Transcriptional Repression of the Human Rhodopsin Gene by Artificial Zinc Finger Protein Recovers Retinal Function in a Model of Autosomal Dominant Retinitis Pigmentosa

Retinitis Pigmentosa (RP) represents a heterogeneous group of severe retinal degenerations. Rhodopsin (Rho) is the gene most commonly associated to the autosomal dominant form of RP (ADRP) with over 150 mutations identifi ed. The objective of this study is to treat ADRP by silencing the Rho gene expression at the transcriptional level in a mutation independent fashion to potentially treat all ADRP due to rhodopsin mutations. To this end we have used artifi cial zinc fi nger transcription factors (ZF-TFs) targeted to human Rhodospin promoter. Through the modular assembly method we generated 10 DNA Binding Domains (DBDs) targeted to hRho promoter and we fused them to the KRAB or VP64 domains to assemble specifi c Rho transcriptional repressors (ZFRs) and activators (ZFAs), respectively. We selected the most effi cient and selective hRho transcription inhibitor using the luciferase reporter system. Twenty percent success rate (2 out of 10) was obtained in generating artifi cial ZF-TFs that robustly and specifi cally repress expression of luciferase driven by hRho promoter in vitro. As further demonstration of specifi city, the 2 selected ZF-TFs, when fused to VP64 domain, are also able to effi ciently transactivate the targeted promoter as well as directly bind it as assessed through Electromobility shift assay (EMSA) experiments. The therapeutic potential of the two selected hRho specifi c repressors (ZFR-2 and ZFR-6) was assessed by transduction experiments on Retinal Stem Cells (RSC) explanted from adult P347S ADRP transgenic mouse model. These cells die once differentiated in vitro because of the expression of the mutated copy of hRho gene. Retroviral delivery of the selected ZFRs to RSC resulted in selective repression of the mutated human Rhodopsin as measured by quantitative Real Time PCR analysis, and remarkably in 90% reduction of apoptotic cells compared to controls. To assess the feasibility of such approach in vivo, we generated an AAV 2/8 harbouring the ZFR-6 driven by a Rhodopsin kinase promoter. One month after vector delivery RT-PCR analysis showed specifi c repression of RHO transcript. Remarkably a significant retinal morphological and functional recovery was observed. Taken together these results demonstrate that artifi cial Zinc Finger transcription factors repress hRho gene in vitro and in vivo, underscoring the potential impact of this novel strategy to treat ADRP