An AAV-CRISPR/Cas9 gene editing approach for GUCY2D-associated cone rod dystrophy (CORD6)

Abstract

Purpose : Autosomal dominant mutations in GUCY2D (GC1) are the leading cause of cone-rod dystrophy (CORD6). Given our proven ability to deliver therapeutic GUCY2D to photoreceptors (PR) via AAV, an attractive treatment approach for CORD6 is to combine AAV-CRISPR/Cas9-based knockout of GUCY2D with supplementation of a ‘hardened’ wtGUCY2D. Here, we evaluate selectivity, efficiency and phenotypic outcomes of CRISPR/Cas9 mediated editing of the GC1 gene in mouse and macaque (M. fascicularis).

Methods : S. aureus Cas9 gRNAs targeting exon 4 of GUCY2D or exon 2 of Gucy2e were designed to recognize GUCY2D, but not Gucy2e, and vice versa. C57Bl/6J (WT) and GC1^+/-:GC2^-/- mice were subretinally injected with AAV5-hGRK1-Cas9 along with either AAV5-Gucy2e-gRNA-hGRK1-GFP or AAV5-GUCY2D-gRNA-hGRK1-GFP control. Fundoscopy, ERG, OCT, IHC, western blot (WB), indel analysis and/or qRT-PCR of transgene transcripts were performed at 6 and 20 weeks post-injection (p.i.). Three macaques received subretinal injections (n=3/eye) of AAV5-GUCY2D-gRNA-hGRK1-GFP + AAV5-hGRK1-Cas9 (right eyes) and AAV5-Gucy2e gRNA-hGRK1-GFP + AAV5-hGRK1-Cas9 (left eyes), except for one animal who received AAV5-hGRK1-GFP in its left eye. hGRK1-GFP in gRNA vectors allowed selective sorting of PRs for evaluating cell selectivity and efficiency of gene editing.

Results : In WT and GC1^+/-:GC2^-/- mice, reductions in both scotopic and photopic a- and b-waves were noted in Gucy2e gRNA-treated eyes relative to controls. Editing of Gucy2e was restricted to PRs with rates of 45% and 36%, respectively. At 20 weeks p.i., Gucy2e gRNA-treated eyes of GC1^+/-:GC2^-/- mice exhibited a significant loss of retinal structure (ONL thinning) and downregulation of GC1 relative to control treated eyes. In macaque, GUCY2D editing was also restricted to PRs. In GUCY2D gRNA-treated eyes, PR outer segments were shortened and GC1 was downregulated in areas exposed to vectors (i.e. expressing GFP). This was not seen in Gucy2e gRNA-injected control eyes.

Conclusions : Our gene editing results in mice and primates establish the feasibility of an AAV-CRISPR/Cas9 therapy for the treatment of CORD6 in human photoreceptors.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.