Gene Augmentation for Autosomal Dominant CRX-Associated Retinopathies

Abstract

The cone-rod homeobox (CRX) protein is a key transcription factor essential for photoreceptor function and survival. Mutations in human CRX gene are linked to a wide spectrum of blinding diseases ranging from mild macular dystrophy to severe Leber congenital amaurosis (LCA), cone-rod dystrophy (CRD), and retinitis pigmentosa (RP). These diseases are still incurable and mostly inherited in an autosomal dominant form. Dysfunctional mutant CRX protein interferes with the function of wild-type CRX protein, demonstrating the dominant negative effect. At present, gene augmentation is the most promising treatment strategy for hereditary diseases. This study aims to review the pathogenic mechanisms of various CRX mutations and propose two therapeutic strategies to rescue sick photoreceptors in CRX-associated retinopathies, namely, Tet-On-hCRX system and adeno-associated virus (AAV)-mediated gene augmentation. The outcome of proposed studies will guide future translational research and suggest guidelines for therapy evaluation in terms of treatment safety and efficacy.

Keywords: AAV gene therapy; CRX mutations; Dominant negative effects; Gene augmentation; Tet-On.

Figure 1.

Components of Tet-On-hCRX system. (A) TRE-hCRX (B) pCAG-LSL-rtTA (C) pCrx-Cre.

Figure 2.

Quantitative PCR (qPCR) analysis of hCrx and Rho expression in untreated Crx−/−, Crx-BAC-Cre⁺, Tet-On-hCRX⁺; doxycycline-treated Crx−/−, Crx-BAC-Cre⁺, Tet-On-hCRX⁺; WT mice. (A) Treatments started at P0, samples were harvested at P5, P14, P21 or P35. (B) Treatments started at P14 or P21, samples were harvested at P35. Results are plotted as relative Log2 expression to untreated samples (n≥4). Asterisks (**, ***, ****) denote p ≤ 0.01, p ≤ 0.001 and p ≤ 0.0001 respectively by T-test.