rAAV-mediated Gene Replacement Therapy Restores Vision and Delays Degeneration in the CNGB1-/- Mouse Model of Retinitis Pigmentosa

Abstract

Purpose: : CNGB1 encodes the B1a subunit of the rod cyclic nucleotide-gated (CNG) channel. Mutations in CNGB1 cause autosomal recessive retinitis pigmentosa. Here, we used "exon 26" CNGB1 knockout (CNGB1^-/-) mice to evaluate rAAV-mediated CNGB1a gene replacement as a potential treatment for this disease.

Methods: : Due to the size of the CNGB1a cDNA (approx. 4 kb) optimization of the regulatory elements within the expression cassette was necessary to comply with the packaging limitations of rAAV vectors. We therefore replaced the BGH polyA site and the WPRE element with a 221bp SV 40 polyA element. A short mouse rhodopsin promoter element was used to drive rod-specific expression. Therapeutic rAAVs (serotype 8) were injected into the subretinal space of 2-week-old CNGB1^-/- mice. The treatment success was monitored using immunohistochemistry, electrophysiology and a water-maze test designed to specifically assess rod-mediated vision.

Results: : Using the optimized rAAV vectors we could successfully express full-length CNGB1a in the knockout retina. Signs of a functional restoration were first observed in the ERG at 6 weeks after injection. The CNGB1a protein was exclusively found in rod photoreceptors and mainly localized to outer segments. The treatment restored the expression of the previously down-regulated endogenous CNGA1 protein, which co-localized with CNGB1a in rod outer segments. In contrast to untreated mice, treated CNGB1^-/- mice became able to generate rod photoreceptor responses. Moreover, treated CNGB1^-/- mice showed a performance similar to wild type control mice in a rod vision behavioral test. In contrast, untreated CNGB1^-/- mice were not able to navigate to the escape platform and showed no learning progress. In addition, the treatment led to a substantial morphological preservation of rods. In particular, the treated area showed significant preservation of rod outer segment structure and outer nuclear layer morphology.

Conclusions: : Using a capacity-optimized rAAV vector for rod-specific expression of large (up to 4.2 kb) transgenes, we were able to successfully restore rod function in CNGB1^-/- mice. This work provides a proof-of-concept for the treatment of retinitis pigmentosa due to a rod channelopathy by rAAV-mediated gene replacement.

Keywords: gene transfer/gene therapy • ion channels • retinitis