IMPROVED RETROVIRAL TRANSDUCTION EFFICIENCY OF VASCULAR CELLS IN-VITRO AND IN-VIVO DURING CLINICALLY RELEVANT INCUBATION PERIODS USING CENTRIFUGATION TO INCREASE VIRAL TITERS

Vascular cells are an important target for gene transfer because of th eir potential to deliver gene products both locally and systemically. Direct retroviral gene transfer to vascular cells in vivo has been lim ited by inefficient rates of transduction. We hypothesized that vascul ar cell transduction efficiency (TE), during shea retroviral incubatio n periods, is significantly improved in vitro and in vivo using centri fugation to increase viral titer. Furthermore, we hypothesized a linea r relationship between concentration of viable viral particles (measur ed as colony-forming units (CFUs)/cell) and retroviral TE during short incubation periods. Cultured rat pulmonary artery endothelial cells ( RPAECs), rat aortic smooth muscle cells (RSMCs), and human iliac arter y endothelial cells (HIAECs) demonstrated a strong correlation between TE and high concentrations of virus (>100 CFU/cell) during retroviral incubation periods of 10 to 60 minutes. High titers, and thereby high concentrations, were achieved by centrifugation and resuspension in a fraction of the original volume. Titer was consistently increased ten fold, for a twentyfold increase in concentration by volume. A 20-minut e incubation with a Moloney murine leukemia-derived retroviral vector coding for human placental alkaline phosphatase, pLJhpAP, at a concent ration of 1150 CFU/cell yielded TEs of 10.6% +/- 0.7%, 40.4% +/- 1.6%, and 15.1% +/- 2.0% for RPAECs, RSMCs, and HIAECs, respectively. A sim ilar effect was shown using the Moloney murine leukemia-derived MFGlac Z retroviral vector, coding for Escherichia coli beta-galactosidase. I ncreased titer and concentration had no effect on target cell viabilit y, as shown by trypan blue exclusion. Although RSMCs had the most cell s transduced in a given incubation period (p < 0.05), RPAECs had the h ighest replication rate (p < 0.05), suggesting the importance of facto rs other than cell cycle on retroviral TEs during short, clinically re levant incubation periods. In subsequent in vivo experiments, gene tra nsfer was achieved in the rat carotid artery during a 20-minute incuba tion period infusing the concentrated pLJhpAP retrovirus after carotid balloon injury. Rats infused with virus 2 days after balloon injury e xhibited hpAP activity (0 to 10 cells/section/rat) in the neointima of five out of six rats. Rats infused 4 days after balloon injury exhibi ted hpAP activity (0 to 25 cells/section/rat) in the media and adventi tia of five out of five rats. Control rats that received the balloon i njury alone or the balloon injury and unconcentrated retrovirus exhibi ted zero hpAP activity. We conclude that the TE of retroviral-mediated gene transfer to vascular cells in vitro and in vivo can be improved during short, clinically relevant incubation periods using centrifugat ion to increase retroviral titer, and thereby concentration of viable viral particles.