CRISPR-Cas9–mediated therapeutic editing of Rpe65 ameliorates the disease phenotypes in a mouse model of Leber congenital amaurosis

Abstract

Leber congenital amaurosis (LCA), one of the leading causes of childhood-onset blindness, is caused by autosomal recessive mutations in several genes including RPE65. In this study, we performed CRISPR-Cas9–mediated therapeutic correction of a disease-associated nonsense mutation in Rpe65 in rd12 mice, a model of human LCA. Subretinal injection of adeno-associated virus carrying CRISPR-Cas9 and donor DNA resulted in >1% homology-directed repair and ~1.6% deletion of the pathogenic stop codon in Rpe65 in retinal pigment epithelial tissues of rd12 mice. The a- and b-waves of electroretinograms were recovered to levels up to 21.2 ± 4.1% and 39.8 ± 3.2% of their wild-type mice counterparts upon bright stimuli after dark adaptation 7 months after injection. There was no definite evidence of histologic perturbation or tumorigenesis during 7 months of observation. Collectively, we present the first therapeutic correction of an Rpe65 nonsense mutation using CRISPR-Cas9, providing new insight for developing therapeutics for LCA.

Therapeutic adenine base editing corrects nonsense mutation and improves visual function in a mouse model of Leber congenital amaurosis

“…Small insertions and deletions (indels) after DNA double-stranded breaks (DSBs) can correct a frame-shift intron variant of the CEP290 gene (IVS26; c.2991+1655 A>G), the most frequent one in patients with LCA type 10 (6). On the other hand, we have shown the therapeutic potential of homology-directed repair (HDR) in the previous study (7). Dual AAVs carrying CRISPR-Cas9 and donor DNA in each vector resulted in ~1.2% of HDR and ~1.6% of in-frame 1-codon deletion when they were administered through subretinal injection in retinal degeneration 12 (rd12) mice (7).…”

Pathways and disease-causing alterations in visual chromophore production for vertebrate vision

“…Thus, recent progress in animal models, human genetics, and structural biology have provided essential information about these processes, as well as innovative approaches to treat human blinding disorders. Because of these discoveries, new opportunities now exist for pharmacological treatments (Kiser and Palczewski, 2016) and gene therapies (Jo et al, 2019;Suh et al, 2020) to prevent the deterioration of vision or even in some cases the restoration of sight.…”

“…Restoration of normal CEP290 expression raises the possibility that this common form of LCA can be rectified by gene editing. In a proof-of-concept study, a mutated Rpe65 was targeted by CRISPR-Cas9mediated homology directed repair (HDR) in a mouse model called rd12, which harbors a naturally occurring Rpe65 mutation (Jo et al, 2019). The rescue was modest, characterized by approximately 1% homology-directed repair and ~1.6% deletion of the pathogenic stop codon in the Rpe65 gene.…”

Delivery Approaches for Therapeutic Genome Editing and Challenges

“…In a study using AAV delivery for CRISPR-Cas9, the IVS26 intronic mutation in the CEP290 that causes Leber congenital amaurosis-10 was removed in a large deletion [ 109 ], demonstrating the applicability of the therapeutic approach for large gene deletions. In a separate study, congenital amaurosis-10 was corrected using CRISPR-Cas9-mediated targeting of the disease-associated nonsense mutation in Rpe65 [ 110 ]. Furthermore, the study by Nishiguchi et al used AAV-mediated delivery of CRISPR-Cas9 and a donor template to correct a mutation causing retinal dystrophy in 10% of photoreceptors with improvement in light sensitivity and enhanced visual acuity [ 111 ].…”