Evaluation of in silico reconstructed ancestral adeno-associated virus for gene augmentation therapy in a mouse model of LCA

Abstract

Purpose : The Retinitis Pigmentosa GTPase Regulator Interacting Protein 1 (RPGRIP1) is an essential protein for the maintenance of photoreceptor outer segments. Mutations in this gene cause severe retinal dystrophy, known as Leber Congenital Amaurosis (LCA). Rpgrip1 knock-out mice show outer nuclear layer (ONL) thinning at 3 months of age, and nearly complete loss after 5 months. This study evaluated the use of in silico reconstructed ancestral adeno-associated virus (Anc80) compared to adeno-associated virus serotype 8 (AAV8), for gene augmentation therapy in Rpgrip1 knock-out mice.

Methods : The cell transduction patterns resulting from sub-retinal injection of Anc80.CMV.EGFP and AAV8.CMV.EGFP in wild-type mice were compared using fundus imaging and immunofluorescence (IF) analyses. Rpgrip1 knock-out mice were treated with sub-retinal injection of either Anc80.RK.hRPGRIP1 or AAV8.RK.hRPGRIP1 at two weeks of age. Expression of the hRPGRIP1 transgene was evaluated using western blot (WB), RT-qPCR and IF analyses, 3 weeks post injection.

Results : Fundus imaging showed transgene expression after subretinal injection of either Anc80.CMV.EGFP or AAV8.CMV.EGFP after 3 days. The GFP intensity and area of transduction continued to expand until 2 weeks, and remained consistent until 5 weeks. By IF, Anc80 sections showed transgene expression in the retinal pigmented epithelium, photoreceptor cells, Müller cells, inner nuclear layer, and the retinal ganglion cell layer. AAV8 injected eyes show similar transduction, but only from the RPE through the ONL, with some Müller cells. hRPGRIP1 expression was evident in knock-out mice by use of either Anc80 or AAV8 by IF, WB and RT-qPCR.

Conclusions : Anc80 mediated robust expression of GFP within the RPE and ONL and extended into the INL and RGC retinal layers with a broad (CMV) promoter. Anc80 also mediated hRPGRIP1 expression within the photoreceptor connecting cilium by use of a photoreceptor specific (RK) promoter. Efficacy of gene delivery will be determined at 20 weeks post-injection by OCT and ERG.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.