Rescue of a panel of splicing mutations causing hemophilia A by engineered U1snRNAs

Background. Hemophilia A (HA) is an X-linked recessive hemorrhagic disorder caused by coagulation factor VIII (FVIII) deficiency. Among all HA-causing mutations, those affecting splicing are relatively frequent, particularly in severe forms. These mutations, often leading to exon skipping, can be potentially rescued by RNA therapeutics based on variants of the key U1snRNA spliceosomal component, as already shown in several human disease models. The aim of this project is to dissect the molecular mechanisms of eight F8 splicing variants identified in HA patients and located at the 5' splice sites (5'ss) of exon 6 (c.787+2T>C; c.787+3A>G; c.787+3A>T; c.787+5G>A; c.787+6T>C) and exon 11 (c.1752+5G>T; c.1752+5G>C; c.1752+5G>A) and to test engineering U1snRNAs to restore the correct pre-mRNA processing. Methods. Molecular cloning of splicing competent vectors containing the F8 exon 6 or exon 11 with the respective surrounding introns, either wild-type or harboring the eight F8 variants (c.787+2T>C; c.787+3A>G; c.787+3A>T; c.787+5G>A; c.787+6T>C; c.1752+5G>T; c.1752+5G>C; c.1752+5G>A). Creation of recombinant vectors expressing engineered U1snRNAs designed to bind to the mutated 5'ss (compensatory U1snRNA) or to less-conserved downstream intronic sequences (Exon Specific U1snRNA, ExSpeU1snRNA). Transient transfection of expression vectors on various human cell lines and RT-PCR with plasmid specific primers to evaluate the splicing patterns and their modulation by U1snRNA variants. Results. Firstly, a Bioinformatic analysis with SpliceRover showed that F8 exon 6 and exon 11 are welldefined exons and that the mutations do not activate cryptic 5'splice sites but rather weaken the canonical 5'ss. In vitro splicing pattern analysis revealed that these mutations lead to exon 6 or exon 11 skipping with low levels (5-30% based on mutation position within the 5'ss) of correctly spliced transcripts. The reduced levels of correctly spliced transcripts are in accordance with the pathological phenotype and observed FVIII antigen levels, whenever available. We also showed that basal levels of correctly spliced transcripts are cell-specific, thus indicating that splicing of these exons is regulated in a cell-dependent manner. Notably, co-transfection of U1snRNA variants significantly improves F8 exon 6 and exon 11 definition and thus inclusion up to 95%. Conclusions. We provided experimental evidence that the analysed mutations alter F8 splicing by impairing proper exon definition. Trace levels of correctly spliced transcripts are regulated in a cell-dependent manner. Importantly, exon definition can be efficiently restored by engineered U1snRNA variants, with one ExSpeU1snRNA able to rescue different splicing mutations located in the same 5'ss. These RNA therapeutics are currently under investigation through lentiviral-mediated delivery in Blood-Born Endothelial Cells (BOECs) isolated from HA patients.