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

biomedicines Review Targeting Alternative Splicing as a Potential Therapy for Episodic Ataxia Type 2 1 2 2,3 4,5, Fanny Jaudon , Simona Baldassari , Ilaria Musante , Agnes Thalhammer y, Federico Zara 2,3 and Lorenzo A. Cingolani 1,4,* 1 Department of Life Sciences, University of Trieste, 34127 Trieste, Italy; [email protected] 2 Unit of Medical Genetics, IRCCS Istituto Giannina Gaslini, 16147 Genoa, Italy; [email protected] (S.B.); [email protected] (I.M.); [email protected] (F.Z.) 3 Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health (DINOGMI), University of Genoa, 16126 Genoa, Italy 4 Center for Synaptic Neuroscience and Technology, Istituto Italiano di Tecnologia (IIT), 16132 Genoa, Italy; [email protected] 5 IRCCS Ospedale Policlinico San Martino, 16132 Genoa, Italy * Correspondence: [email protected] Present address: SISSA-Scuola Internazionale Superiore di Studi Avanzati, 34136 Trieste, Italy. y Received: 21 August 2020; Accepted: 4 September 2020; Published: 5 September 2020 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: episodic ataxia type 2; P/Q-type Ca2+ channels; alternative splicing; antisense oligonucleotides; SMaRT; CRISPR/Cas9 1. Introduction Episodic ataxia type 2 (EA2) is an autosomal dominant neurological disorder characterized by recurrent disabling attacks of imbalance, vertigo, nausea and ataxia, typically lasting hours to days [1,2]. Symptoms may also include fatigue, migraine headaches, and visual disturbances. Nystagmus commonly occurs between attacks and progressive cerebellar atrophy can also be observed. The frequency of attacks ranges from once a year to four times a week. Ataxic episodes can be triggered by emotional stress, physical exercise, alcohol intake, or fever. Although late-onset cases have been reported, the onset is typically in childhood or early adolescence [3,4]. Acetazolamide and 4-aminopyridine can reduce or control the symptoms in some patients. However, treatments are sometimes discontinued because the drugs are either no longer effective or because the patients develop adverse effects to them [5]. Biomedicines 2020, 8, 332; doi:10.3390/biomedicines8090332 www.mdpi.com/journal/biomedicines Biomedicines 2020, 8, 332 2 of 23 EA2 is caused by mutations in CACNA1A, the gene encoding the pore-forming α1 subunit of 2+ P/Q-type voltage-gated Ca channels (VGCCs; CaV2.1) [6,7]. These channels are enriched in the cerebellum where they control neurotransmitter release in cooperation with N-type (CaV2.2) and R-type (CaV2.3) VGCCs. P/Q-type channels are however more efficient than the other two types of VGCCs in supporting synaptic transmission at mature synapses, partly because they are more tightly coupled to the neurotransmitter release machinery [8–10]. The CACNA1A gene, located on chromosome 19p13, contains 47 exons, many of which are subject to alternative splicing (AS). As a consequence, the total number of CACNA1A splice isoforms is estimated to be in the order of thousands. Moreover, functional studies indicate that some of them exhibit differential properties and expression patterns [7,11]. This creates a large molecular variability that is thought to optimize Ca2+ signaling to specific cellular tasks. Over 100 different mutations can cause EA2 (Figure1; Table1). Some of them are nonsense loss-of-function mutations that disrupt the open reading frame of CACNA1A and result in truncated channels generating little or no current. EA2 mutations are found in all regions of the channel but most of them reside in the pore loop region (Figure1)[ 4,6,12]. Interestingly, some mutations are predicted toBiomedicines induce aberrant 2020, 8, x FOR splicing. PEER REVIEW 3 of 23 FigureFigure 1. 1.Scheme Scheme of of human human Ca CaV2.1V2.1 highlighting highlighting the the position position of of 103 103 mutations mutations causing causing EA2. EA2. Topology Topology of Ca 2.1 as for Uniprot entry O00555. The two mutually exclusive exons 37a and 37b are shown in pink ofV CaV2.1 as for Uniprot entry O00555. The two mutually exclusive exons 37a and 37b are shown in and blue, respectively. The 103 mutations are depicted in the scheme and listed in Table1. pink and blue, respectively. The 103 mutations are depicted in the scheme and listed in Table 1. For example, G to A substitutions at position +1 of the donor splice sites in exons 6, 11, 21, 24, 26, and 27 are all predicted to formTable aberrant 1. List mRNAs of CaV2.1 [ 6mutations,13–15]. Likewise,causing EA2. mutations G to A at position +5 of the donor splice site in exon 4, G to A at position 1 in exon 7, T to C at position +2 of the donor No. Amino Acid −DNA Mutation References splice1 site in exonp.(Ala56Serfs*20) 19 and a four-base pair deletion at positionc.165dupA+4 of the donor splice site[4] in exon 41 also impair2 splicingp.Glu147Lys [4,14,16,17 ]. Further, the insertionc.439G>A of a T at position +3 of intron 24[27] creates a cryptic3 splice donorp.Gly162Val site within exon 24, leading to an aberrantc.485G>T splicing event [18]. While all[28] the above mutations4 affectp.(Trp168Glyfs*10) constitutive splicing, defects in AS are alsoc.504delC important for EA2. Specifically,[4] an A to G substitution at position 2 of the acceptor splice site in the mutually exclusive exon 37a has been 5 p.Arg192Trp− c.574C>T [29] found6 in EA2 patientsp.Arg198Gln [19]. This mutation likely impairs thec.593G>A insertion in the final transcript[30] of exon37a while7 not affecting the inclusion of its mutuallyc.868+5G>A; exclusive possible exon 37b.aberrant splicing [16] The8 mutuallyp.Ser218Leu exclusive exons 37a and 37b produce twoc.653C>T major isoforms of the channel, Ca[30]V 2.1[EFa] and Ca 2.1[EFb], which diverge in an EF-hand-like domain located in the proximal intracellular 9 V p.Tyr248Asn/Cys c.742T>A/c.743A>G [31,32] 10 p.Gly250Glufs*60 c.749delG [12] 11 p.His253Tyr c.1032C >T [33] 12 p.(Cys256Arg) c.1041T>C [34] 13 c.983-1G>A; aberrant splicing [4] 14 p.Arg279Cys c.835C>T [28] 15 p.Cys287Tyr c.1096G>A [1] 16 p.Gly293Arg c.1152G>A [35] 17 p.Gly297Arg c.889G>A [36] 18 p.Asp302Asn c.904G>A [28] 19 p.Thr310 fs*5 c.928_931delACTG [28] 20 p.Trp320* c.959G>A [12] 21 p.Arg387Gly c.1159C>G [28] 22 p.Glu388Lys c.1161G>A [37] 23 p.(Leu389Phe) c.1165C>T [4] 24 p.Gly411Trp c.1231G>T [28] 25 c.1253+1G>A; probable aberrant splicing [15] 26 p.Ala454Thr c.1360G>A [38] Biomedicines 2020, 8, 332 3 of 23 C-terminus [20–22]. The ratio between the synaptic expression of CaV2.1[EFa] and CaV2.1[EFb] is key to controlling synaptic efficacy: CaV2.1[EFa] is tightly coupled to the neurotransmitter release machinery and support efficient vesicle release while CaV2.1[EFb] is loosely coupled to the release machinery and participates in vesicle release only upon repetitive stimulations [7,23,24]. The developmental expression pattern of the two isoforms is also markedly different. Whereas, for CaV2.1[EFb], the expression levels do not undergo major developmental changes, being high in most brain regions from the early stages of postnatal development, for CaV2.1[EFa], the expression levels build up during synapse maturation, presumably contributing to the developmental increase in neurotransmitter release efficiency. As a result, both splice isoforms are expressed at similar levels in most regions of the adult brain [20–23,25]. In addition to the aforementioned mutation impairing inclusion of exon 37a [19], four loss-of-function mutations have been identified within exon 37a in four unrelated families [4,26], while none has been found, to date, in exon 37b (Figure1; Table1). The presence of five mutations impairing selectively CaV2.1[EFa] may therefore suggest a major role of this splice isoform in EA2. Table 1. List of CaV2.1 mutations causing EA2. No. Amino Acid DNA Mutation References 1 p.(Ala56Serfs*20) c.165dupA [4] 2 p.Glu147Lys c.439G>A[27] 3 p.Gly162Val c.485G>T[28] 4 p.(Trp168Glyfs*10) c.504delC [4] 5 p.Arg192Trp c.574C>T[29] 6 p.Arg198Gln c.593G>A[30] 7 c.868+5G>A; possible aberrant splicing [16] 8 p.Ser218Leu c.653C>T[30] 9 p.Tyr248Asn/Cys c.742T>A/c.743A>G[31,32] 10 p.Gly250Glufs*60 c.749delG [12] 11 p.His253Tyr c.1032C >T[33] 12 p.(Cys256Arg) c.1041T>C[34] 13 c.983-1G>A; aberrant splicing [4] 14 p.Arg279Cys c.835C>T[28] 15 p.Cys287Tyr c.1096G>A[1] 16 p.Gly293Arg c.1152G>A[35] 17 p.Gly297Arg c.889G>A[36] 18 p.Asp302Asn c.904G>A[28] 19 p.Thr310 fs*5 c.928_931delACTG [28] 20 p.Trp320* c.959G>A[12] 21 p.Arg387Gly c.1159C>G[28] 22 p.Glu388Lys c.1161G>A[37] 23 p.(Leu389Phe) c.1165C>T[4] 24 p.Gly411Trp c.1231G>T[28] 25 c.1253+1G>A; probable aberrant splicing [15] 26 p.Ala454Thr c.1360G>A[38] 27 p.Arg455Gln c.1364G>A[39] Biomedicines 2020, 8, 332 4 of 23 Table 1.

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