738. Optimizing a Lentiviral Vector System for Efficient Transduction of Primary Rat Cardiovascular Cells In Vitro

Abstract

Cardiovascular disease (CVD) is the number one cause of mortality in the United States. However, little progress has been made in the development of effective gene therapy treatments for cardiovascular pathologies. Recently, lentiviral vectors based on HIV-1 have been developed and optimized, and have ideal characteristics for gene therapy applications to treat CVD, particularly in myocardium and vascular smooth muscle. To achieve our goal of efficient gene transfer in vivo, in vitro systems needed to be developed to assay modified lentiviral vector transduction into isolated primary rat cardiomyocytes and vascular smooth muscle cells (VSMCs). In our initial studies, lentiviral vector transduction efficiency was examined using vectors pseudotyped with the vesicular stomatitis virus G protein (VSV-G) and containing the bacterial lacZ gene driven by a number of different viral and cellular promoters, including phosphoglycerokinase (PGK), cytomegalovirus (CMV), ubiquitin (Ub) and elongation factor 1a (EF1a), CMV-EF1a hybrid (CEF) and cardiac-specific promoter α-myosin heavy chain (αMHC). All of the promoters were found to function in the cardiovascular cells, even at MOI's below 1. The promoter with the highest transduction efficiency was CEF (3.37 × 105 T.U./ng p24; n=5 preparations in triplicate) as determined by end-point dilution and p24 Gag ELISA, whereas the PGK promoter resulted in the lowest lentiviral vector titer (7.14 × 102 T.U./ng p24; n=5 preparations in triplicate). The CEF promoter was then used to determine lentiviral vector transduction efficiency in isolated rat primary VSMCs from the jugular vein and thoracic aorta. The VSMCs were difficult to infect, with multiple infections (MOI=10/infection) being necessary to achieve high infectivity (i.e., greater than 85% of the VSMCs). Interestingly, the cells from the aorta were slightly more transducible than those from the jugular vein. The relatively low infectivity in the VSMCs may be one reason that VSV-G pseudotyped lentivectors have been unable to effectively transduce these cells in vivo. We have analyzed each of the promoters to assess their ability to drive gene expression in primary rat cardiomyocytes. Following the infection of the cardiomyocytes at a MOI of 1, we determined the β-gal transgene expression for each of the promoters, and found that the PGK promoter produced the highest level of β-gal protein (1497 ± 129 pg β-gal/mg total protein; n=5 preparations in triplicate) and no β-gal expression was observed in the mock treated cells. No significant differences were observed in lentiviral vector transduction and/or gene expression using lentiviral vectors that either contained or were devoid of the accessory proteins, specifically vif, vpr, vpu and nef. Current studies are being performed with these lentivectors to determine if the in vitro experiments in the primary cardiomyocytes correlate with in vivo transduction using different routes of administration into the heart. These studies should help to better illuminate lentiviral vectors as a gene therapy tool for use in cardiovascular disease in basic and clinical research. Funded by NHLBI 18766–01.