CRISPR/Cas9-based gene editing approaches for the treatment of USH2A-associated diseases

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Project Summary/Abstract Adeno-associated virus (AAV)-mediated gene augmentation is currently the most effective approach for treating recessive inherited retinal degenerations (IRDs). However, due to the limited capacity of AAV vectors, gene augmentation currently is not possible for one third of IRD patients due to mutations in genes that are too large for AAV, e.g., USH2A, which has a coding sequence of 15.6kb. Mutations in the USH2A gene are the leading cause of retinitis pigmentosa (RP) and Usher Syndrome Type II (USH2). These two diseases affect approximately 500,000 people worldwide. Both diseases are characterized by progressive vision loss beginning in early adulthood, with the addition of bilateral hearing loss from birth in USH2. Our long-term goal is to develop therapeutics that slow or prevent vision and hearing loss in USH2A patients. Over 1000 pathogenic and likely pathogenic variants have been identified in the USH2A gene. To date, studies have mainly focused on developing therapeutic strategies for the most prevalent mutation c.2299delG in exon 13 of the USH2A gene, which accounts for approximately one third of all USH2A cases in Europe and the U.S. c.2299delG disrupts the open reading frame of USH2A transcript. Multiple genetic approaches have been and are being tested to repair this genetic defect, including antisense oligonucleotide (ASO)-based exon skipping therapy or CRISPR/Cas9 gene editing. The exon skipping strategy have been extensively pursued in the past several years. Multiple lines of pre-clinical and clinical studies have demonstrated that excision of exon 13 from the USH2A gene can restore the disrupted open reading frame caused by c.2299delG mutation and lead to rescue of the retinal phenotypes in animals and improved vision in human. These findings strongly support the use of exon skipping as a treatment modality for some of the USH2A patients. The specific object of the proposed research is to investigate the potentials of innovative technologies, including exon skipping, base editing and prime editing, for the treatment of USH2A-related diseases. We plan to perform three proof-of- concept studies in three specific aims. In Aim 1, we will identify which other exons in the USH2A gene can serve as the next targets for exon skipping approach. To achieve this goal, we will generate mouse model that lacks an exon of interest and evaluate the ability of the abbreviated forms of the USH2A protein is sufficient to correct USH2A deficient phenotypes in mice. In Aim 2, we will explore the use of base editing to induce exon skipping in cells and human retinal explants, with the hope to circumvent the constraints of ASO, or Cas9 nuclease mediated exon skipping approaches. In Aim 3, we will investigate the feasibility of implementing prime editing to directly correct the mostly reported mutations in the USH2A gene, in particular the c.2299delG. If successful, these studies will open new therapeutic avenues for USH2A-associated disorders. It will also provide a template for similar efforts to develop therapies for other IRDs due to mutations in large genes.