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