from the complex. E2F then binds to the promoter region of the above cell cycle genes and activates their transcription. The consensus sequence TTTCGCGC is the binding site for E2F. Our strategy for inhibition of cell proliferation is the intracellular delivery of double-stranded ODN containing the TTTCGCGC sequence to act as a decoy to trap the released E2F (25). We synthesized a 14-mer as well as a 30-mer double-stranded ODN containing the consensus sequence and demonstrated that both are effective E2F based on competitive gel-shift assay. Using HVJ-liposomes, E2F decoy ODN was then introduced into cultured VSMCs, and it completely inhibited serum-stimulated growth. This growth inhibition was accompanied by reductions in PCNA and cdc2 kinase levels in these VSMCs. In contrast, mismatched decoy showed no inhibitory effect.
Based on these in vitro results, we examined the effect of E2F decoy on the prevention of neointimal hyperplasia in vivo. E2F decoy was transduced into balloon-injured rat carotid arteries using HVJ-liposomes. Our results demonstrated a marked suppression of neointimal formation at 2 weeks after balloon injury. In contrast, mismatched, scrambled, or progesterone responsive element decoy had no effect on neointimal development. Interestingly, we observed that a single administration of E2F decoy resulted in a sustained inhibition of neointimal formation up to 8 weeks after the treatment.
Gene transfer approach. Using HVJ-liposomes, we also attempted to inhibit neointimal formation by plasmid DNA gene transfer (26). Several studies had suggested NO could inhibit neointimal formation. For example, NO inhibited VSMC growth and migration in vitro. Systemic administration of a NO synthase inhibitor accelerated atherosclerotic lesion formation and impaired vascular reactivity. We therefore postulated that overexpression of endothelial cell NO synthase (ec-NOS) is an effective gene therapeutic strategy. Accordingly, we transfected balloon-injured rat carotid arteries with an expression vector containing the ec-NOS gene.
Four days after HVJ-liposome-mediated ec-NOS gene transfer into injured rat carotid arteries, significant levels of ec-NOS protein expression were detected. Consequently, NO production in the injured artery was enhanced by ec-NOS gene transfer. Two weeks after ec-NOS gene transfer, histological analysis revealed a 70% reduction in neointimal area as compared with the nontransfected injured artery (26). In contrast, no inhibition of neointima formation was observed in injured vessels undergoing control vector transfection. Since NO has multiple effects on the vessel wall, including vasorelaxation, inhibition of platelet aggregation, prevention of leukocyte adhesion, and suppression of VSMC growth and migration, we propose that our strategy to augment NO production may be an effective and practical approach to the gene therapy of restenosis.
Another important consideration for the therapy of restenosis is reendothelialization of the injured artery. Although several factors are known to stimulate endothelial cell growth, we have recently found that hepatocyte growth factor is a more potent accelerator of endothelialization than either vascular endothelial cell growth factor or basic fibroblast growth factor. In addition, unlike basic fibroblast growth factor, hepatocyte growth factor does not stimulate VSMC growth. We are therefore developing a strategy to prevent restenosis via the inhibition of VSMC growth using an AS, decoy, or NOS gene transfer approach in combination with the stimulation of endothelial cell growth by hepatocyte growth factor gene transfer.
Genetic engineering of vein grafts resistant to atherosclerosis. Saphenous vein grafts are the most commonly used bypass conduits for the treatment of occlusive vascular disease. However, up to 50% of vein grafts fail within a period of 10 years, primarily as a result of accelerated graft atherosclerosis. When grafted into arteries, veins are subjected to increased intraluminal pressure and undergo adaptive wall thickening. This thickening, however, involves neointimal hyperplasia, and this neointimal layer is believed to form the substrate for the aggressive atherosclerotic disease that eventually causes graft failure. We therefore hypothesized that a cytostatic strategy to prevent the hyperplastic response to the acute injury of grafting would redirect the biology of vein graft adaptation away from neointimal hyperplasia and toward medial hypertrophy (27). Rabbit jugular vein was isolated and transfected with AS ODN against PCNA and cdc2 kinase using HVJ-liposomes. The transfected vein was then grafted into the carotid artery. Neointima formation inhibited in the AS ODN-treated vein grafts for up to 10 weeks after surgery. In response to cell-cycle arrest with AS ODN, the genetically engineered vein grafts developed hypertrophy of the medial layer. When the rabbits were fed a high-cholesterol diet, accelerated atherosclerotic changes, characterized by plaque formation and macrophage infiltration, developed in the untreated and control ODN-treated grafts. In contrast, neither plaque formation nor significant macrophage infiltration was observed in any of the AS ODN-treated grafts, despite cholesterol feeding. These results establish the feasibility of developing genetically engineered bioprostheses that are resistant to failure and better suited to the long-term treatment of occlusive vascular diseases.
Treatment of glomerulosclerosis. We have also used E2F decoy oligonucleotide to ameliorate the changes seen in an animal model of mesangial proliferative nephritis. Injection of anti-Thy-1 antibody, which specifically injures glomerular mesangial cells, results in a proliferative glomerular lesion. We demonstrated that intrarenal arterial perfusion of HVJ-liposome complexes containing 14-mer E2F double-stranded decoy ODN inhibited anti-Thy-1-induced mesangial cells proliferation, as documented by BrdUrd incorporation and total glomerular cell counts. Furthermore, this decoy treatment prevented histopathologic changes in the glomeruli that closely mimic the mesangioproliferative nephritis seen in IgA nephropathy and in some forms of focal glomerular sclerosis.
The fusigenic viral liposome appears to be an effective tool for gene transfer and therapy. Our current system is an HVJ-liposome complex, but other viral fusion proteins may be applicable. In addition, in forming fusigenic liposome complexes, purified or recombinant fusion polypeptides may be used instead of the entire viral envelope. Since the system is a hybrid between viral and nonviral vectors, safety issues must be considered. It will be necessary to test the safety of UV-inactivated HVJ itself, as well as the safety of the liposome and the immunogenicity of the HVJ-liposome complex. HVJ-liposomes may be useful for short-term and local gene therapy. Modifications of this system will be necessary to permit high levels of stable expression of the transgene for clinical therapy.
1. Marshall, E. (1995) Science 265, 1050–1055.
2. Hopkins, N. (1993) Proc. Natl. Acad. Sci. USA 90, 8759–8760.
3. Yang, Y., Nunes, F.A., Berencsi, K., Furth, E.E., Gonczol, E. & Wilson, J.M. (1994) Proc. Natl. Acad. Sci. USA 91, 4407–4411.
4. Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F.H., Verma, I.M. & Trono, D. (1996) Science 272, 263–267.
5. Goyal, K. & Huang, L. (1995) J. Liposome Res. 5, 49–60.
6. Remy, J.-S., Kickler, A., Mordvinov, V., Shuber, F. & Behr, J.-P. (1995) Proc. Natl. Acad. Sci. USA 92, 1744–1748.
7. Wagner, E., Plank, C., Zatloukal, K., Gotten, M. & Birnstiel, M.L. (1992) Proc. Natl. Acad. Sci. USA 89, 6099–6102.
8. Cheng, L., Ziegelhoffer, P.R. & Yang, N.-S. (1993) Proc. Natl. Acad. Sci. USA 90, 4455–4459.
9. Kaneda, Y. (1994) in Cell Biolab: A Laboratory Handbook, ed. Celis, J.E. (Academic, New York) Vol. 3, pp. 50–57.
10. Okada, Y. (1993) Methods Enzymol. 221, 18–41.