Research conducted at the Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, has shown that CRISPR/Cas9 gene editing technology may be capable of correcting dominant forms of retinitis pigmentosa. The research, published in Molecular Therapy (advance online publication January 19th, 2016) claims to be the first in vivo functional correction of an inherited dominant mutation using CRISPR/Cas9. Of the approximate 250 genes associated with inherited ocular disorders to date, an estimated 70 genes appear to be inherited in a dominant fashion and could, at least in theory, benefit from a similar approach. While a detailed analysis of the viable ocular therapeutic targets available for CRISPR correction is awaited, several commercial entities and clinics are already turning their attention to the application of CRISPR/Cas9 within ocular disease.
In testing their CRISPR/Cas9 approach the researchers used a transgenic rat model of autosomal dominant retinitis pigmentosa (Ser344ter) carrying two wild-type copies of the rhodopsin gene, together with a separate mutated rhodopsin allele engineered with a gain-of-function mutation. However, in a more representative clinical situation it is postulated that as little as 10% normal rhodopsin expression in a dominantly mutated phenotype may be sufficient to bring about some degree of functional rescue. A CRISPR/Cas9 cassette was used to selectively disrupt the mutated allele by targeting a protospacer motif (PAM) which differed by only a single nucleotide between the mutated and wild-type alleles. A single subretinal unilateral injection of the guide RNA transcript at P0 transcript, followed by a short electroporation, appeared sufficient to bring about therapeutic benefit.
An assessment of treated animals showed that “extensive and robust retinal preservation” was observed in injected eyes with up to eight layers of rescued photoreceptors while contralateral eyes, used as controls, showed only a single photoreceptor cell layer. Photoreceptor rescue appeared almost exclusively in transfected regions of the retina suggesting that the gain-of-function toxicity of the mutated allele had been extinguished facilitating normal rhodopsin expression and photoreceptor outer segment formation, compared to the expected pathology progression in untreated retinas. In addition, as the team used the contralateral eye of individual animals as internal controls, they were able to show that visual acuity was 35% higher in treated eyes compared with the fellow eye. The authors concluded their study by commenting that, “the challenge of generating targeted therapies for diseases with mutational heterogeneity may be addressed by altering the PAM specificity of Cas through rational-design engineering or by using non-canonical Cas enzymes. Such efforts may broaden the number of targetable mutations and, thereby, expand the treatable pool of patients with degenerative diseases of the retina, and possibly other tissues.”