Japanese researchers at the University of Kyoto have developed a small molecule-based approach to suppressing pseudo exon inclusion, which may lead to the first mechanism-based personalized treatments for pseudo-exon-type genetic diseases, notably cystic fibrosis (CF), they reported in the September 8, 2020, edition of Cell Chemical Biology.

An autosomal recessive disease, CF is caused by deep-intronic splicing mutations in the CF transmembrane conductance regulator (CFTR) gene, causing defects in CFTR protein, which regulates the transepithelial chloride and bicarbonate secretion controlling fluid transport and homeostasis.

"There are currently no medications available that can restore the suppression of CFTR expression by such mutations," said study co-leader Masahiko Ajiro, an assistant professor in the Department of Drug Discovery Medicine in the Graduate School of Medicine at Kyoto University.

Deep-intronic splicing mutations often cause the aberrant inclusion of a pseudo exon, which in turn produces a premature stop codon leading to genetic inactivation.

Recent whole-genome sequencing and transcriptome analysis advances have led to increased numbers of disease-associated pseudo-exonic mutations, but the mechanisms underlying pseudo exon recognition remain poorly understood.

Understanding disease-related splicing mechanisms is crucial for finding effective therapies, such as how characterization of SMN2 exon 7 splicing led to the development of nusinersen (Spinraza; Biogen) for spinal muscular atrophy.

Moreover, splice-targeting therapeutics using small-molecule compounds have been developed for genetic diseases, including Duchenne muscular dystrophy and familial dysautonomia, by targeting splicing regulators.

Therefore, revealing the molecular mechanisms of pseudo exon recognition may provide a therapeutic strategy for pseudo-exonic diseases such as CF.

Cystic fibrosis

In CF, compromised CFTR function results in malfunctioning mucosal surfaces in the lung, pancreas and intestine, limiting patients' survival, with pulmonary infections and respiratory failure being major causes of mortality.

More than 2,000 pathogenic CFTR mutations have been identified and grouped into six classes (class I-VI), according to their functional consequences.

The first drug to treat the cause of CF rather than just its symptoms, Kalydeco (ivacaftor), was developed by Vertex Pharmaceuticals in conjunction with the Cystic Fibrosis Society, and granted FDA approval in January 2012.

Since then a number of other small-molecule CFTR modulators have received FDA approval for treating CF with class II-IV mutations, most recently, in October 2019, Trikafta (elexacaftor/tezacaftor/ivacaftor/ivacaftor).

But as yet there is no treatment for CF patients with class V mutations, which cause CFTR messenger RNA (mRNA) splicing defects.

A class act

The 3,849 + 10 kb C>T deep-intronic mutation is the most common splicing variant, found in more than 60% of CF patients with class V CFTR mutations, which create a pseudo exon with a premature stop codon.

However, despite its pathogenic importance, the pseudo exon recognition mechanism is unclear, and there is no practical strategy to reverse the genetic inactivation of CFTR created by pseudo exon inclusion.

In their new study, researchers co-led by Ajiro and Masatoshi Hagiwara, professor and chairman of the Department of Anatomy and Developmental Biology at Kyoto University, investigated the mechanism underlying CFTR pseudo exon recognition, to develop a small-molecule treatment to release the mutation-driven inactivation of CFTR.

They showed that the mutation-induced CFTR pseudo exon was regulated by phosphorylation of serine/arginine-rich splicing factors, and that their functional inhibition by a CDC-like kinase inhibitor (CLK) restored normal splicing of CFTR.

"We found an essential role for alternative splicing regulator serine/arginine-rich splicing factors, which are activated through phosphorylation by CLKs, prompting our investigation of the effects of CLK inhibition," explained Ajiro.

Subsequent screening of a focused chemical library identified a small molecule designated CaNDY (CDC37 association inhibitor for DYRK1A) as being a rectifier of the aberrant splicing.

"We used a dual fluorescent reporter system for alternative splice detection, in which green fluorescent protein was expressed if the CF pseudo exon was skipped, or red fluorescent protein if it was included," said Ajiro.

CaNDY treatment restored normal splicing of CFTR with the 3,849 + 10 kb C>T mutation in CF patient cells and functional CFTR protein expression in CF model cells.

"CaNDY rescued mutant CFTR channel activity to a comparable extent as normal CFTR in CF model cells, so in future we will investigate CaNDY in CF cells, such as bronchial airway cells and/or intestinal organoids," Ajiro told BioWorld Science.

Together, these findings potentially open the way to the development of a mechanism-based personalized medicine for pseudo-exon-type genetic diseases such as CF.

However, before CaNDY can be tested clinically in humans, "it will be necessary to improve its pharmacokinetics and to analyze its safety range, which will take 2-3 years to complete," said Ajiro.

Meanwhile, "increasing numbers of pseudo exon-related genetic diseases are being reported, and recent advances in transcriptome studies have led to the discovery of more pseudo exonic mutations overlooked in past studies.

"In this regard, CLK inhibitors should provide wider applications for numerous pseudo exonic mutations, so we intend to develop new mechanism-based splicing therapeutics for pseudo exonic diseases including CF." (Shibata, S. et al. Cell Chem Biol 2020, 27: 1).