GTMB 7 - Gene Therapy & Molecular Biology

Martin et al: Advances in cationic lipid-mediated gene deliverywith the evolutionary time-scale upon which these abilitieshave been honed. Although it seemed therefore natural toharness viruses (among them adenoviruses, adenoassociatedviruses and retroviruses) for therapeutic genedelivery (Mulligan, 1993; Kootstra and Verma, 2003), itcan be contested that much of their inconveniences are yetto be discarded. Problems with immunogenicity andtoxicity remain, added to the practical issues of large scaleproduction and quality control.Focus has therefore shifted to a de novo approach invector design, where synthetic organic molecules are usedto bind the transgene and facilitate its passage across thesignificant extracellular and intracellular barriers thatseparate it from the cell nucleus where expression takesplace via the cellular transcription machinery (Crystal,1995; Lehn et al, 1998). Such carriers are termed non-viralvectors and generally take the form of cationic lipids orcationic polymers. In addition to avoiding problemsassociated with the use of recombinant viruses, anadvantage of using synthetic vectors is that there is nolimit on the size of DNA to be delivered. A large numberand wide variety of synthetic non-viral vectors have beenprepared and their transfection efficiency assessed notonly in in vitro and in vivo experimental studies, butfurther, into the clinical setting for treatment in particularof cancer (Roth and Cristiano, 1997; Hersh and Stopeck,1998) and cystic fibrosis (Alton et al, 1999; Boucher,1999; Griesenbach et al, 1999; Davies et al, 2001). Anexhaustive list of clinical gene therapy trials is available atwww.wiley.co.uk/genmed/clinical.Despite some positive results, the overall outcomeindicates that a critical requirement for successful genetherapy is the use of more efficient gene delivery systems,i.e. systems leading to a higher percentage of transfectedcells or an increased amount of transgene protein in thetransfected cells according to the given experimental orclinical situation (Crystal, 1995; Aissaoui et al, 2002;Miller, 2003). This review aims to highlight the recentadvances in improving cationic lipid-mediated genedelivery in terms of overcoming both intracellular andextracellular barriers to gene transfer. This will be dealtwith by firstly surveying the progress made in vectordesign at the molecular level. Structure and functionalityof the cationic lipid/DNA complexes will then bedescribed with special focus placed on our own work withnovel lipids. Finally, the stability of the lipoplex in theextracellular medium and the features of its intracellulartrafficking towards the cell nucleus will be discussed, aswell as the proposal of creating sophisticated modular selfassemblinggene delivery systems incorporating variousfunctional elements to face the barriers encountered. Inshort, the goal is the development of ‘virus-like’ systems,which share the levels of gene delivery efficiency of viralcounterparts, but coupled with the safety of purpose-madeorganic molecules.II. Basic principlesThe first stage in the preparation of particles suitablefor gene delivery is the condensation of the large DNAmolecules by the vectors. The general structure of acationic lipid vector is shown in Figure 1. The cationicnature of the amphiphilic vector drives an electrostaticinteraction in the presence of negatively charged DNA,spontaneously self-assembling into nanometricvector/DNA complexes termed lipoplexes (stage 1, Figure2). This initial compaction step enables protection of theDNA from nucleases which are found in the extracellularmedium. Use of an excess of cationic vector (quantified bythe lipid/DNA ratio resulting in a mean theoretical chargeratio of the lipoplex (+/-)) conveniently decorates the outersurface of the lipoplex with a net positive charge which isgenerally considered to facilitate subsequent cellularuptake by interaction with negative cell surface residuessuch as proteoglycans (Friend et al, 1996; Labat-Moleur etal, 1996). Non-specific endocytosis ensues, encapsulatingthe lipoplex in intracellular vesicular compartments(Zabner et al, 1995) (stage 2), though fusion-based uptakecannot be entirely ruled out (Gao and Huang, 1995).Internalisation achieved, the DNA must avoid degradationin the late endosome and lysosome (barred arrow) byescaping the endosome to the cytoplasm (stage 3) (Zabneret al, 1995; Mukherjee et al, 1997). Trafficking of theDNA to the perinuclear region precedes passage across thenuclear membrane (stage 4) and subsequent expression ofthe transgene (stage 5). When localised within the nucleus,the DNA is already separated from its vector (Hasegawa etal, 2001) and it has been shown by microinjectionexperiments (Zabner et al, 1995) that gene expression doesnot occur if the complex remains intact.Cationic lipids designed to achieve the ambitioustask of gene delivery were first introduced by Felgner etal, whose work was founded on initial attempts to transfernucleic acids via encapsulation into classical liposomes(Nicolau and Sene, 1982; Nicolau et al, 1983).The lipid DOTMA (N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethyl ammonium chloride) resulted, consistingof a quaternary amine connected to two unsaturatedaliphatic hydrocarbon chains via ether groups (Felgner etal, 1987) (Figure 3).Figure 1: Schematic representation of a cationic lipid: lipid moiety, linker and cationic headgroup274