Pathological mechanism and antisense oligonucleotide-mediated rescue of a non-coding variant suppressing factor 9 RNA biogenesis leading to hemophilia B

• Published in: PLoS genetics Vol. 16; no. 4; p. e1008690
• Main Authors: Krooss, Simon, Werwitzke, Sonja, Kopp, Johannes, Rovai, Alice, Varnholt, Dirk, Wachs, Amelie S, Goyenvalle, Aurelie, Aarstma-Rus, Annemieke, Ott, Michael, Tiede, Andreas, Langemeier, Jörg, Bohne, Jens
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 01.04.2020; Public Library of Science (PLoS)
• Subjects: 3' Untranslated Regions; Animals; Antisense oligonucleotides; Binding sites; Biochemistry, Molecular Biology; Biology and Life Sciences; Cell culture; CHO Cells; Coagulation; Coagulation factors; Cricetinae; Cricetulus; Disease; Endocrinology; Evolution; Factor IX - genetics; Factor IX - metabolism; Factor IX deficiency; Gastroenterology; Gene expression; HEK293 Cells; HeLa Cells; Hematology; Hemophilia; Hemophilia B - genetics; Hepatology; Human health and pathology; Humans; Infections; Life Sciences; Loss of Function Mutation; Medical research; Medical schools; Molecular biology; Mutants; Mutation; Oligonucleotides, Antisense - genetics; Oncology; Phenotype; Phenotypes; Poly(A); Research and Analysis Methods; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA, Small Nuclear - genetics; Splicing; Suppression, Genetic; Transcription; Virology; Germany
• ISSN: 1553-7404; 1553-7390; 1553-7404
• DOI: 10.1371/journal.pgen.1008690

Loss-of-function mutations in the human coagulation factor 9 (F9) gene lead to hemophilia B. Here, we dissected the consequences and the pathomechanism of a non-coding mutation (c.2545A>G) in the F9 3' untranslated region. Using wild type and mutant factor IX (FIX) minigenes we revealed that the mutation leads to reduced F9 mRNA and FIX protein levels and to lower coagulation activity of cell culture supernatants. The phenotype could not be compensated by increased transcription. The pathomechanism comprises the de novo creation of a binding site for the spliceosomal component U1snRNP, which is able to suppress the nearby F9 poly(A) site. This second, splicing-independent function of U1snRNP was discovered previously and blockade of U1snRNP restored mutant F9 mRNA expression. In addition, we explored the vice versa approach and masked the mutation by antisense oligonucleotides resulting in significantly increased F9 mRNA expression and coagulation activity. This treatment may transform the moderate/severe hemophilia B into a mild or subclinical form in the patients. This antisense based strategy is applicable to other mutations in untranslated regions creating deleterious binding sites for cellular proteins.