Subretinal gene therapy in Bbs1 mice

Abstract

Purpose: : To test the safety and efficacy of subretinal injection of AAV-WTBbs1 for the treatment of a mouse model of Bardet Biedl syndrome type 1 (BBS1).

Methods: : Bbs1M390R/M390R mice were generated as described previously. Constructs were made of the wild type Bbs1 gene in AAV2/5 vectors under control of the CMV or chicken β-actin promoter both with and without a FLAG tag. Subretinal injections were given through a scleral puncture under the temporal retina at P30 to P60 using AAV-WTBbs1 in the right eyes and sham injection with vehicle in the left. Two or 3 microliters were delivered including 1 part AAV-GFP and 9 parts AAV-WTBbs1 to enable visualization of the transduced area. Transgene expression was analyzed by immunohistochemistry and Western blotting following sucrose gradient ultracentrifugation. Retinal function was analyzed by electroretinogram, and structure was analyzed by optical coherence tomography (OCT). Histology was performed on selected animals at different timepoints.

Results: : Expression of the FLAG-tagged Bbs1 transgene was demonstrated in the inner segments of the injected area of the retina by immunofluorescence using an antibody directed against the FLAG peptide. Western blot demonstrated FLAG-Bbs1 protein expression and also a reconstitution of the BBSome. Two microliter injections were better tolerated than 3 microliter. ERG dark adapted b-wave was higher amplitude in the WTBbs1 injected Bbs1M390R/M390R eyes than in sham injected or uninjected fellow eyes in 7 of 13 mice. Simultaneous injection of dilute AAV-GFP demonstrated that 24-32% of the retina was transduced in each injection.

Conclusions: : BBS is a challenging disorder to treat with gene therapy due to the stoichiometry of the protein products that contribute to the BBSome. In a knock-in model of Bbs1 there is evidence that subretinal delivery of AAV-WTBbs1 causes production of the Bbs1 protein in the inner segment and partially rescues the BBSome formation, however only about 50% of eyes demonstrated an improved ERG. Future studies will be required to determine the optimal dose of virus necessary to achieve rescue.

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