CRISPR/CAS9-Mediated In Vivo Gene Editing Corrects The Retinal Dystrophy Phenotype of S334ter-3 Rats.

Abstract

Purpose: The recent advent of CRISPR/Cas9-mediated gene editing technology has improved the targeting for genome manipulation. However, the feasibility of using CRISPR in vivo to provide gene therapy for retinal degenerative diseases is not yet clear. To this end, we demonstrate the reversal of retinal defects in an animal model of retinitis pigmentosa (RP) through targeted in vivo genome modification using CRISPR.

Methods: Heterozygous S334ter-3 rats received unilateral subretinal injection of CRISPR/Cas9/guide-RNA constructs that were designed to target the mutant rhodopsin gene of S334ter-3 rats. Rhodopsin and scrambled guide-RNA constructs contained the mCherry reporter for later detection of transfected photoreceptors by fluorescence microscopy or for isolation by flow cytometry. Eyes were removed and evaluated for photoreceptor preservation between three and 72 days following treatment. Dissociated S334ter-3 retinal cells were sorted on the basis of mCherry reporter expression for subsequent DNA and protein analyses. Immunohistochemical evaluation of photoreceptor rescue was performed using monoclonal antibodies that exclusively recognize the native rhodopsin isoform.

Results: Correct guide-RNA targeting and Cas9 cleavage were confirmed by genomic DNA sequencing of S334ter-3 rat-derived bone marrow progenitor cells in vitro. Photoreceptors were preserved in rhodopsin-targeted CRISPR/Cas9/guide-RNA injected eyes, which had 4-6 rows of photoreceptors, compared with a single discontinuous layer of photoreceptors in control-injected and untreated eyes. Immunostaining for the functional rhodopsin isoform was observed in the outer segments of preserved photoreceptors, whereas outer segments in this model fail to develop. Visual function in treated S334ter-3 rats is currently being evaluated by electroretinography (ERG), optokinetic response (OKR) and luminescence threshold recording (LTR) from the superior colliculi (SC).

Conclusions: This study demonstrated that CRISPR/Cas9 repaired the retinal defects associated with S334ter-3 rats by deleting the dominant negative function of mutant rhodopsin. Long-term safety and efficacy are currently under investigation.