Pharmacological Premature Termination Codon Readthrough of ABCB11 in Bile Salt Export Pump Deficiency: An In Vitro Study.

Abstract

BACKGROUND AND 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 patients with PFIC2.

APPROACH AND 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 human embryonic kidney 293 (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]-taurocholate transcellular transport in stable Madin-Darby canine kidney clones co-expressing Na+-taurocholate co-transporting polypeptide (Ntcp). Combinations of gentamicin and chaperone drugs (ursodeoxycholic acid, 4-phenylbutyrate [4-PB]) were investigated. In NIH3T3, aminoglycosides significantly increased the readthrough level of all mutations studied, while PTC124 only slightly increased the readthrough of p.E1302X. Gentamicin induced a readthrough of p.R415X, p.R470X, p.R1057X, and p.R1090X in HEK293 cells. The resulting full-length proteins localized within the cytoplasm, except for BsepR1090X , which was also detected at the plasma membrane of human embryonic kidney HEK293 and at the canalicular membrane of Can 10 and HepG2 cells. Additional treatment with 4-PB and ursodeoxycholic acid significantly increased the canalicular proportion of full-length BsepR1090X protein in Can 10 cells. In Madin-Darby canine kidney clones, gentamicin induced a 40% increase of the BsepR1090X [3 H]-taurocholate transport, which was further increased with additional 4-PB treatment.

CONCLUSION:

This study constitutes a proof of concept for readthrough therapy in selected patients with PFIC2 with nonsense mutations.

• Authors: Rachida Amzal, Alice Thébaut, Martine Lapalus, Marion Almes, Brigitte Grosse, Elodie Mareux, Mauricette Collado-Hilly, Anne Davit-Spraul, Laure Bidou, Olivier Namy, Emmanuel Jacquemin, Emmanuel Gonzales
• Journal: Hepatology (Baltimore, Md.) (Hepatology) Vol. 73 Issue 4 Pg. 1449-1463 (04 2021) ISSN: 1527-3350 [Electronic] United States
• PMID: 32702170 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2021 by the American Association for the Study of Liver Diseases.

Chemical References

• ABCB11 protein, human
• ATP Binding Cassette Transporter, Subfamily B, Member 11
• Abcb11 protein, mouse
• Codon, Nonsense
• Gentamicins
• Oxadiazoles
• Phenylbutyrates
• Ursodeoxycholic Acid
• 4-phenylbutyric acid
• ataluren

Topics

• ATP Binding Cassette Transporter, Subfamily B, Member 11 (genetics, metabolism)
• Animals
• Cholestasis, Intrahepatic (genetics, metabolism)
• Codon, Nonsense (drug effects)
• Cohort Studies
• Dogs
• Gentamicins (pharmacology)
• HEK293 Cells
• Hep G2 Cells
• Humans
• Madin Darby Canine Kidney Cells
• Mice
• NIH 3T3 Cells
• Oxadiazoles (pharmacology)
• Phenylbutyrates (pharmacology)
• Signal Transduction (drug effects)
• Transfection
• Ursodeoxycholic Acid (pharmacology)