(684) Correction of the CFTR 1717-1G >A splicing mutation through CRISPR based technology

Background: Almost 13% of mutations causing cystic fibrosis alter the correct splicing of the CFTR gene. Among these, the Class 1 1717-1G >A substitution is one of the most common mutation affecting approximately 1% of cystic fibrosis (CF) patients. This mutation consists in a G to A conversion in intron 11 which results in the inactivation of a conserved AG dinucleotide at the 3’ end acceptor splice site leading to exon 12 skipping or recognition of an alternative cryptic splice site. Both altered splicing products generate a stop codon and consequent lack of CFTR protein synthesis which is associated with CF clinical development not treatable with any currently available pharmacological agent. Gene therapy, in particular CRISPR-Cas derived systems, is an emerging opportunity to permanently cure CF caused by any class type mutations.
Here, we developed a genome editing strategy to correct the 1717-1G >A mutation through the most recent CRISPR-Cas technology, DSB-free.

Methods: We tested the recently developed adenine base-editing (ABE) and prime-editing (PE) technologies capable of promoting precise genome editing in the absence of double-strand breaks (DSB) as opposed to the original CRISPR-Cas strategies thus preventing deleterious genotoxic effects.

Results: We have set up the correction strategy in 1717-1G >A HEK293 models that we have developed by using CFTRconstructs mimicking the splicing defect. We obtained up to 42% of correction using NG-ABE8e and NG-ABE8.20m base-editors through plasmid transfection. Nevertheless, both ABEs were associated with high levels of bystander edits, which resulted in the unwanted modifications near the mutated nucleotide with potential detrimental effects in CFTR protein synthesis. As alternative strategy we tested the most recent prime-editing approach which produced encouraging preliminary results with over 10% scarless correction and no detected indels.

Conclusions: Overall, we obtained results showing that the recent prime-editor CRISPR-Cas approach can be exploited to repair the 1717-1G >A providing precise modification over the bystander unwanted mutations introduced by the base-editors. Further optimization of the prime-editor strategies is ongoing to produce a more efficient correction of the 1717-1G >A splicing mutation.

Acknowledgements: This work was supported by the Cystic Fibrosis Foundation

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