# Antisense-Mediated Down-Regulation of Factor V-Short Splicing in a Liver Cell Line Model
## 4 Discussion
However, splicing regulatory elements are likely to play an important role in the physiological control of FV-short splicing, as suggested by (a) the very low _in vivo_ expression of FV-short despite the relatively high consensus scores (>80%) of the FV-short-specific donor and acceptor splice sites; and (b) the massive up-regulation of FV-short splicing associated with the _F5_-Atlanta deletion [29], which presumably acts by eliminating one or more splicing silencers or by bringing a splicing enhancer closer to the FV-short donor splice site [40]. Therefore, future elucidation of these additional splicing signals may suggest novel and better targets for antisense-mediated FV-short splicing modulation. The ability of our MAOs to decrease FV-short splicing was tested _in vitro_ in HepG2 cells, which express both full-length FV and FV-short mRNA in proportions similar to normal hepatocytes. Both MAOs, alone or in combination, dose-dependently decreased the FV-short/full-length FV ratio down to ~50% of its original value in the absence of treatment. Some scattering in the qPCR data might be attributed to the very low concentration of the FV-short mRNA already before treatment, and the consequent extremely narrow dynamic range following treatment. The effects of the two MAOs were specific, but (contrary to our expectation) not synergistic. To produce an appreciable change in the overall splicing pattern, MAOs need to access a substantial fraction of cells and to end up in the nucleus. Although MAO transfection efficiency was unfortunately not evaluated in our study, previous work from our laboratory suggests that the Endo-Porter reagent is able to effectively deliver MAOs to HepG2 cells, as judged by the high efficiency of splicing correction achieved upon treatment with a mutation-specific MAO [11]. Moreover, the analysis of the _F5_ gene in HepG2 cells excluded _F5_ gene amplification, as well as the presence of sequence variations that could interfere with the annealing of the MAOs to their intended target sites on the _F5_ pre-mRNA. Therefore, we speculate that the inability of our MAOs to reduce FV-short expression below 50% (even at a concentration of 50 \(\upmu\)M) may be related to the complexity of alternative splicing regulation [41], which involves many other mechanisms (epigenetics, transcription rate, pre-mRNA secondary structures, etc.) and regulatory elements (splicing enhancers and silencers, _trans_-acting splicing factors) than just the donor and acceptor splice sites. Since the FV-short concentration in conditioned media was too low for detection by Western blot analysis and no other assay is currently available to measure FV-short, we could unfortunately not verify whether the observed reduction in FV-short mRNA upon MAO treatment also translates in a decrease in FV-short protein expression. This is a major limitation of our study. Moreover, it should be emphasised that, unlike the East Texas [22], Amsterdam [28] and Atlanta [29] patients, the HepG2 cell model employed in our study has a normal _F5_ exon 13 sequence and a very low basal expression of FV-short. Therefore, it remains to be established whether an antisense-mediated approach using mutation-specific MAOs would be able to correct FV-short over-expression in models of these diseases, such as patients' induced pluripotent stem cell (iPSC)-derived hepatocytes. On the other hand, it is worth noting that FV-short levels show major inter-individual differences even in the absence of \(F5\) gene mutations, and that high levels and/or activity of TFPI\(\alpha\) contribute to the haemorrhagic tendency in various coagulopathies, from haemophilia [42] to bleeding arising from unknown causes [43; 44]. Therefore, antisense-mediated strategies aimed at decreasing FV-short splicing might be more widely applicable than just to the East Texas and allied disorders.