Development of an EIAV Lentiviral Based Gene Therapy, RetinoStat®, for the Treatment of CNV Age–Related Macular Degeneration

Abstract

Purpose: : In these studies we aimed to develop an effective sustained gene therapy for patients with CNV AMD based on a recombinant lentiviral vector Equine Infectious Anemia virus (EIAV). Subretinal delivery of a minimal EIAV lentiviral vector leads to efficient, long–term gene expression in the retinal pigment epithelial (RPE) cells, an ideal platform for the secretion of angiostatic proteins.

Methods: : We constructed a series of EIAV vectors expressing different combinations of angiostatic molecules including endostatin, angiostatin and sFlt1. Included in this series of vectors were EIAV vectors expressing both endostatin and angiostatin transcriptionally linked via the EMCV internal ribosome entry site (IRES). We developed a number of in vitro endothelial–based angiogenesis assays in which to investigate the biological effects of these vectors prior to evaluation in the murine laser CNV model.

Results: : We evaluated several in vitro angiogenesis assays. The Matrigel assay in which endothelial cells form tubule–like structures over a Matrigel platform appeared to be optimal in terms of sensitivity, robustness, ease and reproducibility. We used this assay to evaluate EIAV vectors expressing either endostatin (EIAV Endo) alone or in combination with angiostatin (EIAV Endo/Angio) downstream of the strong CMV promoter. The EIAV Endo/Angio vector inhibited tubule formation with statistically greater efficiency than the EIAV Endo vector. Both of these vectors subsequently showed similar relative potency in the murine laser CNV model indicating that the in vitro data were predictive of the relative efficacy in vivo thus underlying their utility as a screening assay.

Conclusions: : The in vitro data confirm the additive effect of endostatin and angiostatin in the EIAV vector platform. We have called this recombinant EIAV vector RetinoStat® and are continuing to develop this product for clinical application.

Keywords: age-related macular degeneration • gene transfer/gene therapy • retinal pigment epithelium