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Splice inducing Antisense Oligonucleotides as a potential gene correction therapy for haemophilia A

Tiao, J., Powell, S., Gilmore, G., Veedu, R., Wilton, S. and Baker, R. (2020) Splice inducing Antisense Oligonucleotides as a potential gene correction therapy for haemophilia A. Haemophilia, 26 (S4). pp. 21-22.

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Introduction: Haemophilia A(HA) is a complex bleeding disorder resultant from mutations in the Factor VIII protein. Factor VIII is encoded by the F8 gene, and mutations within exon14 contribute to the aetiology of HA in a subset of patients (˜14%), resulting in an incorrectly spliced mRNA transcript and subsequent mutant protein. The Factor VIII B‐domain is mostly encoded by exon14, however, there exists a small degree of functional redundancy in the B‐domain which may be potential targets for nucleic acid‐based therapies. This study aims to target non‐canonical splicing sequences in the F8 exon14 sequences using antisense‐oligonucleotides (AON) incorporating mixer chemistry to induce a truncated but functional Factor VIII transcript.

Materials and Method: Splice inducing sequences can be difficult to predict, we targeted predicted spliceosome elements in the F8 exon14 in HuH7 cells using mixmer chemistries. Following 24 h transfection, total RNA was extracted, converted into cDNA and PCR‐assessed using primers spanning exon13&14 and exon 14&15 boundaries. Positive PCR products were sequenced for identity. Factor VIII protein was analysed by immunoblotting with anti‐factor VIII antibodies.

Results: The entire F8 Exon14 can be deleted by targeting the canonical donor and acceptor splice sites. Human splicing finder 3.1 predicted several sequences for mixmer AON targeting the 3‐prime end of the F8 Exon14 sequence. Different mixmer AON could induce novel F8 pre‐mRNA splicing in HuH7 hepatocarcinoma cells. A wild‐type product (3.5Kb) was present in all treatments, and excision events were observed in mixmer AON‐ #7‐17. Three main F8 products were confirmed by Sanger sequencing. All three contained novel splice inducing sites (U1‐ and U12‐like sequences) and the truncated F8 exon14 transcript is in‐frame with F8‐exon15. All novel transcripts retained the furin‐cleavage site.

Conclusion: Gene skipping of mutated regions of the Factor VIII B‐domain at the pre‐mRNA stage is a potential novel treatment strategy for Haemophilia A. This study successfully excised exon14 of the F8 gene to generate a complete B domain deletion, as well as several partially deleted B‐domain Factor VIII transcripts by inducing spliceosome binding in exonic sequences with mixmer antisense‐oligonucleotides.

Item Type: Journal Article
Murdoch Affiliation: Medical, Molecular and Forensic Sciences
Western Australian Centre for Thrombosis and Haemostasis (WACTH)
Publisher: Blackwell Publishing Inc.
Copyright: © 2020 The Authors Haemophilia © 2020 John Wiley & Sons Ltd
Other Information: MED-FP-009(487) Abstracts of the World Federation of Hemophilia, Virtual Summit – Connecting the Global Bleeding Disorders Community, June 2020
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