The successful blockade of multiple cell cycle genes via the delivery of a single decoy ODN directed against E2F, as well as the subsequent inhibition of neointimal hyperplasia observed in a model of arterial injury, prompted us to consider the application of this strategy to human therapy. We selected for our first therapeutic target human bypass vein graft failure, a process characterized by neointimal hyperplasia and accelerated atherosclerosis. Despite long-term failure rates that approach 50%, autologous vein grafts remain the most widely used conduit for surgical revascularization in roughly 600,000 patients annually in the US who suffer from occlusive disease of the coronary or lower extremity circulations. Failures, which can lead to reoperation, amputation, or heart attack, can be traced to the development of a neointimal layer during the postoperative remodeling of the initially thin-walled vein in its new high-pressure arterial environment (6
). Although such neointimal thickening relieves tangential wall stress in the bypass vessel (7
), the expression of cytokines, adhesion molecules, and chemoattractants by the activated neointimal smooth muscle cells is associated with dysfunction of the overlying endothelium, leukocyte invasion of the vessel wall, and the development of an aggressive, accelerated form of atherosclerosis (Figure ) (6
Figure 2 Vein grafts are initially thin-walled vessels that must undergo wall thickening to resist increased stress in the arterial circulation. The neointimal hyperplasia that produces this thickening, however, involves the proliferation of activated smooth muscle (more ...)
We hypothesized that E2F decoy–mediated arrest of cell cycle progression during the early postoperative period would block this neointimal hyperplastic response to the acute injuries associated with grafting. Furthermore, this intervention would allow a more desirable hypertrophic response in the medial layer, which would help the vessel accommodate to the chronic hemodynamic stress of the arterial circulation. Indeed, a single, intraoperative transfection of rabbit vein grafts with E2F decoy provided a long-term inhibition of neointimal hyperplasia and led to a shift toward medial hypertrophy, which succeeded in reducing wall stress to near-arterial levels (9
). Furthermore, the inhibition of neointimal hyperplasia helped preserve vein graft endothelial function and prevented diet-induced graft atherosclerosis for at least 6 months after operation (9
On the strength of these preclinical data, we initiated a clinical program of prospective, double-blind, randomized evaluation of E2F decoy transfection of human bypass vein grafts (11
). The initial study involved a small cohort of infrainguinal bypass patients, including a large proportion of individuals at high risk for neointimal disease and graft failure. Because of the anatomic location of the vein grafts, we were able to use ultrasound (12
) to monitor them for critical lesion formation in a reliable and noninvasive manner. To carry out ex vivo E2F decoy transfection, we exposed the grafts to a “naked” ODN solution under a nondistending pressure of 300 mmHg, in a process that required only 10–15 minutes and could be carried out while the patient’s leg was prepared for grafting. Little, if any, systemic exposure to the E2F decoy occurred during this procedure. Small specimens of transfected vein were collected for laboratory evaluation, which revealed an 89% transfection efficiency and a sequence-specific inhibition both of target cell cycle mRNA expression and of vascular cell replication in an organ culture assay. This ex vivo, pressure-mediated E2F decoy transfection of human vein grafts did not influence either postoperative complication rates or blood counts and chemistries.
Despite the small size of this preliminary, single-center trial, the choice of a high-risk cohort allowed us to compare the occurrence of primary graft failure (graft occlusion or a need for invasive graft revision) between patients who did and those who did not receive the E2F decoy treatment. We found that, whereas failures occurred among the untreated cohort throughout the 12-month follow up period, no failures were observed among the E2F decoy–treated grafts beyond the first 6 months, suggesting that the adaptive remodeling seen in preclinical experiments also occurred in the human grafts (Figure ). Larger-scale clinical trials will be necessary to verify the results, but this work, one of the first attempts to apply an ex vivo gene-based strategy to treat a common cardiovascular disorder, offers the first evidence that human gene expression can be modulated for therapeutic purposes using a transcription factor decoy.
Figure 3 Primary vein graft patency with or without intraoperative treatment of the grafts with E2F decoy oligonucleotides. In a small cohort of patients at high risk for primary graft failure, primary patency at 12 months was improved with E2F decoy blockade (more ...)