Rescue of missense and splicing mutations in Haemophilia A by a unique Exon Specific U1snRNA

Background: In Hemophilia A (HA) patients, splicing mutations account for about 8-10% of all, a significant proportion if considered that they are largely underestimated. In fact, also the abundant missense mutations can trigger, besides potentially influencing protein biology, aberrant splicing by affecting splicing regulatory elements. The knowledge of molecular mechanisms leading to aberrant splicing led us to the development of new RNA-therapeutics, in particular the engineered spliceosomal U1snRNA targeting specific intronic sequences downstream of donor splice sites (Exon Specific U1, ExSpeU1). This approach enabled us to rescue mutations at donor (5’ss) or acceptor (3’ss) splice sites, as well as within exons. Here, the aim is to characterize the impact on splicing of different HA-causing mutations occurring in F8 exon 19 (at splice sites or within the exon) and develop ExSpeU1 able to correct them for therapeutic purposes. Methods: Expression of F8 minigenes in human hepatoma cells and dissection of splicing pattern by RT-PCR. Evaluation of ability of ExSpeU1, or the first generation U1, to rescue splicing. Results: The F8 variants c.6115+1G>A, c.6115+2T>C, c.6115+3G>T, c.6115+4A>G, c.6115+5G>A and c.6115+6T>A, expressed in HepG2 cells, led to complete skipping of exon 19. On the other hand, c.6115+9C>G, c.6113A>G (p.N2038S), c.6037G>A (p.G2013R), c.6053A>G (p.E2018G), c.6108C>T (p.Y2036Y) and c.5999G>C (p.G2000A) impaired to various extent exon 19 inclusion (exon skipping), with variable amounts of correct transcript. The impact of missense changes on protein function showed that the p.N2038S and p.G2013R impair both protein secretion and function (<10% of F8 wt). Notably, the p.E2018G has no effect on protein levels. By screening a panel of ExSpeU1s, we identified a unique ExSpeU1 (U1s1) able to rescue both different splicing-defective mutations, either at splice sites or exonic. In particular, the U1s1 promoted (from 0 to ~40%) exon inclusion in presence of c.6115+3G>T, c.6115+4A>G and c.6115+6T>A variants. Notably, the U1s1-mediated rescue, in presence of p.G2013R, p.N2038S and p.E2018G, was more robust (from ~30 to ~80%). Conclusions: We provide insights in the molecular mechanisms causing aberrant splicing in presence of splicing and missense changes in HA. We also provided the proof that a single ExSpeU1 can efficiently rescue multiple mutations and its effect, if translated into patients, could have therapeutic implication also for those missense changes with negligible impact on protein levels.