Rescue of coagulation factor VII mRNA processing and protein function by engineered U1+5A snRNA

Changes affecting mRNA processing represent a frequent cause of severe coagulation factor defects and of all inherited human diseases. We extensively investigated the IVS7+5G/A mutation (9726+5A) in the coagulation factor VII (FVII) gene, occurring in the IVS7 donor splice site (5ss) in the first of six highly homologous 37bp repeats containing several cryptic splice sites. This mutation is the most frequent cause of severe FVII deficiency in Central Italy. A cellular model of this deficiency was created by producing a full length FVII splicing competent construct (pSCFVII-wt). This minigene drove in COS-1 cells the synthesis of properly processed FVII transcripts and of secreted functional FVII (23 ±4 ng/mL), which were virtually undetectable upon introduction of the mutation (pSCFVII-9726+5A). At the mRNA level the mutation caused exon 7 skipping and, to a less extent, activation of the 37-bp downstream cryptic site. To attempt rescue of FVII expression, we have engineered the U1-snRNA, the spliceosome component selectively recognizing donor splice sites (5'ss), to re-direct recognition of the mutated donor splice site. Upon cotrasfection of the engineered U1- snRNA (pU1+5A) with pSCFVII-9726+5A, the splicing pattern and protein level were evaluated. At RNA level, the expression of U1+5A reduced from 80% to 40% the exon 7 skipping and increased recognition of the correct 5'ss, resulting in appreciable synthesis of normal transcripts (from hardly detectable to 20%). At protein level, we observed an increase of secreted protein levels in medium (5.0±2.8 ng/mL) and of the FVII coagulant activity, which reached 9.5±3.2% of pSCFVII-wt. The effects of engineered U1-snRNA were dose-dependent. Altogether these results demonstrate for the first time the U1- snRNAmediated rescue at the mRNA and protein levels, thus highlighting its therapeutic implications in bleeding disorders, which would benefit even from tiny increase of functional levels