RESUMEN
In cystic fibrosis (CF), the correction of splicing defects represents an interesting therapeutic approach to restore normal CFTR function. In this study, we focused on 10 common mutations/variants 711+3A>G/C, 711+5G>A, TG13T3, TG13T5, TG12T5, 1863C>T, 1898+3A>G, 2789+5G>A, and 3120G>A that induce skipping of the corresponding CFTR exons 5, 10, 13, 16, and 18. To rescue the splicing defects we tested, in a minigene assay, a panel of modified U1 small nuclear RNAs (snRNAs), named Exon Specific U1s (ExSpeU1s), that was engineered to bind to intronic sequences downstream of each defective exon. Using this approach, we show that all 10 splicing mutations analyzed are efficiently corrected by specific ExSpeU1s. Using complementary DNA-splicing competent minigenes, we also show that the ExspeU1-mediated splicing correction at the RNA level recovered the full-length CFTR protein for 1863C>T, 1898+3A>G, 2789+5G>A variants. In addition, detailed mutagenesis experiments performed on exon 13 led us to identify a novel intronic regulatory element involved in the ExSpeU1-mediated splicing rescue. These results provide a common strategy based on modified U1 snRNAs to correct exon skipping in a group of disease-causing CFTR mutations.
Asunto(s)
Fibrosis Quística/genética , Exones/genética , Mutación/genética , ARN Nuclear Pequeño/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células HeLa , Humanos , Intrones/genética , Conformación de Ácido Nucleico , Empalme del ARN/genética , ARN Nuclear Pequeño/químicaRESUMEN
BACKGROUND: We analyzed the CFTR response to VX-809/VX-770 drugs in conditionally reprogrammed cells (CRC) of human nasal epithelium (HNE) from F508del/F508del patients based on SNP rs7512462 in the Solute Carrier Family 26, Member 9 (SLC26A9; MIM: 608481) gene. METHODS: The Isc-eq measurements of primary nasal epithelial cells from F508del/F508del patients (nâ¯=â¯12) for CFTR function were performed in micro Ussing chambers and compared with non-CF controls (nâ¯=â¯2). Data were analyzed according to the rs7512462 genotype which were determined by real-time PCR. RESULTS: The CRC-HNE cells from F508del/F508del patients evidenced high variability in the basal levels of CFTR function. Also, the rs7512462*C allele showed an increased basal CFTR function and higher responses to VX-809â¯+â¯VX-770. The rs7512462*CCâ¯+â¯CT genotypes together evidenced CFTR function levels of 14.89% relatively to wt/wt (rs7512462*CT alone-15.29%) i.e., almost double of rs7512462*TT (7.13%). Furthermore, sweat [Cl-] and body mass index of patients also evidenced an association with the rs7512462 genotype. CONCLUSION: The CFTR function can be performed in F508del/F508del patient-derived CRC-HNEs and its function and responses to VX-809â¯+â¯VX-770 combination as well as clinical data, are all associated with the rs7512462 variant, which partially sheds light on the generally inter-individual phenotypic variability and in personalized responses to CFTR modulator drugs.
