Gene-targeted transcriptional silencing by DNA-binding

Abstract

Purpose : Silencing and replacement strategy is a promising approach to overcome mutational heterogeneity of genetic defects. In autosomal dominant retinitis pigmentosa (adRP) due to rhodopsin gene (RHO) approximately 200 different mutations have been described, posing a challenge for the design of effective therapeutics. We generate a transcriptional silencer through the development of a zinc finger based synthetic DNA-binding protein lacking effectors domain (ZF6-DBD) that targets rhodopsin promoter. We combined the ZF6-DBD with a human RHO gene for silencing and replacement experiment in porcine retina.

Methods : To assess the efficacy of ZF6-DBD transcriptional interference we generated an Adeno-associated virus (AAV) vector (AAV8-CMV-ZF6DBD) and delivered by sub-retinal injection to adult porcine retina. Fifteen days after ZF6-DBD retinal gene transfer RHO transcript levels were assessed by qReal Time PCR on both whole retina and FACS sorted rods. To determine the RHO silencing specificity genome wide and the morphological impact we applied RNA sequencing (RNAseq) and, histological analysis, respectively. Silencing and replacement strategy was assessed upon delivery of a single vector carrying both ZF6-DBD and human RHO (AAV8-hRHO-ZF6DBD-GNAT1-hRHO) under rod-specific promoter elements. Two moths after delivery RT-PCR, western blot and histological analysis were performed.

Results : Retinal delivery of AAV8-CMV-ZF6DBD (n=6), demonstrated 45% decrease of RHO transcript and protein compared with control retina (n=7). On rod population, the ZF6DBD showed complete ablation of RHO (90%). Histological analysis showed collapse of the rod outer segment (OS) and depletion of RHO content, while maintaining the integrity of the outer nuclear layer (ONL). The RNAseq showed that ZF6-DBD perturbed 19 genes (differentially expressed genes DEGs), including RHO (n=6) demonstrating high selectivity (on-target). Silencing and replacement generated a repression of the 30% of endogenous porcine RHO expression and the simultaneous replacement of the exogenous human rhodopsin (45%; n=3).

Conclusions : We demonstrate that the synthetic DNA-binding protein ZF6-DBD, without effector domains, enables selective and specific RHO transcriptional silencing with negligible off-target effects in porcine retina. These data support ZF6-DBD use as therapeutic, for the treatment of gain of function mutations of RHO in adRP patients.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.