Artificial Zinc Finger Transcription Factor-mediated Repression of Mutated Human Rhodopsin Improves Visual Function in Autosomal Dominant Model of Retinitis Pigmentosa (p347s)

Abstract

Purpose: : Retinitis pigmentosa (RP) is the most common inherited retinal degeneration affecting about one in 4000 individuals worldwide. Rhodopsin (Rho) is the gene most commonly associated with dominant RP (25-50% of cases) and over 150 mutations have been identified in its sequence. The aim of the project is to specifically repress human Rho using allele independent Zinc Finger based transcriptional repressors (ZFRs) and to test the efficacy in the P347S ADRP mouse model

Methods: : ZFRs were first selected by a Luciferase reporter assay. Retroviral vectors were used to deliver ZFRs to Cultured Retinal Stem Cells (RSC) explanted by the P347S mouse model to validate their functionality. Repression of the human mutated rho was measured by Real Time (RT) PCR and protection from apoptosis was evidenced by Tunel staining. To test in vivo the efficacy of the strategy, AAV2/8 containing the selected ZFRs was subretinally injected in P347S newborn mice. RT- PCR was used to measure Rho levels. Visual function of the treated and control eyes were measured by Electroretinograms. The photoreceptor protection from degeneration was evaluated histologically by counting nuclei in ONL

Results: : 2 ZFRs were able to selectively bind the human Rho promoter and repress the expression of the reporter gene (Luciferase) driven by the human Rho promoter. The delivery of the 2 ZFRs to cultured RSC resulted in protection from apoptosis through the selective repression of the human P347S Rho expression. The transfer of the artificial ZFRs to photoreceptors of P347S mice resulted in 40% reduction of human Rho expression and in a significant morphological and functional recovery of the treated retinae compared to controls

Conclusions: : Taken together these results demonstrate that artificial Zinc Finger transcription factors repress human Rho gene in vitro and in vivo and ameliorate the phenotype of the treated P347S mouse model. These results underscore the impact of this novel mutation independent strategy to treat ADRP in a future clinical setting

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