EVALUATION OF SEQUENCE-SPECIFIC RNA EDITING TOOLS FOR CORRECTING CFTR NONSENSE MUTATIONS IN CYSTIC FIBROSIS THERAPY

Background. Premature-termination codons (PTCs) in the CFTR mRNA cause theproduction of truncated, non-functional CFTR proteins accounting for approximately 8% ofcystic fibrosis (CF) cases worldwide. Despite the intensive research in the field, these patientscannot benefit from specific and approved therapies yet. To address this issue, we haveexplored RNA-editing approaches to correct PTCs in the CFTR mRNA of CFF-16HBEgehuman bronchial epithelial cells. Methods. These systems exploit ADAR (adenosinedeaminase acting on RNA) enzymes to convert the adenosine (A) within the PTC into inosine(I). As the ribosome reads the inosine as guanosine (G), the stop codon could be recoded as asense codon, thereby allowing the synthesis of a full-length CFTR protein. We used twodifferent approaches according to the use of exogenous or endogenous ADARs. In theminixABE (mini-dCas13X-mediated RNA adenine base editor) system, the ADAR2deaminase domain (ADAR2DD), which is fused to a truncated dCAS13x.1, is recruited to theadenosine within the PTC by specifically designed guide RNAs. Instead, with the RESTORE(Recruiting endogenous ADAR to specific transcripts for oligonucleotide-mediated RNAediting) approach, linear or circular specific antisense RNA oligonucleotides (arRNAs) targetthe CFTR mRNA region surrounding the PTC to promote the endogenous ADAR recruitment.Results. Our results show the rescue of CFTR protein on the cell’s plasma membrane byimmunofluorescence, and the increase of CFTR full-length transcript expression by RT-qPCRin the mutated cell lines when both systems were applied. Moreover, ADAR-mediated RNAediting tools induced the functional rescue of CFTR protein in these cultures, which wasmeasured by electrophysiological methods. Conclusions. The obtained results suggest thatthese RNA editing-based tools mediated by ADARs might be explored as a promising strategyto treating nonsense mutations in CFTR, potentially contributing to novel therapeutic optionsfor CF patients that have nonsense mutations.