Abstract
Supplementing wildtype copies of functionally defective genes with adeno-associated virus (AAV) is a strategy being explored clinically for various retinal dystrophies. However, the low cargo limit of this vector allows its use in only a fraction of patients with mutations in relatively small pathogenic genes. To overcome this issue, we developed a single AAV platform that allows local replacement of a mutated sequence with its wildtype counterpart, based on combined CRISPR-Cas9 and micro-homology-mediated end-joining (MMEJ). In blind mice, the mutation replacement rescued approximately 10% of photoreceptors, resulting in an improvement in light sensitivity and an increase in visual acuity. These effects were comparable to restoration mediated by gene supplementation, which targets a greater number of photoreceptors. This strategy may be applied for the treatment of inherited disorders caused by mutations in larger genes, for which conventional gene supplementation therapy is not currently feasible.
Highlights
Supplementing wildtype copies of functionally defective genes with adeno-associated virus (AAV) is a strategy being explored clinically for various retinal dystrophies
We aim to develop a single AAV vector platform for mutation replacement genome editing using micro-homology-mediated end-joining (MMEJ)
Six guide RNAs (gRNAs) designed to flank the mutation were assessed with a T7 endonuclease 1 (T7E1) assay (Supplementary Fig. 2b, c and Supplementary Table 2)
Summary
Supplementing wildtype copies of functionally defective genes with adeno-associated virus (AAV) is a strategy being explored clinically for various retinal dystrophies. The low cargo limit of this vector allows its use in only a fraction of patients with mutations in relatively small pathogenic genes To overcome this issue, we developed a single AAV platform that allows local replacement of a mutated sequence with its wildtype counterpart, based on combined CRISPR-Cas[9] and micro-homology-mediated end-joining (MMEJ). Genome editing for loss-of-function mutations in larger genes that require local replacement of the mutated sequence with a wildtype counterpart (mutation replacement) has not been successful in treatment of neuronal disorders primarily affecting neurons, due to its low editing efficiency[9,10,11,12] This could be partly attributed to the requirement of two separate vectors for this approach, in which various components including Cas[9], two guide RNAs (gRNAs) and U6 promoters, and DNA template and flanking homology arms all needs to be contained. Through application of the platform in mouse models of retinal dystrophy, we show that a robust restoration of the visual function can be achieved, supported by an improved genome editing efficacy
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