GTMB 7 - Gene Therapy & Molecular Biology

George et al: Gene therapy for vascular diseasesA. Biological processes involved inrestenosis and molecular targets in vein graftfailureA complex series of biological events is initiated inthe vein immediately after implantation into the arterialcirculation. Within the first few days after implantationmany vein grafts fail due to thrombosis, stimulated byendothelial injury (Bryan et al, 1994). Furthermore, in thefirst 24 hours vein grafts undergo a period of ischemiafollowed by reperfusion, which leads to the generation ofsuperoxide and other reactive oxygen species that triggerscytoxicity of endothelial and smooth muscle cells (Shi etal, 2001; West et al, 2001). The grafted vein is thentargeted by an acute inflammatory response involvingneutrophil and mononuclear cell recruitment and oxidativestress persists (West et al, 2001). In the first week afterimplantation matrix remodelling and migration of smoothmuscle cells into the intima takes place; once in the intimathe smooth muscle cells proliferate contributing further tothe intimal thickening (Newby et al, 1996). Each of theseprocesses offers a set of potential molecular targets forgene therapyapy.B. Anti-thrombotic and accelerated reendothelializationstrategiesAnti-thrombotic strategies have been investigated asa relevant target for gene transfer to reduce thrombosis invarious models of arterial injury and thrombosisformation. Thrombosis is dramatically reduced usingnatural anti-thrombotic, anti-aggregatory, and fibrinolyticpathways such as overexpression of thrombomodulin(Waugh et al, 1999), tissue factor pathway inhibitor(Nishida et al, 1999; Zoldhelyi et al, 2000), CD39(Gangadharan et al, 2001) and tissue plasminogenactivator (Waugh et al, 1999). Despite their provensuccess, the potential of these anti-thrombotic strategieshas not been widely tested in vein graft models perhapsdue to the availability of pharmacological treatments.However, acceleration of re-endothelialization by genetransfer of C-type natriuretic peptide in rabbit jugular veingrafts reduced both thrombosis and intimal thickening(Ohno et al, 2002). This illustrates that promoting reendothelializationand reducing thrombosis is a promisingstrategy to circumvent vein graft failure.C. Anti-proliferative strategyIn an attempt to inhibit VSMC proliferation in veingrafts both overexpression of cell cycle inhibitory proteinsand inhibition of cell cycle promontory genes usingantisense has been investigated in arterial injury and veingraft models. In fact it is thought that strategies targetingmultiple cell cycle genes offer greater potential than singletargets. Rabbit vein grafts treated simultaneously withantisense oligonucleotides to proliferating cell nuclearantigen (PCNA) and cell division cycle-2 kinase showedreduced intimal thickening and diet inducedatherosclerosis (Mann et al, 1995).Recently, transfection of cis-element double-strandedoligonucleotides (decoy ODNs) has been reported as anew powerful tool in a new class of anti-gene strategiesfor gene therapyapy. Transfection of double-strandedODN corresponding to the cis sequence will result inattenuation of the authentic cis-trans interaction, leading toremoval of trans-factors from the endogenous cis-elementswith subsequent modulation of gene expression. A decoyto E2F, which induces the coordinated expression of anumber of critical cell cycle genes, including PCNA,cyclin-dependent kinase-1, cell division cycle-2 kinase, c-myc, c-myb, was used successfully. This E2F decoy ODNnot only almost completely inhibited intimal thickeningafter balloon injury of the rat carotid at two weeks afterinjury (Morishita et al, 1995), but sustained inhibition wasobserved after eight weeks. This inhibition of intimalthickening was also observed using a porcine coronaryartery model (Nakamura et al, 2002). Furthermore, asingle intraoperative pressure-mediated delivery of E2Fdecoy effectively provided vein grafts with long-term (upto 6 months) resistance to intimal thickening andatherosclerosis (Ehsan et al, 2001). Interestingly, it hasbeen demonstrated that although E2F decoy ODNtreatment of vascular grafts inhibits VSMC proliferationand activation, it spares the endothelium, thereby allowingnormal endothelial healing (Ehsan et al, 2002). A clinicaltrial (PREVENT) using intraoperative delivery of E2Fdecoy ODN to infrainguinal arterial bypass graftsdemonstrated fewer graft occlusions, revisions, or criticalstenoses in the E2F-treated group (Mann et al, 1999).Recently, a corporate-sponsored (Corgentech, Inc, PaloAlto, Calif) phase II trial of E2F decoy treatment ofcoronary vein grafts was completed (SoRelle 2001). Thisstudy, which involved 200 patients revealed a 30%reduction in critical stenosis and has formed the basis fordesign of a phase III trial in coronary bypass grafting.Furthermore, on the basis of this combination ofpreclinical and phase I/II clinical data, a phase III trial ofE2F decoy ODN for the prevention of lower extremityvein graft failure involving 1400 patients was initiated inDecember 2001.D. Pro-apoptotic strategyIn addition to the above-mentioned cytostaticapproaches, cytotoxic strategies have also beenconsidered. Delivery of TIMP-3, which in addition toinhibiting MMP activity and VSMC migration promotesVSMC apoptosis significantly reduced intimal thickeningin a porcine vein graft model (George et al, 2000).Adenoviral delivery of wild type p53 which promotesVSMC apoptosis has also been studied in humansaphenous vein in vitro studies (George et al, 2001).Induction of VSMC apoptosis by overexpression of p53,without a detectable reduction in VSMC proliferation, ledto a significant reduction, >70%, in intimal thickening(George et al, 2001). Studies using a porcine arteriovenousbypass model are currently been underway todetermine if this cytostatic strategy reduces intimalthickening in vivo. Despite initial concerns, this proapoptoticstrategy with TIMP-3 and p53 did not lead to a138