Development of an AAV-CRISPR-Cas9-based treatment for dominant cone-rod dystrophy 6

Russell W Mellen; Kaitlyn R Calabro; K Tyler McCullough; Sean M Crosson; Alejandro de la Cova; Diego Fajardo; Emily Xu; Sanford L Boye; Shannon E Boye

doi:10.1016/j.omtm.2023.05.020

Development of an AAV-CRISPR-Cas9-based treatment for dominant cone-rod dystrophy 6

Mol Ther Methods Clin Dev. 2023 Jun 1:30:48-64. doi: 10.1016/j.omtm.2023.05.020. eCollection 2023 Sep 14.

Authors

Russell W Mellen¹, Kaitlyn R Calabro¹, K Tyler McCullough¹, Sean M Crosson¹, Alejandro de la Cova¹, Diego Fajardo¹, Emily Xu¹, Sanford L Boye², Shannon E Boye¹

Affiliations

¹ Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida, Gainesville, FL, USA.
² Powell Gene Therapy Center, Department of Pediatrics, University of Florida, Gainesville, FL, USA.

Abstract

Cone-rod dystrophy 6 (CORD6) is caused by gain-of-function mutations in the GUCY2D gene, which encodes retinal guanylate cyclase-1 (RetGC1). There are currently no treatments available for this autosomal dominant disease, which is characterized by severe, early-onset visual impairment. The purpose of our study was to develop an adeno-associated virus (AAV)-CRISPR-Cas9-based approach referred to as "ablate and replace" and evaluate its therapeutic potential in mouse models of CORD6. This two-vector system delivers (1) CRISPR-Cas9 targeted to the early coding sequence of the wild-type and mutant GUCY2D alleles and (2) a CRISPR-Cas9-resistant cDNA copy of GUCY2D ("hardened" GUCY2D). Together, these vectors knock out ("ablate") expression of endogenous RetGC1 in photoreceptors and supplement ("replace") a healthy copy of exogenous GUCY2D. First, we confirmed that ablation of mutant R838S GUCY2D was therapeutic in a transgenic mouse model of CORD6. Next, we established a proof of concept for "ablate and replace" and optimized vector doses in Gucy2e^+/-:Gucy2f^-/- and Gucy2f^-/- mice, respectively. Finally, we confirmed that the "ablate and replace" approach stably preserved retinal structure and function in a novel knockin mouse model of CORD6, the RetGC1 (hR838S, hWT) mouse. Taken together, our results support further development of the "ablate and replace" approach for treatment of CORD6.

Keywords: AAV; CORD6; GUCY2D; adeno-associated virus; cone-rod dystrophy; gene therapy; inherited retinal disease; ophthalmology; retina.

Abstract

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