U1-snRNA-mediated rescue of mRNA processing in severe factor VII deficiency

Factor VII (FVII) is the plasma protease triggering coagulation, and its absence is lethal. Life-threatening hemorrhagic symptoms in severe FVII deficiency are prevented by frequent administration of fresh frozen plasma or recombinant FVIIa. Studies in animal and cellular models of human diseases showed that modified small nuclear RNAs (snRNAs) can promote changes in mRNA splicing and thus in gene expression. Splicing mutations in clotting factors, a relatively frequent cause of severe bleeding, represent ideal models to test this strategy, because tiny increases in functional full-length protein levels in patients significantly ameliorate hemorrhagic phenotypes. We explored the snRNA-mediated rescue of coagulation factor VII (FVII) expression impaired by the IVS7+5 g/a mutation, which is associated to life-threatening bleeding in homozygous patients. This change occurs in the first of six homologous 37bp repeats containing cryptic donor splice site (5'ss) identical to the normal one. Expression of extended FVII minigenes in human hepatoma cells (Hep3B) and studies at the mRNA level (RT-PCR, fluorescent labeling and capillary electrophoresis) indicated that the IVS7+5g/a induces exon 7 skipping and activation of the first downstream cryptic 5'ss, thus generating frameshifts. Levels of normal transcripts were barely detectable (<0.2%). To restore correct mRNA processing we engineered the U1-snRNA, the spliceosome components selectively recognizing 5’ss. Vectors for three U1-snRNAs, complementary to the mutated 5’ss (U1+5a) or to neighbouring sequences, were created and co-expressed with FVII minigenes in Hep3B. The U1-snRNAs reduced from 80-40% the exon 7 skipping, thus increasing exon definition. The U1+5a construct also dramatically increased recognition of the correct 5’ss over the 37bp-downstream cryptic site preferentially activated by the mutation, thus inducing appreciable synthesis of normal transcripts (from barely detectable to 50%). This effect, which was dose-dependent, clearly demonstrated that impaired recognition by the U1-snRNA was the mechanism responsible for FVII deficiency. These findings suggest compensatory U1-snRNAs as therapeutic tools in coagulation factor deficiencies caused by mutations at 5’ss, a frequent cause of severe defects.