**7. Hybrid vectors**

42 Non-Viral Gene Therapy

antigen-specific CD8+ T cells *in vitro* [Kim et al., 1997; Riedl et al., 2004]. A fusion protein containing the carboxy-terminal end of Tat (amino acids: 49–86) linked to the HPV16 E7 oncoprotein enhanced tumor specific immune responses *in vivo* [Giannouli et al., 2003]. In C57BL/6 mice, E7-Tat mixed with Quil A generated efficient prophylactic and therapeutic suppression of HPV16-positive C3 tumor outgrowth. This study offers a new strategy for improving subunit cancer vaccines [Giannouli et al., 2003]. Particularly, a Tat-derived peptide in combination with a PEG-PEI copolymer could be a promising candidate as gene delivery vehicle intended for pulmonary administration. Tat-PEG-PEI represents a new approach to non-viral gene carrier for lung therapy, comprising protection for plasmid DNA, low toxicity and significantly enhanced transfection efficiency under *in vivo*

It has been shown that covalent attachment of low molecular weight polyethyleneimine (PEI) improves Tat peptide mediated gene delivery *in vitro* [Alexis et al., 2006; Putnam et al., 2001; Wang, 2006]. In our recent study, two delivery systems including polymer PEI 25 kDa and polymer peptide hybrid as PEI600-Tat conjugate were used to compare their efficiency for HPV16 E7 DNA transfection *in vitro*. Our data indicated that both delivery systems including PEI 25 kDa and PEI600-Tat conjugate are efficient tools for E7 gene transfection. In fact, PEI potency for E7 gene transfection is higher than PEI600-Tat *in vitro*, but its toxicity is obstacle *in vivo* [Bolhassani et al., 2008]. Using HPV16 E7 as a model antigen, the effect of PEI600-Tat conjugate has been evaluated on the potency of antigen-specific immunity in mice model. Assessment of lymphoproliferative and cytokine responses against recombinant E7 protein (rE7) showed that PEI600-Tat/E7DNA complex at certain ratio induces Th1 response. This study has demonstrated that PEI600-Tat conjugate is efficient to

Moreover, synthetic peptides containing a nuclear localization signal (NLS) can be bound to the DNA and the resulting DNA-NLS complexes can be recognized as a nuclear import substrate by specific intracellular receptor proteins [53]. For example, conjugation of an NLS to a Minimalistic Immunogenically Defined Gene Expression (MIDGE) vector encoding a truncated and secreted form of BHV-1 glycoprotein D (tgD) improved the tgD expression *in vitro* and induced both humoral and cellular immune responses in mice [Zheng et al., 2006]. This strategy could be applied as an efficient pathway in enhancement of DNA vaccine

On the other hand, one of the CPPs that have currently received extensive attention in the field of DNA vaccination is the herpes simplex virus (HSV-1) protein VP22 [Brooks et al., 2010]. VP22 can form compacted complexes with short oligonucleotides and form particles of spherical nature with a size range of 0.3 to 1 μm in diameter. These particles entered cells efficiently within 2 to 4 hours. Furthermore, VP22 enables spreading of the antigenic peptide to the cells surrounding the transfected cells [Brooks et al., 2010]. Efforts have been made to increase the potency of DNA vaccines by exploiting the cell-to-cell spreading capabilities of the HSV-1 VP22 protein or the analogous protein from bovin herpesvirus 1 [Ulmer et al., 2006]. The significance of VP22 in intercellular spreading has been demonstrated through *in vitro* studies linking VP22 to p53, thymidine kinase, cytosine deaminase and Green Fluorescent Protein (GFP). These proteins were observed to be distributed to nuclei of surrounding cells [Lin et al., 2010]. Furthermore, vaccination with DNA encoding HPV16E7 linked to the HSV type 1 VP22 elicited the enhanced E7 specific memory CD8+ T lymphocytes and anti-tumor effects against E7-expressing tumor cells [Michel et al., 2002]. Also, VP22 has been used for HPV DNA vaccines targeting the

conditions [Kleemann et al., 2005].

potency against cancer.

improve immune responses *in vivo* [Bolhassani et al., 2009].

A promising approach to overcome the limitations and develop the advantages of the individual types of vectors is their combination. Several types of hybrid vectors have been known: A) Virosomes are produced by the fusion of lipoplexes (liposomes with DNA) with inactivated HVJ virus (hemagglutinating virus of Japan) or influenza virus [Gardlik et al., 2005]. It was shown that the efficiency of gene transfer into the respiratory tract is higher than cationic liposomes or viral vectors. In addition, they are very well tolerated from the immunological view, so even repeated injection does not influence the efficiency and safety of transfer; B) The second type is represented by hybrids that were generated by mixing cationic liposomes or polymers with adenoviral vector. These are effective mainly in cells which do not have viral receptors. In addition, it was proved that an inactivated adenovirus attachment improves the efficiency of the transfer mediated by cationic liposomes or polymers [Gardlik et al., 2005]; C) Hybrid viruses can be produced by a combination of various types of viral vectors, and they represent a system which employs the main advantages of both viruses [Gardlik et al., 2005].

Non-Viral Delivery Systems in Gene Therapy and Vaccine Development 45

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

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

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

al, 2002; Breton et al., 2005].

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

production [Maleki et al., 2010; Bian et al., 2010].

**10. Conclusion** 
