GTMB 7 - Gene Therapy & Molecular Biology

Gene Therapy and Molecular Biology Vol 7, page 2751 Spontaneous self-assembly of vector/DNA complex (lipoplex)2 Endocytosis – encapsulation of lipoplex into endosome3 Endosomal escape before degradation of DNA4 Trafficking of DNA to perinuclear region and nuclear ingress5 Nuclear expression of the transfected DNAFigure 2: Schematic representation of lipoplex formation and trafficking to the target cell nucleus.A multivalent lipid soon followed in the form of thelipopolyamine DOGS (dioctadecylamido-glycylspermine)(Behr et al, 1989) and use of cholesterol as thehydrophobic portion was subsequently validated by thevector DC-Chol (3‚-[_N-(-(N’,N’-dimethylaminoethyl)carbamoyl] cholesterol) (Gao andHuang, 1991) (Figure 3). It is of particular importancethat the transfection efficiencies of many cationic lipidscan be enhanced by their formulation as stable cationicliposomes (Farhood et al, 1995). This is achieved bymixing cationic lipids, especially those which areincapable of forming bilayers alone, with the neutralcolipid DOPE (dioleoyl phosphatidylethanolamine) priorto complexation with DNA. For example, DOTMA/DOPEliposomes are commercially available as Lipofectin(Gibco BRL). Also, the structural analogues DDAB(dimethyldioctadecyl ammonium bromide) (Gibco BRL)and DOTAP (1,2-dioleoyloxy-3-[trimethylammonio]-propane, where ester groups replace ethers of DOTMA)(Boehringer Mannheim) (Figure 4), are commerciallyavailable alone, as well as formulated with DOPE.In addition to stabilisation properties, DOPE is alsothought to have fusogenic properties which are expected toplay a role in endosomal membrane disruption and soenhance escape of the lipoplexes into the cytoplasm(Ellens et al, 1986; Farhood et al, 1995; Vidal andHoekstra, 1995). However, because a selection oflipoplexes formed in the absense of DOPE are also able toescape the endosome (Behr et al, 1989; Vigneron et al,1996), these cationic lipids are also credited with intrinsicmembrane destabilisation properties.Since the initial ‘proof of principle’ period whichconfirmed the ability of cationic lipids to protect, transferand release DNA for cellular expression, a challengingperiod has followed. Indeed slow progress has been madein improving the level of transfection efficiency up to thatrequired for the potential therapeutic use of non-viralvectors, and this is largely attributed to an unclearstructure-activity relationship in vector design. Thus, thedevelopment of novel vectors is justified as the highlycomplex series of steps that connect the DNA outside thecell to its expression in the nucleus are not fullyunderstood and so a novel cationic lipid may not just be a‘me too’ addition to an already extended list, but ratheropen new possibilites for differently influencing thosesteps (Lehn, 1999). As a result, a diverse library of vectorsexists, representing a wide variety in structures and thusnumerous potential mechanisms by which bettertransfection levels might be obtained. Highlights of itscontents will be discussed in the following section.III. Design of the basic domains of acationic lipidAll cationic lipids are positively charged amphiphilescontaining the three following functional domains: i) apolar hydrophilic headgroup which is positively charged,generally via the protonation of one (monovalent lipid) orseveral (multivalent lipid) amino groups; ii) a linker whoselength and nature may influence the stability and thebiodegradability of the vector; and iii) a hydrophobicportion composed of alkyl chains (saturated orunsaturated) or of a steroid (Figure 1).275