Background: Coagulation factor V (FV) is a liver-derived protein encoded by the F5 gene. Recently, a low-abundance splicing variant of FV lacking 702 amino acids (FV-short) has been identified. Both activated FV and FV-short express procoagulant activity as cofactors of factor Xa (FXa) in prothrombin activation. However, FV-short also binds tissue factor pathway inhibitor (TFPIα) with high affinity, stabilizing it in the circulation and potently enhancing its anti-FXa activity. Accordingly, genetic mutations that up-regulate FV-short expression have been associated with markedly elevated TFPIα levels and a bleeding tendency (East Texas and Amsterdam bleeding disorders). These disorders may be amenable to splicing modulation therapy using specific antisense oligonucleotides that decrease the relative expression of FV-short.

Aims: To design and test specific morpholino antisense oligonucleotides (MAOs) able to decrease the relative expression of FV-short, as a potential “molecular therapy” for the East Texas and Amsterdam bleeding disorders.

Methods: MAOs targeting the donor and acceptor splice sites of the FV-short-specific intron were designed and tested on a liver cell line (HepG2) that naturally expresses FV and FV-short. After treating the cells with 0-20 µM MAOs, total RNA was isolated and reverse-transcribed. FV and FV-short mRNAs were amplified using transcript-specific primers and quantified by real-time qPCR.

Results: Untreated HepG2 cells expressed 2-3 orders of magnitude more FV than FV-short mRNA. Cell treatment with both MAOs (alone or in combination) decreased FV-short mRNA expression in a dose-dependent manner down to 30-40% of the level in untreated cells (p < 0.01, n=3 biological replicates). Differently, treatment with a control MAO did not decrease the FV-short expression level.

Conclusions: We have designed MAOs that decrease the relative expression of FV-short mRNA in HepG2 cells. Once fully validated, these molecules may form the basis for a “molecular therapy” for the East Texas and Amsterdam bleeding disorders, and possibly also haemophilia.

« Back to ISTH 2020 Congress

ISTH Congress Abstracts - https://abstracts.isth.org/abstract/modulation-of-alternative-splicing-of-the-f5-gene-using-morpholino-antisense-oligonucleotides/