329. Using CRISPR/Cas9 as a Therapeutic Approach for Leber Congenital Amaurosis 10 (LCA10)

Introduction: Leber congenital amaurosis (LCA) is the most severe form of inherited retinal diseases with early onset of symptoms in the first year of life. The most frequent genetic cause of LCA, accounting for approximately 15% of all LCA cases in western countries, is a deep-intronic mutation c. 2991+1655A>G located in the intron 26 of human CEP290 gene. LCA caused by CEP290 mutation is known as LCA10. The intronic mutation of CEP290 generates a cryptic splice donor site, resulting in the inclusion of a pseudoexon that leads to a premature stop codon and a truncated protein. The size of human CEP290 cDNA (~7.4 kb) exceeds the cargo size (~4.8 kb) of recombinant adeno-associated viral vectors (rAAVs), which makes this gene challenging for gene replacement therapy. In this regard, we tested if we can use the new genome editing technology CRISPR/Cas9 as an alternative strategy for LCA10 by removing the intronic mutation of CEP290 and preventing the cryptic splicing.Methods: CRISPR/SpCas9 was employed to introduce the intronic splice mutation c. 2991+1655 A>G into HEK 293FT cells to create a cellular model for LCA10/CEP290. In this cellular model, we used guide RNA pairs coupled with SpCas9 to delete the intronic region flanking the intronic mutation.Results: The deep-intronic mutation c. 2991+1655 A>G was successfully introduced into 293FT cells through the homology directed repair (HDR) pathway of CRISPR/spCas9. CEP290 expression levels were markedly reduced in the mutant cell line. Using this cellular model, we identified three sgRNA pairs that could efficiently (>50%) delete the intronic mutation, significantly rescue wild-type CEP290 expression levels and reduce mutant CEP290 expression.Conclusions: Our results demonstrate that the paired sgRNAs coupled with SpCas9 are capable of removing the c. 2991+1655A>G mutation and are highly efficient in preventing the splicing of the mutant cryptic exon and restoring wild-type CEP290 expression.