Abstract
Inherited retinal dystrophies (IRDs) are a large and heterogeneous group of degenerative diseases caused by mutations in various genes. Given the favorable anatomical and immunological characteristics of the eye, gene therapy holds great potential for their treatment. Our goal is to validate the preservation of visual functions by viral-free homology directed repair (HDR) in an autosomal recessive loss of function mutation. We used a tailored gene editing system based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to prevent retinal photoreceptor death in the retinal degeneration 10 (Rd10) mouse model of retinitis pigmentosa. We tested the gene editing tool in vitro and then used in vivo subretinal electroporation to deliver it to one of the retinas of mouse pups at different stages of photoreceptor differentiation. Three months after gene editing, the treated eye exhibited a higher visual acuity compared to the untreated eye. Moreover, we observed preservation of light-evoked responses both in explanted retinas and in the visual cortex of treated animals. Our study validates a CRISPR/Cas9-based therapy as a valuable new approach for the treatment of retinitis pigmentosa caused by autosomal recessive loss-of-function point mutations.
Highlights
Retinitis pigmentosa is a group of Inherited retinal dystrophies (IRDs) that cause the progressive death of retinal photoreceptors and eventually blindness (Ferrari et al, 2011)
We designed for the final editing tool guide RNA (gRNA) #4 which differ from gRNA #2 only in a single base pair (Figure 1D), corresponding to the C to T mutation found in the mutated Pde6b gene of retinal degeneration 10 (Rd10) mice
The transfected Rd10 cells were selected for green fluorescent protein (GFP)-expression with fluorescent activated cell sorting (FACS) and the editing efficiency was evaluated by BanI restriction assay, as described above for Neuro 2A (N2A) cells
Summary
Retinitis pigmentosa is a group of IRDs that cause the progressive death of retinal photoreceptors and eventually blindness (Ferrari et al, 2011). The treatment of retinitis pigmentosa is still a major challenge because of the early death of rod photoreceptors and the late onset of the symptoms. Vision in humans mainly depends on cone photoreceptors, which in retinitis pigmentosa degenerate only at a late stage: likely because cones metabolically depend on rods, which provide them nutrients (Narayan et al, 2016). Acting on the principal cause of degeneration, namely at the level of rod photoreceptors, would be an effective therapeutic approach to preserve vision in retinitis pigmentosa. Mutations in the β-domain of the phosphodiesterase 6 (PDE6B) gene, which hydrolyses cyclic guanosine monophosphate (cGMP) and initiates phototransduction, are among the most commonly identified causes of autosomal recessive retinitis pigmentosa
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