**1. Introduction**

26 Non-Viral Gene Therapy

Xing, L., Salas, M., Lin, C.-S., Zigman, W., Silverman, W., Subramaniyam, S., et al. (2007).

Yamashita, Y.-ichi, Shimada, M., Tachibana, K., Harimoto, N., Tsujita, E., Shirabe, K., et al.

Yang, N. S., Burkholder, J., Roberts, B., Martinell, B., & McCabe, D. (1990). In vivo and in

Zeira, E., Manevitch, A., Khatchatouriants, A., Pappo, O., Hyam, E., Darash-Yahana, M., et

Zhang, XM., & Huang, JD. (2003). Combination of overlapping bacterial artificial

system for prolonged expression. *Molecular therapy*, 8(2), pp. 342-50. Zhang, G., Budker, V., & Wolff, JA. (1999). High levels of foreign gene expression in

*Proceedings of the National Academy of Sciences USA*, 87(24), pp. 9568-72. Yates, J. L., Warren, N., & Sugden, B. (1985.). Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. *Nature*, 313(6005), pp. 812-5. Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J. L., Tian, S., et al.

gene in BAC-transgenic mice. *Mammalian genome*, 18(2), pp. 113-22.

*therapy,* 13(17), pp. 2079-84.

*Science*, 318(5858), pp. 1917-20.

10(10), pp. 1735-7.

*research*, 31(15), e81.

Faithful tissue-specific expression of the human chromosome 21-linked COL6A1

(2002). In vivo gene transfer into muscle via electro-sonoporation. *Human gene* 

vitro gene transfer to mammalian somatic cells by particle bombardment.

(2007). Induced pluripotent stem cell lines derived from human somatic cells.

al. (2003). Femtosecond infrared laser-an efficient and safe in vivo gene delivery

hepatocytes after tail vein injections of naked plasmid DNA. *Human gene therapy*,

chromosomes by a two-step recombinogenic engineering method. *Nucleic acids* 

Gene therapy is the process of treating a particular disease through the introduction of genetic material in order to elicit a therapeutic benefit [Stone, 2010]. The defective gene of a diagnosed patient can be corrected by a number of different strategies such as "gene replacement", "gene correction", and "gene augmentation" [Katare and Aeri, 2010]. In replacement therapy, a normal gene is inserted somewhere in the genome so that its product could replace that of a defective gene. This approach may be suitable for recessive disorders, which are marked by deficiency of an enzyme or other proteins. Although, the gene functions in the genome providing an appropriate regulatory sequence, the approach may not be successful in treating dominant disorders associated with the production of an abnormal gene product, which interferes with the product of normal gene [Katare and Aeri, 2010]. Corrective gene therapy requires replacement of a mutant gene or a part of it with a normal sequence. This can be achieved by using recombinant technology. Another form of corrective therapy involves the suppression of a particular mutation by a transfer RNA that is introduced into a cell [Katare and Aeri, 2010]. In gene augmentation, introducing a normal genetic sequence into a host genome modifies the expression of mutant gene in defective cell and the defective host gene remains unaltered. In general, the gene therapy recipient cells may be germline cells or somatic cells. Germline cells therapy involves modifying the genes in germ cells which will pass these genetic changes to the future generations. Somatic cells therapy involves the insertion of genes into specific somatic cells like the bone marrow stem cells, fibroblasts, hepatocytes or mycocytes [Katare and Aeri, 2010]. This form of gene therapy is being used at most genetic engineering laboratories throughout the world.

Clearly, gene therapy provides great opportunities for treating diseases from genetic disorders, infections and cancer [Park et al., 2006]. While the genetic mutations underlying various diseases are well understood, delivering a corrective gene to the unhealthy organs/tissues remains a remarkable challenge [Stone, 2010]. To achieve successful gene therapy, development of proper gene delivery systems could be one of the most important factors. Gene delivery systems should be designed to protect the genetic materials from premature degradation in systemic blood stream and to efficiently transfer the therapeutic genes to target cells. Intracellular delivery systems will be required for all molecules that have intracellular function. For example, nucleic acid molecules including encoding genes, oligonucleotides and RNA molecules must enter cells and target the nucleus when transcription is the target. Regardless of the molecules for delivery, a common requirement

Non-Viral Delivery Systems in Gene Therapy and Vaccine Development 29

produced in large amounts of virus particles. For this purpose, there are specialized cell lines called "packaging cell lines" (PCLs) engineered to replace a function of a deleted viral gene and for the production of recombinant viruses [Gardlik et al., 2005]. However, the interaction between a vector and a host-cell genome cannot be completely eliminated. Some

<sup>1</sup>Generation of an immune response to expressed viral proteins that subsequently

<sup>4</sup>Difficulties in engineering viral envelopes or capsids to achieve specific delivery to

7 High costs in producing large amounts of high-titer viral stocks for use in the clinic

Table 1. Viral delivery system disadvantages [Templeton and Lasic, 1999; Gupta et al., 2004] Currently, developed Viruses as transfer vectors are divided into two classes, following their different strategies for replication and survival: a) Non-lytic viruses, including *retroviruses* and *lentiviruses*, produce virions from the cellular membrane of an infected cell, leaving the host cell relatively intact; b) Lytic viruses, including human *adenovirus* and *herpes simplex virus* families, destroy the infected cell after replication and virion production. This native nature of the original viruses determines the use of each recombinant replicationdefective viral vector in clinical applications [Table 2]. Despite some limitations on the use of viral vectors regarding safety and reproducibility, they are still the most used gene transfer

<sup>5</sup>Possible recombination of the viral vector with DNA sequences in the host chromosome that generates a replication-competent, infectious virus

<sup>8</sup>Limited size of the nucleic acid that can be packaged and used for viral gene

disadvantages of viral delivery are addressed in Table 1.

**No. Disadvantage** 

3 Clearance of viruses delivered systemically

therapy

*Adeno-associated* 

*viruses (AAV)* ssDNA Capsid

kill the target cells producing a therapeutic gene product 2 Random integration of some viral vectors into the host chromosome

cells other than those with natural tropism for the virus

6 Inability to administer certain viral vectors more than once

vehicles [Gardlik et al., 2005; Katare, 2010; Gupta et al., 2004].

**Vector Genome Structure Properties** 

Table 2. Utilization of viral vectors for gene delivery [Katare and Aeri, 2010]

*Adenoviruses* dsDNA Capsid Transient expression, strong immunogenicity *Alphaviruses* RNA Envelope Transient, but extreme, expression levels; low

*HSV* dsDNA Envelope Latent infection, long-term expression, low

*Lentiviruses* RNA Envelope Genome integration, long term expression,

*Retroviruses* RNA Envelope Genome integration, long-term expression

immunogenicity

toxicity (mutant)

safety concerns low titers, inefficient production

Slow expression onset, genome integration, long term expression, inefficient large-scale virus production

is the avoidance of endosomal uptake that may cause degradation and denaturation [Gould and Chernajovsky, 2007]. Several approaches are being developed that can be applied to the delivery of all these types of molecules at disease sites. For the goal to be fully achieved, celltargeting strategies require still further development.

Currently, a number of older and more recently discovered techniques have been developed for therapeutic gene transfer. A variety of viral and non-viral possibilities are available for basic and clinical researches [Gardlik et al., 2005]. Among these studies, DNA based vaccines are becoming popular. They stimulate the CD4+ T cells of Th1 subset and thereby mediate cellular immune response, which is effective against pathogens. On the other hand, the recombinant protein vaccines stimulate Th2 subset of T cells thereby eliciting a humoral response. The studies showed that DNA vaccines have been successful in protecting animals against influenza, herpes, rabies, malaria and leishmaniasis [Katare and Aeri, 2010]. However, the potential disadvantages of DNA vaccine have reduced the value of the approach. To optimize antigen delivery efficiency as well as vaccine efficacy, the non-viral vector as vaccine carrier has shown particular benefits to avoid the obstacles that both peptide/protein and gene-based vaccines have encountered [Chen and Huang, 2005]. For example, the success of the liposome-based vaccine has been demonstrated in clinical trials and further human trials are also in progress. This chapter summarizes the non-viral delivery routes and methods for gene transfer used in gene therapy and vaccine development.

### **2. Gene delivery systems**

The simplest way of gene delivery is injecting naked DNA encoding the therapeutic protein, but because of low efficiency, there is a need to use special molecules and methods to improve gene delivery. A vector can be described as a system fulfilling several functions, including (a) enabling delivery of genes into the target cells and their nucleus, (b) providing protection from gene degradation, and (c) ensuring gene transcription in the cell. The ideal DNA vehicle should also be suitable for clinical application. It has to be inexpensive and easy to produce and purify in large amounts [Gardlik et al., 2005]. Two kinds of vectors have been employed as vehicles for gene transfer: 1) Viral vectors for gene transduction (e.g., *retroviral*, *adenoviral*, *adeno-associated viral* and *lentiviral* vectors), and 2) Non-viral vectors for gene transfection based on lipids, water soluble polycations, non-condensing polymers and nano/ micro-particles [Gardlik et al., 2005; Katare, 2010]. However, each vector has its own advantages and disadvantages.

#### **2.1 Viral vectors (biological delivery systems)**

Viral techniques use various classes of viruses as a tool for gene delivery [Gardlik et al., 2005; Stone, 2010]. Viruses introduce their DNA into the cells with high efficiency. Therefore, it is possible to take advantage of this system by introducing a foreign gene into the virus and then using the properties of the virus to deliver this gene with high efficiency into the target cells [Gardlik et al., 2005]. Gene therapy vectors are being developed by genetic modification of *retroviruses*, *adenoviruses*, *poxviruses*, *parvoviruses* (*adeno-associated viruses*), *herpesviruses* etc. [Gardlik et al., 2005; Stone, 2010]. Unlike wild type viruses, these vectors are used to transfer therapeutic genes into target cells and thus are engineered by deleting the essential genes which allow replication, assembling or infection. Replication deficiency ensures the safety of viral vectors, but on the other hand, vectors need to be

is the avoidance of endosomal uptake that may cause degradation and denaturation [Gould and Chernajovsky, 2007]. Several approaches are being developed that can be applied to the delivery of all these types of molecules at disease sites. For the goal to be fully achieved, cell-

Currently, a number of older and more recently discovered techniques have been developed for therapeutic gene transfer. A variety of viral and non-viral possibilities are available for basic and clinical researches [Gardlik et al., 2005]. Among these studies, DNA based vaccines are becoming popular. They stimulate the CD4+ T cells of Th1 subset and thereby mediate cellular immune response, which is effective against pathogens. On the other hand, the recombinant protein vaccines stimulate Th2 subset of T cells thereby eliciting a humoral response. The studies showed that DNA vaccines have been successful in protecting animals against influenza, herpes, rabies, malaria and leishmaniasis [Katare and Aeri, 2010]. However, the potential disadvantages of DNA vaccine have reduced the value of the approach. To optimize antigen delivery efficiency as well as vaccine efficacy, the non-viral vector as vaccine carrier has shown particular benefits to avoid the obstacles that both peptide/protein and gene-based vaccines have encountered [Chen and Huang, 2005]. For example, the success of the liposome-based vaccine has been demonstrated in clinical trials and further human trials are also in progress. This chapter summarizes the non-viral delivery routes and methods for gene transfer used in gene therapy and vaccine

The simplest way of gene delivery is injecting naked DNA encoding the therapeutic protein, but because of low efficiency, there is a need to use special molecules and methods to improve gene delivery. A vector can be described as a system fulfilling several functions, including (a) enabling delivery of genes into the target cells and their nucleus, (b) providing protection from gene degradation, and (c) ensuring gene transcription in the cell. The ideal DNA vehicle should also be suitable for clinical application. It has to be inexpensive and easy to produce and purify in large amounts [Gardlik et al., 2005]. Two kinds of vectors have been employed as vehicles for gene transfer: 1) Viral vectors for gene transduction (e.g., *retroviral*, *adenoviral*, *adeno-associated viral* and *lentiviral* vectors), and 2) Non-viral vectors for gene transfection based on lipids, water soluble polycations, non-condensing polymers and nano/ micro-particles [Gardlik et al., 2005; Katare, 2010]. However, each

Viral techniques use various classes of viruses as a tool for gene delivery [Gardlik et al., 2005; Stone, 2010]. Viruses introduce their DNA into the cells with high efficiency. Therefore, it is possible to take advantage of this system by introducing a foreign gene into the virus and then using the properties of the virus to deliver this gene with high efficiency into the target cells [Gardlik et al., 2005]. Gene therapy vectors are being developed by genetic modification of *retroviruses*, *adenoviruses*, *poxviruses*, *parvoviruses* (*adeno-associated viruses*), *herpesviruses* etc. [Gardlik et al., 2005; Stone, 2010]. Unlike wild type viruses, these vectors are used to transfer therapeutic genes into target cells and thus are engineered by deleting the essential genes which allow replication, assembling or infection. Replication deficiency ensures the safety of viral vectors, but on the other hand, vectors need to be

targeting strategies require still further development.

development.

**2. Gene delivery systems** 

vector has its own advantages and disadvantages.

**2.1 Viral vectors (biological delivery systems)** 

produced in large amounts of virus particles. For this purpose, there are specialized cell lines called "packaging cell lines" (PCLs) engineered to replace a function of a deleted viral gene and for the production of recombinant viruses [Gardlik et al., 2005]. However, the interaction between a vector and a host-cell genome cannot be completely eliminated. Some disadvantages of viral delivery are addressed in Table 1.


Table 1. Viral delivery system disadvantages [Templeton and Lasic, 1999; Gupta et al., 2004]

Currently, developed Viruses as transfer vectors are divided into two classes, following their different strategies for replication and survival: a) Non-lytic viruses, including *retroviruses* and *lentiviruses*, produce virions from the cellular membrane of an infected cell, leaving the host cell relatively intact; b) Lytic viruses, including human *adenovirus* and *herpes simplex virus* families, destroy the infected cell after replication and virion production. This native nature of the original viruses determines the use of each recombinant replicationdefective viral vector in clinical applications [Table 2]. Despite some limitations on the use of viral vectors regarding safety and reproducibility, they are still the most used gene transfer vehicles [Gardlik et al., 2005; Katare, 2010; Gupta et al., 2004].


Table 2. Utilization of viral vectors for gene delivery [Katare and Aeri, 2010]

Non-Viral Delivery Systems in Gene Therapy and Vaccine Development 31

One of the methods that improve DNA penetration of the cell is electroporation [Lee et al., 2009; , Harrison et al., 1998; Rossini et al., 2002; Ahmad et al., 2009; Collins et al., 2006; Kang et al., 2011;]. *In vivo* use of electroporation is done by injecting naked DNA followed by electric pulses from electrodes that are located *in situ* in the target tissues. Successful use of electroporation was observed in transfecting muscles, brain, skin, liver, and tumors [Gardlik et al., 2005; Garcia-Frigola et al., 2007; Umeda et al., 2004; Babiuk et al., 2006; Harrison et al., 1998; Kang et al., 2011]. Since every tissue is specific and has its own characteristics, there are no generally accepted optimal conditions of electroporation that are suitable for effective transfection. These are dependent both on the amplitude and duration of the electric pulses and on the amount and concentration of DNA [Gardlik et al., 2005]. The generated pulse may be either a high voltage (1.5 kV) rectangular wave pulse for a short duration or a low

Up to now, several clinical trials have been planned using the electroporation with DNA vaccines for cancer therapy such as: a) Intra-tumoral IL-12 DNA plasmid (pDNA) [ID: NCT00323206, phase I clinical trials in patients with malignant melanoma]; 2) Intratumoral VCL-IM01 (encoding IL-2) [ID: NCT00223899; phase I clinical trials in patients with metastatic melanoma]; 3) Xenogeneic tyrosinase DNA vaccine [ID: NCT00471133, phase I clinical trials in patients with melanoma]; 4) VGX-3100 [ID: NCT00685412, phase I clinical trials for HPV infections], and 5) IM injection prostate-specific membrane antigen (PSMA)/ pDOM fusion gene [ID: UK-112, phase I/II clinical trials for prostate cancer] [Bodles-

Furthermore, Hepatitis C virus DNA vaccine showed acceptable safety when delivered by Inovio Biomedical's electroporation delivery system in phase I/II clinical study at Karolinska University Hospital. ChronVac-C is a therapeutic DNA vaccine being given to

Fig. 1. Summary of the main methods of gene delivery systems

voltage (350 V) pulse for a longer duration [Katare and Aeri, 2010].

Brakhop and Draghia-Akli, 2008; Bodles-Brakhop et al., 2009].

**3.1 Electroporation** 

In clinical studies, a recombinant *vaccinia* virus vector has been developed to express single or multiple T cell co-stimulatory molecules as a vector for local gene therapy in patients with malignant melanoma. This approach generated local and systemic tumor immunity and induced effective clinical responses in patients with metastatic disease [Kim-Schulze and Kaufman, 2009]. Furthermore, PSA-TRICOM vaccine (prostate-specific antigen plus a TRIad of co-stimulatory molecules; PROSTVAC) includes a priming vaccination with recombinant *vaccinia* (rV)-PSA-TRICOM and booster vaccinations with recombinant fowlpox (rF)-PSA-TRICOM. Each vaccine consists of the transgenes for PSA, including an agonist epitope, and three immune co-stimulatory molecules (B7.1, ICAM-1, and LFA3; designated TRICOM) [Kaufman, 2002]. The efficacy of PSA-TRICOM has been evaluated in phase II clinical trials in patients with metastatic hormone-refractory prostate cancer (mHRPC). PANVAC-VF, another poxviral-based vaccine, consists of a priming vaccination with rV encoding CEA (6D), MUC1 (L93), and TRICOM plus booster vaccinations with rF expressing the identical transgenes. CEA (6D) and MUC1 (L93) represent carcinoembryonic antigen and mucin 1 glycoprotein, respectively, with a single amino acid substitution designed to enhance their immunogenicity. This vaccine is currently under evaluation in several different types of CEA or MUC1-expressing carcinomas and in patients with a life expectancy more than three months [Vergati et al., 2010].

#### **2.2 Non-viral vectors (Non-biological gene delivery systems)**

In comparison with virus-derived vectors, non-viral vectors have several advantages, such as the safety of administration without immunogenicity, almost unlimited transgene size and the possibility of repeated administration [Gardlik et al., 2005]. Non-viral gene delivery systems generally consist of three categories: (a) naked DNA delivery, (b) lipid-based and (c) polymer-based delivery [Park et al., 2006]. Therapeutic gene can be introduced into the target cell either as an insert in plasmid with regulation sequences, what enables the regulation control of expression (inducible promoter) or as a PCR product. The simplest way of gene introduction is an injection of naked DNA into target cells. Such a naked plasmid DNA was used in several pre-clinical and clinical trials [Gardlik et al., 2005]. For example, some positive results were gained in cancer therapy by intra-tumoral injection of tumor suppressor genes or cytokines. However, this approach does not have the transfection efficiency of viral vectors. Low transfection efficacy and short-term expression still remain the main disadvantages of naked DNA gene transfer compared with viral vectors. Thus, many techniques have been developed to improve the introduction of therapeutic gene. Physical methods like electroporation and gene gun increase the entry of transgene into target cells. In addition, chemical methods like lipoplexes (DNA-liposomes complexes) and polyplexes (DNA-polymers complexes) improve the stability of DNA and also facilitate the entry into the cell [Gardlik et al., 2005]. Main methods of gene delivering systems are summarized in figure 1.

#### **3. Physical delivery systems**

A number of methods utilizing various physical techniques have been developed to facilitate the transfer of foreign genes into the host cells [Katare and Aeri, 2010]. Among them, electroporation and gene gun are further involved in preclinical and clinical trials.

Fig. 1. Summary of the main methods of gene delivery systems

#### **3.1 Electroporation**

30 Non-Viral Gene Therapy

In clinical studies, a recombinant *vaccinia* virus vector has been developed to express single or multiple T cell co-stimulatory molecules as a vector for local gene therapy in patients with malignant melanoma. This approach generated local and systemic tumor immunity and induced effective clinical responses in patients with metastatic disease [Kim-Schulze and Kaufman, 2009]. Furthermore, PSA-TRICOM vaccine (prostate-specific antigen plus a TRIad of co-stimulatory molecules; PROSTVAC) includes a priming vaccination with recombinant *vaccinia* (rV)-PSA-TRICOM and booster vaccinations with recombinant fowlpox (rF)-PSA-TRICOM. Each vaccine consists of the transgenes for PSA, including an agonist epitope, and three immune co-stimulatory molecules (B7.1, ICAM-1, and LFA3; designated TRICOM) [Kaufman, 2002]. The efficacy of PSA-TRICOM has been evaluated in phase II clinical trials in patients with metastatic hormone-refractory prostate cancer (mHRPC). PANVAC-VF, another poxviral-based vaccine, consists of a priming vaccination with rV encoding CEA (6D), MUC1 (L93), and TRICOM plus booster vaccinations with rF expressing the identical transgenes. CEA (6D) and MUC1 (L93) represent carcinoembryonic antigen and mucin 1 glycoprotein, respectively, with a single amino acid substitution designed to enhance their immunogenicity. This vaccine is currently under evaluation in several different types of CEA or MUC1-expressing carcinomas and in patients with a life expectancy more than three months [Vergati et

In comparison with virus-derived vectors, non-viral vectors have several advantages, such as the safety of administration without immunogenicity, almost unlimited transgene size and the possibility of repeated administration [Gardlik et al., 2005]. Non-viral gene delivery systems generally consist of three categories: (a) naked DNA delivery, (b) lipid-based and (c) polymer-based delivery [Park et al., 2006]. Therapeutic gene can be introduced into the target cell either as an insert in plasmid with regulation sequences, what enables the regulation control of expression (inducible promoter) or as a PCR product. The simplest way of gene introduction is an injection of naked DNA into target cells. Such a naked plasmid DNA was used in several pre-clinical and clinical trials [Gardlik et al., 2005]. For example, some positive results were gained in cancer therapy by intra-tumoral injection of tumor suppressor genes or cytokines. However, this approach does not have the transfection efficiency of viral vectors. Low transfection efficacy and short-term expression still remain the main disadvantages of naked DNA gene transfer compared with viral vectors. Thus, many techniques have been developed to improve the introduction of therapeutic gene. Physical methods like electroporation and gene gun increase the entry of transgene into target cells. In addition, chemical methods like lipoplexes (DNA-liposomes complexes) and polyplexes (DNA-polymers complexes) improve the stability of DNA and also facilitate the entry into the cell [Gardlik et al., 2005]. Main methods of gene delivering

A number of methods utilizing various physical techniques have been developed to facilitate the transfer of foreign genes into the host cells [Katare and Aeri, 2010]. Among them, electroporation and gene gun are further involved in preclinical and clinical

**2.2 Non-viral vectors (Non-biological gene delivery systems)** 

systems are summarized in figure 1.

**3. Physical delivery systems** 

trials.

al., 2010].

One of the methods that improve DNA penetration of the cell is electroporation [Lee et al., 2009; , Harrison et al., 1998; Rossini et al., 2002; Ahmad et al., 2009; Collins et al., 2006; Kang et al., 2011;]. *In vivo* use of electroporation is done by injecting naked DNA followed by electric pulses from electrodes that are located *in situ* in the target tissues. Successful use of electroporation was observed in transfecting muscles, brain, skin, liver, and tumors [Gardlik et al., 2005; Garcia-Frigola et al., 2007; Umeda et al., 2004; Babiuk et al., 2006; Harrison et al., 1998; Kang et al., 2011]. Since every tissue is specific and has its own characteristics, there are no generally accepted optimal conditions of electroporation that are suitable for effective transfection. These are dependent both on the amplitude and duration of the electric pulses and on the amount and concentration of DNA [Gardlik et al., 2005]. The generated pulse may be either a high voltage (1.5 kV) rectangular wave pulse for a short duration or a low voltage (350 V) pulse for a longer duration [Katare and Aeri, 2010].

Up to now, several clinical trials have been planned using the electroporation with DNA vaccines for cancer therapy such as: a) Intra-tumoral IL-12 DNA plasmid (pDNA) [ID: NCT00323206, phase I clinical trials in patients with malignant melanoma]; 2) Intratumoral VCL-IM01 (encoding IL-2) [ID: NCT00223899; phase I clinical trials in patients with metastatic melanoma]; 3) Xenogeneic tyrosinase DNA vaccine [ID: NCT00471133, phase I clinical trials in patients with melanoma]; 4) VGX-3100 [ID: NCT00685412, phase I clinical trials for HPV infections], and 5) IM injection prostate-specific membrane antigen (PSMA)/ pDOM fusion gene [ID: UK-112, phase I/II clinical trials for prostate cancer] [Bodles-Brakhop and Draghia-Akli, 2008; Bodles-Brakhop et al., 2009].

Furthermore, Hepatitis C virus DNA vaccine showed acceptable safety when delivered by Inovio Biomedical's electroporation delivery system in phase I/II clinical study at Karolinska University Hospital. ChronVac-C is a therapeutic DNA vaccine being given to

Non-Viral Delivery Systems in Gene Therapy and Vaccine Development 33

neutral and anionic liposomes suitable for *in vivo* gene therapy are being constructed [Gardlik et al., 2005; Gupta et al., 2004]; 2) Cationic liposomes: These lipids are naturally produced complexes with negatively charged DNA. Moreover, their positive charge allows interactions with the negatively charged cell membrane and thus penetration into the cell is permitted [Gardlik et al., 2005]. Cationic liposomes ensure effective protection against the degradation of the foreign DNA by the cell. The interactions of liposomes with DNA and the subsequent lipoplex formation are dependent on several physical conditions (pH, charge) as well as structural characteristics of the liposomes. The most frequent use of DNA-liposome complexes is in gene transfer into cancer cells, where the applied genes stimulate anti-tumor immune responses or genes decreasing the activity of oncogenes [Gardlik et al., 2005]. Recent studies revealed the ability of lipoplex gene transfer into the epithelial cells of the respiratory tract, which supports their usage in the therapy of respiratory diseases and cystic fibrosis. Their expression in all main organs, mostly in lungs, was observed after intravenous administration of lipoplexes. Targeted transfection can be gained, to some extent, by the addition of tissue-specific target ligand. It is suggested that the transfection is

The advantages of using liposomes for gene therapy are included as: 1) lack of immunogenicity; 2) lack of clearance by complement system using improved formulations; 3) unlimited size of nucleic acids that can be delivered, from single nucleotides up to large mammalian artificial chromosomes containing several thousand kilobases; 4) ability to perform repeated administrations *in vivo* without adverse consequences; 5) low cost and relative ease of generating nucleic acid: liposome complexes that deliver therapeutic gene products in large scale; 6) safety, because plasmids used for non-viral delivery contain noviral sequences, thereby precluding generation of an infectious virus; 7) naked DNA carried by liposome increases its uptake by antigen-presenting cells (APCs); 8) naked DNA carried by liposome enhances both humoral and cellular immunity; 9) naked DNA carried by

For vaccine development, a general overview of different lipid-based particulate delivery systems, their composition, preparation methods, typical size, route of administration and model antigens has been listed by Myschik J. *et al.*, 2009 [Myschik et al., 2009]. Stimuvax (BLP25 liposome vaccine, L-BLP25, Oncothyreon partnered with Merck KGaA) is a cancer vaccine designed to induce an immune response against the extracellular core peptide of MUC1, a type I membrane glycoprotein widely expressed on many tumors (i.e., lung cancer, breast cancer, prostate cancer and colorectal cancer) [Vergati et al., 2010]. Stimuvax consists of MUC1 lipopeptide BLP25 [STAPPAHGVTSAPDTRPAPGSTAPPK (Pal) G], an immunoadjuvant monophosphoryl lipid A, and three lipids (cholesterol, dimyristoyl phosphatidylglycerol, and dipalmitoyl phosphatidylcholine), capable of enhancing the delivery of the vaccine to APCs. A randomized phase II B clinical trial evaluated the effect of Stimuvax on survival and toxicity in 171 patients with stage III B and IV non-small cell lung cancer (NSCLC), after stable disease or response to first-line chemotherapy. Based on these data, Merck is currently conducting three large phase III clinical trials of Stimuvax. This

Furthermore, a cationic lipid DNA complex (CLDC) consisting of DOTIM/cholesterol liposomes and plasmid DNA, containing immunostimulatory CpG and non-CpG motifs has been designed, with potential immunostimulating and anti-neoplastic activities. Upon systemic administration, TLR-directed cationic lipid-DNA complex JVRS-100 enters dendritic cells (DCs) and macrophages; immunostimulatory DNA binds to and activates

based on endocytosis of the host cell [Gardlik et al., 2005].

liposome induces cytotoxic T lymphocyte response [Trimble et al., 2003].

study will involve more than 1300 patients [Vergati et al., 2010].

individuals already infected with hepatitis C virus with the aim to clear the infection by boosting a cell-mediated immune response against the virus. This vaccination was among the first infectious disease DNA vaccine to be delivered in humans using electroporationbased DNA delivery [Bodles-Brakhop et al., 2009].
