Restoration of RPGR expression in vivo using CRISPR/Cas9 gene editing

Abstract

Mutations in the gene for Retinitis Pigmentosa GTPase Regulator (RPGR) cause the X-linked form of inherited retinal degeneration, and the majority are frameshift mutations in a highly repetitive, purine-rich region of RPGR known as the OFR15 exon. Truncation of the reading frame in this terminal exon ablates the functionally important C-terminal domain. We hypothesized that targeted excision in ORF15 by CRISPR/Cas9 and the ensuing repair by non-homologous end joining could restore RPGR reading frame in a portion of mutant photoreceptors thereby correcting gene function in vivo. We tested this hypothesis in the rd9 mouse, a naturally occurring mutant line that carries a frameshift mutation in RPGRORF15, through a combination of germline and somatic gene therapy approaches. In germline gene-edited rd9 mice, probing with RPGR domain-specific antibodies demonstrated expression of full length RPGRORF15 protein. Hallmark features of RPGR mutation-associated early disease phenotypes, such as mislocalization of cone opsins, were no longer present. Subretinal injections of the same guide RNA (sgRNA) carried in AAV sgRNA and SpCas9 expression vectors restored reading frame of RPGRORF15 in a subpopulation of cells with broad distribution throughout the retina, confirming successful correction of the mutation. These data suggest that a simplified form of genome editing mediated by CRISPR, as described here, could be further developed to repair RPGRORF15 mutations in vivo.

CRISPR/Cas9—A Promising Therapeutic Tool to Cure Blindness: Current Scenario and Future Prospects

“…The same approach has been used successfully in human cells to resolve the RHO gene mutation. This study confirms CRISPR/Cas9 as an effective system to target gene/alleles in a well-organized way indicating that it may be cast off for the treatment of RP and further dominant human genetic disorders [ 83 ].…”

Update of application of olfactory ensheathing cells and stem cells/exosomes in the treatment of retinal disorders

“…Rhodopsin (RHO) gene mutations had been widely used in mice and dogs as animal models of autosomal dominant RP [ 53 ]. Gumerson et al established an X-linked RP animal model, which was a mouse model of retinitis pigmentosa GTPase regulator (RPGR) deficiency similar to human RP3 [ 54 ]. Researchers used drug injection to create animal models of RP.…”

X-Linked Retinitis Pigmentosa Gene Therapy: Preclinical Aspects

“…Superior effects of rAAV2tYF-GRK1-hRPGRco and clues for reaching the appropriate dose for human studies were demonstrated [77]. Also, using CRISPR/Cas9 for repairing the RPGR gene with a pattern DNA strand in the process of HDR (homology-directed repair) in the RPGR -/y mouse model has been associated with good therapeutic effects for 6 to 12 months after treatment [78,79].…”