**6. Cationic peptides/cell-penetrating peptides (CPP)/trojan peptides**

The studies have shown that a number of peptides and proteins are able to penetrate the cell membrane and enter the cell. It has been observed that many cargo molecules that are covalently attached to these peptides will be translocated into the cell. Recently, various natural and/ or synthetic cell-penetrating peptides (CPP) have known as efficient tools in vaccine design as they are capable of delivering therapeutic targets into cellular compartments. In fact, the cell membrane is impermeable to hydrophilic substances and delivery into cells could be facilitated by linking to CPP. Different cargos such as drugs, peptide/ protein, oligonucleotide/ DNA/ RNA, nanoparticles, liposomes, bacteriophages, fluorescent dyes and quantum dots have been linked to CPPs for intracellular delivery with possible use in future vaccine design [Brooks et al., 2010]. Two applications of CPP already validated in vaccine studies are delivery of tumor-associated antigens into antigenpresenting cells (APCs) and use as a non-viral gene delivery vehicle in DNA vaccines. There are two methods for designing CPP incorporating immunogenic antigens: A) chemical linking via covalent bonds B) coupling via recombinant fusion constructs produced by bacterial expression vectors. The orientation of the peptide and cargo and the type of linkage are likely important [Brooks et al., 2010]. In addition, the utilized CPP, attached cargo, concentration and cell type, all significantly affect the mechanism of internalization. The mechanism of cellular uptake and subsequent processing still remains controversial. It is now apparent that CPP mediate intracellular delivery via both endocytic and non-endocytic pathways [Brooks et al., 2010; Jarver and Langel, 2004; Wagstaff and Jans, 2006]. An attractive feature of using polypeptides as gene delivery vectors is incorporating multiple functional domains into one polypeptide chain, such as a DNA-binding domain linked with a receptor-targeting domain. This kind of polypeptides will recognize and bind to cell surface receptors that are unique to target cells and deliver the bound DNA into the cells through receptor-mediated endocytosis. Therefore, this process may ensure the therapeutic effect in desired cells and limit the potential side effects caused by transgene expression into the non-targeted cells [Zeng and Wang, 2005].

Several studies have shown that oligo-deoxynucleotides (ODN) with immune-stimulating sequences (ISS) containing CpG motifs facilitate the priming of MHC class I- restricted CD8+ T cell responses to proteins or peptides. Therefore, ODN/cationic peptide complexes are potent tools for priming CD8+ T cell immunity [Schirmbeck et al., 2003]. The complex formation required electrostatic linkage of the positively charged peptide to the negatively charged ODN. Conjugation of immunostimulatory DNA or ODN to protein antigens facilitates the rapid, long-lasting, and potent induction of cell-mediated immunity. It was shown that ODN (with or without CpG-containing sequences) are potent Th1-promoting adjuvants when bound to cationic peptides covalently linked to antigenic epitopes, a mode of antigen delivery existing in many viral nucleocapsids [Schirmbeck et al., 2003]. Table 3

Non-Viral Delivery Systems in Gene Therapy and Vaccine Development 41

Fluo-RQIKIFFQNRRMKFKK-NH2

KLALKLALKALKAALKL A-NH2

Fluo-RQIRIWFQNRRMRWRR-NH2

Fluo-KQIKIWFQNKKMKWKK -NH2

ALAALAKKIL

KLALKLALKALKAALKL A amide

KKKRKV

Ac-GLWRALWRLLRSLWRL LWRA-cysteamide

APKKKRKV

MPG GALFLGWLGAAGSTMG

Furthermore, the HIV Tat derived peptide is a small basic peptide that has been successfully shown to deliver a large variety of cargoes, from small particles to proteins, peptides and nucleic acids. The "transduction domain" or region conveying the cell penetrating properties is clearly confined to a small stretch of basic amino acids, with the sequence RKKRRQRRR (residues 49–57) [Riedl et al., 2004; Brooks et al., 2005]. This polycationic nanopeptide is known to be a transfection enhancer of plasmid DNA. The conditions of DNA-peptide complex formation and DNA/Tat ratio have significant impact on the level of transgene expression and degree of DNA protection from nuclease attack [Hellgren et al., 2004]. The conjugation of this peptide to ovalbumin (OVA) resulted in efficient stimulation of MHC class I-restricted T cell responses *in vitro* and, more importantly, the generation of CTLs *in vivo* [Kim et al., 1997]. Also, soluble Tat-antigen conjugates can deliver the antigen directly to the MHC class I processing pathway and thereby increase the generation of

144) TRRNKRNRIQEQLNRK + Futaki,

153) SQMTRQARRLYV - Futaki,

QWKAAN + Futaki,

AAVALLPAVLLALLP Rojas et

R + Futaki,

E N-(1-22) MDAQTRRRERRAEKQA

B21 N-(12-29) TAKTRYKARRAELIAER

**Mastoparan)** Transportan GWTLNSAGYLLGKINLK

**NLS** Pep-1 KETWWETWWTEWSQP

Model amphipathic peptide (MAP)

Membrane translocating sequence peptide (MTS)

Futaki, 2005

Futaki, 2005

Futaki, 2005

Futaki, 2005

2005

2005

2005

2005

Pooga et al., 1998

Oehlke et al., 1998

Morris et al., 2001

Crombez et al., 2009

al., 2008

Morris et al., 2008

Pen2W2F

PenArg

PenLys

Yeast PRP6-(129-

Hum U2AF-(142-

**NLS** CADY

**Galanin/** 

**Galanin/ Mastoparan)** 

**Hydrophobic-**

**Hydrophobic-**

**Hydrophobic-NLS** 

**HIVGp41-NLS of SV40 T-antigen** 

Table 3. Protein transduction domains (PTD)

Model amphipathic peptide

**Chimera (synthetic)** 

contains a list of peptides that have been investigated for their ability to penetrate the cell [Futaki, 2005; Brooks et al., 2010].


contains a list of peptides that have been investigated for their ability to penetrate the cell

HIV-1 Tat (48-60) GRKKRRQRRRPPQ +++

R9-Tat GRRRRRRRRRPPQ +++ Futaki,

R7W Fluo-RRRRRRRW-NH2 +++ Futaki,

BMV Gag-(7-25) KMTRAQRRAAARRNR

CCMV Gag-(7-25) KLTRAQRRAAARKNKR

**Calcitonin** hCT (9-32) LGTYTQDFNKFHTFPQT

peptide (SAP) (VRLPPP)3

**Basic VP22 [HSV]** VP22 DAATATRGRSAASRPTE

Sweet arrow

Antennapedia (43-58) [penetratin]

**Arginine-rich RNA binding peptides** 

TatP59W Fluo-

HTLV-II REX-(4-

**Human** 

**Vascular endothelial cadherin [Mouse]**

**Related to γ-Zein [Maize]** 

**Related to γ-**

**Antennapedia homeodomain [***Drosophila***]** 

Fluoro-Penetratin

**Basic/ amphiphilic**

**peptide Sequence Translocation** 

HIV-1 Rev-(34-50) TRQARRNRRRRWRERQR +++ Futaki,

GRKKRRQRRRPWQ-NH2

16) TRRQRTRRARRNR +++ Futaki,

RPRAPARSASRPRRPVE +++

pVEC LLIILRRRIRKQAHAHSK Elmquist

RQIKIWFQNRRMKWKK +++ Derossi et

FHV Coat-(35-49) RRRRNRTRRNRRRVR Futaki,

P22 N-(14-30) NAKTRRHERRRKLAIER ++ Futaki,

AIGVGAP

**Zein [Maize]** (Tyr-ZnDPA)n Johnson et

Fluo-RQIKIWFQNRRMKWKK -NH2

**peptides** Vives et

**efficiency**

**Reference** 

al., 1997

Futaki, 2005; Brooks et al., 2010

2005

Futaki, 2005; Brooks et al., 2010

2005

2005

2005

2005

2005

2005

2005

2005

Cashman et al., 2002

Trehin et al., 2004

et al., 2001

Fernandez -Carneado et al., 2004

al., 2008

al., 1994

Futaki, 2005

+++ Futaki,

WTAR +++ Futaki,

NTR ++ Futaki,

[Futaki, 2005; Brooks et al., 2010].

**Basic** 

**Class Protein Functional** 

**Basic TAT [HIV] Tat and related** 


Table 3. Protein transduction domains (PTD)

Furthermore, the HIV Tat derived peptide is a small basic peptide that has been successfully shown to deliver a large variety of cargoes, from small particles to proteins, peptides and nucleic acids. The "transduction domain" or region conveying the cell penetrating properties is clearly confined to a small stretch of basic amino acids, with the sequence RKKRRQRRR (residues 49–57) [Riedl et al., 2004; Brooks et al., 2005]. This polycationic nanopeptide is known to be a transfection enhancer of plasmid DNA. The conditions of DNA-peptide complex formation and DNA/Tat ratio have significant impact on the level of transgene expression and degree of DNA protection from nuclease attack [Hellgren et al., 2004]. The conjugation of this peptide to ovalbumin (OVA) resulted in efficient stimulation of MHC class I-restricted T cell responses *in vitro* and, more importantly, the generation of CTLs *in vivo* [Kim et al., 1997]. Also, soluble Tat-antigen conjugates can deliver the antigen directly to the MHC class I processing pathway and thereby increase the generation of

Non-Viral Delivery Systems in Gene Therapy and Vaccine Development 43

E6 protein [Lin et al., 2010]. Various groups have demonstrated that DNA constructs which encode fusion proteins of VP22 linked to an antigen increase the immune responses in mice and cattle. Bovine herpesvirus VP22 (BVP22) and Marek's disease virus VP22 (MVP-1) are both closely related by their structural homology to HSV-1 VP22, and can also have a significant role in intercellular spreading. Hung *et al.* has demonstrated that mice vaccinated with DNA encoding MVP22/E7 significantly increased numbers of IFNγ-secreting, E7-specific CD8+ T cell precursors compared to mice vaccinated with wildtype E7 DNA alone, which directly lead to a stronger tumor prevention response. Similarly, immunization of mice and cattle with DNA vaccine coding for BVP22 linked to truncated glycoprotein D (BVP-tgD) was shown to generate a stronger tgD-specific immune response compared to animals vaccinated with tgD alone. Taken together, DNA vaccine encoding VP22 linked to antigens represents a promising approach to enhance

To evaluate the VP22 role in gene therapy of hepatocellular carcinomas (HCCs), the expression vectors were constructed for N- and C-terminal fragments of VP22-p53 fusion proteins and investigated the VP22-mediated shuttle effect in hepatoma cells by cotransfection experiments. VP22-mediated trafficking was not detectable in hepatoma cells *in vitro* by fluorescence microscopy [Zender et al., 2002]. For *in vivo* experiments, the recombinant adenoviruses Ad5CMVp53 and Ad5CMVp53-VP22 were constructed. In contrast to the *in vitro* experiments, intercellular trafficking of VP22-p53 could be observed in subcutaneous tumors of hepatoma cells by fluorescence microscopy, indicating a stronger shuttle effect in solid tumors compared to cell culture experiments [Zender et al., 2002]. In our current study, Herpes simplex virus type 1 (HSV-1) VP22 protein was employed to enhance DNA vaccine potency of *Leishmania major* amastin antigen in BALB/c mice model. Vaccination with the VP22-amastin-EGFP fusion construct elicited significantly higher IFN-gamma response upon antigen stimulation of splenocytes from immunized mice compared to amastin as a sole antigen. These results suggest that the development of DNA vaccines encoding VP22 fused to a target *Leishmania* antigen would be a promising strategy to improve immunogenicity and DNA

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

DNA vaccine potency [Lin et al., 2010].

vaccine potency [Bolhassani et al., 2011].

advantages of both viruses [Gardlik et al., 2005].

**7. Hybrid vectors** 

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* conditions [Kleemann et al., 2005].

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

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 potency against cancer.

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 E6 protein [Lin et al., 2010]. Various groups have demonstrated that DNA constructs which encode fusion proteins of VP22 linked to an antigen increase the immune responses in mice and cattle. Bovine herpesvirus VP22 (BVP22) and Marek's disease virus VP22 (MVP-1) are both closely related by their structural homology to HSV-1 VP22, and can also have a significant role in intercellular spreading. Hung *et al.* has demonstrated that mice vaccinated with DNA encoding MVP22/E7 significantly increased numbers of IFNγ-secreting, E7-specific CD8+ T cell precursors compared to mice vaccinated with wildtype E7 DNA alone, which directly lead to a stronger tumor prevention response. Similarly, immunization of mice and cattle with DNA vaccine coding for BVP22 linked to truncated glycoprotein D (BVP-tgD) was shown to generate a stronger tgD-specific immune response compared to animals vaccinated with tgD alone. Taken together, DNA vaccine encoding VP22 linked to antigens represents a promising approach to enhance DNA vaccine potency [Lin et al., 2010].

To evaluate the VP22 role in gene therapy of hepatocellular carcinomas (HCCs), the expression vectors were constructed for N- and C-terminal fragments of VP22-p53 fusion proteins and investigated the VP22-mediated shuttle effect in hepatoma cells by cotransfection experiments. VP22-mediated trafficking was not detectable in hepatoma cells *in vitro* by fluorescence microscopy [Zender et al., 2002]. For *in vivo* experiments, the recombinant adenoviruses Ad5CMVp53 and Ad5CMVp53-VP22 were constructed. In contrast to the *in vitro* experiments, intercellular trafficking of VP22-p53 could be observed in subcutaneous tumors of hepatoma cells by fluorescence microscopy, indicating a stronger shuttle effect in solid tumors compared to cell culture experiments [Zender et al., 2002]. In our current study, Herpes simplex virus type 1 (HSV-1) VP22 protein was employed to enhance DNA vaccine potency of *Leishmania major* amastin antigen in BALB/c mice model. Vaccination with the VP22-amastin-EGFP fusion construct elicited significantly higher IFN-gamma response upon antigen stimulation of splenocytes from immunized mice compared to amastin as a sole antigen. These results suggest that the development of DNA vaccines encoding VP22 fused to a target *Leishmania* antigen would be a promising strategy to improve immunogenicity and DNA vaccine potency [Bolhassani et al., 2011].
