Usher syndrome is a devastating inherited disease with vision and hearing impairment. The most common genetic mutation to cause Usher syndrome is a single nucleotide deletion in the USH2A gene. The USH2A protein is expressed in rod and cone photoreceptors in the retina and in sensory hair cells of the inner ear. Affected patients have retinal degeneration with progressively constricting visual fields and moderate non-progressive hearing loss. Currently, there is no therapy available for vision loss caused by mutations in USH2A.

We are developing a gene correction strategy based on prime editing, a novel technology. Prime editing can precisely insert the missing G nucleotide into the USH2A sequence, in order to restore expression of the functional protein. To facilitate translation of this concept to the clinic, we will apply models that are closely related to the living human eye, including human retinal explants, human engineered retinal organoids and humanised mouse models.

• We are developing an adeno-associated vector (AAV) platform for prime editing. AAVs will be tested and optimized on cell lines using a disease-specific reporter system. We will also perform prime editing in human retinal explants, using the disease-specific reporter. This system will reveal which cells in the retina are corrected and provide proof-of-concept that prime editing can successfully correct the mutation in the retina.
• We are creating mutation-carrying human retinal organoids, and we are testing prime editing on them. Retinal organoids are three-dimensional and they exhibit the presence of the two types of photoreceptors along with downstream retinal neurons. We will engineer the del2299G mutation into induced pluripotent stem cells and derive retinal organoids form them. We will analyze whether we can detect a disease-associated phenotype in organoids and test prime editing to normalize this.
• We are creating a humanized mouse model that will allow testing of AAV vectors carrying prime editors on the human mutation in vivo. Importantly, we expect to see retinal degeneration and hearing loss in this mouse model. We anticipate that using our therapeutic strategy, we can halt the degeneration of photoreceptors. Such results would provide in vivo proof-of-concept for prime editing in the diseased retina.

Our experimental plan aims at testing feasibility of gene correction of USH2A del2299G by prime editing. Prime editing can potentially correct 90% of all human mutations. If this project is successful, theoretically other mutations can be corrected with only slight changes in the therapeutic construct.

The idea of potential correction of an underlying genetic problem in vivo is unprecedented in biomedicine and the fact that this strategy could reasonably reach the clinic within 5 years or so provides an exceptional opportunity.

Project Leader: Bence György, Head of the IOB Clinical Translation Group