Pharmacological premature termination codon readthrough of ABCB11 in bile salt export pump deficiency: an in vitro study
Résumé
BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis type 2 (PFIC2) is a severe hepatocellular cholestasis due to biallelic mutations in ABCB11 encoding the canalicular bile salt export pump (BSEP). Nonsense mutations are responsible for the most severe phenotypes. The aim was to assess the ability of drugs to induce readthrough of six nonsense mutations (p.Y354X, p.R415X, p.R470X, p.R1057X, p.R1090X and p.E1302X) identified in PFIC2 patients. APPROACH & RESULTS: The ability of G418, gentamicin and PTC124 to induce readthrough was studied using a dual gene reporter system in NIH3T3 cells. The ability of gentamicin to induce readthrough and to lead to the expression of a full-length protein was studied in HEK293, HepG2 and Can 10 cells, using immunodetection assays. The function of the gentamicin-induced full-length protein was studied by measuring the [3 H[3H] ]-taurocholate transcellular transport in stable MDCK clones co-expressing Ntcp. Combinations of gentamicin and chaperone drugs (UDCA, 4-PB) were investigated. In NIH3T3, aminoglycosides significantly increased readthrough level of all mutations studied, while PTC124 only slightly increased readthrough of p.E1302X. Gentamicin, induced a readthrough of p.R415X, p.R470X, p.R1057X, p.R1090X in HEK293 cells. The resulting full-length proteins localized within the cytoplasm, except for BsepR1090X that was also detected at the plasma membrane of HEK293 and at the canalicular membrane of Can 10 and HepG2 cells. Additional treatment with 4-PB and UDCA significantly increased the canalicular proportion of full-length BsepR1090X protein in Can 10 cells. In MDCK clones, gentamicin induced a 40% increase of the BsepR1090X [3 H[3H] ] [3H] -taurocholate transport, which was further increased with additional 4-PB treatment. CONCLUSION: This study constitutes a proof of concept for readthrough therapy in selected PFIC2 patients with nonsense mutations.