GTMB 7 - Gene Therapy & Molecular Biology

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Martin et al: Advances in cationic lipid-mediated gene deliveryFigure 7: The use of redox-sensitive linkers in cationic lipid design.algorithm that it follows being stored in the componentsand operating via selective molecular recognition events(Lehn, 1993).IV. Structural features ofsupramolecular assemblies formed bylipoplexesAs gene transfection by cationic lipids involves thespontaneous formation of discrete lipid/DNA particles, itmay be expected that the transfection efficiency of a givencationic lipid system depends not only on the properties (atthe molecular level) of the cationic lipid itself, but also onthe structural and functional characteristics of the selfassembledsupramolecular assemblies formed by thelipoplexes. Because of the extensive characterisation thatwe have undertaken on the BGTC-based lipoplexesprepared by our group (Pitard et al, 1999), the structuralfeatures of these assemblies will be summarised here andcompared with the results obtained by others when usingother cationic lipid systems.The structural features of BGTC-based lipoplexeswere visualised by cryotransmission electron microscopy(cryo-TEM) which has the advantage of imagingbioassemblies close to their native state. With theadditional perspective given by data from synchrotronsmall-angle x-ray scattering (SAXS), precise structuraldetails could be resolved. The results obtained indicatedthe presence of highly ordered multilamellar domains witha regular spacing of 70 _ and 68 _ in BGTC/DOPE/DNAand BGTC/DNA lipoplexes, respectively (Pitard et al,1999). It can be assumed from this data that the DNA isintercalated between the lipid bilayers. Interestingly, DNAlipoplexes with similar lamellar patterns were alsodetected inside transfected HeLa cells by conventionalelectron microscopy and as such may be the “active”lipoplexes in the process of trafficking. Further, theselamellar lipoplexes were mainly detected in intracellularvesicles, a finding suggesting that endocytosis was themajor route of uptake.Characterisation of the structural features oflipoplexes used for gene transfection has always been anactive area of research. A hypothetical “bead on thestring” model in which unmodified cationic liposomeswere distinctly attached to the DNA was originallyproposed (Felgner and Ringold, 1989). Over the years,various electron microscopy techniques have then beenused to visualise the structures of the lipoplexes formed bydifferent cationic lipids. These studies essentiallysuggested that the DNA was entrapped in condensedstructures formed by interrelated lipid fusion and DNAcollapse for which thermodynamic models have beenproposed in order to facilitate analysis and understandingof the process of particle formation (Gershon et al, 1993;Ahearn and Malone, 1999). These condensed structureswere found to exhibit various ordered patterns ofsupramolecular organisation, including multilamellarstructures and direct or inverted hexagonal packing(Gustafsson et al, 1995; Labat-Moleur et al, 1996; Lasic etal, 1997; Lasic et al, 1998). Of particular note is the studyof DC-Chol/DOPE/DNA lipoplexes where in addition toconcentric lamellar structures, tubular “spaghetti-like”structures consisting of DNA rods coated by a single lipidbilayer were observed (Sternberg et al, 1994). As concernsx-ray diffraction studies, lamellar domains with aperiodicity similar to that found in BGTC lipoplexes wereobserved with DOTAP/DOPC (dioleoylphosphatidylcholine, a structural analogue of DOPE),DDAB and lipopolyamines (Lasic et al, 1997; Pitard et al,1997; Radler et al, 1997; Safinya and Koltover, 1999).However, a study has shown that DOTAP/DOPE/DNA282
Gene Therapy and Molecular Biology Vol 7, page 283lipoplexes have an inverted hexagonal organization whichwas attributed to the presence of DOPE (as a multilamellarordering was found when DOPC was used instead ofDOPE) and it was further suggested that such an invertedhexagonal arrangement favoured endosomal escape of thelipoplexes via its bilayer-destabilising activity (Koltover etal, 1998).In conclusion, it should be emphasised here thatelucidation of the structural and functional features of the“active” lipoplexes may require the determination not onlyof their initial features but also of the structural changesthat may be induced in the lipoplex by the environmentsthat are encountered during trafficking to the nucleus. Forexample, it has been demonstrated that an interaction withthe microtubules is involved in the cellular trafficking ofthe lipoplex. Fluorescently tagged lipid I/DOPE lipoplexes(Figure 4) were found to be co-localised withmicrotubules using laser scanning confocal microscopy incells expressing fluorescently tagged tubulin (Hasegawa etal, 2001). Equally, live-cell real-time multiple particletracking has been used to observe the trafficking of thecationic polymer polyethylenimine (PEI) (Boussif et al,1995) through the cell, presumably by the same process(Suh et al, 2003). These results provide a striking parallelwith virus-mediated uptake which is known to be‘directed’ through the cytoplasm (Tang et al, 1999; Goff,2001; Seisenberger et al, 2001) rather than relying onrandom diffusion, which was expected to be the onlymode of transport available to non-viral vector/DNAcomplexes.V. Inclusion of ligands for nuclearuptakeAs vectors have become increasingly sophisticated,it has become clearer that significant advances can only bemade if cationic lipid design also addresses the mostconsiderable barrier to gene delivery: the passage acrossthe nuclear membrane. Indeed, experiments comparingtransgene expression after microinjection of plasmid DNAeither into the cytoplasm or directly into the nucleus haveidentified the nuclear membrane as being a severelylimiting step in transgene delivery (Capecchi, 1980;Zabner et al, 1995; Escriou et al, 1998). Accordingly, thesuccess of the current non-viral vectors may at least partlyrely on the disappearance of the nuclear membrane duringmitosis, as transfection is generally found to depend on theproliferation status of the cells (Fasbender et al, 1997;Oudrhiri et al, 1997; Mortimer et al, 1999; Escriou et al,2001). As such it is suggested that gene delivery duringinterphase is likely to be low. When adding to this therelatively instability of plasmid DNA in the cytoplasm(probably due to degradation by Ca 2+ -sensitive nucleases(Lechardeur et al, 1999; Pollard et al, 2001), the need foran efficient method of crossing the nuclear membranebecomes evident.The nuclear membrane consists of two concentriclipid bilayers which make contact at numerous points,forming aqueous pores through the two membranes, whichare termed the nuclear pore complexes (NPCs) and havean internal diameter of roughly 9 nm (Mazzanti et al,2001). These are the only known route for direct exchangeof substances between the cytoplasm and the intranuclearcompartment. Consequently, the transport of largemolecules such as plasmid DNA is non-passive andrequires signal mediation by shuttling molecules(importins) (Boulikas, 1997; Escriou et al, 2003). Thebasic peptide from the SV40 large tumor antigencharacterised by a PKKKRL amino acid sequence acts as anuclear localisation signal (NLS) and has been used inattempts to induce nuclear uptake of plasmid DNA.Binding of multiple NLSs to plasmid DNA (Collas et al,1996; Ciolina et al, 1999; Neves et al, 1999) was found tobe bettered by attachment of just a single NLS peptide toone end of a capped linear DNA fragment (Zanta et al,1999). However, although transfection (mediated by PEIor DOGS) was enhanced by an impressive 10-1000 foldwith the single NLS-DNA conjugate, the techniqueremains only a proof of principle as preparation of such aDNA fragment requires complex engineering and istherefore relatively impractical for gene therapyapplications.An alternative approach to the use of NLSs fornuclear uptake of plasmids is steroid-mediated genedelivery. This technique relies on binding of thetransfected plasmid to the glucocorticoid receptors (GRs),which thus stimulated, actively transport the boundplasmid into the nucleus. The steroid dexamethasone wasused as a GR-binding motif and so attached to a DNAbinder (psoralen) via a short spacer (Rebuffat et al, 2001).The small increase in transfection activity seen incomparison to lipofection of unmodified DNA in dividingcells, became more pronounced (15-40 fold) in nondividingcells. Importantly, increasing the number of GRtargeting molecules attached to the DNA (via psoralencoupling and thus without regiospecificity) led to loss oftransgene expression probably due to covalent damage tothe reporter gene sequence in the plasmid. These studieswere therefore extended, and the preparation of a peptidenucleic acid (PNA) ‘clamp’ followed, which wasdemonstrated to link dexamethasone to the DNA at adefined position where transgene expression remainedunhindered (Rebuffat et al, 2002).VI. Extracellular barriers andrequirements of in vivo lipofectionThe applicability of cationic lipids for in vivo genedelivery was investigated following their provenefficiency for in vitro gene transfection into a greatdiversity of established cell lines and primary cell cultures.Although some promising results have been reported, suchas the transfection of the airway epithelium via the airwaypassages, numerous studies in animals yielded much lesssatisfactory results, especially as regards systemicadministration (Gao and Huang, 1995; Miller, 1998).Accordingly, in the first gene therapy clinical trials in manwith cationic lipids, the lipoplexes were applied to thepatients via in situ administration such as intranasalinstillation or direct intratumoral injection (Hersh and283
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GTMB 7 - Gene Therapy & Molecular Biology

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