Antisense-Mediated Down-Regulation of Factor V-Short Splicing in a Liver Cell Line Model

Abstract

Coagulation factor V (FV) is a liver-derived protein encoded by the F5 gene. Alternative splicing of F5 exon 13 produces a low-abundance splicing isoform, known as FV-short, which binds the anticoagulant protein tissue factor pathway inhibitor (TFPIα) with high affinity, stabilising it in the circulation and potently enhancing its anticoagulant activity. Accordingly, rare F5 gene mutations that up-regulate FV-short splicing are associated with bleeding. In this study we have explored the possibility of decreasing FV-short splicing by antisense-based splicing modulation. To this end, we have designed morpholino antisense oligonucleotides (MAOs) targeting the FV-short-specific donor and acceptor splice sites and tested their efficacy in a liver cell line (HepG2) that naturally expresses full-length FV and FV-short. Cells were treated with 0–20 µM MAO, and full-length FV and FV-short mRNA expression was analysed by RT-(q)PCR. Both MAOs, alone or in combination, decreased the FV-short/full-length FV mRNA ratio down to ~50% of its original value in a specific and dose-dependent manner. This pilot study provides proof-of-principle for the possibility to decrease FV-short expression by antisense-mediated splicing modulation. In turn, this may form the basis for novel therapeutic approaches to bleeding disorders caused by FV-short over-expression and/or elevated TFPIα (activity) levels.

A Strategy for Gene Knockdown in Dinoflagellates

“…The sequence of the initiation factor eIF4E-1a was found by our lab previously [22]. Morpholino antisense oligonucleotides (MOs) are nucleic acid analogues in which DNA bases are bound to a non-charged backbone (morpholine rings linked by phosphorodiamidate bonds) [51]. For our purposes, a translation-blocking MO was created that covered the eIF4E-1a translational start site (5 -TCATTGAAGCTCAAACAAGCCATTG-3 ).…”