# Antisense-Mediated Down-Regulation of Factor V-Short Splicing in a Liver Cell Line Model
## 4 Discussion  
_F5_ gene mutations that up-regulate FV-short splicing are associated with a bleeding tendency, such as in the recently characterised East Texas [22], Amsterdam [28] and Atlanta [29] bleeding disorders. This is due to the major increase in the level and anticoagulant activity of plasma TFPI\(\alpha\) that accompanies FV-short over-expression [22]. Currently, there is no specific treatment for these bleeding disorders and their clinical management  
Figure 4: **Qualitative analysis of the full-length FV and FV-short transcripts in untreated and treated HepG2 cells. ** HepG2 cells were treated for 48 h with increasing concentrations (0–20 uM) of a morpholino antisense oligonucleotide (MAO) targeting the FV-short-specific donor splice site (Donor-MAO), a MAO targeting the FV-short-specific acceptor splice site (Acceptor-MAO), a 1:1 mix of Donor-MAO and Acceptor-MAO, (MAO-mix), or a control MAO with an irrelevant sequence. Total RNA was isolated and reverse transcribed into cDNA. Amplicons corresponding to the full-length FV (top) and FV-short transcripts (bottom) were amplified in separate PCR reactions and PCR products were analysed by gel electrophoresis. M: molecular weight marker, B: blank. Sequencing chromatograms of the full-length FV and FV-short amplicons are shown in Supplementary Figure S1. Figure 5: **Quantitative analysis of full-length FV and FV-short transcripts in untreated and treated HepG2 cells. ** HepG2 cells were treated with 0–20 uM morpholino antisense oligonucleotides (MAOs) for 48 h. Total RNA was isolated and reverse-transcribed into cDNA, and the full-length FV and FV-short transcripts were quantified by real-time qPCR analysis. The FV-short/full-length FV mRNA ratio, normalised to the FV-short/full-length FV ratio of untreated cells, was plotted as a function of the concentration of (**A**) a MAO targeting the FV-short-specific donor splice site (Donor-MAO), (**B**) a MAO targeting the FV-short-specific acceptor splice site (Acceptor-MAO), (**C**) a 1:1 mix of Donor-MAO and Acceptor-MAO (MAO-mix), and (**D**) a control MAO with an irrelevant sequence. Results are reported as mean \(\pm\) S.E.M. of three biological replicates (two for the Control-MAO). The numbers in the top right corner of each graph represent the p-for-trend (Jonckheere-Terpstra trend test) of the respective titrations. Note the different \(y\)-axis scale for Control-MAO. remains challenging. Affected patients have been treated with plasma and/or prothrombin complex concentrates [22, 28], but therapies specifically targeting TFPI\(\alpha\) (such as those used as bypassing agents for haemophilia) might be more appropriate. Although several aptamers, antibodies and peptides against TFPI\(\alpha\) are already in various stages of development as therapeutic agents [37], in this study we propose an alternative strategy based on the use of antisense oligonucleotides to down-regulate FV-short splicing. The rationale for this approach is that elevated FV-short is the primary abnormality in the East Texas and allied bleeding disorders, and that FV-short is both the carrier of TFPI\(\alpha\) in plasma [22] and a potent cofactor for its activity [25]. Therefore, down-regulating FV-short might be even more effective than antagonising TFPI\(\alpha\) directly. Moreover, the liver, which is the main site of FV biosynthesis, is considered an optimal target for splicing modulation therapies [38], and MAOs generally show high bioavailability and low toxicity profiles in _in vivo_ studies [39]. In order to decrease FV-short splicing, we have targeted the donor and acceptor splice sites that define the FV-short-specific intron. This was a somewhat necessary choice, because enhancers and silencers potentially regulating FV-short splicing are presently unknown and, due to their rather degenerate consensus sequences, cannot be easily predicted _in silico_.