Efficient workflow for validating homology-independent targeted integration-mediated gene insertion in rod photoreceptor cells to treat dominant-negative mutations causing retinitis pigmentosa

Among the genome-editing methods for repairing disease-causing mutations resulting in dominant inhibition, homology-independent targeted integration (HITI)-mediated gene insertion of the normal form of the causative gene is useful because it allows the development of mutation-agnostic therapeutic products. For the rapid optimization and validation of highly effective HITI-treatment gene constructs against dominant-negative inheritance of inherited retinal dystrophy, we improved the gene constructs available in both plasmid and adeno-associated virus (AAV) vectors, and established a workflow that uses in vivo electroporation to verify the in vivo efficacy. By targeting the mouse Rhodopsin gene, we derived a construct in which HITI-mediated gene insertion occurs in 80%-90% of transduced mouse rod photoreceptor cells. This construct suppressed degeneration and induced visual restoration in the mutant mice. The HITI-treatment constructs for the rhodopsin gene were shown to be effective in AAV vectors, and this construction is available for the mouse Peripherin 2 gene. These findings suggest that the workflow reported here may be useful for the generation of HITI-treatment constructs for various target genes and for the development of gene therapy products.