Didier Branellec

The DispatchTM catheter as a delivery tool for arterial gene transfer

OBJECTIVEnMost currently available percutaneous delivery methods for arterial gene therapy are limited by the need for a long incubation period, which may lead to unacceptable tissue ischemia, especially in the coronary vasculature. Conversely, shorter incubation times may result in inefficient gene transfer, especially in atheromatous arteries. A new local delivery autoperfusion multichamber catheter is now available which permits local delivery in the coronary arterial system without inducing myocardial ischemia. The present study aimed at evaluating the performance of this catheter for achieving arterial gene transfer using replication-defective adenoviral vectors in normal and atheromatous arteries.nnnMETHODSnA replication-defective adenoviral vector carrying a nuclear-targeted beta-galactosidase reporter gene (Ad-RSV beta gal, 5.10(9) plaque-forming units [pfu]) was delivered to the iliac arteries of normal (n = 7) and atheromatous (1% cholesterol diet + arterial abrasion) (n = 6) rabbits, via a multichamber autoperfusion balloon catheter (Dispatch, SciMed). Duration of gene delivery was 60 min.nnnRESULTSnThree days later, marked expression of the reporter gene was detected by histochemistry in the endothelium at the delivery site (percentage of transfected cells: 16 +/- 8% / artery (range 11-25%). There was a low transduction rate in medial smooth muscle cells 0.7 +/- 0.4%/artery (range 0.3-1.1%). In atheromatous arteries, transduction was consistently achieved in the superficial layers of the neointima but was lower (1.1 +/- 0.5%/artery, range 0.3-1.7%). Transgene expression was detected by histochemistry in the liver of 3/13 animals, suggesting that there is a substantial risk of systemic dissemination of the viral vectors.nnnCONCLUSIONnEfficient arterial gene delivery to endothelial and superficial smooth muscle cells is feasible using local delivery of adenoviral vectors via the Dispatch autoperfusion catheter, in both normal and atheromatous arteries. This perfusion catheter may be a useful tool for coronary artery gene transfer.

Adenovirus-mediated delivery of the Gax transcription factor to rat carotid arteries inhibits smooth muscle proliferation and induces apoptosis

Adenovirus-mediated gene delivery in animal models of vascular injury has provided insights into the mechanisms underlying vessel wall pathologies. We have previously demonstrated that overexpression of the Gax transcription factor inhibits neointimal formation in rat and rabbit models of arterial injury. Here, we evaluate potential mechanisms for the reduction in stenotic lesion size due to Gax overexpression. At 3, 7 and 14 days after injury the Ad-Gax-infected arteries displayed a marked decrease in medial vascular smooth muscle cell number (3 days, 54% reduction P < 0.01; 7 days, 41% reduction p < 0.003; 14 days, 49% reduction p < 0.02). at 3 days after injury, pcna expression was attenuated in the ad-gax-treated vessels compared with control vessels (65% reduction p < 0.02), indicating a reduction in cellular proliferation. at 7 days and 14 days after injury ad-gax-infected arteries exhibited elevated number of tunel-positive medial vsmcs compared with control-treated arteries (7 days, 9.2-fold increase p < 0.03; 14 days, 17.2-fold increase p < 0.03), indicating an induction of apoptotic cell death. these data suggest that deregulated gax expression induces first cell cycle arrest and then apoptosis in the vascular smooth muscle cells that contribute to the neointimal layer. therefore, the efficacy of this therapeutic strategy appears to result from the ability of the gax transcriptional regulator to modulate multiple cellular responses.

Pre-treatment with elastase improves the efficiency of percutaneous adenovirus-mediated gene transfer to the arterial media.

The endothelium and internal elastic lamina (IEL) appear to be the main barriers to adenovirus-mediated gene transfer to medial smooth muscle cells (SMC). The present randomized study tested whether controlled incubation with elastase enhanced the efficiency of catheter-based gene transfer to medial SMC by adenoviral vectors. After an initial safety dose ranging study, rabbits underwent balloon abrasion of the iliac endothelium followed by local incubation of either elastase (2u2009×u200910−7 IU over 5u2009min) or saline using a double balloon catheter (DBC). Then, adenoviral vectors (5u2009×u2009109 p.f.u.) carrying Cmv-Luc or RSV-βgal reporter genes were instilled for 30 min. Three days later, the number of medial SMC expressing lacZ was increased in the elastase-treated arteries compared with saline-treated arteries (7.2u2009±u20092.5 versus 2.3u2009±u20090.9 cells per section, Pu2009=u20090.003). Likewise, the amount of luciferase protein product was increased (70u2009±u200932 versus 36u2009±u200915u2009pg luciferase/mg tissue, Pu2009=u20090.03). No vessel enlargement, light or electron microscopic evidence of injury or inflammation was seen in elastase-treated arteries up to 7 weeks. Preincubation with elastase increased transduction efficiency of catheter-based gene delivery of replication-defective adenoviral vectors to rabbit iliac arteries without detectable arterial damage.

Intramuscular gene transfer of fibroblast growth factor-1 using improved pCOR plasmid design stimulates collateral formation in a rabbit ischemic hindlimb model.

Fibroblast growth factor 1 (FGF1) is an angiogenic factor known to play a role in the growth of arteries. The purpose of this study was to evaluate the usefulness of direct intramuscular injection of an optimized expression plasmid encoding FGF1 to augment collateral formation and tissue perfusion in a rabbit ischemic hindlimb model. Truncated FGF1 fused to the human fibroblast interferon (FIN) signal peptide was expressed from a newly designed plasmid backbone with an improved safety profile for gene therapy applications. In vitro, optimization of plasmid design yielded in a dramatic increase in expression efficiency for FGF1, independent of the presence of a signal peptide, as analyzed by Western Blotting. In vivo, successful transgene expression could be demonstrated by FGF1 immunostaining after gene application. FGF1 plasmid containing FIN signal peptide (100, 500, and 1,000xa0μg), when injected into ischemic muscle areas of rabbits 10 days after ligation of the external iliac artery, exhibited a pronounced therapeutic effect on collateral formation to the ischemic hindlimb in a dose-depending manner, as assessed by physiological (blood pressure ratio, maximal intra-arterial Doppler flow) and anatomical (angiographic score, histologic evaluation of capillary density) measurements 30 days after therapy, compared to saline or lacZ control plasmid. FGF1 plasmid without a signal peptide sequence resulted in a comparable therapeutic effect on collateral formation at comparable doses (500 and 1,000xa0μg). Our results indicate that intramuscular FGF1 gene application could be useful to stimulate collateral formation in a situation of chronic peripheral ischemia. The presence of a signal peptide does not seem to be obligatory to achieve bioactivity of intramuscular transfected FGF1. An optimized vector design improved both biosafety of gene transfer and expression efficiency of the transgene, rendering this vector highly suitable for human gene therapy. Therefore, this new generation vector encoding FGF1 might be useful as an alternative treatment for patients with chronic ischemic disorders not amenable to conventional therapy.