**8. Bacterial delivery systems**

The most recent approach in targeting the gene therapy is the use of bacterial systems as vectors for transfer and gene expression in tumor cells. The principle lies in the ability of some anaerobic bacteria to selectively colonize hypoxic areas (e.g., tumors) and replicate there. Therefore, it is possible to achieve selective expression of therapeutic genes specifically in tumors. Currently known and tested bacterial vectors have been divided into two groups: A) Strictly anaerobic bacteria (the species *Clostridium* and *Bifi dobacterium*) are used in *in vivo*  experiments. *Clostridium* is the most important bacterial species for use as a vector. On the other hand, the non-pathogenic *Bifi dobacterium*, is naturally present in the human gastrointestinal tract and provides higher safety [Gardlik et al., 2005]. B) The second group consists of attenuated auxotrophic strains of *Salmonella typhimurium* that require the presence of tumor specific nutrition factors for selective replication. They use these factors for their own metabolism, thus prohibiting the tumor cells from utilizing them and growing. In *in vivo*  experiments, high levels of *Salmonella* (109 bacteria/g tissue) were obtained. A similar tumor inhibitory effect was shown by application of *Salmonella* producing tymidin-kinase [Gardlik et al., 2005]. Although the above bacteria are characterized by high selectivity in tissue colonization, it is necessary to ensure the maximum level of specificity and therapeutic efficiency of bacterial vectors. For this purpose, promoters inducible by radiation were constructed. The transcription of such promoters is conditioned by irradiation with visible light. Bacteria are known for their resistance to irradiation (having a relatively small genome and effective DNA repair mechanisms). Therefore the localization of a therapeutic gene under the control of a radiation-inducible promoter ensures that cytotoxic proteins are expressed only in bacteria colonizing currently irradiated tissues. This strategy allows eliminating the possibility of expression in non-tumor and hypoxic tissues. The latest approach using prokaryotes in gene therapy is a system of transformed bacteria producing a therapeutic protein *in situ* under exogenous induction regulation [Gardlik et al., 2005].

### **9. Eukaryotic delivery systems**

#### **9.1** *Leishmania tarentolae* **as a novel live vector**

Although live recombinant vectors (bacterial or viral recombinant vectors) have been known to develop new vaccine strategies against pathogens (e.g., HIV-1), their use as vaccine candidates in human is delayed due to problems related to pre-existing immunity, inefficient antigen delivery or presentation and toxicity issues. Therefore, it is necessary to develop new live-vaccine vectors that are able to enhance antigen presentation and elicit potent immune responses without the risk of developing disease in humans. Recently, a lizard parasitic protozoan that is not pathogenic to humans, *Leishmania tarentolae* (*L. tarentolae)*, has been used as a candidate vaccine against visceral leishmaniasis [Breton et al., 2005], and HIV-1 [Breton et al., 2007]. *L. tarentolae* can elicit T-cell proliferation and the production of gamma interferon (IFN-γ), skewing the T-cell response towards a Th1-cell phenotype, and it provides inflammatory responses for the APC and acts as an immunostimulatory adjuvant. Unlike other pathogenic *Leishmania* strains, *L.tarentolae* lacks the potential to replicate within the targeted APCs and is eliminated after several days from the infected murine host [Breton et al., 2005; Breton et al., 2007]. It has been shown that a single intraperitoneal injection of *L. tarentolae* could elicit a protective immune response against infectious challenge with *L. donovani* in susceptible BALB/c mice [Breton et al., 2005]. Similarly, a single intraperitoneal administration of the A2-recombinant *L. tarentolae*

strain could induce high levels of IFN-gamma and protect BALB/c mice against *L. infantum* challenge [Mizbani et al., 2009]. Interestingly, a recombinant *L. tarentolae* vaccine expressing high levels of full-length HIV-1 Gag elicited cell-mediated immunity in mice model and decreased HIV-1 replication in human tonsillar tissue following exposure to HIV-1 infection [Breton et al., 2007]. These data suggest that the use of *L. tarentolae* as a live vaccine vector may represent a promising approach for improving immunity and safety of candidate live vaccines against *Leishmania* infections and likely other intracellular pathogens for which Tcell mediated responses are critical for the development of protective immunity [Breitling et al, 2002; Breton et al., 2005].

#### **9.2 Yeast as an efficient tool in vaccine development**

Recent studies have indicated that yeast cell wall components possess multiple adjuvant properties. Interactions between yeast and DCs result in DC maturation, and whole recombinant yeast internalized by DCs can deliver heterologous antigens to both MHC class I and class II pathways and induce potent cell-mediated immunity [Capilla et al., 2009; Bian et al., 2010; Haller et al., 2007]. Vaccination with *Saccharomyces cerevisiae* (*S.cerevisiae)* expressing tumor-associated antigens can induce antigen-specific T-cell responses and protect animals against tumor challenge. In addition, *S. cerevisiae* is inherently nonpathogenic and heat-killed recombinant *S. cerevisiae* shows no toxicity in clinical studies. Yeast can be easily engineered to express multiple antigens and the inherent adjuvant properties of *S. cerevisiae* avoid the need for additional adjuvants. These characteristics make *S. cerevisiae* a potential vaccine vehicle for cancer and infectious diseases [Capilla et al., 2009; Bian et al., 2010; Haller et al., 2007]. There are some limitations and drawback in *S. cerevisiae* expression systems. For example, *S. cerevisiae* has a tendency to hyperglycosylate recombinant proteins, N-linked carbohydrate chains are terminated with alpha-1, 3-linked mannose residues which is considered to be allergenic. Other restriction is that the varieties of carbon sources that can be utilized by this species are limited [Bian et al., 2010]. Currently, two other species including *Hansenula polymorpha* and *Pichia pastoris* belonging to the *Saccharomycetaceae* family, could potentially overcome the described limitations of *S. cerevisiae* [Bazan et al., 2009; Bian et al., 2010]. On the other hand, these two species are broadly used as industrial platforms for heterologous protein production [Maleki et al., 2010; Bian et al., 2010].

#### **10. Conclusion**

44 Non-Viral Gene Therapy

The most recent approach in targeting the gene therapy is the use of bacterial systems as vectors for transfer and gene expression in tumor cells. The principle lies in the ability of some anaerobic bacteria to selectively colonize hypoxic areas (e.g., tumors) and replicate there. Therefore, it is possible to achieve selective expression of therapeutic genes specifically in tumors. Currently known and tested bacterial vectors have been divided into two groups: A) Strictly anaerobic bacteria (the species *Clostridium* and *Bifi dobacterium*) are used in *in vivo*  experiments. *Clostridium* is the most important bacterial species for use as a vector. On the other hand, the non-pathogenic *Bifi dobacterium*, is naturally present in the human gastrointestinal tract and provides higher safety [Gardlik et al., 2005]. B) The second group consists of attenuated auxotrophic strains of *Salmonella typhimurium* that require the presence of tumor specific nutrition factors for selective replication. They use these factors for their own metabolism, thus prohibiting the tumor cells from utilizing them and growing. In *in vivo*  experiments, high levels of *Salmonella* (109 bacteria/g tissue) were obtained. A similar tumor inhibitory effect was shown by application of *Salmonella* producing tymidin-kinase [Gardlik et al., 2005]. Although the above bacteria are characterized by high selectivity in tissue colonization, it is necessary to ensure the maximum level of specificity and therapeutic efficiency of bacterial vectors. For this purpose, promoters inducible by radiation were constructed. The transcription of such promoters is conditioned by irradiation with visible light. Bacteria are known for their resistance to irradiation (having a relatively small genome and effective DNA repair mechanisms). Therefore the localization of a therapeutic gene under the control of a radiation-inducible promoter ensures that cytotoxic proteins are expressed only in bacteria colonizing currently irradiated tissues. This strategy allows eliminating the possibility of expression in non-tumor and hypoxic tissues. The latest approach using prokaryotes in gene therapy is a system of transformed bacteria producing a therapeutic

protein *in situ* under exogenous induction regulation [Gardlik et al., 2005].

Although live recombinant vectors (bacterial or viral recombinant vectors) have been known to develop new vaccine strategies against pathogens (e.g., HIV-1), their use as vaccine candidates in human is delayed due to problems related to pre-existing immunity, inefficient antigen delivery or presentation and toxicity issues. Therefore, it is necessary to develop new live-vaccine vectors that are able to enhance antigen presentation and elicit potent immune responses without the risk of developing disease in humans. Recently, a lizard parasitic protozoan that is not pathogenic to humans, *Leishmania tarentolae* (*L. tarentolae)*, has been used as a candidate vaccine against visceral leishmaniasis [Breton et al., 2005], and HIV-1 [Breton et al., 2007]. *L. tarentolae* can elicit T-cell proliferation and the production of gamma interferon (IFN-γ), skewing the T-cell response towards a Th1-cell phenotype, and it provides inflammatory responses for the APC and acts as an immunostimulatory adjuvant. Unlike other pathogenic *Leishmania* strains, *L.tarentolae* lacks the potential to replicate within the targeted APCs and is eliminated after several days from the infected murine host [Breton et al., 2005; Breton et al., 2007]. It has been shown that a single intraperitoneal injection of *L. tarentolae* could elicit a protective immune response against infectious challenge with *L. donovani* in susceptible BALB/c mice [Breton et al., 2005]. Similarly, a single intraperitoneal administration of the A2-recombinant *L. tarentolae*

**8. Bacterial delivery systems** 

**9. Eukaryotic delivery systems** 

**9.1** *Leishmania tarentolae* **as a novel live vector** 

A number of methods have been and are being invented for the efficient and safe delivery of therapeutic DNA. The perspectives and hopes that are associated with gene therapy support research in this field of molecular biology. Although, clinical trials have already started, there are still various limitations that must be solved before routine clinical use. The major aim in gene therapy is to develop efficient, non-toxic gene carriers that can encapsulate and deliver foreign genetic materials into specific cell types including cancerous cells. Both viral and non-viral vectors were developed and evaluated for delivering therapeutic genes into cancer cells. Many viruses such as *retroviru*s, *adenovirus*, *herpes simplex virus*, *adeno-associated virus* and *pox virus* have been modified to eliminate their toxicity and maintain their high gene transfer capability. Due to the limitations correlated to viral vectors, non-viral vectors have been further focused as an alternative in delivery systems. The main non-viral vectors include cationic polymers, cationic peptides and cationic liposomes. Currently, many modifications to the current delivery systems and novel carrier systems have been

Non-Viral Delivery Systems in Gene Therapy and Vaccine Development 47

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developed to optimize the transfection efficiency. Furthermore, the route of immunization can influence the outcome of the immune response through altering the interaction between the vaccine and different APCs at the site of injection. Hence, the routes of administration and formulation of DNA clearly affect the therapeutic response by altering immune pathway. Among the commonly used methods of DNA vaccination, the highest efficacy was achieved after *in vivo* electroporation and gene gun delivery. However, it is critical to further analyze the results of ongoing clinical trials, specifically, in the aspect of their success or failure of certain delivery methodologies for gene therapies.
