Evaluation of novel gene therapy approaches for USH2A

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Usher syndrome (USH) is characterized by retinal defects due to retinitis pigmentosa and congenital or progressive deafness with occasional vestibular dysfunction. Mutations in the USH2A gene are by far the most common cause of USH syndrome, but there is currently no therapy. Because of the size of the USH2A gene, classical gene-supplementation therapies using the gold standard adeno-associated viral (AAV) vectors require at least four vectors (quadruple AAV vector strategy), and it is not clear whether this approach works in principle. In this project, to establish appropriate therapeutic strategies for USH2A, we will pursue three approaches: i) CRISPR/Cas-mediated genome editing using prime and base editors to correct three most common mutations in USH2A (c.2299delG and c.2276G>T) ii) Correction of mutant USH2A transcripts using mRNA trans-splicing iii) A mutation-independent quadruple AAV vector strategy using a method developed in our preliminary work to reconstitute split gene fragments at the transcript level. All approaches will first be tested and optimized in different human cell lines suitable for the respective therapeutic strategies. The procedures established in vitro will subsequently be further validated in human retinal organoids and (if available) in explants from human donor retinas. The expression and localization of USH2A will additionally be investigated in the retina of wild-type mice and pigs injected using the mutation-independent quadruple AAV vector strategy at the transcript and protein levels. In addition, we will also investigate the structural and functional effects of the quadruple AAV vector strategy using the ush2a zebrafish model and a humanized c.2299delG mouse model. Finally, this mouse model will also allow us to analyze the therapy success of the prime editing strategy in vivo. If successful, these experiments will pave the way for future USH2A clinical trials using innovative technologies that would benefit many patients worldwide.