Therapeutic manipulation of IKBKAP mis-splicing with a small molecule to cure familial dysautonomia

Masahiko Ajiro,Saiko Shibata,Nobuo Tanaka,Ryo Kurosawa,Adrian R Krainer,Tomonari Awaya,Masatoshi Hagiwara,Kei Iida,Lorenz Studer,Masatsugu Denawa,Young Jin Kim,Tetsunori Sakamoto,Shingo Matsushima

doi:10.1038/s41467-021-24705-5

Masahiko Ajiro, Saiko Shibata + Show 11 more

Open Access

https://doi.org/10.1038/s41467-021-24705-5

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Abstract

Approximately half of genetic disease-associated mutations cause aberrant splicing. However, a widely applicable therapeutic strategy to splicing diseases is yet to be developed. Here, we analyze the mechanism whereby IKBKAP-familial dysautonomia (FD) exon 20 inclusion is specifically promoted by a small molecule splice modulator, RECTAS, even though IKBKAP-FD exon 20 has a suboptimal 5′ splice site due to the IVS20 + 6 T > C mutation. Knockdown experiments reveal that exon 20 inclusion is suppressed in the absence of serine/arginine-rich splicing factor 6 (SRSF6) binding to an intronic splicing enhancer in intron 20. We show that RECTAS directly interacts with CDC-like kinases (CLKs) and enhances SRSF6 phosphorylation. Consistently, exon 20 splicing is bidirectionally manipulated by targeting cellular CLK activity with RECTAS versus CLK inhibitors. The therapeutic potential of RECTAS is validated in multiple FD disease models. Our study indicates that small synthetic molecules affecting phosphorylation state of SRSFs is available as a new therapeutic modality for mechanism-oriented precision medicine of splicing diseases.

Highlights

Half of genetic disease-associated mutations cause aberrant splicing
It lacks complementarity to U1 snRNA at the −1 and +6 positions (Fig. 1a), and such suboptimal splice sites are more prone to be regulated by alternative splicing factors[11,12,13,26,27], as we reported for promotion of spliceosomal recognition of this 5′ splice site by RBM24 in skeletal muscle tissue[25]
The RNA sequencing (RNA-Seq) evaluation of the transcriptome of familial dysautonomia (FD) patient fibroblasts homozygous for the IKBKAP IVS20 + 6 T > C mutation following RECTAS treatment revealed a highly selective action of RECTAS on IKBKAP-FD exon 20, with the secondhighest ΔPSI of 618 altered splicing events with ≥0.1 of | ΔPSI | (Fig. 1b, c and Supplementary Data 1)

Summary

Introduction

Half of genetic disease-associated mutations cause aberrant splicing. a widely applicable therapeutic strategy to splicing diseases is yet to be developed. Several approaches have been taken to amend mutation-derived splicing defects by targeting either cis-regulatory elements or trans-acting splice factors The former approach includes the antisense-oligonucleotide drug nusinersen, which rescues deficient SMN2 exon 7 inclusion for spinal muscular atrophy therapy[1,2,3], and small-molecule compounds, such as SMN-C3 and RG7916, that promote SMN2 exon 7 inclusion, in part by directly binding to an imperfect RNA helix formed by the exon 7 5′-splice site and U1 snRNA acting for bulge repair[4,5,6,7,8]. We synthesized CLK inhibitors[10] and demonstrated their therapeutic potential in disease models of cystic fibrosis (c.3849 + 10 kb C > T mutation of cystic fibrosis transmembrane conductance regulator (CFTR))[11], Duchenne muscular dystrophy (c.4303 G > T mutation of dystrophin)[12], and anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID; IVS6 + 866 C > T mutation of NF-kappa-B essential modulator (NEMO))[13]

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