Partial cDNA instertion
Genome editing is a therapeutic approach particularly well suited to target large genes, which cannot be treated by gene addition therapy due to the packaging limits associated with the AAV vector. This project aims at employing genome editing tools and DNA double strand break (DSB) repair mechanisms in order to integrate minigene sequences into the target genes. The majority of EYS and USH2A mutations is spread over the entire gene. Recent data suggest that the DSB repair pathways non-homologous end joining (NHEJ), which is independent of homologous sequences in a repair template, and microhomology-mediated end joining (MMEJ), which depends on the presence of micro-homologous sequences (MHS), are both active in mature photoreceptors, and can be modelled in human 2D neuronal cell culture systems. By providing a template without or with MHS, together with the nuclease, to a photoreceptor cell, targeted integration or replacement of large DNA sequences is possible based either on homology-independent targeted integration (HITI) or microhomology-mediated targeted integration (MMTI). Here, we aim to design templates comprising partial cDNA sequences covering a large number of exons at the 3’-end of both genes plus a poly adenylation signal (polyA) in order to integrate them into the genomic locus in photoreceptor cells. This would make the genomic exons downstream of the integration site inactive and disease-causing mutations in these exons irrelevant. We will test the approach in human cell lines, in patient-derived retina-like cells, and in a human organotypic retina culture system.
