer [Fig. 8(B)].

attached to the hydrochloride salts of amines in BDA-AEPZ (B). [Source from Ref. [50]].

BDA-AEPZ was around 5126 with a polydispersity index of 1.52 as determined by GPC.

[Source from Ref. [37]].

molecular weight [37].

[Source from Ref. [50]].

hyperbranched sturctures [Fig. 6(B)].

[51]].

in Fig. 7(B).

solutions (B). [Source from Ref. [51]].

and PCL/PEI-1.8) (B). [Source from Ref. [40]].

the half life of 4-4.5 days as shown in Fig. 7(B).

**2.1.2 Branched poly(amino ester)s**

Park et al. reported the synthesis of linear poly(amino ester)s from three different molecular weights of PEG diacrylate and low molecular weight PEI As shown in Fig. 4 [37].

[Source from Ref. [37]]. It was found that molecular weights of poly(amino ester)s were maintained relatively constant at about 4kDa during the degradation aftger 72 h regardless of molecular weight of PEG diacylate. **Figure 4.** Proposed reaction scheme for copolymer formation (A) and degradation of copolymers (B). [Source from Ref. [37]].

However, half life was observed depending on molecular weight of PEG diacrylate. Poly(β-amino ester)s composed of PEG diacylate (Mn: 575) showed an half life of 8 h while those of 25 h for poly(β-amino ester)s with PEG diacylate (Mn: 700). This rapid degradation in case of linear poly(β-

amino ester)s is plausible as even few cleavages may reduce chain length rapidly with quick drop in molecular weight [37]. **2.1.2 Branched poly(amino ester)s** Liu et al. synthesized the branched poly(amino ester)s by the polymerization of 1-(2 aminoethyl)piperazine (AEPZ) with 1,4-butanediol diacrylate (BDA), which was carried out by adding BDA dropwise to an equimolar solution of AEPZ in chloroform at 45 °C as shown in Fig. 5 [50]. After the polymerization was performed for around 72 h, a water-soluble polymer, BDA-AEPZ, was obtained by precipitating the solution into acetone containing HCl. The molecular weight of BDA-AEPZ was around 5126 with a polydispersity index of 1.52 as determined by GPC. It was found that molecular weights of poly(amino ester)s were maintained relatively constant at about 4kDa during the degradation aftger 72 h regardless of molecular weight of PEG diacylate. However, half life was observed depending on molecular weight of PEG diacrylate. Poly(β-amino ester)s composed of PEG diacylate (Mn: 575) showed an half life of 8 h while that of 25 h for poly(β-amino ester)s with PEG diacylate (Mn: 700). This rapid degradation in case of linear poly(β-amino ester)s is plausible as even few cleavages may reduce chain length rapidly with quick drop in molecular weight [37].

#### *2.1.2. Branched poly(amino ester)s*

(A) (B) Fig. 5. Possible routes of the Michael addition polymerization of trifunctional amine monomers and diacrylates (A) and the structure of poly(amino ester) obtained and enlarged 13C-NMR (INVGATE) spectrum of methylene carbons attached to the hydrochloride salts of amines in BDA-AEPZ (B). [Source from Ref. [50]]. Liu et al. synthesized the branched poly(amino ester)s by the polymerization of 1-(2-amino‐ ethyl)piperazine (AEPZ) with 1,4-butanediol diacrylate (BDA), which was carried out by adding BDA dropwise to an equimolar solution of AEPZ in chloroform at 45 °C as shown in Fig. 5 [50]. After the polymerization was performed for around 72 h, a water-soluble polymer, BDA-AEPZ, was obtained by precipitating the solution into acetone containing HCl. The molecular weight of BDA-AEPZ was around 5126 with a polydispersity index of 1.52 as determined by GPC.

The polymerization of AEPZ with diacylate monomers was reported but branched poly(amino ester)s with primary, secondary and tertiary amines were supposed to be formed based on un-solidified experimental conditions, suggesting that secondary amines are more reactive than primary ones in case of trifunctional amines [50]. Wu et al. also synthesized protonated hyperbranched poly(amino ester)s and characterized as gene delivery carriers as shown in Fig. 6(A) [51]. It was found that all

(A) (B)

Fig. 4. Proposed reaction scheme for copolymer formation (A) and degradation of copolymers (B).

It was found that molecular weights of poly(amino ester)s were maintained relatively constant at about 4kDa during the degradation aftger 72 h regardless of molecular weight of PEG diacylate. However, half life was observed depending on molecular weight of PEG diacrylate. Poly(β-amino ester)s composed of PEG diacylate (Mn: 575) showed an half life of 8 h while those of 25 h for poly(β-amino ester)s with PEG diacylate (Mn: 700). This rapid degradation in case of linear poly(βamino ester)s is plausible as even few cleavages may reduce chain length rapidly with quick drop in

Liu et al. synthesized the branched poly(amino ester)s by the polymerization of 1-(2-

[50]. After the polymerization was performed for around 72 h, a water-soluble polymer, BDA-AEPZ, was obtained by precipitating the solution into acetone containing HCl. The molecular weight of

[Source from Ref. [37]].

molecular weight [37].

**2.1.2 Branched poly(amino ester)s**

hyperbranched sturctures [Fig. 6(B)].

Based high throughput methods, in 2003, Anderson synthesized over 2,350 poly(β-amino es‐ ter)s as shown in Fig. 3 [48]. Polymerization reactions were performed in 1.6M DMSO at 56 °C for 5 days. Anderson et al. observed that reaction conditions such as optimum tempera‐ ture and solvent play an important role during the synthesis of poly(β-amino ester)s. Even though maximizing monomer concentration in reaction is desirable to obtain high molecular

Park et al. reported the synthesis of linear poly(amino ester)s from three different molecular

Fig. 4. Proposed reaction scheme for copolymer formation (A) and degradation of copolymers (B).

It was found that molecular weights of poly(amino ester)s were maintained relatively constant at about 4kDa during the degradation aftger 72 h regardless of molecular weight of PEG diacylate. However, half life was observed depending on molecular weight of PEG diacrylate. Poly(β-amino ester)s composed of PEG diacylate (Mn: 575) showed an half life of 8 h while those of 25 h for poly(β-amino ester)s with PEG diacylate (Mn: 700). This rapid degradation in case of linear poly(βamino ester)s is plausible as even few cleavages may reduce chain length rapidly with quick drop in

**Figure 4.** Proposed reaction scheme for copolymer formation (A) and degradation of copolymers (B). [Source from

Liu et al. synthesized the branched poly(amino ester)s by the polymerization of 1-(2 aminoethyl)piperazine (AEPZ) with 1,4-butanediol diacrylate (BDA), which was carried out by adding BDA dropwise to an equimolar solution of AEPZ in chloroform at 45 °C as shown in Fig. 5 [50]. After the polymerization was performed for around 72 h, a water-soluble polymer, BDA-AEPZ, was obtained by precipitating the solution into acetone containing HCl. The molecular weight of

It was found that molecular weights of poly(amino ester)s were maintained relatively constant at about 4kDa during the degradation aftger 72 h regardless of molecular weight of PEG diacylate. However, half life was observed depending on molecular weight of PEG diacrylate. Poly(β-amino ester)s composed of PEG diacylate (Mn: 575) showed an half life of 8 h while that of 25 h for poly(β-amino ester)s with PEG diacylate (Mn: 700). This rapid degradation in case of linear poly(β-amino ester)s is plausible as even few cleavages may reduce chain length

Fig. 5. Possible routes of the Michael addition polymerization of trifunctional amine monomers and diacrylates (A) and the structure of poly(amino ester) obtained and enlarged 13C-NMR (INVGATE) spectrum of methylene carbons attached to the hydrochloride salts of amines in BDA-AEPZ (B).

Liu et al. synthesized the branched poly(amino ester)s by the polymerization of 1-(2-amino‐ ethyl)piperazine (AEPZ) with 1,4-butanediol diacrylate (BDA), which was carried out by adding BDA dropwise to an equimolar solution of AEPZ in chloroform at 45 °C as shown in Fig. 5 [50]. After the polymerization was performed for around 72 h, a water-soluble polymer, BDA-AEPZ, was obtained by precipitating the solution into acetone containing HCl. The molecular weight of BDA-AEPZ was around 5126 with a polydispersity index of 1.52 as

The polymerization of AEPZ with diacylate monomers was reported but branched poly(amino ester)s with primary, secondary and tertiary amines were supposed to be formed based on un-solidified experimental conditions, suggesting that secondary amines are more reactive than primary ones in case of trifunctional amines [50]. Wu et al. also synthesized protonated hyperbranched poly(amino ester)s and characterized as gene delivery carriers as shown in Fig. 6(A) [51]. It was found that all

BDA-AEPZ was around 5126 with a polydispersity index of 1.52 as determined by GPC.

(A) (B)

(A) (B)

weight poly(β-amino ester)s and it leads to insoluble gel formation [49].

[Source from Ref. [37]].

Ref. [37]].

380 Novel Gene Therapy Approaches

molecular weight [37].

*2.1.2. Branched poly(amino ester)s*

[Source from Ref. [50]].

determined by GPC.

**2.1.2 Branched poly(amino ester)s**

rapidly with quick drop in molecular weight [37].

weights of PEG diacrylate and low molecular weight PEI As shown in Fig. 4 [37].

diacrylates (A) and the structure of poly(amino ester) obtained and enlarged 13C-NMR (INVGATE) spectrum of methylene carbons attached to the hydrochloride salts of amines in BDA-AEPZ (B). [Source from Ref. [50]]. **Figure 5.** Possible routes of the Michael addition polymerization of trifunctional amine monomers and diacrylates (A) and the structure of poly(amino ester)s obtained and enlarged 13C-NMR (INVGATE) spectrum of methylene carbons attached to the hydrochloride salts of amines in BDA-AEPZ (B). [Source from Ref. [50]].

The polymerization of AEPZ with diacylate monomers was reported but branched poly(amino ester)s

Fig. 5. Possible routes of the Michael addition polymerization of trifunctional amine monomers and

with primary, secondary and tertiary amines were supposed to be formed based on un-solidified experimental conditions, suggesting that secondary amines are more reactive than primary ones in case of trifunctional amines [50]. Wu et al. also synthesized protonated hyperbranched poly(amino ester)s and characterized as gene delivery carriers as shown in Fig. 6(A) [51]. It was found that all The polymerization of AEPZ with diacylate monomers was reported but branched poly(amino ester)s with primary, secondary and tertiary amines were supposed to be formed based on unsolidified experimental conditions, suggesting that secondary amines are more reactive than primary ones in case of trifunctional amines [50]. Wu et al. also synthesized protonated hyperbranched poly(amino ester)s and characterized as gene delivery carriers as shown in Fig. 6(A) [51]. It was found that all these hyperbranched poly(amino ester)s degraded in a controlled manner within 50 days and it was speculated that this phenomenon may be due to the lesser water accessibility of the ester groups in hyperbranched sturctures [Fig. 6(B)]. these hyperbranched poly(amino ester)s degraded in a controlled manner within 50 days and it was

speculated that this phenomenon may be due to the lesser water accessibility of the ester groups in

polymerization of trifunctional amines with diacrylates and triacrylates (A) and comparison of the hydrolysis profiles of protonated hyperbranched poly(BDA2-AEPZ1)-MPZ, hyperbranched poly(TMPTA1-AEPZ2), and linear poly(BDA-AEPZ) in aqueous solutions (B). [Source from Ref. [51]]. **Figure 6.** Structure of linear and hyperbranched poly(amino ester)s obtained via Michael addition polymerization of trifunctional amines with diacrylates and triacrylates (A) and comparison of the hydrolysis profiles of protonated hy‐ perbranched poly(BDA2-AEPZ1)-MPZ, hyperbranched poly(TMPTA1-AEPZ2), and linear poly(BDA-AEPZ) in aqueous solutions (B). [Source from Ref. [51]].

Cho's group also reported the synthesis of branch poly(amino ester)s by Michael addition, based on

Fig. 6. Structure of linear and hyperbranched poly(amino ester)s obtained via Michael addition

hydrophobic polycaprolactone diacrylate and low molecular weight PEI [Fig. 7(A)] [40]. It was simply an indication of application of ester linkage which supports the easy degradation leaving nontox building blocks, thereby increased transfection efficiency and reduced cytotoxicity. The branched poly(amino ester)s showed controlled degradation with the half life of 4-4.5 days as shown in Fig. 7(B). Cho's group also reported the synthesis of branch poly(amino ester)s by Michael addition, based on hydrophobic polycaprolactone diacrylate and low molecular weight PEI [Fig. 7(A)] [40]. It was simply an indication of application of ester linkage which supports the easy degradation leaving nontoxic building blocks, thereby increased transfection efficiency and reduced cytotoxicity. The branched poly(amino ester)s showed controlled degradation with the half life of 4-4.5 days as shown in Fig. 7(B).

Fig. 7. The synthetic scheme of PEA by Michael addition (A) and degradation of PEAs (PCL/PEI-1.2

Same group also reported another degradable branched poly(amino ester)s based on poloxamer diacrylate and low molecular weight PEI [52]. These hyperbranched poly(amino ester)s can be easily synthesized by Michael type addition reaction between poloxamer diacrylate and low molecular weight PEI [Fig. 8(A)] and the hyperbranched poly(amino ester)s showed slow degradation at physiological conditions which was greatly dependent on hydrophilicity of poloxamer [Fig. 8(B)].

and PCL/PEI-1.8) (B). [Source from Ref. [40]].

(A) (B)

[51]].

hyperbranched sturctures [Fig. 6(B)].

these hyperbranched poly(amino ester)s degraded in a controlled manner within 50 days and it was speculated that this phenomenon may be due to the lesser water accessibility of the ester groups in

Fig. 6. Structure of linear and hyperbranched poly(amino ester)s obtained via Michael addition polymerization of trifunctional amines with diacrylates and triacrylates (A) and comparison of the hydrolysis profiles of protonated hyperbranched poly(BDA2-AEPZ1)-MPZ, hyperbranched poly(TMPTA1-AEPZ2), and linear poly(BDA-AEPZ) in aqueous solutions (B). [Source from Ref.

simply an indication of application of ester linkage which supports the easy degradation leaving nontox building blocks, thereby increased transfection efficiency and reduced cytotoxicity. The branched poly(amino ester)s showed controlled degradation with the half life of 4-4.5 days as shown

(A) (B)

by cells via natural processes such as adsorptive endocytosis, pinocytosis and phagocytosis [32]. DNA in the complexes was protected from nuclease attack whereas the naked DNA was degraded. This result suggests that intact DNA could be delivered by poly(β-amino ester)s

Poly(amino ester)s-Based Polymeric Gene Carriers in Cancer Gene Therapy

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383

**Figure 9.** DNA condensation and protection study. Agarose gel electrophoresis of poly(β-amino ester)s (GPT– SPE)/DNA complexes at various weight ratios (A) and DNA protection and release assay (B). [Source from Ref. [34]].

Surface properties, such as particle size and surface charge of the complex, are necessary to assure its uptake by cells [53]. In particular, the particle size of a complex is an important factor that influences the access and passage of the complex through the targeting site. Successful gene carrier depends on its ability to condense negatively charged DNA into nanosized particles with positive charges so as to enter into the cells [54]. Compact particles of small size are usually obtained only at higher N/P ratios, resulting in complexes with a strong positive

*2.2.2. Particle sizes and surface charges of poly(amino ester)s/DNA complexes*

into cells without degradation [Fig. 9(B)] [34].

Fig. 7. The synthetic scheme of PEA by Michael addition (A) and degradation of PEAs (PCL/PEI-1.2 and PCL/PEI-1.8) (B). [Source from Ref. [40]]. Same group also reported another degradable branched poly(amino ester)s based on poloxamer **Figure 7.** The synthetic scheme of PEAs by Michael addition (A) and degradation of PEAs (PCL/PEI-1.2 and PCL/ PEI-1.8) (B). [Source from Ref. [40]].

diacrylate and low molecular weight PEI [52]. These hyperbranched poly(amino ester)s can be easily synthesized by Michael type addition reaction between poloxamer diacrylate and low molecular weight PEI [Fig. 8(A)] and the hyperbranched poly(amino ester)s showed slow degradation at physiological conditions which was greatly dependent on hydrophilicity of poloxamer [Fig. 8(B)]. Same group also reported another degradable branched poly(amino ester)s based on polox‐ amer diacrylate and low molecular weight PEI [52]. These hyperbranched poly(amino ester)s can be easily synthesized by Michael type addition reaction between poloxamer diacrylate and low molecular weight PEI [Fig. 8(A)] and the hyperbranched poly(amino ester)s showed slow degradation at physiological conditions which was greatly dependent on hydrophilicity of poloxamer [Fig. 8(B)].

PEAs were dissolved in 0.1 M PBS, and incubated at 37 °C and 100 rpm. [Source from Ref. [52]]. All together, poly(amino ester)s can be easily synthesized by Michael type addition reaction and **Figure 8.** Synthetic scheme of PEA by Michael addition reaction (A) and degradation of PEAs (B). PEAs were dissolved in 0.1 M PBS, and incubated at 37 °C with 100 rpm. [Source from Ref. [52]].

Fig. 8. Synthetic scheme of PEA by Michael addition reaction (A) and degradation of PEAs (B).

showed good degradation profiles due to the hydrolysis of the ester bonds in the polymer backbones.

**2.2 Characterization of poly(amino ester)s/DNA complexes 2.2.1 DNA condensation and protection** One prerequisite of a polymeric gene carrier is DNA condensation [53]. Polycation-mediated gene delivery is based on the electrostatic interactions between the positive charged polycation and All together, poly(amino ester)s can be easily synthesized by Michael type addition reaction and showed good degradation profiles due to the hydrolysis of the ester bonds in the polymer backbones.

negatively charged phosphate groups of DNA [32]. As shown in Fig. 9(A), retardation of DNA

the DNA from degradation by nucleases, and the compact particles can be taken up by cells via

#### migration begins at poly(β-amino ester)/DNA ratios as low as 0.1:1 (w/w) and migration is completely retarded at poly(β-amino ester)/DNA ratios above 5:1 (w/w) [34]. Condensation protects **2.2. Characterization of poly(amino ester)s/DNA complexes**

#### natural processes such as adsorptive endocytosis, pinocytosis and phagocytosis [32]. DNA in the complexes was protected from nuclease attack whereas the naked DNA was degraded. This result *2.2.1. DNA condensation and protection*

[Source from Ref. [34]].

suggests that intact DNA could be delivered by poly(β-amino ester) into cells without degradation [Fig. 9(B)] [34]. One prerequisite of a polymeric gene carrier is DNA condensation [53]. Polycation-mediated gene delivery is based on the electrostatic interactions between the positive charged polycation and negatively charged phosphate groups of DNA [32]. As shown in Fig. 9(A), retardation of DNA migration begins at poly(β-amino ester)s/DNA ratios as low as 0.1:1 (w/w) and migration is completely retarded at poly(β-amino ester)s/DNA ratios above 5:1 (w/w) [34]. Condensation protects the DNA from degradation by nucleases, and the compact particles can be taken up

Fig. 9. DNA condensation and protection study. Agarose gel electrophoresis of poly(β-amino ester) (GPT–SPE)/DNA complexes at various weight ratios (A) and DNA protection and release assay (B).

Surface properties, such as particle size and surface charge of the complex, are necessary to assure its uptake by cells [53]. In particular, the particle size of a complex is an important factor that influences

**2.2.2 Particle sizes and surface charges of poly(amino ester)s/DNA complexes** 

by cells via natural processes such as adsorptive endocytosis, pinocytosis and phagocytosis [32]. DNA in the complexes was protected from nuclease attack whereas the naked DNA was degraded. This result suggests that intact DNA could be delivered by poly(β-amino ester)s into cells without degradation [Fig. 9(B)] [34].

these hyperbranched poly(amino ester)s degraded in a controlled manner within 50 days and it was speculated that this phenomenon may be due to the lesser water accessibility of the ester groups in

Fig. 6. Structure of linear and hyperbranched poly(amino ester)s obtained via Michael addition polymerization of trifunctional amines with diacrylates and triacrylates (A) and comparison of the hydrolysis profiles of protonated hyperbranched poly(BDA2-AEPZ1)-MPZ, hyperbranched poly(TMPTA1-AEPZ2), and linear poly(BDA-AEPZ) in aqueous solutions (B). [Source from Ref.

Cho's group also reported the synthesis of branch poly(amino ester)s by Michael addition, based on hydrophobic polycaprolactone diacrylate and low molecular weight PEI [Fig. 7(A)] [40]. It was simply an indication of application of ester linkage which supports the easy degradation leaving nontox building blocks, thereby increased transfection efficiency and reduced cytotoxicity. The branched poly(amino ester)s showed controlled degradation with the half life of 4-4.5 days as shown

Fig. 7. The synthetic scheme of PEA by Michael addition (A) and degradation of PEAs (PCL/PEI-1.2

Same group also reported another degradable branched poly(amino ester)s based on poloxamer diacrylate and low molecular weight PEI [52]. These hyperbranched poly(amino ester)s can be easily synthesized by Michael type addition reaction between poloxamer diacrylate and low molecular weight PEI [Fig. 8(A)] and the hyperbranched poly(amino ester)s showed slow degradation at physiological conditions which was greatly dependent on hydrophilicity of poloxamer [Fig. 8(B)].

Same group also reported another degradable branched poly(amino ester)s based on polox‐ amer diacrylate and low molecular weight PEI [52]. These hyperbranched poly(amino ester)s can be easily synthesized by Michael type addition reaction between poloxamer diacrylate and low molecular weight PEI [Fig. 8(A)] and the hyperbranched poly(amino ester)s showed slow degradation at physiological conditions which was greatly dependent on hydrophilicity of

Fig. 8. Synthetic scheme of PEA by Michael addition reaction (A) and degradation of PEAs (B). PEAs were dissolved in 0.1 M PBS, and incubated at 37 °C and 100 rpm. [Source from Ref. [52]]. All together, poly(amino ester)s can be easily synthesized by Michael type addition reaction and showed good degradation profiles due to the hydrolysis of the ester bonds in the polymer backbones.

**Figure 8.** Synthetic scheme of PEA by Michael addition reaction (A) and degradation of PEAs (B). PEAs were dissolved

All together, poly(amino ester)s can be easily synthesized by Michael type addition reaction and showed good degradation profiles due to the hydrolysis of the ester bonds in the polymer

One prerequisite of a polymeric gene carrier is DNA condensation [53]. Polycation-mediated gene delivery is based on the electrostatic interactions between the positive charged polycation and negatively charged phosphate groups of DNA [32]. As shown in Fig. 9(A), retardation of DNA migration begins at poly(β-amino ester)s/DNA ratios as low as 0.1:1 (w/w) and migration is completely retarded at poly(β-amino ester)s/DNA ratios above 5:1 (w/w) [34]. Condensation protects the DNA from degradation by nucleases, and the compact particles can be taken up

One prerequisite of a polymeric gene carrier is DNA condensation [53]. Polycation-mediated gene delivery is based on the electrostatic interactions between the positive charged polycation and negatively charged phosphate groups of DNA [32]. As shown in Fig. 9(A), retardation of DNA migration begins at poly(β-amino ester)/DNA ratios as low as 0.1:1 (w/w) and migration is completely retarded at poly(β-amino ester)/DNA ratios above 5:1 (w/w) [34]. Condensation protects the DNA from degradation by nucleases, and the compact particles can be taken up by cells via natural processes such as adsorptive endocytosis, pinocytosis and phagocytosis [32]. DNA in the complexes was protected from nuclease attack whereas the naked DNA was degraded. This result suggests that intact DNA could be delivered by poly(β-amino ester) into cells without degradation

Fig. 9. DNA condensation and protection study. Agarose gel electrophoresis of poly(β-amino ester) (GPT–SPE)/DNA complexes at various weight ratios (A) and DNA protection and release assay (B).

Surface properties, such as particle size and surface charge of the complex, are necessary to assure its uptake by cells [53]. In particular, the particle size of a complex is an important factor that influences

**2.2.2 Particle sizes and surface charges of poly(amino ester)s/DNA complexes** 

**2.2 Characterization of poly(amino ester)s/DNA complexes**

**2.2. Characterization of poly(amino ester)s/DNA complexes**

in 0.1 M PBS, and incubated at 37 °C with 100 rpm. [Source from Ref. [52]].

**2.2.1 DNA condensation and protection**

[Fig. 9(B)] [34].

*2.2.1. DNA condensation and protection*

backbones.

[Source from Ref. [34]].

**Figure 7.** The synthetic scheme of PEAs by Michael addition (A) and degradation of PEAs (PCL/PEI-1.2 and PCL/

(A) (B)

(A) (B)

(A) (B)

hyperbranched sturctures [Fig. 6(B)].

[51]].

382 Novel Gene Therapy Approaches

in Fig. 7(B).

PEI-1.8) (B). [Source from Ref. [40]].

poloxamer [Fig. 8(B)].

and PCL/PEI-1.8) (B). [Source from Ref. [40]].

**Figure 9.** DNA condensation and protection study. Agarose gel electrophoresis of poly(β-amino ester)s (GPT– SPE)/DNA complexes at various weight ratios (A) and DNA protection and release assay (B). [Source from Ref. [34]].

#### *2.2.2. Particle sizes and surface charges of poly(amino ester)s/DNA complexes*

Surface properties, such as particle size and surface charge of the complex, are necessary to assure its uptake by cells [53]. In particular, the particle size of a complex is an important factor that influences the access and passage of the complex through the targeting site. Successful gene carrier depends on its ability to condense negatively charged DNA into nanosized particles with positive charges so as to enter into the cells [54]. Compact particles of small size are usually obtained only at higher N/P ratios, resulting in complexes with a strong positive net charge. For most cell types, the poly(amino ester)s/DNA complexes size requirement is on the order of 200 nm or less [55]. As shown in Fig. 10(A), poly(β-amino ester)s formed complexes with diameters in the range of 50-150 nm at DNA/polymer ratios above 1:2. A positive surface charge of polyplexes is necessary for binding to anionic cell surfaces, which consequently facilitates uptake by the cell [30, 56]. The surface charge of poly(β-amino ester)s/DNA com‐ plexes has been examined in terms of ζ-potential. The ζ-potentials for complexes were on the order of +10 to +15 mV at DNA/polymer ratios above 1:1, and the complexes did not aggregate extensively over an 18h period as shown in Fig. 10(B). the access and passage of the complex through the targeting site. Successful gene carrier depends on its ability to condense negatively charged DNA into nanosized particles with positive charges so as to enter into the cells [54]. Compact particles of small size are usually obtained only at higher N/P ratios, resulting in complexes with a strong positive net charge. For most cell types, the poly(amino ester)s/DNA complexes size requirement is on the order of 200 nm or less [55]. As shown in Fig. 10(A), poly(β-amino ester)s formed complexes with diameters in the range of 50-150 nm at DNA/polymer ratios above 1:2. A positive surface charge of polyplexes is necessary for binding to anionic cell surfaces, which consequently facilitates uptake by the cell [30, 56]. The surface charge of poly(β-amino ester)s/DNA complexes has been examined in terms of ζ-potential. The ζ-potentials for complexes were on the order of +10 to +15 mV at DNA/polymer ratios above 1:1, and the complexes

with poly(amino ester)s obtained from R117 to R121. Slight cytotoxicity was observed at higher mass ratios (90:1 and 110:1) (viability above 80% in all ratios) indicating that cytotoxicity was highly sensitive to monomer ratio and varied drastically even with small change in monomer concentration. It was reported that the cytotoxicity of cationic polymers is probably caused by polymer aggregation on cell surfaces, impairing important membrane functions. Also, the cationic polymers may interfere with critical intracellular processes of cells: in particular, the primary amine was reported to disrupt PKC function through disturbance of protein kinase activity [60, 61]. On the other hands, in 293T cells, poly(amino ester)s obtained from R106 to R113 showed some transfection at lower weight ratios but it was suddenly decreased with increased weight ratios which may be because of low cell viability at these ratios [Fig. 11(C)]. Poly(amino ester)s obtained from R114 to R119 showed intermediate transfection while poly(amino ester)s obtained from R120 and R121 gave good transfection. However, in HeLa cells slightly different transfection pattern was observed as shown in Fig. 11(D)]. Poly(amino ester)s obtained from R106 to R115 failed to give significant transfection. On the other hand, poly(amino ester)s obtained from R116 to R119 showed intermediate transfection which was slowly increased from R116 to R119. Transfection was highest with poly(amino ester)s obtained from R120 and R121 and it was increased with increasing weight ratios till 90:1 after that it again decreased due to increased cytotoxicity. It was also reported that in addition to factors such as chemical structure and polymer molecular weight, either amine or acrylate terminated also plays a significant role in determining transfection efficiency of poly(amino ester)s [46]. Excess of amine monomers results into amine terminated polymer which effec‐ tively binds with cell membrane and promotes its uptake whereas acrylate terminated

Poly(amino ester)s-Based Polymeric Gene Carriers in Cancer Gene Therapy

http://dx.doi.org/10.5772/54740

385

cytotoxicity of cationic polymers is probably caused by polymer aggregation on cell surfaces, impairing important membrane functions. Also, the cationic polymers may interfere with critical intracellular processes of cells: in particular, the primary amine was reported to disrupt PKC function through disturbance of protein kinase activity [60, 61]. On the other hands, in 293T cells, poly(amino ester) obtained from R106 to R113 showed some transfection at lower weight ratios but it was suddenly decreased with increased weight ratios which may be because of low cell viability at these ratios [Fig. 11(C)]. Poly(amino ester) obtained from R114 to R119 showed intermediate transfection while poly(amino ester) obtained from R120 and R121 gave good transfection. However, in HeLa cells slightly different transfection pattern was observed as shown in Fig. 11(D)]. Poly(amino ester) obtained from R106 to R115 failed to give significant transfection. On the other hand, poly(amino ester) obtained from R116 to R119 showed intermediate transfection which was slowly increased from R116 to R119. Transfection was highest with poly(amino ester) obtained from R120 and R121 and it was increased with increasing weight ratios till 90:1 after that it again decreased due to increased cytotoxicity. It was also reported that in addition to factors such as chemical structure and polymer molecular weight, eigher amine or acrylate terminated also plays a significant role in determining transfection efficiency of poly(amino ester)s [46]. Excess of amine monomers results into amine terminated polymer which effectively binds with cell membrane and promotes its uptake

where as acrylate terminated polymers has poor cellular entry and transfection efficiency.

Fig. 11. Cytotoxicity of PAEs at various concentrations in 293T cell line (A) and HeLa cell line (B); and transfection efficiency of PAE/DNA complexes in serum free-media at various mass ratios in

**Figure 11.** Cytotoxicity of PAEs at various concentrations in 293T cell line (A) and HeLa cell line (B); and transfection efficiency of PAE/DNA complexes in serum free-media at various mass ratios in 293T cells (C) and HeLa cells (D).

**2.4 Toxicity and transfection considerations of poly(amino ester)s/DNA complexes in vivo** Intravenous administration as one of the most commonly used methods in gene therapy area even most gene therapy vectors, as well as other biomolecules and potential engineered drugs, has short elimination half-lives due to the serum proteins in the blood stream. In vivo transfection efficiency of the poly(ester amine) was studied after intravenous administration into mice [62]. As shown in Fig. 12, the quantity of luciferase was determined in lung, liver, spleen, kidney and heart after 24 h intravenous administration of polymer/DNA complexes. Fig. 12 shows luciferase gene expression in various mouse organs after intravenous administration of the polymer/DNA complexes via the tail

(A) (B)

(C) (D)

polymers has poor cellular entry and transfection efficiency.

293T cells (C) and HeLa cells (D). [Source from Ref. [59]].

[Source from Ref. [59]].

did not aggregate extensively over an 18-h period as shown in Fig. 10(B).

from pCMV-Luc plasmid and poly(β-amino ester) (polymer 3) (Mn = 31000) as a function of polymer concentration. [Source from Ref. [44]]. **2.3 Toxicity and transfection considerations of poly(amino ester)s/DNA complexes in vitro Figure 10.** Average effective diameters (A) and ζ-potentials (B) of DNA/polymer complexes formed from pCMV-Luc plasmid and poly(β-amino ester)s (polymer 3) (Mn = 31000) as a function of polymer concentration. [Source from Ref. [44]].

Fig. 10. Average effective diameters (A) and ζ-potentials (B) of DNA/polymer complexes formed

#### Safe and efficient delivery of genes is critical for the successful application of gene therapy. In fact, it is the only major obstacle in the expansion of gene therapy from bench to beside. Many vectors with high transfection efficiency are highly toxic while vectors with low toxicity are poor in transfecting **2.3. Toxicity and transfection considerations of poly(amino ester)s/DNA complexes** *in vitro*

cells. Optimum balance between these two parameters is a key to the success in gene therapy [57-59]. As biodegradable polymers are designed to contain a combination of various functional components, it is likely that engineered systems for non-viral gene delivery, especially with the application of biodegradable ester linkage will eventually be constructed. This biodegradable linkage approach to vector development is giving way to a safety profile where low molecular weight amine contain monomers are couples with acylate linkers to yield high molecular weight poly(amino ester)s with reduced toxicity and enhanced transfection efficiency. Jere et al. evaluated cytotoxicity of mini-library of poly(amino ester)s in 293T and HeLa cells by MTS assay [59]. In order to measure maximum possible cytotoxicity, poly(amino ester)s were administered in increasing concentrations to 293T cells as shown in Figs. 11(A) and (B). In both cell lines, poly(amino ester)s obtained from R106 to R113 exhibited very high cytotoxicity which further increased with increase in weight ratios, while poly(amino ester)s obtained from R114, R115 and R116 showed good cell viability at lower ratios but significant cytotoxicity at higher weight ratios. Excellent cell viability and uniform transfection pattern were observed with poly(amino ester)s obtained from R117 to R121. Slight cytotoxicity was observed at higher mass ratios (90:1 and 110:1) (viability above 80% in all ratios) indicating that cytotoxicity was highly sensitive to monomer ratio and varied drastically even with small change in monomer concentration. It was reported that the Safe and efficient delivery of genes is critical for the successful application of gene therapy. In fact, it is the only major obstacle in the expansion of gene therapy from bench to beside. Many vectors with high transfection efficiency show high toxicity while vectors with low toxicity are poor in transfecting cells. Optimum balance between these two parameters is a key to the success in gene therapy [57-59]. As biodegradable polymers are designed to contain a combi‐ nation of various functional components, it is likely that engineered systems for non-viral gene delivery, especially with the application of biodegradable ester linkage will eventually be constructed. This biodegradable linkage approach to vector development is giving way to a safety profile where low molecular weight amine contain monomers are couples with acylate linkers to yield high molecular weight poly(amino ester)s with reduced toxicity and enhanced transfection efficiency.

Jere et al. evaluated cytotoxicity of mini-library of poly(amino ester)s in 293T and HeLa cells by MTS assay [59]. In order to measure maximum possible cytotoxicity, poly(amino ester)s were administered in increasing concentrations to 293T cells as shown in Figs. 11(A) and (B). In both cell lines, poly(amino ester)s obtained from R106 to R113 exhibited very high cytotox‐ icity which further increased with increase in weight ratios, while poly(amino ester)s obtained from R114, R115 and R116 showed good cell viability at lower ratios but significant cytotoxicity at higher weight ratios. Excellent cell viability and uniform transfection pattern were observed with poly(amino ester)s obtained from R117 to R121. Slight cytotoxicity was observed at higher mass ratios (90:1 and 110:1) (viability above 80% in all ratios) indicating that cytotoxicity was highly sensitive to monomer ratio and varied drastically even with small change in monomer concentration. It was reported that the cytotoxicity of cationic polymers is probably caused by polymer aggregation on cell surfaces, impairing important membrane functions. Also, the cationic polymers may interfere with critical intracellular processes of cells: in particular, the primary amine was reported to disrupt PKC function through disturbance of protein kinase activity [60, 61]. On the other hands, in 293T cells, poly(amino ester)s obtained from R106 to R113 showed some transfection at lower weight ratios but it was suddenly decreased with increased weight ratios which may be because of low cell viability at these ratios [Fig. 11(C)]. Poly(amino ester)s obtained from R114 to R119 showed intermediate transfection while poly(amino ester)s obtained from R120 and R121 gave good transfection. However, in HeLa cells slightly different transfection pattern was observed as shown in Fig. 11(D)]. Poly(amino ester)s obtained from R106 to R115 failed to give significant transfection. On the other hand, poly(amino ester)s obtained from R116 to R119 showed intermediate transfection which was slowly increased from R116 to R119. Transfection was highest with poly(amino ester)s obtained from R120 and R121 and it was increased with increasing weight ratios till 90:1 after that it again decreased due to increased cytotoxicity. It was also reported that in addition to factors such as chemical structure and polymer molecular weight, either amine or acrylate terminated also plays a significant role in determining transfection efficiency of poly(amino ester)s [46]. Excess of amine monomers results into amine terminated polymer which effec‐ tively binds with cell membrane and promotes its uptake whereas acrylate terminated polymers has poor cellular entry and transfection efficiency. cytotoxicity of cationic polymers is probably caused by polymer aggregation on cell surfaces, impairing important membrane functions. Also, the cationic polymers may interfere with critical intracellular processes of cells: in particular, the primary amine was reported to disrupt PKC function through disturbance of protein kinase activity [60, 61]. On the other hands, in 293T cells, poly(amino ester) obtained from R106 to R113 showed some transfection at lower weight ratios but it was suddenly decreased with increased weight ratios which may be because of low cell viability at these ratios [Fig. 11(C)]. Poly(amino ester) obtained from R114 to R119 showed intermediate transfection while poly(amino ester) obtained from R120 and R121 gave good transfection. However, in HeLa cells slightly different transfection pattern was observed as shown in Fig. 11(D)]. Poly(amino ester) obtained from R106 to R115 failed to give significant transfection. On the other hand, poly(amino ester) obtained from R116 to R119 showed intermediate transfection which was slowly increased from R116 to R119. Transfection was highest with poly(amino ester) obtained from R120 and R121 and it was increased with increasing weight ratios till 90:1 after that it again decreased due to increased cytotoxicity. It was also reported that in addition to factors such as chemical structure and polymer molecular weight, eigher amine or acrylate terminated also plays a significant role in

net charge. For most cell types, the poly(amino ester)s/DNA complexes size requirement is on the order of 200 nm or less [55]. As shown in Fig. 10(A), poly(β-amino ester)s formed complexes with diameters in the range of 50-150 nm at DNA/polymer ratios above 1:2. A positive surface charge of polyplexes is necessary for binding to anionic cell surfaces, which consequently facilitates uptake by the cell [30, 56]. The surface charge of poly(β-amino ester)s/DNA com‐ plexes has been examined in terms of ζ-potential. The ζ-potentials for complexes were on the order of +10 to +15 mV at DNA/polymer ratios above 1:1, and the complexes did not aggregate

the access and passage of the complex through the targeting site. Successful gene carrier depends on its ability to condense negatively charged DNA into nanosized particles with positive charges so as to enter into the cells [54]. Compact particles of small size are usually obtained only at higher N/P ratios, resulting in complexes with a strong positive net charge. For most cell types, the poly(amino ester)s/DNA complexes size requirement is on the order of 200 nm or less [55]. As shown in Fig. 10(A), poly(β-amino ester)s formed complexes with diameters in the range of 50-150 nm at DNA/polymer ratios above 1:2. A positive surface charge of polyplexes is necessary for binding to anionic cell surfaces, which consequently facilitates uptake by the cell [30, 56]. The surface charge of poly(β-amino ester)s/DNA complexes has been examined in terms of ζ-potential. The ζ-potentials for complexes were on the order of +10 to +15 mV at DNA/polymer ratios above 1:1, and the complexes

Fig. 10. Average effective diameters (A) and ζ-potentials (B) of DNA/polymer complexes formed from pCMV-Luc plasmid and poly(β-amino ester) (polymer 3) (Mn = 31000) as a function of

**Figure 10.** Average effective diameters (A) and ζ-potentials (B) of DNA/polymer complexes formed from pCMV-Luc plasmid and poly(β-amino ester)s (polymer 3) (Mn = 31000) as a function of polymer concentration. [Source from Ref.

**2.3. Toxicity and transfection considerations of poly(amino ester)s/DNA complexes** *in vitro* Safe and efficient delivery of genes is critical for the successful application of gene therapy. In fact, it is the only major obstacle in the expansion of gene therapy from bench to beside. Many vectors with high transfection efficiency show high toxicity while vectors with low toxicity are poor in transfecting cells. Optimum balance between these two parameters is a key to the success in gene therapy [57-59]. As biodegradable polymers are designed to contain a combi‐ nation of various functional components, it is likely that engineered systems for non-viral gene delivery, especially with the application of biodegradable ester linkage will eventually be constructed. This biodegradable linkage approach to vector development is giving way to a safety profile where low molecular weight amine contain monomers are couples with acylate linkers to yield high molecular weight poly(amino ester)s with reduced toxicity and enhanced

Jere et al. evaluated cytotoxicity of mini-library of poly(amino ester)s in 293T and HeLa cells by MTS assay [59]. In order to measure maximum possible cytotoxicity, poly(amino ester)s were administered in increasing concentrations to 293T cells as shown in Figs. 11(A) and (B). In both cell lines, poly(amino ester)s obtained from R106 to R113 exhibited very high cytotoxicity which further increased with increase in weight ratios, while poly(amino ester)s obtained from R114, R115 and R116 showed good cell viability at lower ratios but significant cytotoxicity at higher weight ratios. Excellent cell viability and uniform transfection pattern were observed with poly(amino ester)s obtained from R117 to R121. Slight cytotoxicity was observed at higher mass ratios (90:1 and 110:1) (viability above 80% in all ratios) indicating that cytotoxicity was highly sensitive to monomer ratio and varied drastically even with small change in monomer concentration. It was reported that the

Jere et al. evaluated cytotoxicity of mini-library of poly(amino ester)s in 293T and HeLa cells by MTS assay [59]. In order to measure maximum possible cytotoxicity, poly(amino ester)s were administered in increasing concentrations to 293T cells as shown in Figs. 11(A) and (B). In both cell lines, poly(amino ester)s obtained from R106 to R113 exhibited very high cytotox‐ icity which further increased with increase in weight ratios, while poly(amino ester)s obtained from R114, R115 and R116 showed good cell viability at lower ratios but significant cytotoxicity at higher weight ratios. Excellent cell viability and uniform transfection pattern were observed

**2.3 Toxicity and transfection considerations of poly(amino ester)s/DNA complexes in vitro** Safe and efficient delivery of genes is critical for the successful application of gene therapy. In fact, it is the only major obstacle in the expansion of gene therapy from bench to beside. Many vectors with high transfection efficiency are highly toxic while vectors with low toxicity are poor in transfecting cells. Optimum balance between these two parameters is a key to the success in gene therapy [57-59]. As biodegradable polymers are designed to contain a combination of various functional components, it is likely that engineered systems for non-viral gene delivery, especially with the application of biodegradable ester linkage will eventually be constructed. This biodegradable linkage approach to vector development is giving way to a safety profile where low molecular weight amine contain monomers are couples with acylate linkers to yield high molecular weight poly(amino ester)s with

(A) (B)

extensively over an 18h period as shown in Fig. 10(B).

384 Novel Gene Therapy Approaches

polymer concentration. [Source from Ref. [44]].

[44]].

transfection efficiency.

reduced toxicity and enhanced transfection efficiency.

did not aggregate extensively over an 18-h period as shown in Fig. 10(B).

determining transfection efficiency of poly(amino ester)s [46]. Excess of amine monomers results into amine terminated polymer which effectively binds with cell membrane and promotes its uptake

where as acrylate terminated polymers has poor cellular entry and transfection efficiency.

and transfection efficiency of PAE/DNA complexes in serum free-media at various mass ratios in 293T cells (C) and HeLa cells (D). [Source from Ref. [59]]. **2.4 Toxicity and transfection considerations of poly(amino ester)s/DNA complexes in vivo Figure 11.** Cytotoxicity of PAEs at various concentrations in 293T cell line (A) and HeLa cell line (B); and transfection efficiency of PAE/DNA complexes in serum free-media at various mass ratios in 293T cells (C) and HeLa cells (D). [Source from Ref. [59]].

Intravenous administration as one of the most commonly used methods in gene therapy area even most gene therapy vectors, as well as other biomolecules and potential engineered drugs, has short elimination half-lives due to the serum proteins in the blood stream. In vivo transfection efficiency of the poly(ester amine) was studied after intravenous administration into mice [62]. As shown in Fig. 12, the quantity of luciferase was determined in lung, liver, spleen, kidney and heart after 24 h intravenous administration of polymer/DNA complexes. Fig. 12 shows luciferase gene expression in various mouse organs after intravenous administration of the polymer/DNA complexes via the tail

Fig. 11. Cytotoxicity of PAEs at various concentrations in 293T cell line (A) and HeLa cell line (B);

#### **2.4. Toxicity and transfection considerations of poly(amino ester)s/DNA complexes** *in vivo*

Intravenous administration as one of the most commonly used methods in gene therapy area even most gene therapy vectors, as well as other biomolecules and potential engi‐ neered drugs, has short elimination half-lives due to the serum proteins in the blood stream. In vivo transfection efficiency of the poly(amino ester)s was studied after intrave‐ nous administration into mice [62]. As shown in Fig. 12, the quantity of luciferase was de‐ termined in lung, liver, spleen, kidney and heart after 24 h intravenous administration of polymer/DNA complexes. Fig. 12 shows luciferase gene expression in various mouse or‐ gans after intravenous administration of the polymer/DNA complexes via the tail vein. As shown in Fig. 12, injection of polymer/DNA complexes resulted in transfection primarily in the lung which is in agreement with previous results [63, 64]. Verbaan et al. suggest‐ ed two mechanisms regarding this phenomenon of predominant gene expression in the lung; firstly, because the lung is the first organ encountered by polyplexes after tail vein injection, the positively charged polyplexes may electrostatically interact with the nega‐ tively charged membranes of the endothelial cells in the lung, secondly, the physical trap‐ ping of large aggregates formed by the interaction of polyplexes with blood components like serum proteins and erythrocytes [63, 64]. Also, the poly(amino ester)s/DNA com‐ plexes showed the highest transfection activity in the lung regardless of N/P ratio. This may be caused by the positive charge of the poly(amino ester)s/DNA complexes like PEI 25K/DNA complexes. In contrast to PEI 25K/DNA complexes, the poly(amino ester)s/DNA complexes had high transfection in the liver because the liver is the main or‐ gan for gene accumulation and subsequent degradation [62]. Plank et al. reported that op‐ sonization of the polyplexes led to a rapid clearance by the mononuclear phagocytic system (MPS) [65]. Uptake by the MPS would be in agreement with the observed liver and spleen accumulations. In addition, the presence of discontinuous or fenestrated endo‐ thelia in the vascularization of the liver and spleen may facilitate the gene accumulation in these tissues [66]. The poly(amino ester)s/DNA complexes showed higher transfection efficiency than golden standard PEI 25K/DNA ones, and the luciferase activity was in‐ creased in all organs except kidney with increase of N/P ratio indicating that poly(amino ester)s/DNA complexes function efficiently after intravenous administration.

**Figure 12.** Tissue distribution of poly(amino ester)s/DNA (gWIZ-Luc) complexes administered by intravenous injection and inhalation at various N/P ratios. (∗p < 0.1; ∗∗p < 0.05, Student's t-test, two-tailed). [Source from Ref. [62]].

Fig. 13. Intraperitoneal gene delivery in mice (A). (a) Whole-body optical images of luciferase expression in FVB/J mice 6 hours after intraperitoneal injection of polymer/DNA complexes. Images show the highest expression obtained for each polymer. The control mouse was injected with 120 μl of 50 mM NaAc buffer, pH 5.2. Pseudocolor images representing emitted bioluminescence are superimposed over grayscale images. Relative light units (RLUs)/pixel are indicated in the color scale bar on the left. (b) Quantification of whole-body luciferase expression at various times after intraperitoneal injection of C32- (hatched) and C32-117-delivered (solid) DNA. Statistically significant differences between C32 and C32-117 at a given time point are indicated. n = 4 for each treatment group. \*P <0.05; \*\*P < 0.01; \*\*\*P < 0.001. Organ distribution of gene expression (B). Quantification of luciferase expression in whole body and individual organs 6 hours after intraperitoneal injection of polymer/DNA complexes in FVB/J mice. Results are expressed as mean transfection levels (± SD) for a buffer control (white), C32-103 (yellow), C32-116 (red), C32-117 (green), C32-122 (blue), C32 (pink), and jet-polyethylenimine (jet-PEI) (black). n ≥ 3 for each

**Figure 13.** Intraperitoneal gene delivery in mice (A). (a) Whole-body optical images of luciferase expression in FVB/J mice 6 hours after intraperitoneal injection of polymer/DNA complexes. Images show the highest expression obtained for each polymer. The control mouse was injected with 120 μl of 50 mM NaAc buffer, pH 5.2. Pseudocolor images representing emitted bioluminescence are superimposed over grayscale images. Relative light units (RLUs)/pixel are indicated in the color scale bar on the left. (b) Quantification of whole-body luciferase expression at various times af‐ ter intraperitoneal injection of C32- (hatched) and C32-117-delivered (solid) DNA. Statistically significant differences between C32 and C32-117 at a given time point are indicated. n = 4 for each treatment group. \*P <0.05; \*\*P < 0.01; \*\*\*P < 0.001. Organ distribution of gene expression (B). Quantification of luciferase expression in whole body and in‐ dividual organs 6 hours after intraperitoneal injection of polymer/DNA complexes in FVB/J mice. Results are expressed as mean transfection levels (± SD) for a buffer control (white), C32-103 (yellow), C32-116 (red), C32-117 (green), C32-122 (blue), C32 (pink), and jet-polyethylenimine (jet-PEI) (black). n ≥ 3 for each treatment group. [Source from

High transfection levels were observed after intraperitoneal injection of polymer/DNA complexes. End-modified polymers resulted in whole-body reporter protein expression more than an order-of magnitude higher than that for jet-PEI. They also outperformed the best poly(β-amino ester) synthesized to date, C32, with overall expression levels 4- to 12-fold higher. They found that sustained expression past 1 week both with modified C32 and with C32, but modified C32 was expressed at significantly higher levels, reflecting its enhanced delivery capabilities. This effect was most evident between the C32-116 and C32-117 polymers, where the latter displayed 5- to 65-fold higher delivery to the bladder, spleen, liver, and kidney. The only difference between these two

One of the most non-invasive approaches to drug/gene delivery is via inhalation. Gene therapy to the lung can potentially be exploited for the treatment of both genetic and acquired diseases. However, any therapeutic approach for the respiratory tract must take into account the heterogeneity of the cellular targets in the lung: epithelial cell, alveolar cells, vascular cells, serous cells in the submucosal glands and a number of other cell types [69]. Our group developed spermine-based biocompatible poly(β-amino ester) as an aerosol delivery gene carrier [34]. As shown in Fig. 14 (A-A), GFP signal was dominant in the lungs with GPT–SPE/GFP complexes-exposed group compared to the control and naked GFP-exposed groups. No necrosis, degeneration, metaplasia, anaplasia in pneumocytes, atelectasis, or emphysema were detected [Fig. 14 (A-B)]. These results indicate that GPT–SPE functions safely and efficiently in aerosol delivery system. Significant anticancer effects of GPT–SPE/Akt1 shRNA complexes in the lungs through aerosol inhalation were observed in lung tumor bearing K-*ras*LA1 mice [Fig. 14 (BA-E)] without toxicity [Table 2-1]. These result indicating

diaminopropane end-capping reagents is the ethyl versus dimethyl branching.

treatment group. [Source from Ref. [68]].

Ref. [68]].

(A) (B)

Poly(amino ester)s-Based Polymeric Gene Carriers in Cancer Gene Therapy

http://dx.doi.org/10.5772/54740

387

Implantable infusion pumps have been developed as an one of therapy methods for a num‐ ber of diseases, and there has been remarkable progress in endoscopic and laparoscopic sur‐ gical techniques. This progress in surgical techniques and devices could make intraperitoneal administration a conventional and feasible approach for future clinical appli‐ cations [67]. Intraperitoneal gene delivery may provide a strategy for the treatment of a vari‐ ety of diseases, including cancer. Zugates et al. synthesized parallel end-modification of poly(β-amino ester)s by the conjugate addition of amines to diacrylate monomers as shown in Fig. 13 [68].

**2.4. Toxicity and transfection considerations of poly(amino ester)s/DNA complexes** *in*

Intravenous administration as one of the most commonly used methods in gene therapy area even most gene therapy vectors, as well as other biomolecules and potential engi‐ neered drugs, has short elimination half-lives due to the serum proteins in the blood stream. In vivo transfection efficiency of the poly(amino ester)s was studied after intrave‐ nous administration into mice [62]. As shown in Fig. 12, the quantity of luciferase was de‐ termined in lung, liver, spleen, kidney and heart after 24 h intravenous administration of polymer/DNA complexes. Fig. 12 shows luciferase gene expression in various mouse or‐ gans after intravenous administration of the polymer/DNA complexes via the tail vein. As shown in Fig. 12, injection of polymer/DNA complexes resulted in transfection primarily in the lung which is in agreement with previous results [63, 64]. Verbaan et al. suggest‐ ed two mechanisms regarding this phenomenon of predominant gene expression in the lung; firstly, because the lung is the first organ encountered by polyplexes after tail vein injection, the positively charged polyplexes may electrostatically interact with the nega‐ tively charged membranes of the endothelial cells in the lung, secondly, the physical trap‐ ping of large aggregates formed by the interaction of polyplexes with blood components like serum proteins and erythrocytes [63, 64]. Also, the poly(amino ester)s/DNA com‐ plexes showed the highest transfection activity in the lung regardless of N/P ratio. This may be caused by the positive charge of the poly(amino ester)s/DNA complexes like PEI 25K/DNA complexes. In contrast to PEI 25K/DNA complexes, the poly(amino ester)s/DNA complexes had high transfection in the liver because the liver is the main or‐ gan for gene accumulation and subsequent degradation [62]. Plank et al. reported that op‐ sonization of the polyplexes led to a rapid clearance by the mononuclear phagocytic system (MPS) [65]. Uptake by the MPS would be in agreement with the observed liver and spleen accumulations. In addition, the presence of discontinuous or fenestrated endo‐ thelia in the vascularization of the liver and spleen may facilitate the gene accumulation in these tissues [66]. The poly(amino ester)s/DNA complexes showed higher transfection efficiency than golden standard PEI 25K/DNA ones, and the luciferase activity was in‐ creased in all organs except kidney with increase of N/P ratio indicating that poly(amino

ester)s/DNA complexes function efficiently after intravenous administration.

Implantable infusion pumps have been developed as an one of therapy methods for a num‐ ber of diseases, and there has been remarkable progress in endoscopic and laparoscopic sur‐ gical techniques. This progress in surgical techniques and devices could make intraperitoneal administration a conventional and feasible approach for future clinical appli‐ cations [67]. Intraperitoneal gene delivery may provide a strategy for the treatment of a vari‐ ety of diseases, including cancer. Zugates et al. synthesized parallel end-modification of poly(β-amino ester)s by the conjugate addition of amines to diacrylate monomers as shown

*vivo*

386 Novel Gene Therapy Approaches

in Fig. 13 [68].

**Figure 12.** Tissue distribution of poly(amino ester)s/DNA (gWIZ-Luc) complexes administered by intravenous injection and inhalation at various N/P ratios. (∗p < 0.1; ∗∗p < 0.05, Student's t-test, two-tailed). [Source from Ref. [62]].

expression in FVB/J mice 6 hours after intraperitoneal injection of polymer/DNA complexes. Images show the highest expression obtained for each polymer. The control mouse was injected with 120 μl of 50 mM NaAc buffer, pH 5.2. Pseudocolor images representing emitted bioluminescence are superimposed over grayscale images. Relative light units (RLUs)/pixel are indicated in the color scale bar on the left. (b) Quantification of whole-body luciferase expression at various times after intraperitoneal injection of C32- (hatched) and C32-117-delivered (solid) DNA. Statistically significant differences between C32 and C32-117 at a given time point are indicated. n = 4 for each treatment group. \*P <0.05; \*\*P < 0.01; \*\*\*P < 0.001. Organ distribution of gene expression (B). Quantification of luciferase expression in whole body and individual organs 6 hours after intraperitoneal injection of polymer/DNA complexes in FVB/J mice. Results are expressed as mean transfection levels (± SD) for a buffer control (white), C32-103 (yellow), C32-116 (red), C32-117 (green), C32-122 (blue), C32 (pink), and jet-polyethylenimine (jet-PEI) (black). n ≥ 3 for each treatment group. [Source from Ref. [68]]. **Figure 13.** Intraperitoneal gene delivery in mice (A). (a) Whole-body optical images of luciferase expression in FVB/J mice 6 hours after intraperitoneal injection of polymer/DNA complexes. Images show the highest expression obtained for each polymer. The control mouse was injected with 120 μl of 50 mM NaAc buffer, pH 5.2. Pseudocolor images representing emitted bioluminescence are superimposed over grayscale images. Relative light units (RLUs)/pixel are indicated in the color scale bar on the left. (b) Quantification of whole-body luciferase expression at various times af‐ ter intraperitoneal injection of C32- (hatched) and C32-117-delivered (solid) DNA. Statistically significant differences between C32 and C32-117 at a given time point are indicated. n = 4 for each treatment group. \*P <0.05; \*\*P < 0.01; \*\*\*P < 0.001. Organ distribution of gene expression (B). Quantification of luciferase expression in whole body and in‐ dividual organs 6 hours after intraperitoneal injection of polymer/DNA complexes in FVB/J mice. Results are expressed as mean transfection levels (± SD) for a buffer control (white), C32-103 (yellow), C32-116 (red), C32-117 (green), C32-122 (blue), C32 (pink), and jet-polyethylenimine (jet-PEI) (black). n ≥ 3 for each treatment group. [Source from Ref. [68]].

High transfection levels were observed after intraperitoneal injection of polymer/DNA complexes. End-modified polymers resulted in whole-body reporter protein expression more than an order-of magnitude higher than that for jet-PEI. They also outperformed the best poly(β-amino ester) synthesized to date, C32, with overall expression levels 4- to 12-fold higher. They found that sustained expression past 1 week both with modified C32 and with C32, but modified C32 was expressed at significantly higher levels, reflecting its enhanced delivery capabilities. This effect was most evident between the C32-116 and C32-117 polymers, where the latter displayed 5- to 65-fold higher delivery to the bladder, spleen, liver, and kidney. The only difference between these two

One of the most non-invasive approaches to drug/gene delivery is via inhalation. Gene therapy to the lung can potentially be exploited for the treatment of both genetic and acquired diseases. However, any therapeutic approach for the respiratory tract must take into account the heterogeneity of the cellular targets in the lung: epithelial cell, alveolar cells, vascular cells, serous cells in the submucosal glands and a number of other cell types [69]. Our group developed spermine-based biocompatible poly(β-amino ester) as an aerosol delivery gene carrier [34]. As shown in Fig. 14 (A-A), GFP signal was dominant in the lungs with GPT–SPE/GFP complexes-exposed group compared to the control and naked GFP-exposed groups. No necrosis, degeneration, metaplasia, anaplasia in pneumocytes, atelectasis, or emphysema were detected [Fig. 14 (A-B)]. These results indicate that GPT–SPE functions safely and efficiently in aerosol delivery system. Significant anticancer effects of GPT–SPE/Akt1 shRNA complexes in the lungs through aerosol inhalation were observed in lung tumor bearing K-*ras*LA1 mice [Fig. 14 (BA-E)] without toxicity [Table 2-1]. These result indicating

diaminopropane end-capping reagents is the ethyl versus dimethyl branching.

lung cancer gene therapy.

High transfection levels were observed after intraperitoneal injection of polymer/DNA complexes. End-modified polymers resulted in whole-body reporter protein expression more than an order-of magnitude higher than that for jet-PEI. They also outperformed the best poly(β-amino ester)s synthesized to date, C32, with overall expression levels 4- to 12-fold higher. They found that sustained expression past 1 week both with modified C32 and with C32, but modified C32 was expressed at significantly higher levels, reflecting its enhanced delivery capabilities. This effect was most evident between the C32-116 and C32-117 polymers, where the latter displayed 5- to 65-fold higher delivery to the bladder, spleen, liver, and kidney. The only difference between these two diaminopropane end-capping reagents is the ethyl versus dimethyl branching.

shRNA complexes in the lungs through aerosol inhalation were observed in lung tumor bearing K-*ras*LA1 mice [Fig. 14 (B)] without toxicity [Table 2]. These result indicating that poly(β-amino ester)s (GPT-SPE) could be a safe and efficient gene carrier in aerosol-adminis‐

Poly(amino ester)s-Based Polymeric Gene Carriers in Cancer Gene Therapy

http://dx.doi.org/10.5772/54740

389

CBC, complete blood count; WBC, white blood cell; RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit; MCV, mean cell volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; CHCM, mean cell hemoglobin concentration; RDW, red cell distribution width; PLT, platelets; MPV, mean platelet volume; PDW, platelet distribution width; PCT, plateletcrit; MPC, mean platelet component; MPM, mean platelet mass; Large Pit, large platelets.

**Table 2.** Toxicological analysis. Blood samples were collected for routine examination and to assess the potential

Targeting confers another important criterion in gene delivery. To increase specificity and safety of gene therapy further, the expression of the therapeutic gene needs to be tightly controlled within the target tissue. Targeted gene expression has been analyzed using tissuespecific promoters (breast-, prostate-, and melanoma-specific promoters) and disease-specific promoters (carcinoembryonic antigen, HER-2/neu, Myc-Max response elements, DF3/MUC). Alternatively, expression could be regulated externally with the use of radiation-induced promoters or tetracycline-responsive elements [70]. Recently, Arote et al. coupled folic acid moiety to the poly(amino ester)s backbone using PEG (MW: 5000 Da) as a linker for targeting of folate receptor, a tumor associated glycosylphosphatidylinositol anchored protein [71]. As shown in Fig. 15, folate-PEG-poly(amino ester)s (FP-PAEs) showed marked anti-tumor activity against folate receptor-positive human KB tumors in nude mice with no evidence of toxicity

tered lung cancer gene therapy.

toxicity of GTP–SPE. [Source from Ref. [34]].

**2.5. Targeting considerations**

that poly(β-amino ester) (GPT-SPE) could be a safe and efficient gene carrier in aerosol-administered

Fig. 14. (A) In vivo analysis after aerosol administration to lungs. Two days after exposure, mice

were sacrificed and lungs were collected for the detection of GFP signal and Hematoxylin & Eosin staining. (A) Transfection efficiency study: GFP expression analysis (magnification: 200×). (B) Lung histopathology study: Hematoxylin & Eosin staining (magnification: 200×, scale bar represents 50 μm). (B) Therapeutic efficiency of GPT–SPE as aerosol gene delivery carrier in lung tumor bearing K-*ras*LA1 mice: aerosol delivery of GPT–SPE/Akt1 shRNA significantly inhibited lung tumor numbers: (A) Lungs showing numerous visible lesions (red circle represents tumor tissues). (B) Total tumor numbers (n = 4, \*p < 0.05, \*\*p < 0.01). (C) Tumor size over 1 mm tumor numbers (n = 4, \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001). Aerosol delivery of GPT–SPE/Akt1 shRNA significantly suppressed lung tumor progression through the Akt signaling pathway. (D) Histopathological characteristics. Red circle indicates the incidence in the lungs (magnification: 200×, scale bar represents 50 μm). (E) Western blot analysis of Akt1 protein expression in the lungs and bands-of-interest were further analyzed by densitometer (n = 4, \*\*p < 0.01, \*\*\*p < 0.001). **Figure 14.** A) In vivo analysis after aerosol administration to lungs. Two days after exposure, mice were sacrificed and lungs were collected for the detection of GFP signal and Hematoxylin & Eosin staining. (A) Transfection efficiency study: GFP expression analysis (magnification: 200×). B) Lung histopathology study: Hematoxylin & Eosin staining (magnification: 200×, scale bar represents 50 μm). (B) Therapeutic efficiency of GPT–SPE as aerosol gene delivery carri‐ er in lung tumor bearing K-*ras*LA1 mice: aerosol delivery of GPT–SPE/Akt1 shRNA significantly inhibited lung tumor numbers: (A) Lungs showing numerous visible lesions (red circle represents tumor tissues). (B) Total tumor numbers (n = 4, \*p < 0.05, \*\*p < 0.01). (C) Tumor size over 1 mm tumor numbers (n = 4, \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001). Aero‐ sol delivery of GPT–SPE/Akt1 shRNA significantly suppressed lung tumor progression through the Akt signaling path‐ way. (D) Histopathological characteristics. Red circle indicates the incidence in the lungs (magnification: 200×, scale bar represents 50 μm). (E) Western blot analysis of Akt1 protein expression in the lungs and bands-of-interest were further analyzed by densitometer (n = 4, \*\*p < 0.01, \*\*\*p < 0.001).

Table 2-1. Toxicological analysis. Blood samples were collected for routine examination and to

assess the potential toxicity of GTP–SPE. [Source from Ref. [34]]. CBC, complete blood count; WBC, white blood cell; RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit; MCV, mean cell volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; CHCM, mean cell hemoglobin concentration; RDW, red cell One of the most non-invasive approaches to drug/gene delivery is via inhalation. Gene ther‐ apy to the lung can potentially be exploited for the treatment of both genetic and acquired diseases. However, any therapeutic approach for the respiratory tract must take into account the heterogeneity of the cellular targets in the lung: epithelial cells, alveolar cells, vascular cells, serous cells in the sub-mucosal glands and a number of other cell types [69]. Our group developed spermine-based biocompatible poly(β-amino ester)s as an aerosol delivery gene carrier [34]. As shown in Fig. 14 (A-A), GFP signal was dominant in the lungs with GPT–SPE/GFP complexes-exposed group compared to the control and naked GFP-exposed groups. No necrosis, degeneration, metaplasia, anaplasia in pneumocytes, atelectasis, or em‐ physema were detected [Fig. 14 (A-B)]. These results indicate that GPT–SPE functions safely and efficiently in aerosol delivery system. Significant anticancer effects of GPT–SPE/Akt1

distribution width; PLT, platelets; MPV, mean platelet volume; PDW, platelet distribution width;

shRNA complexes in the lungs through aerosol inhalation were observed in lung tumor bearing K-*ras*LA1 mice [Fig. 14 (B)] without toxicity [Table 2]. These result indicating that poly(β-amino ester)s (GPT-SPE) could be a safe and efficient gene carrier in aerosol-adminis‐ tered lung cancer gene therapy.


CBC, complete blood count; WBC, white blood cell; RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit; MCV, mean cell volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; CHCM, mean cell hemoglobin concentration; RDW, red cell distribution width; PLT, platelets; MPV, mean platelet volume; PDW, platelet distribution width; PCT, plateletcrit; MPC, mean platelet component; MPM, mean platelet mass; Large Pit, large platelets.

**Table 2.** Toxicological analysis. Blood samples were collected for routine examination and to assess the potential toxicity of GTP–SPE. [Source from Ref. [34]].

#### **2.5. Targeting considerations**

High transfection levels were observed after intraperitoneal injection of polymer/DNA complexes. End-modified polymers resulted in whole-body reporter protein expression more than an order-of magnitude higher than that for jet-PEI. They also outperformed the best poly(β-amino ester)s synthesized to date, C32, with overall expression levels 4- to 12-fold higher. They found that sustained expression past 1 week both with modified C32 and with C32, but modified C32 was expressed at significantly higher levels, reflecting its enhanced delivery capabilities. This effect was most evident between the C32-116 and C32-117 polymers, where the latter displayed 5- to 65-fold higher delivery to the bladder, spleen, liver, and kidney. The only difference between these two diaminopropane end-capping reagents is the ethyl

that poly(β-amino ester) (GPT-SPE) could be a safe and efficient gene carrier in aerosol-administered

Fig. 14. (A) In vivo analysis after aerosol administration to lungs. Two days after exposure, mice were sacrificed and lungs were collected for the detection of GFP signal and Hematoxylin & Eosin staining. (A) Transfection efficiency study: GFP expression analysis (magnification: 200×). (B) Lung histopathology study: Hematoxylin & Eosin staining (magnification: 200×, scale bar represents 50 μm). (B) Therapeutic efficiency of GPT–SPE as aerosol gene delivery carrier in lung tumor bearing K-*ras*LA1 mice: aerosol delivery of GPT–SPE/Akt1 shRNA significantly inhibited lung tumor numbers: (A) Lungs showing numerous visible lesions (red circle represents tumor tissues). (B) Total tumor numbers (n = 4, \*p < 0.05, \*\*p < 0.01). (C) Tumor size over 1 mm tumor numbers (n = 4, \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001). Aerosol delivery of GPT–SPE/Akt1 shRNA significantly suppressed lung tumor progression through the Akt signaling pathway. (D) Histopathological characteristics. Red circle indicates the incidence in the lungs (magnification: 200×, scale bar represents 50 μm). (E) Western blot analysis of Akt1 protein expression in the lungs and bands-of-interest were further

**Figure 14.** A) In vivo analysis after aerosol administration to lungs. Two days after exposure, mice were sacrificed and lungs were collected for the detection of GFP signal and Hematoxylin & Eosin staining. (A) Transfection efficiency study: GFP expression analysis (magnification: 200×). B) Lung histopathology study: Hematoxylin & Eosin staining (magnification: 200×, scale bar represents 50 μm). (B) Therapeutic efficiency of GPT–SPE as aerosol gene delivery carri‐ er in lung tumor bearing K-*ras*LA1 mice: aerosol delivery of GPT–SPE/Akt1 shRNA significantly inhibited lung tumor numbers: (A) Lungs showing numerous visible lesions (red circle represents tumor tissues). (B) Total tumor numbers (n = 4, \*p < 0.05, \*\*p < 0.01). (C) Tumor size over 1 mm tumor numbers (n = 4, \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001). Aero‐ sol delivery of GPT–SPE/Akt1 shRNA significantly suppressed lung tumor progression through the Akt signaling path‐ way. (D) Histopathological characteristics. Red circle indicates the incidence in the lungs (magnification: 200×, scale bar represents 50 μm). (E) Western blot analysis of Akt1 protein expression in the lungs and bands-of-interest were

Table 2-1. Toxicological analysis. Blood samples were collected for routine examination and to

One of the most non-invasive approaches to drug/gene delivery is via inhalation. Gene ther‐ apy to the lung can potentially be exploited for the treatment of both genetic and acquired diseases. However, any therapeutic approach for the respiratory tract must take into account the heterogeneity of the cellular targets in the lung: epithelial cells, alveolar cells, vascular cells, serous cells in the sub-mucosal glands and a number of other cell types [69]. Our group developed spermine-based biocompatible poly(β-amino ester)s as an aerosol delivery gene carrier [34]. As shown in Fig. 14 (A-A), GFP signal was dominant in the lungs with GPT–SPE/GFP complexes-exposed group compared to the control and naked GFP-exposed groups. No necrosis, degeneration, metaplasia, anaplasia in pneumocytes, atelectasis, or em‐ physema were detected [Fig. 14 (A-B)]. These results indicate that GPT–SPE functions safely and efficiently in aerosol delivery system. Significant anticancer effects of GPT–SPE/Akt1

CBC, complete blood count; WBC, white blood cell; RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit; MCV, mean cell volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; CHCM, mean cell hemoglobin concentration; RDW, red cell distribution width; PLT, platelets; MPV, mean platelet volume; PDW, platelet distribution width;

analyzed by densitometer (n = 4, \*\*p < 0.01, \*\*\*p < 0.001).

further analyzed by densitometer (n = 4, \*\*p < 0.01, \*\*\*p < 0.001).

assess the potential toxicity of GTP–SPE. [Source from Ref. [34]].

(A) (B)

versus dimethyl branching.

388 Novel Gene Therapy Approaches

lung cancer gene therapy.

Targeting confers another important criterion in gene delivery. To increase specificity and safety of gene therapy further, the expression of the therapeutic gene needs to be tightly controlled within the target tissue. Targeted gene expression has been analyzed using tissuespecific promoters (breast-, prostate-, and melanoma-specific promoters) and disease-specific promoters (carcinoembryonic antigen, HER-2/neu, Myc-Max response elements, DF3/MUC). Alternatively, expression could be regulated externally with the use of radiation-induced promoters or tetracycline-responsive elements [70]. Recently, Arote et al. coupled folic acid moiety to the poly(amino ester)s backbone using PEG (MW: 5000 Da) as a linker for targeting of folate receptor, a tumor associated glycosylphosphatidylinositol anchored protein [71]. As shown in Fig. 15, folate-PEG-poly(amino ester)s (FP-PAEs) showed marked anti-tumor activity against folate receptor-positive human KB tumors in nude mice with no evidence of toxicity platelets.

**2.5 Targeting considerations** 

during and after therapy using the TAM67 gene. Anti-tumor activity with PAEs without folic acid moiety (PEGylated-PAEs, P-PAEs) proved ineffective against a xenograft mice model than that with FP-PAEs at the same dose, suggesting that FP-PAEs is a highly effective gene carrier capable of producing a therapeutic benefit in a xenograft mice model without any signs of toxicity. antigen, HER-2/neu, Myc-Max response elements, DF3/MUC). Alternatively, expression could be regulated externally with the use of radiation-induced promoters or tetracycline-responsive elements [70]. Recently, Arote et al. coupled folic acid moiety to the poly(amino ester) backbone using PEG (MW: 5000 Da) as a linker for folate receptor, a tumor associated glycosylphosphatidylinositol anchored protein, targeting [71]. As shown in Fig. 15, folate-PEG-poly(amino ester) (FP-PAE) showed marked anti-tumor activity against folate receptor-positive human KB tumors in nude mice with no evidence of toxicity during and after therapy using the TAM67 gene. Anti-tumor activity with PAE without folic acid moiety (PEGylated-PAE, P-PAE) proved ineffective against a xenograft mice model when administered at the same dose as that of FP-PAE, suggesting that FP-PAE is a highly

**Author details**

You-Kyoung Kim1

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Poly(amino ester)s-Based Polymeric Gene Carriers in Cancer Gene Therapy

and Hu-Lin Jiang1

http://dx.doi.org/10.5772/54740

391

effective gene carrier capable of producing a therapeutic benefit in a xenograft mice model without

PCT, plateletcrit; MPC, mean platelet component; MPM, mean platelet mass; Large Pit, large

target tissue. Targeted gene expression has been analyzed using tissue-specific promoters (breast-, prostate-, and melanoma-specific promoters) and disease-specific promoters (carcinoembryonic

Fig. 17. Effect of FP-PEA/TAM67 complexes on tumor growth. The tumor volume in BALB/c mice

bearing KB cells was recorded every 3 d. Tumor tissue homogenates were subjected to western blot analysis. Blots were probed with antibodies as indicated. (A) Expression level of phospho-c-Jun. (B) The bands-of-interest were further analyzed by densitometer. (C) Immunohistochemical analysis of phospho-c-Jun in the tumors. Dark brown color indicates the phospho-c-Jun expression (magnification, X 400; bar = 20 μm). (D) Comparison of phospho-c-Jun labeling index in tumors. phospho-c-Jun positive staining was determined by counting 10 randomly chosen fields per section, determining the percentage of DAB positive cell per 100 cells at X 400 magnification. (E) Suppression of tumor growth by FP-PEA/TAM67 complexes (F) Expression level of PCNA. (G) The bands-of-interest were further analyzed by densitometer. (H) Immunohistochemical analysis of PCNA in the tumors. Dark brown color indicates the PCNA expression (magnification, X 400; bar = 20 μm). (I) Comparison of PCNA labeling index in tumors. PCNA positive staining was determined by counting 10 randomly chosen fields per section, determining the percentage of DAB positive cell per 100 cells at X 400 magnification. (\*, P < 0.05; \*\*, P < 0.01 compared with control; #, P < 0.05; ##, p < 0.01 compared with vector control; n = 4). [Source from Ref. [71]].  **Figure 15.** Effect of FP-PEAs/TAM67 complexes on tumor growth. The tumor volume in BALB/c mice bearing KB cells was recorded every 3 d. Tumor tissue homogenates were subjected to western blot analysis. Blots were probed with antibodies as indicated. (A) Expression level of phospho-c-Jun. (B) The bands-of-interest were further analyzed by den‐ sitometer. (C) Immunohistochemical analysis of phospho-c-Jun in the tumors. Dark brown color indicates the phos‐ pho-c-Jun expression (magnification, X 400; bar = 20 μm). (D) Comparison of phospho-c-Jun labeling index in tumors. phospho-c-Jun positive staining was determined by counting 10 randomly chosen fields per section, determining the percentage of DAB positive cell per 100 cells at X 400 magnification. (E) Suppression of tumor growth by FP-PEAs/ TAM67 complexes (F) Expression level of PCNA. (G) The bands-of-interest were further analyzed by densitometer. (H) Immunohistochemical analysis of PCNA in the tumors. Dark brown color indicates the PCNA expression (magnifica‐ tion, X 400; bar = 20 μm). (I) Comparison of PCNA labeling index in tumors. PCNA positive staining was determined by counting 10 randomly chosen fields per section, determining the percentage of DAB positive cell per 100 cells at X 400 magnification. (\*, P < 0.05; \*\*, P < 0.01 compared with control; #, P < 0.05; ##, p < 0.01 compared with vector control; n = 4). [Source from Ref. [71]].

## **3. Conclusion**

Gene therapy shows tremendous promise for a broad spectrum of clinical applications. Development of a safe and efficient gene delivery system is one of the main challenges to be solved before this strategy can be adopted for routine use in clinical trails. As a degradable cationic polymeric gene carrier, poly(amino ester)s comprise many desirable properties in the context of gene delivery, including condensation of DNA into nanoscale-size particles and protects DNA from endogenous nucleases and efficiently deliver DNA with low toxicity. The need for clinical application of poly(amino ester)s, more comprehensive preclinical investiga‐ tions such as exact quality control (QC) of polymer, pharmacokinetics and toxicological studies should be performed.

## **Author details**

during and after therapy using the TAM67 gene. Anti-tumor activity with PAEs without folic acid moiety (PEGylated-PAEs, P-PAEs) proved ineffective against a xenograft mice model than that with FP-PAEs at the same dose, suggesting that FP-PAEs is a highly effective gene carrier capable of producing a therapeutic benefit in a xenograft mice model without any signs of

Fig. 17. Effect of FP-PEA/TAM67 complexes on tumor growth. The tumor volume in BALB/c mice bearing KB cells was recorded every 3 d. Tumor tissue homogenates were subjected to western blot analysis. Blots were probed with antibodies as indicated. (A) Expression level of phospho-c-Jun. (B) The bands-of-interest were further analyzed by densitometer. (C) Immunohistochemical analysis of phospho-c-Jun in the tumors. Dark brown color indicates the phospho-c-Jun expression (magnification, X 400; bar = 20 μm). (D) Comparison of phospho-c-Jun labeling index in tumors. phospho-c-Jun positive staining was determined by counting 10 randomly chosen fields per section, determining the percentage of DAB positive cell per 100 cells at X 400 magnification. (E) Suppression of tumor growth by FP-PEA/TAM67 complexes (F) Expression level of PCNA. (G) The bands-of-interest were further analyzed by densitometer. (H) Immunohistochemical analysis of PCNA in the tumors. Dark brown color indicates the PCNA expression (magnification, X 400; bar = 20 μm). (I) Comparison of PCNA labeling index in tumors. PCNA positive staining was determined by counting 10 randomly chosen fields per section, determining the percentage of DAB positive cell per 100 cells at X 400 magnification. (\*, P < 0.05; \*\*, P < 0.01 compared with control; #, P < 0.05; ##,

**Figure 15.** Effect of FP-PEAs/TAM67 complexes on tumor growth. The tumor volume in BALB/c mice bearing KB cells was recorded every 3 d. Tumor tissue homogenates were subjected to western blot analysis. Blots were probed with antibodies as indicated. (A) Expression level of phospho-c-Jun. (B) The bands-of-interest were further analyzed by den‐ sitometer. (C) Immunohistochemical analysis of phospho-c-Jun in the tumors. Dark brown color indicates the phos‐ pho-c-Jun expression (magnification, X 400; bar = 20 μm). (D) Comparison of phospho-c-Jun labeling index in tumors. phospho-c-Jun positive staining was determined by counting 10 randomly chosen fields per section, determining the percentage of DAB positive cell per 100 cells at X 400 magnification. (E) Suppression of tumor growth by FP-PEAs/ TAM67 complexes (F) Expression level of PCNA. (G) The bands-of-interest were further analyzed by densitometer. (H) Immunohistochemical analysis of PCNA in the tumors. Dark brown color indicates the PCNA expression (magnifica‐ tion, X 400; bar = 20 μm). (I) Comparison of PCNA labeling index in tumors. PCNA positive staining was determined by counting 10 randomly chosen fields per section, determining the percentage of DAB positive cell per 100 cells at X 400 magnification. (\*, P < 0.05; \*\*, P < 0.01 compared with control; #, P < 0.05; ##, p < 0.01 compared with vector

Gene therapy shows tremendous promise for a broad spectrum of clinical applications. Development of a safe and efficient gene delivery system is one of the main challenges to be solved before this strategy can be adopted for routine use in clinical trails. As a degradable cationic polymeric gene carrier, poly(amino ester)s comprise many desirable properties in the context of gene delivery, including condensation of DNA into nanoscale-size particles and protects DNA from endogenous nucleases and efficiently deliver DNA with low toxicity. The need for clinical application of poly(amino ester)s, more comprehensive preclinical investiga‐ tions such as exact quality control (QC) of polymer, pharmacokinetics and toxicological studies

p < 0.01 compared with vector control; n = 4). [Source from Ref. [71]].

PCT, plateletcrit; MPC, mean platelet component; MPM, mean platelet mass; Large Pit, large

Targeting confers another important criterion in gene delivery. To increase specificity and safety of gene therapy further, the expression of the therapeutic gene needs to be tightly controlled within the target tissue. Targeted gene expression has been analyzed using tissue-specific promoters (breast-, prostate-, and melanoma-specific promoters) and disease-specific promoters (carcinoembryonic antigen, HER-2/neu, Myc-Max response elements, DF3/MUC). Alternatively, expression could be regulated externally with the use of radiation-induced promoters or tetracycline-responsive elements [70]. Recently, Arote et al. coupled folic acid moiety to the poly(amino ester) backbone using PEG (MW: 5000 Da) as a linker for folate receptor, a tumor associated glycosylphosphatidylinositol anchored protein, targeting [71]. As shown in Fig. 15, folate-PEG-poly(amino ester) (FP-PAE) showed marked anti-tumor activity against folate receptor-positive human KB tumors in nude mice with no evidence of toxicity during and after therapy using the TAM67 gene. Anti-tumor activity with PAE without folic acid moiety (PEGylated-PAE, P-PAE) proved ineffective against a xenograft mice model when administered at the same dose as that of FP-PAE, suggesting that FP-PAE is a highly effective gene carrier capable of producing a therapeutic benefit in a xenograft mice model without

toxicity.

**3. Conclusion**

should be performed.

control; n = 4). [Source from Ref. [71]].

platelets.

390 Novel Gene Therapy Approaches

**2.5 Targeting considerations** 

any signs of toxicity.

You-Kyoung Kim1 , Can Zhang1 , Chong-Su Cho2 , Myung-Haing Cho3 and Hu-Lin Jiang1

1 School of Pharmacy, China Pharmaceutical University, Nanjing,, P. R. China

2 Department of Agricultural Biotechnology, Seoul National University, Seoul,, Korea

3 College of Veterinary Medicine, Seoul National University, Seoul,, Korea

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## *Edited by Ming Wei and David Good*

Gene therapy has the potential to be a tailor-made therapeutic with increased specificity and decreased side effects that can offer a "cure" for many disorders. The aim of this book is to provide up-to-date reviews of the rapidly growing field of gene therapy. Chapters cover a large range of topics including methods of gene delivery, and identification of targets with several papers on cancer gene therapy. If more people become aware of the true nature and potential of gene therapy, perhaps we can achieve the full benefit of such an innovative approach for the treatment of a range of diseases, including cancer.

Novel Gene Therapy Approaches

Novel Gene Therapy

Approaches

*Edited by Ming Wei and David Good*

Photo by ktsimage / iStock