Rescue of blood coagulation Factor VIII exon-16 mis-splicing by antisense oligonucleotides
The human Factor VIII (F8) protein is essential for the blood coagulation cascade and specific F8 mutations cause the rare bleeding disorder Hemophilia A (HA). Here, we investigated the impact of HA-causing single-nucleotide mutations on F8 pre-mRNA splicing. We found that 14/97 (~14.4%) coding sequence mutations tested in our study induced exon skipping. Splicing patterns of 4/11 (~36.4%) F8 exons tested were especially sensitive to the presence of common disease-causing mutations. RNA-chemical probing analyses revealed a three-way junction structure at the 3' end of intron 15 (TWJ-3-15). TWJ-3-15 sequesters the polypyrimidine tract, a key determinant of 3' splice site strength. Using exon-16 of the F8 gene as a model, we designed specific antisense oligonucleotides (ASOs) that target TWJ-3-15 and identified three that promote the splicing of F8 exon-16. Interaction of TWJ-3-15 with ASOs increases accessibility of the polypyrimidine tract and inhibits the binding of hnRNPA1-dependent splicing silencing factors. Moreover, ASOs targeting TWJ-3-15 rescue diverse splicing-sensitive HA-causing mutations, most of which are distal to the 3' splice site being impacted. The TWJ-3-15 structure and its effect on mRNA splicing provide a model for HA etiology in patients harboring specific F8 mutations and provide a framework for precision RNA-based HA therapies. Overall design: F8 Exon16 RNAs (WT & MTs of interest) were chemically probed using a SHAPE reagent, 2-aminopyridine-3-carboxylic acid imidazolide (2A3 which creates chemical adducts in the backbone of the RNA that can be readout as mismatches at the DNA level at positions where these adducts were made, or DMSO a non-modifying agent as a control. Replicates of the same experimental condition in these experiments are labeled as "A" or "B" (32 different experiments, 64 total samples sequenced), ex: F8Exon16_A_DMSO > F8Exon16 Replicate A, DMSO control; F8Exon16_A_2A3 > F8Exon16 Replicate A, 2A3 treated.