Optimizing cardiovascular gene therapy - Increased vascular gene transfer with modified adenoviral vectors

Background: Adenovirus is widely used as a vector for gene transfer to the vasculature. However, the efficiency of these vectors can be limited by ine ffective viral-target cell interactions. Viral attachment, which largely de termines adenoviral tropism, is mediated through binding of the adenoviral fiber coat protein to the Coxsackievirus and adenovirus receptor, while int ernalization follows binding of the adenoviral RGD motif to ol,-integrin re ceptors. Modifications of the fiber coat protein sequence have been success ful for targeting the adenovirus to more prevalent receptors in the vascula ture, including heparan sulfate-containing receptors and alpha-integrin rec eptors. Hypothesis: Modified adenoviral vectors targeted to receptors more prevalen t in the vasculature result in an increased transfer efficiency of the viru s in vitro and in vivo even in the presence of clinically relevant doses of heparin. Design: We tested 2 modified E1- and E3-deleted Ad5 type adenoviral vectors containing the beta-galactosidase gene. AdZ.F(pK7) contains multiple posit ively charged lysines in the fiber coat protein that target the adenovirus to heparan sulfate receptors, while AdZ.F(RGD) contains an RGD integrin-bin ding sequence in the fiber coat protein that allows binding to alpha-integr in receptors. The gene transfer efficiency of these modified viruses was co mpared in rat aortic smooth muscle cells in vitro and in an in vivo porcine model of balloon-induced arterial injury. Because of the use of heparin du ring most vascular surgical procedures and the concern that heparin might i nterfere with the binding of AdZ.F(pK7) to heparan sulfate receptors, the e ffect of heparin on the in vitro and in vivo transfer efficiency of these 2 modified adenoviruses was evaluated. Results: In vitro infection of rat aortic smooth muscle cells with AdZ.F(pK 7) and AdZ.F(RGD) resulted in significantly higher levels of beta-galactosi dase expression compared with the unmodified adenovirus (mean +/- SEM, 1766 .3 +/- 89.1 and 44.8 +/- 3.4 vs 10.1 +/- 0.7 mU per milligram of protein; P < .001). Following heparin administration, the gene transfer efficiency ac hieved with AdZ.F(pK7) diminished slightly in a concentration-dependent man ner. However, the transfer efficiency was still greater than with the unmod ified virus (mean +/- SEM, 1342.3 +/- 101.8 vs 4.8 +/- 0.4 mU per milligram of protein; P < .001). In vivo, following injury to the pig iliac artery w ith a 4F Fogarty balloon catheter, we found that AdZ.F(pK7) transduced the artery approximately 35-fold more efficiently than AdZ.F and 3-fold more ef ficiently than AdZ.F(RGD) following the administration of intravenous hepar in, 100 U/kg body weight, and heparinized saline irrigation. Conclusions: Modifications of the adenovirus that lead to receptor targetin g resulted in significantly improved gene transfer efficiencies. These impr ovements in transfer efficiencies observed with the modified vectors decrea sed slightly in the presence of heparin. However, AdZ.F(pK7) was still supe rior to AdZ.F(RGD) and AdZ.F despite heparin administration. These data dem onstrate that modifications of adenoviral vectors that enhance binding to h eparan sulfate receptors significantly improve gene transfer efficiency eve n in the presence of heparin and suggest an approach to optimize gene trans fer into blood vessels.