Branchpoints as potential targets of exon-skipping therapies for genetic disorders

Hiroaki Ohara; Motoyasu Hosokawa; Tomonari Awaya; Atsuko Hagiwara; Ryo Kurosawa; Yukiya Sako; Megumu Ogawa; Masashi Ogasawara; Satoru Noguchi; Yuichi Goto; Ryosuke Takahashi; Ichizo Nishino; Masatoshi Hagiwara

doi:10.1016/j.omtn.2023.07.011

Branchpoints as potential targets of exon-skipping therapies for genetic disorders

Mol Ther Nucleic Acids. 2023 Jul 17:33:404-412. doi: 10.1016/j.omtn.2023.07.011. eCollection 2023 Sep 12.

Authors

Affiliations

¹ Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
² Department of Neurology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan.
³ Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo 187-8502, Japan.
⁴ Department of Drug Discovery for Intractable Diseases, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
⁵ Laboratory of Tumor Microenvironment and Immunity, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
⁶ Department of Mental Retardation and Birth Defect Research, National Institute of Neurology, National Center of Neurology and Psychiatry, Tokyo 187-8502, Japan.

Abstract

Fukutin (FKTN) c.647+2084G>T creates a pseudo-exon with a premature stop codon, which causes Fukuyama congenital muscular dystrophy (FCMD). We aimed to ameliorate aberrant splicing of FKTN caused by this variant. We screened compounds focusing on splicing regulation using the c.647+2084G>T splicing reporter and discovered that the branchpoint, which is essential for splicing reactions, could be a potential therapeutic target. To confirm the effectiveness of branchpoints as targets for exon skipping, we designed branchpoint-targeted antisense oligonucleotides (BP-AONs). This restored normal FKTN mRNA and protein production in FCMD patient myotubes. We identified a functional BP by detecting splicing intermediates and creating BP mutations in the FKTN reporter gene; this BP was non-redundant and sufficiently blocked by BP-AONs. Next, a BP-AON was designed for a different FCMD-causing variant, which induces pathogenic exon trapping by a common SINE-VNTR-Alu-type retrotransposon. Notably, this BP-AON also restored normal FKTN mRNA and protein production in FCMD patient myotubes. Our findings suggest that BPs could be potential targets in exon-skipping therapeutic strategies for genetic disorders.

Keywords: MT: Oligonucleotides: Therapies and Applications; antisense oligonucleotide; branchpoint; exon-skipping therapy; muscular dystrophy; splice-switching; splicing regulatory elements.