Somatic Gene Editing of GUCY2D by AAV-CRISPR/Cas9 Alters Retinal Structure and Function in Mouse and Macaque

Abstract

Mutations in GUCY2D, the gene encoding retinal guanylate cyclase-1 (retGC1), are the leading cause of autosomal dominant cone-rod dystrophy (CORD6). Significant progress toward clinical application of gene replacement therapy for Leber congenital amaurosis (LCA) due to recessive mutations in GUCY2D (LCA1) has been made, but a different approach is needed to treat CORD6 where gain of function mutations cause dysfunction and dystrophy. The CRISPR/Cas9 gene editing system efficiently disrupts genes at desired loci, enabling complete gene knockout or homology directed repair. Here, adeno-associated virus (AAV)-delivered CRISPR/Cas9 was used specifically to edit/disrupt this gene's early coding sequence in mouse and macaque photoreceptors in vivo, thereby knocking out retGC1 expression and demonstrably altering retinal function and structure. Neither preexisting nor induced Cas9-specific T-cell responses resulted in ocular inflammation in macaques, nor did it limit GUCY2D editing. The results show, for the first time, the ability to perform somatic gene editing in primates using AAV-CRISPR/Cas9 and demonstrate the viability this approach for treating inherited retinal diseases in general and CORD6 in particular.

Gene editing and CRISPR in the clinic: current and future perspectives

“…Despite these differences, it is clear that a portion of the human population has pre-existing antibodies to the two most commonly used Cas9 enzymes, which could affect the use of these Cas9 molecules in vivo. Furthermore, studies in animal models have shown that delivery of SaCas9 by AAV results in the development of anti-SaCas9 antibodies in adult mice after systemic delivery and in non-human primates after injection into the eye [115,143]. Similar to the findings with SaCas9 by AAV, systemic delivery of SpCas9 by adenovirus also resulted in development of anti-SpCas9 antibodies in adult mice [164].…”

“…The first Cas9 clinical trial for blindness, treatment of LCA10, has already been registered (NCT03872479) and was discussed in the current trials section. Also under development is Cas9 therapy involving the knockout of the endogenous gene copies and replacement with a functional copy for autosomal dominant cone-rod dystrophy (CORD6)-mediated blindness which has progressed to use in non-human primates [115]. Beyond the initiated LCA10 trial and CORD6 experiments, CRISPR-based therapeutics are being developed for a range of other inherited retinal disorders and multifactorial retinal diseases which has been reviewed elsewhere [116,117].…”

“…Similar to the humoral immunity studies, there is variability in the percentage of responding humans, but it is clear that many people may have cells capable of killing Cas9 transfected cells in vivo. In addition, animal studies have shown the development of cellular immunity to Cas9 after AAV delivery of SaCas9 and adenoviral delivery of SpCas9 in mouse models and AAV delivery of SaCas9 in non-human primates [115,143,164]. As seen with antibody development, the age of the animal appears to be important, as systemic delivery of AAV-SaCas9 to neonatal mice did not cause the development of a reactive T-cell response [143].…”

Gene editing prospects for treating inherited retinal diseases

“…This strategy could be in principle proposed for the treatment of all mutations in RHO gene related to RP, thus circumventing the allelic heterogeneity of the disease. A similar approach has been also applied to treat the autosomal dominant cone-rod dystrophy (CORD6) in mice and primates 84. This disease is caused by a dominant mutation in the GUCY2D gene encoding the retinal guanylate cyclase‐1 (retGC1), a protein expressed in photoreceptor and involved in the resynthesis of cGMP required for recovery of the dark state after phototransduction.…”

“…This disease is caused by a dominant mutation in the GUCY2D gene encoding the retinal guanylate cyclase‐1 (retGC1), a protein expressed in photoreceptor and involved in the resynthesis of cGMP required for recovery of the dark state after phototransduction. McCullough et al 84 proposed to ablate the expression of WT and mutant endogenous GUCY2D alleles by mutation-independent Cas9/gRNA approach and complement back the WT GUCY2D cDNA, resistant to gene editing, availing AAV vectors. In this study, however, the authors reported only the first part of the strategy consisting of the ablation of endogenous GUCY2D alleles in macaque photoreceptors by subretinal injection of two AAVs, one expressing SpCas9 and the second carrying a gRNA tailored to the exon 4 of GUCY2D gene.…”