Zinc‐finger‐based transcriptional repression of rhodopsin in a model of dominant retinitis pigmentosa

Claudio Mussolino,Germana Meroni,Umberto Di Vicino,Alberto Auricchio,Ciro Bonetti,Daniela Sanges,Valeria Marigo,Elena Marrocco,Enrico Maria Surace

doi:10.1002/emmm.201000119

Abstract

Despite the recent success of gene-based complementation approaches for genetic recessive traits, the development of therapeutic strategies for gain-of-function mutations poses great challenges. General therapeutic principles to correct these genetic defects mostly rely on post-transcriptional gene regulation (RNA silencing). Engineered zinc-finger (ZF) protein-based repression of transcription may represent a novel approach for treating gain-of-function mutations, although proof-of-concept of this use is still lacking. Here, we generated a series of transcriptional repressors to silence human rhodopsin (hRHO), the gene most abundantly expressed in retinal photoreceptors. The strategy was designed to suppress both the mutated and the wild-type hRHO allele in a mutational-independent fashion, to overcome mutational heterogeneity of autosomal dominant retinitis pigmentosa due to hRHO mutations. Here we demonstrate that ZF proteins promote a robust transcriptional repression of hRHO in a transgenic mouse model of autosomal dominant retinitis pigmentosa. Furthermore, we show that specifically decreasing the mutated human RHO transcript in conjunction with unaltered expression of the endogenous murine Rho gene results in amelioration of disease progression, as demonstrated by significant improvements in retinal morphology and function. This zinc-finger-based mutation-independent approach paves the way towards a ‘repression–replacement’ strategy, which is expected to facilitate widespread applications in the development of novel therapeutics for a variety of disorders that are due to gain-of-function mutations.

Highlights

Despite the recent success of gene-based complementation approaches for genetic recessive traits, the development of therapeutic strategies for gain-offunction mutations poses great challenges
We have demonstrated that photoreceptor delivery of an AAV2/8 vector containing artificial ZF transcriptional repressors (ZF-R6) targeted to the human rhodopsin (hRHO) promoter can repress hRHO transgene over-expression, and this, in turn, was associated with improvement in photoreceptor disease in the P347S mouse model of Autosomal dominant forms of Retinitis Pigmentosa (adRP)
A different technological platform based on RNA interference (RNAi) was first used in vivo to silence a dominant allele of ataxin-1 for the treatment of spinocerebellar ataxia type 1 in a mutationalindependent manner; this approach was applied to other mouse models of human disease, including the P347S adRP mouse (O’Reilly et al, 2007; Xia et al, 2004)

Summary

Introduction

Despite the recent success of gene-based complementation approaches for genetic recessive traits, the development of therapeutic strategies for gain-offunction mutations poses great challenges. We show that decreasing the mutated human RHO transcript in conjunction with unaltered expression of the endogenous murine Rho gene results in amelioration of disease progression, as demonstrated by significant improvements in retinal morphology and function. This zinc-fingerbased mutation-independent approach paves the way towards a ‘repression– replacement’ strategy, which is expected to facilitate widespread applications in the development of novel therapeutics for a variety of disorders that are due to gain-of-function mutations. In the case of adRP, due to rhodopsin mutations, more than 150 allele-specific silencing molecules would be required to silence each specific mutation identified far (RHO gain-of-function mutations account for 25–50% of the total autosomal dominant adRP cases; (Inglehearn et al, 1998; Sohocki et al, 2001)

Methods

Results

Conclusion