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Guoxiang Ruan, Elizabeth Barry, Dan Yu, Michael Lukason, Seng Cheng, Abraham Scaria; CRISPR/Cas9-Mediated Genome Editing as a Therapeutic Approach for Leber Congenital Amaurosis 10. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4468.
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As the most common subtype of Leber congenital amaurosis (LCA), LCA10 is a severe retinal dystrophy caused by mutations in the CEP290 gene. The most frequent mutation found in LCA10 patients is a deep intronic mutation in CEP290 that generates a cryptic splice donor site. The large size of the CEP290 gene prevents its use in AAV-mediated gene augmentation therapy. Here, we test the hypothesis that targeted genomic deletion using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system can be used as a therapeutic approach for the treatment of LCA10 patients bearing the CEP290 splice mutation.
A cellular model of LCA10 was developed by introducing the CEP290 splice mutation into human embryonic kidney (HEK) 293FT cells using CRISPR/Cas9-mediated homology-directed repair (HDR). Paired sgRNAs and Cas9 were employed to excise the intronic fragment containing the CEP290 splice mutation. Dual AAV vectors (AAV5-SpCas9 and AAV5-sgRNA pairs) were co-injected into the subretinal space of C57BL/6J mice to delete an intronic fragment in the Cep290 gene of mouse photoreceptors. A self-limiting CRISPR/Cas9 system was developed to limit the expression of SpCas9 by incorporating sgRNA recognition sites into the SpCas9 plasmid.
The LCA10 cellular model recapitulated the CEP290 expression pattern observed in LCA10 patient fibroblasts. In this model, guide RNA pairs coupled with SpCas9 were highly efficient at removing the intronic splice mutation and restoring the expression of wild-type CEP290. In addition, we demonstrated that the dual AAV system could effectively delete an intronic fragment of the Cep290 gene in the mouse retina. The self-limiting CRISPR/Cas9 system was effective at minimizing the duration of SpCas9 expression and rescuing the levels of wild-type CEP290 mRNA.
These results support further studies to determine the therapeutic potential of CRISPR/Cas9-based strategies for the treatment of LCA10 patients bearing the CEP290 splice mutation.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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