Reversal of Cystic Fibrosis Phenotype in a Cultured Δ508 Cystic Fibrosis Transmembrane Conductance Regulator Cell Line by Oligonucleotide Insertion

Cystic fibrosis (CF) is a lethal genetic disorder that is due to mutations in the gene encoding the cAMP-activated anion CF transmembrane conductance regulator (CFTR) channel. A three-nucleotide base deletion (TTT), encoding phenylalanine in position 508 of the translatable CFTR sequence (accompanied by a C to T replacement immediately 5′ to the deletion), accounts for ≈75% of cases of the disease. In the present study, an oligonucleotide complex (CF4-CF6, 2′-0-methyl RNA-unmodified RNA oligonucleotide duplex, respectively) was used to restore CFTR function by insertion of missing bases in Δ508 CFTR mRNA from a cultured (Δ508) cell line. cAMP-activated whole-cell currents and Cl-transport were detected in CF4-CF6-treated, but not control Δ508, cells by patch-clamp and 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence (SPQ) quenching analyses, respectively. Further, the nucleotide addition in the deleted region of Δ508 CFTR was determined after amplification by RT-PCR. Insertion of UGU and replacement of U by C immediately 5′ to the deletion site in Δ508 mRNA appear to have taken place, with phenotypic but not genotypic reversion in tissue culture of treated cells. The mechanism of insertion of nucleotides has yet to be determined.