Rescue of blood coagulation Factor VIII exon-16 mis-splicing by antisense oligonucleotides

Tse, V.; Chacaltana, G.; Gutierrez, M.; Nicholas, F. M.; Jimenez, A. G.; Tao, H.; Do, P. H.; Oh, C.; Chary, P.; Quesada, I.; Hamrick, A.; Lee, S.; Stone, M. D.; Sanford, J. R.

doi:10.1101/2023.03.31.535160

bioRxiv · 10.1101/2023.03.31.535160

Rescue of blood coagulation Factor VIII exon-16 mis-splicing by antisense oligonucleotides

Tse, V.·Chacaltana, G.·Gutierrez, M.·Nicholas, F. M.·Jimenez, A. G.·Tao, H.·Do, P. H.·Oh, C.·Chary, P.·Quesada, I.·Hamrick, A.·Lee, S.·Stone, M. D.·Sanford, J. R.

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Abstract

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.

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Tse, V., Chacaltana, G., Gutierrez, M., Nicholas, F. M., Jimenez, A. G., Tao, H., Do, P. H., Oh, C., Chary, P., Quesada, I., Hamrick, A., Lee, S., Stone, M. D., Sanford, J. R.. 2023-04-01. Rescue of blood coagulation Factor VIII exon-16 mis-splicing by antisense oligonucleotides. https://doi.org/10.1101/2023.03.31.535160

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