Efficient Delivery of Genes Into Photoreceptors by Lentiviral Vectors: Application for Treatment of Stargardt Disease

Abstract

Purpose:: Stargardt disease (STGD) is a macular dystrophy caused by mutations in the ABCA4 (ABCR) gene. The most recognized phenotypic feature of STGD patients and the mouse model, the Abca4^-/- mice, is lipofuscin (A2E) accumulation in retinal pigment epithelium (RPE). We tested whether we could efficiently deliver lentiviral vectors which carry either the normal (wt) human ABCA4 gene, or the LacZ reporter gene, to photoreceptors (PR) of the Abca4^-/- mice and non-human primates (NHP), and if the delivery of human ABCA4 would reduce A2E accumulation in Abca4^-/- mice.

Methods:: Equine infectious anemia virus (EIAV)-derived lentiviral vectors were constructed carrying the human ABCA4 gene (or the LacZ reporter gene) under the control of constitutive (CMV) or photoreceptor-specific (Rho, PDE) promoters. Abca4^-/- mice were injected subretinally with 1-5x10⁵ IU of each virus at P4-5 in one eye; the mock-injected contralateral eye served as a control. Mice were sacrificed at: a) 2 weeks after injection for X-gal reaction or ABCA4 antibody (3F4) staining; b) at several time points (for up to one year) after injection to determine the A2E content by HPLC. Baboons and cynomolgus macaques were injected subretinally with 1-5x10⁶ IU of each LacZ virus and sacrificed 2 weeks after injection for X-gal reaction.

Results:: Subretinal injections of EIAV-CMV-ABCR and EIAV-Rho-ABCR substantially reduced A2E accumulation in Abca4^-/- mice compared to the mock-injected controls. The reduction was sustained for up to 1 year after the injection. The immunolabeling of EIAV-CMV-ABCR injected mouse eyes with ABCA4 3F4 antibody showed high protein expression in both PR and RPE. The analysis of the reporter gene expression of injected mouse and non-human primate eyes demonstrated transduction of 30-100% photoreceptors in the injected area.

Conclusions:: The excessive A2E (lipofuscin) accumulation in the mouse model of STGD was substantially reduced, and efficiently sustained, at levels comparable to wt animals by lentivirus-based gene therapy. These data, coupled with a robust expression of the human ABCR protein in PR of Abca4^-/- mice and efficient transduction of photoreceptors in NHP, suggest that lentiviral gene therapy is a potentially efficient tool for treating ABCA4-associated diseases in humans.

Keywords: gene transfer/gene therapy • retinal degenerations: hereditary • photoreceptors