736. Silencing Substituting the RHODODPSIN Gene to Treat Blinding Disorders

Abstract

Silencing and replacement strategy is a promising strategy to overcome mutational heterogeneity of genetic defects. In autosomal dominant retinitis pigmentosa (adRP) approximately 200 different mutations have been associated with rhodopsin gene (RHO). We recently described a system based on the delivery of a synthetic transcriptional repressor (DNA-binding-mediated silencing; ZF6-DBD), which upon Adeno-associated viral (AAV) vector delivery to the porcine retina, demonstrated selective somatic ablation of RHO in mutation independent manner, thus silencing both the wild-type and mutated alleles. To determine the actual therapeutic impact of DNA-binding-mediated silencing, we carried out the silencing-replacement strategy by coupling ZF6-DBD with RHO replacement (human RHO, hRHO CDS). To warrant simultaneous photoreceptor transduction of both ZF6-DBD and hRHO, we enclosed two expression cassettes into a single vector (DNA-binding repression and replacement, DBR-R). To generate the construct we evaluated key variables for balanced simultaneous RHO repression and replacement, which are the vector dose and promoter strength. To ensure high and rod-specific hRHO replacement, we opted for a high vector dose and the strength of the GNAT1 promoter elements. To decrease ZF6-DB expression levels at high vector dose, while keeping rod-specificity, we both shortened the human RHO promoter and deleted the 5’ sequence of the ZF6-DB target. We used 1×1012 gc of vector of DBR-R (AAV8-RHOΔ-ZF6-DB-GNAT1-hRHO) to administer to porcine retina. Administration of the DBR-R vector resulted in concomitant rod-specific transcriptional repression of the porcine Rho (35%) and in balanced replacement with the exogenous hRHO (45%), as assessed by transcripts levels, protein expression and integrity of photoreceptor outer segments. The data support pre-clinical development including a dose-response and long-term efficacy and toxicity.