RNA editing applied to cystic fibrosis: RESTORE can target G542X CFTR mRNA and revert the nonsense mutation.

Background: Nonsense mutations in the CFTR gene are responsible for approximately 8 % of cystic fibrosis (CF) cases worldwide. The consequent premature termination of translation leads to the production of a truncated and non-functional CFTR protein. Despite the intensive research in the field, these patients cannot benefit from specific and approved therapies yet. To address this issue, in this study we evaluated a potential therapeutic strategy to overcome the nonsense G542X (UGG > UGA) mutation in the CFF-16HBEge human bronchial epithelial cells by restoring the full-length CFTR protein.

Methods: We applied the RESTORE (Recruiting endogenous ADAR to specific transcripts for oligonucleotide-mediated RNA editing) approach, based on specifically designed antisense RNA oligonucleotides (ASOs) to recruit endogenous ADAR (adenosine deaminase acting on RNA) enzymes. The ADAR's recruitment to the target CFTR mRNA is expected to promote the deamination of adenosine (A) into inosine (I) within the premature termination codon (UGA). As the ribosome reads the inosine as guanosine (G), the stop codon could be recoded as a tryptophan (UGG), thereby allowing the synthesis of a full-length CFTR protein, albeit with a different amino acid.

Results: Our results indicate that in the CFF-16HBEge G542X cell line, the transfection of a specific ASO allows the rescue of the CFTR transcript and protein expression, compared to the untransfected mutated cells. Next generation sequencing of CFTR cDNA also confirmed the occurrence of the expected RNA editing outcome.

Conclusions: The obtained results suggest that the RESTORE approach might be explored as a promising strategy to treating nonsense mutations in CFTR, potentially contributing to novel therapeutic options for CF patients.