Ross River Virus Pseudotyped HIV–1 Efficiently and Stably Transduces Rodent Müller Cells In Vitro

Abstract

Abstract: : Purpose: Efficient transduction and subsequent expression of a transgene in rodent Müller cells may be a valuable tool for both therapeutic and physiological investigations of the retina. However, previous studies using both lentiviral and adeno–associated viral vectors have shown extremely limited Müller cell transduction following either subretinal or intravitreal injection in rodents. This study demonstrates that primary isolated rat Müller cells are efficiently transduced by a lentiviral vector when pseudotyped with the envelope glycoprotein from the Ross River Virus. Methods:Recombinant HIV–1 pseudotyped with the Ross River Virus (RRV) envelope glycoprotein E2E1A was packaged through calcium phosphate transient transfection of four plasmids into 293T cells. High titer viral stocks of 10¹⁰ infectious units/ml, were obtained by ultracentrifugal concentration of the cell supernatant. Primary Müller cell cultures were established from excised retinas of Fisher rats and purity was confirmed by immunostaining with antibodies to CRALBP, Vimentin, and GFAP. Müller cell cultures were infected with virus at various multiplicities of infection (MOI), and viral transduction was determined 4 days to 60 days post infection by quantitative PCR of integrated viral genomes using a Taqman probe. RNA particle titers were determined by RT–Q–PCR of cell supernatants and concentrated vector stocks. Results:Rat Müller cells are efficiently transduced by RRV pseudotyped HIV–1 after infection with virus at MOIs as low as 10 particles per cell. Transduction was detected by Q–PCR up to 60 days post infection, suggesting stable integration of the virus. This vector transduces Müller cells 20 times more readily than HEK 293T cells, which are typically used for functional titer determination. Conclusion:These results indicate HIV–1 can be pseudotyped by envelope glycoproteins derived from RRV, and that this vector construct may be concentrated by ultracentrifugation with minimal loss in titer. Furthermore, this vector readily transduces primary rat Müller cells in vitro. We are currently testing several RRV–pseudotyped vectors containing both Müller cell specific and ubiquitous promoters to drive GFP reporter expression in vitro and in vivo.

Keywords: Muller cells • AIDS/HIV • gene transfer/gene therapy