**4.1 Lipoplexes (cationic lipids/liposomes)**

Plasmid DNA can be covered by lipids into organized structures such as liposomes or micelles [Templeton and Lasic, 1999]. The complex of DNA with lipids is called lipoplex. Lipoplexes can be divided into two types: 1) Anionic and neutral liposomes: At first, these kinds of lipids were used for the construction of synthetic vectors. Although, they were characterized by safety, compatibility with body fluids and the possibility of tissue-specific gene transfer, but the level of transduced cell expression was relatively low. At present, new

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 electroporation-

Similar to electroporation, another method, called "gene-gun", does not require the presence of complicated and potentially toxic delivery systems [Gardlik et al., 2005]. The gene transfer is mediated by small particles of gold on which the DNA is bounded. These particles are then shot into the cell under great pressure and speed (with the help of compressed helium) and so pass the membrane barrier [Katare and Aeri, 2010]. At first, the gene-gun was developed for gene transfer into plant cells; then, its use has expanded to gene transfer into the mammalian cells. Effective development of the gene-gun was also achieved in the field of DNA vaccination. The latest clinical experiments focus on cancer vaccines against various human tumors [Gardlik et al., 2005]. This method has been successfully used to deliver DNA *in vivo* into liver, skin, pancreas, muscle, spleen and tumors. Expression of reporter genes (e.g. firefly luciferase and β- galactosidase) or therapeutic genes (human growth hormone) have also been reported by this method [Gardlik et al., 2005]. Recently, gene gun-mediated transgene delivery system has been used for skin vaccination against melanoma using tumor-associated antigen (TAA) human gpl00 and reporter gene assays as experimental systems [Aravindaram and Yang, 2009 S]. In addition, the delivery of HPV DNA vaccines using intradermal administration through gene gun was shown to be the most efficient method of vaccine administration in comparison

Currently, a HPV16 DNA vaccine encoding a signal sequence linked to an attenuated form of HPV16 E7 (E7 detox) and fused to heat shock protein 70 [(Sig/E7detox/HSP70)] has been used in clinical trials. In a previous study, the immunologic and anti-tumor responses have been evaluated by the pNGVL4a-Sig/ E7 (detox)/ HSP70 vaccine administered using three different delivery methods including needle intramuscular, biojector and gene gun. According to obtained results, DNA vaccine administered via gene gun generated the highest number of E7-specic CD8+ T cells as compared to needle intramuscular and

In order to facilitate the effective transfer of non-viral DNA into the cells, synthetic vectors improving the DNA admission into the cell and protecting it from undesirable degradation were designed. The most chemical delivery systems were derived from lipids or synthetic

Plasmid DNA can be covered by lipids into organized structures such as liposomes or micelles [Templeton and Lasic, 1999]. The complex of DNA with lipids is called lipoplex. Lipoplexes can be divided into two types: 1) Anionic and neutral liposomes: At first, these kinds of lipids were used for the construction of synthetic vectors. Although, they were characterized by safety, compatibility with body fluids and the possibility of tissue-specific gene transfer, but the level of transduced cell expression was relatively low. At present, new

based DNA delivery [Bodles-Brakhop et al., 2009].

with routine intramuscular injection [Ogris and Wagner, 2002].

biojector administrations in mice model [Trimble et al., 2003].

**4. Chemical delivery systems** 

polymers [Templeton and Lasic, 1999].

**4.1 Lipoplexes (cationic lipids/liposomes)** 

**3.2 Gold bullet/gene gun** 

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

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

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

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

Non-Viral Delivery Systems in Gene Therapy and Vaccine Development 35

• **Arterial-wall binding peptide (AWBP)-conjugated PLL:** Arterial-wall binding peptide (AWBP) is a peptide containing the arterial-wall binding domain (1000-1016 amino acids) of apoB-100 protein, a major protein component of LDL. The AWBP was conjugated to PLL via a PEG linkage (AWBP-PEG-g-PLL). When interacted with a plasmid DNA, AWBP-PEG-g-PLL could form spherical shaped complexes with a size of 100 nm and showed dramatic increase of transfection efficiency (150-180-fold), compared to PLL and PEG-g-PLL, in bovine aorta endothelial cells and smooth muscle cells. The presence of free AWBP in the transfection medium reduced the transfection efficiency of AWBP-PEG-g-PLL, suggesting that AWBP-PEG-g-PLL could be used as a

• **Antibody-PLL conjugates:** Antibody-antigen interaction is one of the most specific interactions in biological systems. A monoclonal antibody against leukemia-specific JL-1 antigen (anti-JL-1-Ab) was conjugated with PLL by periodate-mediated oxidation of carbohydrate moiety in the Fc domain of the antibody, followed by reaction with PLL. The anti-JL-1-Ab-PLL conjugate demonstrated significantly higher transfection

• **Folate-conjugated PLL:** Folate receptor has been identified as a potential target molecule of various cancer cells. The receptor is up-regulated and over-expressed in a number of rapidly growing malignant tumor cells, resulting in a dramatic promotion of the cellular uptake of folate. Therefore, the conjugation of folate to a variety of polymeric carriers has been chosen as a popular strategy for the target-specific delivery of anti-cancer therapeutics to the folate receptor-bearing tumor cells. A folate-PLL conjugate that incorporates a PEG spacer between folate and PLL (Fol-PEG-PLL) was also synthesized. The Fol-PEG-PLL was coated onto the complexes of PEI/DNA for a receptor-mediated gene transfer. The formulated complexes exhibited much higher transfection efficiency than PEI/DNA or lipofectamine/DNA complexes in the presence of 10% serum, suggesting that the PEG segment of Fol-PEG-PLL could increase the cellular uptake by receptor-mediated endocytosis and efficiently stabilize the complexes by increasing their solubility as well as by reducing the non-specific adsorption of serum proteins. The formulation also showed much lower cytotoxicity

• **The Terplex system:** The Terplex system is a low-density lipoprotein (LDL)-mediated targeting system, where the LDL specifically interacts with the LDL receptors on the cell surface. LDL receptors are membrane-anchored proteins present in many cell types including hepatocytes, endothelial cells and myocytes. The stearyl-PLL conjugate synthesized by N-alkylation of PLL with stearyl bromide interacts with a plasmid DNA to form complexes. The stearyl group could then bind to LDL via hydrophobic interaction to form the supramolecular gene carrier, the terplex. The Terplex system showed efficient *in vitro* transfection in a variety of cells including smooth muscle cells (A7R5), and human lung fibroblasts (CCD-32 Lu). The systemic administration of the Terplex system demonstrated prolonged circulation time compared to naked DNA

• PEI has been one of the most popularly employed cationic gene carriers due to its superior transfection efficiency in many different types of cells. The buffering property of PEI leads to protect the DNA from degradation in the endosomal compartment

efficiency than PLL or lipofectin in leukemia (Molt 4) cells [Park et al., 2006].

tissue-selective gene carrier [Park et al., 2006].

than PEI/DNA complexes [Park et al., 2006].

[Park et al., 2006].

**Polyethylenimine (PEI)-based gene carriers** 

Toll-like receptors (TLRs), which may result in the generation of anti-tumor natural killer (NK) cell and T-cell responses by the innate immune system. In addition, as a vaccine adjuvant, this agent may induce a strong cytotoxic T-lymphocyte (CTL) response to co-administered antigen. The efficacy of JVRS-100 has been evaluated in phase I clinical trials for the treatment of patients with Relapsed or Refractory Leukemia [ID: NCT00860522].
