CRX gene therapy targeting mouse models of dominant CRX-associated retinopathies.

Abstract

Purpose : Efficacy and safety of gene therapies for recessive retinal diseases (RDs) have been proven with preclinical success translated into clinical effectiveness. However, this approach is rarely chosen for dominant forms. Based on our published work, we decided to focus on dominant-negative mutations in the transcription factor CRX. We developed a mutation-independent AAV vector that could circumvent the clinical and genetic heterogeneity of CRX mutations.

Methods : To express CRX specifically in photoreceptors, the Rhodopsin Kinase 1 promoter and the AAV vector serotype AAV2/5 were chosen (AAV-CRX). To assess the efficacy of AAV-CRX for different CRX-associated diseases, we used Crx^Rip/+ mice, a model of Leber Congenital Amaurosis and we established a new model for cone dystrophy, Tg(CRX^R41W) carrying the human CRX^R41W mutation driven by Crx promoter. Subretinal injection was done in PN30 animals. Immunohistochemistry, immunoblot, and electroretinogram (ERG) were used for Tg(CRX^R41W) characterization and gene therapy efficacy assessment.

Results : Tg(CRX^R41W) characterization revealed a dose-dependent deleterious effect of CRX^R41W. Indeed, heterozygous Tg(CRX^R41W) carrying a single insertion displayed a functional retina while homozygous Tg(CRX^R41W) exhibited reduced cone function after 3 months. Presence of multiple copies led to rapid and severe photoreceptor degeneration after 2 months. Our results corroborated the relevance of increasing the amount of CRX^WT to counteract the dominant-negative effect of mutant CRX. We first verified that AAV-CRX injection led to specific and high expression of CRX in Crx^Rip/+ immature photoreceptors with no toxicity. In AAV-GFP Crx^Rip/+ injected retina, photoreceptors still lacked outer segments and visual transduction protein expression. In contrast, 3 months after injection, AAV-CRX led to i) rod and cone opsin expression, ii) outer segment formation, iii) some degree of ERG response whereas it remained flat in controls iv) fully restore behavior response to light stress using a Dark/Light box test. Homozygous Tg(CRX^R41W) mutant mice are also currently being tested.

Conclusions : Overall, our gene therapy approach shows promising results for treating CRX-associated RDs. It also highlights the potential interest of gene therapy to treat patients with RD carrying dominant-negative mutations.

This is a 2021 ARVO Annual Meeting abstract.