Long-term Transduction of Retinal Pigmented Epithelial (RPE) Cells With a Bovine Immunodeficiency Viral (BIV) Vector

Abstract

Abstract: : Purpose: Human immunodeficiency viral (HIV) vectors mediate long-term transduction of many types of nondividing cells, but have safety concerns. Bovine immunodeficiency virus is a lentivirus that shares many characteristics with HIV, but does not cause human disease. In this study, we investigated the potential of BIV vector for ocular gene therapy. Methods: A green fluorescent protein (eGFP) reporter gene was packaged in recombinant BIV vector. The BIV.eGFP was grown in 293T cells, concentrated from cell supernatants, and titered. Adult C57BL/6 mice were given either an intravitreous injection of 5 x 10₄ or 5 x 10₅ viral particles, or a subretinal injection of 5 x 10₅ particles, and then GFP expression was assessed at several time points. Results: Examination of mice by indirect ophthalmoscopy with a blue filter or by in vivo fluorescence microscopy showed that subretinal injection of BIV.eGFP resulted in strong expression of GFP from the first examination at 1 week through the final examination at 20 weeks. Detection of GFP expression by ocular examination of living mice was possible in only a few mice that received an intravitreous injection of BIV.eGFP. Post mortem examinations showed prominent, but not completely uniform, GFP expression in RPE cells throughout the region of subretinal injection of vector. Occasional Muller cells and rarely other retinal cells in the overlying retina expressed GFP. The expression was stable from the first (2 weeks) to the last (20 weeks) time point. Postmortem examination of eyes given an intravitreous injection of BIV.eGFP showed transduction of cells in the ciliary epithelium, corneal endothelium, and a few scattered retinal cells. There was no evidence of inflammation or toxicity in any eyes. Conclusions: There is rapid onset, long duration transduction of RPE cells after subretinal injection of BIV.eGFP, suggesting that BIV vectors may be useful for ocular gene therapy targeting RPE cells.

Keywords: 419 gene transfer/gene therapy • 567 retinal pigment epithelium • 554 retina