488. In Vitro and In Vivo Genome Editing of the RHO Gene to Downregulate Dominant Mutations

Although progresses have been made in the understanding of the genetic basis for Retinitis Pigmentosa (RP), the development of therapeutic intervention is still lagging behind. Gene therapy was successfully applied to retina degeneration but only to recessive mutations. Rhodopsin (RHO) mutations represent a common cause of RP, accounting for 25% of adRP and 8 to 10% of all RP (Hartong et al., 2006) with more that 100 different mutations identified so far. RHO is a G-protein coupled receptor with localization restricted to rod outer segments where the phototransduction cascade initiates. Data on the pathogenic mechanism of mutant RHO are still controversial. Accumulation of mutant RHO in different subcellular compartments, and among those the ER, may trigger unfolded protein response (UPR) devoted to cytoprotective outputs to permit cells to reduce protein synthesis and up-regulate chaperons to cope with stress (Lin et al., 2007). In the autosomal dominant RP the mutation in only one allele is sufficient to the onset of retinal degeneration. To treat this kind of disorder a gene addition therapy is not suitable, as the mutant allele, mRNA, or protein product must be silenced beforehand. We aimed at developing genome editing tools to knock out the RHO defective alleles by introducing a double strand breaks (DSB) into the target gene. Two gRNAs were designed in the first exon on the RHO gene encompassing the P23H mutation. The two gRNAs were tested singularly or together in vitro on HeLa clones stably expressing P23H RHO. We demonstrated insertions or deletions (indels) in the genomic DNA specifically in the RHO gene by Cel I assay, TIDE and sequencing. Indels caused strong reduction of the RHO mRNA and of RHO protein up to 90%. The higher effect was obtained with the two gRNAs together. The two gRNAs were then in vivo expressed with Cas9 in photoreceptors of transgenic mice expressing the human P23H Rho gene by electroporation. Targeted cells were tracked by co-expression of EGFP. EGFP+ cells were FACS-sorted and indels in the human P23H RHO gene were analyzed by sequencing. We were able to detect up to 30% of genome editing in vivo. No editing was scored on murine Rho allele. We also detected reduction of RHO mRNA expression as well as RHO protein. Finally, we developed new tools to downregulate mutant RHO in dominant forms of RP. The CRISPR/Cas9 system reveled a high efficiency and should be tested for knock-down followed by gene replacement approaches.