Abstract
Supplementing wildtype copies of functionally defective genes (gene supplementation) with adeno-associated virus (AAV) is a strategy being explored clinically for various retinal dystrophies. However, the low cargo limit of this vector (∼5,000 bps) 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 ∼10% of photoreceptors, resulting in an incredible ∼10,000-fold improvement in light sensitivity and increasing visual acuity to ∼60% of the controls. Surprisingly, these effects were comparable to restoration mediated by gene supplementation, which targets ∼70% of photoreceptors. This strategy paves the way for treatment of inherited disorders caused by mutations in larger genes, for which conventional gene supplementation therapy is not currently feasible.
Footnotes
The manuscript has been revised during the process of review.