**1. Introduction**

168 Non-Viral Gene Therapy

Walkley, S.U. (2009) Pathogenic cascades in lysosomal disease-Why so complex? *J Inherit* 

Wasungu, L., Hoekstra, D. (2006) Cationic lipids, lipoplexes and intracellular delivery of genes.

Williams, D.A. (2008). Sleeping beauty vector system moves toward human trials in the United

Wilson JM. (2009) Lessons learned from the gene therapy trial for ornithine transcarbamylase

Worgall, S., Sondhi, D., Hackett, N.R., Kosofsky, B., Kekatpure, M.V., Neyzi, N., Dyke, J.P.,

Zhang, Y., Wang, Y., Boado, R.J., Pardridge, W.M. (2007) Lysosomal enzyme replacement of

expressing CLN2 cDNA. *Hum Gene Ther*, Vol 19, No 5, pp 463-474.

Ballon, D., Heier, L., Greenwald, B.M., Christos, P., Mazumdar, M., Souweidane, M.M., Kaplitt, M.G., Crystal, R.G. (2008) Treatment of late infantile neuronal ceroid lipofuscinosis by CNS administration of a serotype 2 adeno-associated virus

the brain with intravenous non-viral gene transfer. *Pharm Res,* Vol 25, No 2, pp 400-

*Metab Dis*, Vol 32, No 2, pp 181-189.

406.

*Journal of Controlled Release*, Vol. 116, No 2, pp 255-264.

deficiency. *Mol Genet Metab,*Vol 96, No 4, pp 151-157.

States. *Mol Ther*, Vol 16, No 9, pp 1515-1516.

#### **Vaccination: Traditional and new generation vaccines**

Vaccination is historically one of the most important methods for the prevention of infectious diseases in humans and animals. When Edward Jenner inoculated James Phipps with a bovine poxvirus to induce protection against the closely related human pathogen smallpox virus in 1796 and then, almost a century later, Pasteur developed a live attenuated vaccine against rabies, the basic principles for vaccine development were established (Fraser and Rappuoli 2005). Traditionally, a vaccine is known as a preparation of attenuated or killed microorganisms or of subunit vaccines (purified components of a pathogen including the protein-conjugated capsular polysaccharides, toxoids, cell-free extracts, recombinant proteins and stand-alone capsular polysaccharides) administered for inducing active immunity to a specific disease.

Two types of immunization exist with intrinsic differences between them: prophylactic vaccination initiates a response against an antigen to which the immune response is naïve, leading to a long-term memory cell maintenance and protective efficacy; therapeutic vaccination stimulates the immune system to a chronically displayed antigen, leading to a clearance of an established infection.

Several infectious diseases can be prevented by vaccines produced with conventional approaches. These methods are based on the cultivation in laboratory conditions of the microorganism from which single components are isolated individually by using biochemical, microbiological and serological techniques. Each antigen is produced in pure form either directly from the bacterium or using the DNA recombinant technology, and finally tested for its ability to induce an immune response (Serruto and Rappuoli 2006).

Conventional approaches provided the basis of vaccinology and led to great achievements such as the eradication of smallpox and the virtual disappearance of diseases like diphtheria, tetanus, poliomyelitis, pertussis, measles, mumps, rubella and invasive *Haemophilus influenzae* B, increasing the life quality and expectancy (Andre 2003). Nevertheless, they present major disadvantages such as to be time-consuming and, more important, to be impractical in some circumstances due to the difficulty in cultivating some microorganisms *in vitro* and to the fact that even attenuation may result in detrimental or unwanted immune responses (Purcell et al. 2007). Moreover, in many cases the antigens

DNA Vaccination by Electrogene Transfer 171

T-cell epitopes can overlap substantially within the sequence of an antigen and, in others, they might be present in separate discrete regions of the antigen or present in different antigens from the targeted pathogen. The simplicity of producing clinical grade peptides allows swift changes in the design of peptide vaccines and, therefore, rapid translation of new immunological concepts, which represent a great advantage for the development of vaccines against rapidly changing viruses such as influenza (Brun et al. 2011). Despite the potential advantages of this approach, the development of successful peptide vaccines has been limited mainly by difficulties associated with stability, poor immunogenicity of simple

Antigen Presenting Cells-based vaccines represent another explored field in vaccine research. With this approach, DC are harvested from the patient, pulsed with antigens or transfected with genes encoding these antigens, and readministered to the patient. This vaccine strategy has the potential to augment presentation through the MHC-class I pathway and subsequently drive the expansion of tumour-specific CTLs. In translational studies with melanoma patients, DC vaccines have demonstrated a keen ability to elicit detectable immune responses. However, such responses often fail to elicit substantial clinical responses. As it is often difficult to discern the relative contributions of DCs and effector T cells in these situations, a thorough investigation of the *in vivo* interactions between these immune cell populations may be required before a complete understanding

Recently, new methods of vaccination such as those based on gene transfer have emerged. Genetic vaccination originates from gene therapy. The objective of genetic vaccination is to transfer in the host a gene encoding for the disease target antigen with the aim to induce a specific immune response, whereas the goal of gene therapy is to ensure production of a protein which is lacking or defective in the host. To date, the vast majority of gene therapy clinical trials have addressed cancer (66.5%), cardiovascular diseases (9.1%) and infectious diseases (6.5%). For infectious diseases, a total of 85 gene therapy trials have been carried out, the majority of these trials being performed on human immunodeficiency virus

infection, tetanus, cytomegalovirus and adenovirus infections (Chiarella et al. 2008a).

cellular compartments assuring good and long-lasting expression levels.

Current techniques of gene transfer in mammals include packaging the DNA into carriers for gene delivery. The ideal carriers for gene delivery should be safe and yet ensure that the DNA survives the extra and intracellular environment, efficiently transfer to the appropriate

Presently, viral vectors are more efficient than non-viral systems, achieving high levels of efficiency, estimated around 90%, for both gene delivery and expression. However, immunogenicity, inflammatory reactions, problems associated with scale-up costs and, more important, the risk of integration in the host genome, are limiting their clinical use in preclinical and clinical protocols respect to the past: i.e. during 2000 year, around 75% of clinical protocols involving gene therapy used recombinant virus-based vectors for DNA delivery (Chiarella et al. 2008a). In the last years, lot of clinical trials pointed out that the use of viral vectors as antigen delivery systems has numerous other drawbacks such as toxicity, recombination, precedent host immunity, higher immunogenicity in comparison to the

peptides and by the MHC polymorphism of the host species (Tam 1996).

**1.3 Ex-vivo loaded dendritic cells** 

of DC role (Palucka et al. 2007).

**1.4 Genetic vaccination** 

expressed during infection are not produced in laboratory conditions, as well as the proteins that are most abundant and easily purified are not necessarily protective antigens and, in any case, only few molecules can be isolated and tested simultaneously (Serruto and Rappuoli 2006).

The last decade has witnessed a revolution in the approach to vaccine design and development. These advances include new delivery technologies aimed at improving the safety and immunogenicity of traditional vaccines, new strategies to identify protective antigens, generation of improved adjuvants.

Considering that new diseases are sure to emerge through evolution by mutation and gene exchange, interspecies transfer or human exposure to novel environments, more reliable approaches must be available to promptly respond to those threats (Plotkin 2005). Thanks to sophisticated technologies such as genomics, proteomics, functional genomics and synthetic chemistry, the rational identification of antigens, the synthesis of complex glycans, the generation of engineered carrier proteins are possible. This leads to identify, generate and test new vaccines, to use not only against infectious diseases but also in the treatment of autoimmune disorders, allergies, chronic inflammatory diseases and cancer. There are several vaccine modalities currently under investigation, including subunit vaccines, syntetic peptide vaccines, ex-vivo loaded dendritic cells (DCs) and genetic vaccination, that will be here discussed.

#### **1.1 Subunit vaccines**

Subunit vaccines have improved conventional attenuated or killed vaccines in many aspects, including safety and production. The systems mostly used to produce these vaccines are based on bacteria, yeast, insect or mammalian cells. However, production of recombinant vaccine proteins in these expression systems is expensive in many cases, requiring large scale fermenters and stringent purification protocols. Worldwide, only a small number of facilities exists with capabilities to produce kilograms of a specific protein to be used as immunogen, and the construction, validation, and final approval of new production facilities take many years implying important investments in capital and human resources. Additionally, some antigens require post-translational modifications that cannot be achieved using all expression systems. In the last decade, non-fermentative alternatives based on living organisms have been developed to solve such problems and provide lowcost technologies for vaccine production. Insects and plants have been adapted for subunit vaccine production with clear advantages to conventional fermentative systems, especially in terms of time of development, scaling-up production and cost-efficiency (Brun et al. 2011). Despite the improvements in the recombinant technology, these vaccines remain hard to produce due to their inherent toxicity for the bacterial/viral expression system (e.g., Human Papilloma Virus type 16-E2, wild-type p53) (van der Burg et al. 2006).

#### **1.2 Syntetic peptide vaccines**

Identification of individual epitopes within protective proteins allows the development of peptide vaccines as alternative approach respect to using a whole protein as a vaccine. Selected peptide epitopes represent the minimal immunogenic region of a protein antigen and allow for precise direction of immune responses aiming at the induction of T-cell immunity. A peptide vaccine should ideally include epitopes recognized both by B and T cells, and take into account the MHC restriction of the T-cell response. In some cases B and

expressed during infection are not produced in laboratory conditions, as well as the proteins that are most abundant and easily purified are not necessarily protective antigens and, in any case, only few molecules can be isolated and tested simultaneously (Serruto and

The last decade has witnessed a revolution in the approach to vaccine design and development. These advances include new delivery technologies aimed at improving the safety and immunogenicity of traditional vaccines, new strategies to identify protective

Considering that new diseases are sure to emerge through evolution by mutation and gene exchange, interspecies transfer or human exposure to novel environments, more reliable approaches must be available to promptly respond to those threats (Plotkin 2005). Thanks to sophisticated technologies such as genomics, proteomics, functional genomics and synthetic chemistry, the rational identification of antigens, the synthesis of complex glycans, the generation of engineered carrier proteins are possible. This leads to identify, generate and test new vaccines, to use not only against infectious diseases but also in the treatment of autoimmune disorders, allergies, chronic inflammatory diseases and cancer. There are several vaccine modalities currently under investigation, including subunit vaccines, syntetic peptide vaccines, ex-vivo loaded dendritic cells (DCs) and genetic vaccination, that

Subunit vaccines have improved conventional attenuated or killed vaccines in many aspects, including safety and production. The systems mostly used to produce these vaccines are based on bacteria, yeast, insect or mammalian cells. However, production of recombinant vaccine proteins in these expression systems is expensive in many cases, requiring large scale fermenters and stringent purification protocols. Worldwide, only a small number of facilities exists with capabilities to produce kilograms of a specific protein to be used as immunogen, and the construction, validation, and final approval of new production facilities take many years implying important investments in capital and human resources. Additionally, some antigens require post-translational modifications that cannot be achieved using all expression systems. In the last decade, non-fermentative alternatives based on living organisms have been developed to solve such problems and provide lowcost technologies for vaccine production. Insects and plants have been adapted for subunit vaccine production with clear advantages to conventional fermentative systems, especially in terms of time of development, scaling-up production and cost-efficiency (Brun et al. 2011). Despite the improvements in the recombinant technology, these vaccines remain hard to produce due to their inherent toxicity for the bacterial/viral expression system (e.g.,

Human Papilloma Virus type 16-E2, wild-type p53) (van der Burg et al. 2006).

Identification of individual epitopes within protective proteins allows the development of peptide vaccines as alternative approach respect to using a whole protein as a vaccine. Selected peptide epitopes represent the minimal immunogenic region of a protein antigen and allow for precise direction of immune responses aiming at the induction of T-cell immunity. A peptide vaccine should ideally include epitopes recognized both by B and T cells, and take into account the MHC restriction of the T-cell response. In some cases B and

Rappuoli 2006).

will be here discussed.

**1.1 Subunit vaccines** 

**1.2 Syntetic peptide vaccines** 

antigens, generation of improved adjuvants.

T-cell epitopes can overlap substantially within the sequence of an antigen and, in others, they might be present in separate discrete regions of the antigen or present in different antigens from the targeted pathogen. The simplicity of producing clinical grade peptides allows swift changes in the design of peptide vaccines and, therefore, rapid translation of new immunological concepts, which represent a great advantage for the development of vaccines against rapidly changing viruses such as influenza (Brun et al. 2011). Despite the potential advantages of this approach, the development of successful peptide vaccines has been limited mainly by difficulties associated with stability, poor immunogenicity of simple peptides and by the MHC polymorphism of the host species (Tam 1996).
