Cell-Based Gene Therapy Using Transfected Muller Glia to Deliver Reagents to the Outer Retina in Vldlr-/- Mice

Abstract

Purpose: : We have demonstrated that subretinal NV leads to photoreceptor degeneration and attenuation of ERG signals in mice deficient for the very low density lipoprotein receptor (VLDLR). To protect against these degenerative effects we tested cell-based gene therapy using AAV2 viral vectors to transfect Muller glia and deliver neurotrophic reagents to degenerating photoreceptors in Vldlr-/- retinas.

Methods: : Retinal expression of green fluorescent protein (GFP) was analyzed after intravitreal injection of AAV2 viral vectors expressing GFP behind a CAG (AAV2-CAG-GFP) or Glial Fibrillary Acidic Protein (GFAP) promoter (AAV2-GFAP-GFP). Vectors containing neurotrophin-4 (AAV-GFAP-NT4) were also injected intravitreally. Photoreceptor degeneration was analyzed by comparing levels of opsin-1 or rhodopsin mRNA in whole retinas from AAV2-GFAP-NT4 treated- , control treated-, or untreated Vldlr-/- mice, as well as age-matched C57Bl6/J wild-type (WT) controls. Visual function was analyzed by electroretinograms (ERGs).

Results: : Muller glia became activated and specifically expressed GFAP in proximity of subretinal NV in Vldlr-/- retinas. Following intravitreal delivery of AAV2-GFAP-GFP vectors, GFP expression was limited to the inner retina in WT mice while expression was observed throughout the trans-retinal processes of GFAP activated Muller glia in Vldlr-/- retinas. Control CAG-driven vector expression was limited to the inner retina in both Vldlr-/- and WT mice. Intravitreal injection of AAV2-GFAP-NT4 viral vectors resulted in NT4 expression by activated Muller glia, accumulation of NT4 at the photoreceptor segments, protection of the photoreceptors, and maintenance of normal ERGs.

Conclusions: : We have demonstrated viral transfection of GFAP activated Muller glia in Vldr-/- retinas. This leads to expression of gene product in the outer retina following intravitreal injection, and attenuation of neuronal degeneration after delivery of neurotrohic factors. These studies provide data supporting the use of activated Müller glia for viral-mediated delivery of therapeutic gene products to the outer retina using intravitreal injections.

Keywords: gene transfer/gene therapy • neuroprotection • retinal glia