**1. Introduction**

348 Non-Viral Gene Therapy

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Gene therapy as an alternative to conventional medicine implies the elimination of the cause of a disease via the introduction of therapeutic nucleic acids (antisense oligonucleotide, or siRNA, or transgene expressing plasmid DNA, or aptamers, etc) into the cells of the organism (Blau & Springer, 1995; Lv et al., 2006; Karmali & Chaudhuri, 2007; Rayburn & Zhang R., 2008; and references in it). However, efficient delivery of genes at the physiological level into cells of living organism (''transfection'') is always more simple in theory than in practice. Both the therapeutic nucleic acids and cell membrane are negatively charged and therefore the spontaneous entry of naked nucleic acid inside cells is unlikely to be an efficient process. In other words, the development of clinically viable gene-targeted therapeutics and the design of safe and efficient gene delivery reagents (''transfection vectors'') are inseparable problems. (Templeton, Ed. 2010).

Prior to reaching the nucleus, the therapeutic nucleic acids must overcome a number of biological barriers, in particular, the cellular, endosomal, and nuclear membranes. This process is achieved by the utilization of appropriate delivery systems, that protect the genetic material from the destructive action of enzymes and encourage their penetration into the intracellular space, transfer through the nuclear membrane, and further expression in the nucleus (Eliyahu et al., 2005). In addition, these delivery systems should be nontoxic, non-immunogenic and biocompatible.

The administration of genes for therapeutic purposes can be achieved using one of three different approaches. The first approach consists of a direct injection of naked nonprotected DNA into the cell resulting in a high level of transgene expression. The simplicity of this approach ensures it can be reasonably applied in a number of experimental protocols (Huang et al., Eds.; 2005). However, therapeutic application of unprotected naked nucleic acids is limited by the easily accessible organs (skin or muscles) for direct injection and is not applicable for systemic delivery due to a number of factors, the most important of which being extracellular nucleases. Gene-modified viruses and virus like particles represent the second approach for the cellular delivery of therapeutic nucleic acids. Viruses are efficient in transducing cells. However, the safety concerns regarding the use of viruses in humans make non-viral delivery systems an attractive alternative.

Non-Viral Gene Delivery Systems Based on

(siRNAs, transgene expressing DNA, etc).

**2. Cholesterol-containing cationic lipids and polymers** 

pertaining to the properties and structure of the resulting lipoplex.

environment.

**2.1 DC-Chol** 

et al., 2003).

et al., 2003).

Cholesterol Cationic Lipids: Structure-Activity Relationships 351

their potential applications in gene therapy (Zhdanov et al., 1998; Gao & Hui, 2001; Choi et al., 2001; Geall et al., 2000; Percot et al., 2004; Ding et al., 2008; Medvedeva et al., 2009; Maslov et al., 2010). The cationic amphiphiles containing cholesterol as a hydrophobic residue possess a high transfection activity and a low toxicity, finding use in the studies of both the structure–activity relationships and membrane fusion mechanisms (Noguchi et al, 1998; Kearns et al., 2008). Found within the synthesized cholesterol derivatives are commercially available lipids, (DC-Chol) and *N*1-cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine (CDAN). The liposomes prepared with DC-Chol or CDAN and DOPE lipids are widely used to deliver the plasmid DNA for tumor immunotherapy or artificial

The efficiency of liposome mediated gene delivery is ascertained not only by the structure of cationic and helper lipids, properties of the nucleic acid, but also by the size and ζ-potential of the lipoplex formed with the nucleic acid. Therefore, the efficiency of the lipid vehicles for nucleic acid delivery is dependent upon the structure of cationic amphiphile whose chemical design requires a tailored approach, taking into account the lipid composition of the membranes, the nature of cell receptors, and the chemical processes in the intracellular

Thus, in this chapter we summarize recent results on the design and structure-activity relationships of cholesterol-based cationic lipids with multiple architectures. We show that some of the designed cationic lipids (liposomal formulations) mediate efficient cellular accumulation, endosomal escape and the biological activity of the delivered nucleic acids

Cationic lipid DC-Chol (**1a**) was originally synthesized by Gao et al. (Gao & Huang, 1991) and is available commercially. The DC-Chol was formulated as a cationic liposome with the helper lipid DOPE which promotes the fusion of lipoplexes with the cell membrane resulting in an increase of the DNA transfection efficiency (Zuidam & Barenholz, 1998; Lin

Cationic liposomes DC-Chol/DOPE have been used extensively both *in vitro* and *in vivo,* displaying high transfection efficiencies (TE) (Litzinger et al., 1996; Song et al., 1997; Porter et al., 1998). There are documented reports that cationic liposomes DC-Chol/DOPE work well in various cell lines e.g. A431 human epithermoid carcinoma cells, A549 human lung carcinoma cells, L929 mouse fibroblast cells, YPT minipig primary endothelial cells (Gao & Huang, 1991), COS-7 cells, CFNPE-9o and 16HBE14o epithelial cell lines (Caplen et al., 1995), SKnSH and the primary rat neuronal cells (Ajmani et al.,1999), glioma cells (Esposito

An experimental study of DNA compaction with the liposomes DC-Chol/DOPE that covered the whole range of mixed lipid composition and several lipid/DNA charge ratios was published (Rodriguez-Pulido et al., 2008; Munoz-Ubeda et al., 2010). A series of experimental techniques (electrophoretic mobility, SAXS, and fluorescence anisotropy), together with a theoretical aggregation-disaggregation model, has attested to the fact that DC-Chol/ DOPE cationic liposomes, with an average hydrodynamic diameter of (120+/-10) nm, properly condense and compact DNA and the liposomes composition is a key factor

immunization and are under clinical trials for the therapy of mucoviscidosis.

The third approached is focused upon the use of non-viral vectors. Non-viral vectors are particularly suitable with respect to their simplicity of use, large-scale production and lack of specific immune response. Non-viral vectors can be grouped into three main categories: cationic lipids, cationic polymers, and peptides. In comparison with their viral counterparts, these vectors are currently considerably less efficient, but their well-defined physical and chemical composition coupled with their reduced immunogenicity and toxicity make them promising candidates for gene delivery (El-Aneed, 2004; Dass, 2002; Verma & Weitzman, 2005).

Another important method of gene delivery is lipofection, a method based on the use of cationic lipids/cationic liposomes for gene transfer (Templeton, Ed. 2010; Huang, et al., Eds.; 2005; Karmali & Chaudhuri, 2007; Tseng et al., 2009). Cationic lipids have many potential advantages and have thus been viewed favorably in comparison with other non-viral vectors, including the significant simplicity and ease of production, good repeatability and biodegradability, potential commercial value, and their wide range of clinical application and safety.

The cationic liposomes are formed using cationic lipids, comprising a wide range of chemical compounds with a common structural feature, namely, the presence of both positively charged hydrophilic and hydrophobic domains. Of the most significant interest are biodegradable cationic lipids of natural origin (Lv et al., 2006). Long-chain hydrocarbons, steroids, and diglycerides are used as the hydrophobic domains (Zhi et al., 2010). The cationic hydrophilic domain can be represented by one (monocationic lipids) or more (polycationic lipids) positively charged groups. Monocationic lipids are most often secondary, tertiary or quaternary derivatives of aliphatic or heterocyclic nitrogen bases. In polycationic lipids, natural or synthetic polyamines or amino acids are used as the hydrophilic domains. The stability and toxicity of cationic lipids in biological systems, are determined by the type of bond connecting hydrophobic and hydrophilic domains.

In addition to cationic lipids, the zwitterionic helper lipid has a major impact upon the structure and activity of lipoplexes. A helper lipid can improve the ability of cationic liposomes to transfect cells. *In vitro* studies show that liposomes composed of an equimolar mixture of dioleoylphosphatidylethanolamine (DOPE) and cationic lipids (DOTMA, DOTAP) can mediate higher levels of transfection than those containing only the cationic lipid (Hui et al., 1996; Mok et al, 1997; Kerner et al., 2001). This fact has been attributed to the ability of DOPE tendency to undergo a transition from a bilayer to a hexagonal configuration under acidic pH, possibly facilitating fusion with, or destabilization of target membranes, in particular endosomal membranes (Zuidam & Barenholz, 1998; Zuidam et al., 1999).

Cholesterol initially used as a helper lipid form more stable but less efficient complexes than those containing DOPE *in vitro*. However, cholesterol containing lipoplexes have shown a higher rate of biological activity when compared to lipoplexes with DOPE, when these complexes were utilized *in vivo* (Liu et al., 1997; Sternberg et al., 1998; Smith et al., 1998; Simberg et al., 2003). The significant transfection activity attained was attributed to an improved cell binding and uptake of the lipoplexes promoted by the presence of cholesterol (Crook et al., 1998) and/or better stability of the lipoplex in serum (Simberg et al., 2003).

In 1991, Gao et al. reported the synthesis and application of the cholesterol-based cationic lipid 3-β-[*N*-(*N*′*,N*′-dimethylaminoethyl)carbamoyl]-cholesterol (DC-Chol, **1a**), which was combined with DOPE to transfect mammalian cells (Gao & Huang, 1991). Since then, considerable endeavors have been made in the synthesis of steroidal cationic lipids, due to their potential applications in gene therapy (Zhdanov et al., 1998; Gao & Hui, 2001; Choi et al., 2001; Geall et al., 2000; Percot et al., 2004; Ding et al., 2008; Medvedeva et al., 2009; Maslov et al., 2010). The cationic amphiphiles containing cholesterol as a hydrophobic residue possess a high transfection activity and a low toxicity, finding use in the studies of both the structure–activity relationships and membrane fusion mechanisms (Noguchi et al, 1998; Kearns et al., 2008). Found within the synthesized cholesterol derivatives are commercially available lipids, (DC-Chol) and *N*1-cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine (CDAN). The liposomes prepared with DC-Chol or CDAN and DOPE lipids are widely used to deliver the plasmid DNA for tumor immunotherapy or artificial immunization and are under clinical trials for the therapy of mucoviscidosis.

The efficiency of liposome mediated gene delivery is ascertained not only by the structure of cationic and helper lipids, properties of the nucleic acid, but also by the size and ζ-potential of the lipoplex formed with the nucleic acid. Therefore, the efficiency of the lipid vehicles for nucleic acid delivery is dependent upon the structure of cationic amphiphile whose chemical design requires a tailored approach, taking into account the lipid composition of the membranes, the nature of cell receptors, and the chemical processes in the intracellular environment.

Thus, in this chapter we summarize recent results on the design and structure-activity relationships of cholesterol-based cationic lipids with multiple architectures. We show that some of the designed cationic lipids (liposomal formulations) mediate efficient cellular accumulation, endosomal escape and the biological activity of the delivered nucleic acids (siRNAs, transgene expressing DNA, etc).
