DEVELOPMENT OF AAV-CRISPR/CAS9-BASED THERAPIES FOR CONE ROD DYSTROPHY

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ABSTRACT Mutations in GUCY2D, the gene encoding retinal guanylate cyclase-1 (retGC1), are the leading cause of autosomal dominant cone-rod dystrophy. GUCY2D-CORD6 patients present with loss of visual acuity, abnormal color vision, photophobia, visual field loss and macular atrophy within the first decade. Rod degeneration and peripheral visual field loss follow. Significant progress towards clinical application of gene replacement therapy for LCA due to recessive mutations in GUCY2D (LCA1) has been made, but a different approach is needed to treat CORD6 where gain of function mutations cause dysfunction and dystrophy. Our preliminary data show that 1) selective and efficient somatic knock-out of GUCY2D and Gucy2e (murine homologue) with AAV- CRISPR/Cas9 results in a subsequent loss of retinal structure/function that manifests from reduced retGC1 expression in macaque and mouse, respectively, 2) a ‘knock-out + complementation in trans’ approach (wherein complementation is performed with ‘hardened’ Gucy2e not recognized by Gucy2e gRNA) preserves retinal function in mice, 3) AAV-CRISPR/Cas9- based editing of GUCY2D is therapeutic in a R838S transgenic (Tg) mouse model of CORD6, and 4) Cas9 variants identified by directed evolution exhibit allele specificity for GUCY2D(R838S). We will build upon these results in the following Aims. Aim 1 will establish the optimal parameters for AAV-CRISPR/Cas9-based gene editing in two R838S CORD6 Tg mouse lines. We will establish the optimal AAV capsid/dose, durability of therapy, treatment window, and feasibility of transient Cas9 expression systems. Aim 2 will evaluate safety/efficacy of AAV-CRISPR/Cas9-based gene editing in macaque by looking for off-target editing and assessing the potential impact of AAV vector insertions and long-term Cas9 expression. We will also evaluate regional differences in editing efficiency and conduct dose-ranging studies. Aim 3 will compare ‘knock out + complementation in trans’ vs. ‘allele-targeted’ approaches for treating CORD6. The optimal AAV capsids and Cas9 expression system from Aim 1 will be used to test KO + complementation in GC2-/- mice. Allele-targeted editing will be performed in humanized R838S CORD6 mice that carry both the wt and R838S- containing exon 13 of GUCY2D. Approaches will include a novel, engineered Cas9 that specifically edits the R838S mutant allele, gRNAs containing mismatches to mutant but not wt allele, or utilization of an alternative PAM site found in the mutant, but not wt allele. Successful approaches will be tested in macaque for efficiency/safety. Our findings will identify the optimal capsid/dose, and treatment age for therapeutic AAV- CRISPR/Cas9-based disruption of R838S GUCY2D in vivo. We will establish the safety profile, and regional efficiencies of gene editing by AAV-CRISPR/Cas9 in a species with both genomic and clinical relevance. In addition, we will identify materials and approaches that will allow clinical application of AAV-CRISPR/Cas9 therapies for CORD6 as well as other dominantly inherited retinal diseases.