Project 2 - Evaluation of novel gene therapy approaches for USH2A

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, c.2276G>T and c.11864 G ii) Correction of mutant USH2A transcripts using mRNA trans-splicing iii) A mutation-independent quadruple AAV vector strategy using a method developed in our previous work.

All approaches will first be tested and optimized in different human cell lines. Gene expression will be addressed in human retinal organoids and in the retina of wild-type mice and pigs. Functional analyses will be performed in the ush2a zebrafish model and in a humanized c.2299delG mouse model. If successful, these experiments will pave the way for future USH2A clinical trials using innovative technologies that would benefit many patients worldwide.

Overview of the different strategies for treatment of USH2A. A, Type and positions of the USH2A mutations to be targeted using the prime editing approaches shown in B. gDNA, genomic DNA. C, Principle of the mRNA trans-splicing approach designed for replacement of exon 13. D, Quadruple vector strategy for reconstitution of the full-length USH2A cDNA. It requires three different binding domains (Bd1-3) to bind simultaneously to their complementary counterparts as indicated.