Retinitis pigmentosa (RP) is a common but untreatable inherited retinal degeneration, affecting approximately 1.5 million people worldwide. RP is a disease of high genetic heterogeneity. More than 100 genes and 3000 mutations associated with RP have been identified to date. Many RP diseases genes play vital roles in the phototransduction cascade, which converts the light signals into neuron signals in the photoreceptors. In rods, the type of photoreceptors that are extremely sensitive to light and responsible for scotopic vision, RHODOPSIN is the light- sensitive receptor protein in the phototransduction cascade. Unfortunately, more than 150 mutations found in the RHO gene, which encodes the RHODOPSIN protein, can lead to RP. Among those, the missense P23H (p.Pro23His, c.68C>A) mutation is the most common genetic cause of autosomal dominant RP (adRP). Although gene therapy has become a promising treatment for inherited eye diseases, it is challenging to develop a therapy for adRP that is caused by missense mutations.

In the present study, I developed a mutation-independent gene knock-in (KI) therapy, which is based on the CRISPR/Cas9-mediated homology-independent targeted integration (HITI) method, to treat RP associated with dominant Rho mutations in mouse models. The KI therapy inserts the wild type Rho coding sequence (WT Rho CDS) into the Rho locus, by which the expression of the mutant gene would be replaced by that of the inserted gene. The efficiency of AAV-CRISPR/Cas9-mediated KI of a GFP reporter in the Rho locus was about 50% of the transduced rods. Rho knock-in (Rho KI) into the exon 2 or into the 5’UTR region of exon 1 of the Rho locus were subsequently evaluated for the therapeutic efficacy in three mouse models of RP caused by Rho mutations. In the Rho null (Rho^-/-) mice, the Rho KI into the 5’UTR successfully resulted in the RHO protein expression in a subset of rods. In the fast degenerating P23H homozygous (Rho^P23H/P23H) mouse model, Rho KI into the Rho exon 2 greatly rescued visual function and prevented photoreceptor loss, likely attributed to both knocking out the P23H alleles and expressing the wild type RHO protein. In the slow degenerating P23H heterozygous (Rho^P23H/wt) mouse model, the Rho KI into the 5’UTR region of Rho resulted in small but significant improvements in the scotopic ERG, photopic ERG, and retina structure compared to the control groups at the later stage of retinal degeneration.

In this thesis, I proved the concept and technical feasibility of CRISPR/Cas9 mediating gene knock-in therapy to correct the mutant Rho in a mutant-independent manner in the RP mouse models. The success of this mutant-independent gene editing therapy would decrease the cost and effort of treating RP and other inherited diseases with the multiplicity of mutations.