Targeting Alternative Splicing as a Potential Therapy for Episodic Ataxia Type 2

Abstract

Episodic ataxia type 2 (EA2) is an autosomal dominant neurological disorder characterized by paroxysmal attacks of ataxia, vertigo, and nausea that usually last hours to days. It is caused by loss-of-function mutations in CACNA1A, the gene encoding the pore-forming α₁ subunit of P/Q-type voltage-gated Ca²⁺ channels. Although pharmacological treatments, such as acetazolamide and 4-aminopyridine, exist for EA2, they do not reduce or control the symptoms in all patients. CACNA1A is heavily spliced and some of the identified EA2 mutations are predicted to disrupt selective isoforms of this gene. Modulating splicing of CACNA1A may therefore represent a promising new strategy to develop improved EA2 therapies. Because RNA splicing is dysregulated in many other genetic diseases, several tools, such as antisense oligonucleotides, trans-splicing, and CRISPR-based strategies, have been developed for medical purposes. Here, we review splicing-based strategies used for genetic disorders, including those for Duchenne muscular dystrophy, spinal muscular dystrophy, and frontotemporal dementia with Parkinsonism linked to chromosome 17, and discuss their potential applicability to EA2.

Keywords: CRISPR/Cas9; P/Q-type Ca2+ channels; SMaRT; alternative splicing; antisense oligonucleotides; episodic ataxia type 2.

Figure 1

Scheme of human Ca_V2.1 highlighting the position of 103 mutations causing EA2. Topology of Ca_V2.1 as for Uniprot entry O00555. The two mutually exclusive exons 37a and 37b are shown in pink and blue, respectively. The 103 mutations are depicted in the scheme and listed in Table 1.

Figure 2

CRISPR-based splicing therapies (A) CRISPR-mediated non-homologous end joining (NHEJ) disrupts intronic splicing silencer (ISS) sites in intron 7 of SMN2 preventing binding of hnRNPs, thus favoring inclusion of exon 7, normally not present in SMN2. This helps compensate for mutated SMN1. (B) Deletion of exon 51 of DMD leads to a frameshift resulting in a truncated nonfunctional dystrophin. CRISPR-guided cytidine deaminase mutates the 5′ splice site of exon 50, leading to skipping of exon 50, which restores the reading frame. The resulting internally truncated dystrophin is able to restore partially WT function. (C) AS of MAPT exon 10 leads to formation of 4R and 3R tau isoforms. The FTDP-17-associated IVS 10 + 16 mutation results in increased exon 10 inclusion and higher 4R tau levels. Expression of dCas13 together with three gRNAs targeting the exon 10 splice acceptor site and two putative exonic splicing enhancers (ESEs) promotes skipping of exon 10, thus restoring a balanced 4R/3R tau ratio. Schemes adapted from [149,161,164].