Ludovic Arandel

Magdalena Matloka

Arnaud F Klein

Frédérique Rau

Joelle Marie

Micaela Polay-Espinoza

Naira Naouar

Arnaud Ferry

Séverine Begard

Morvane Colin

Hélène Tran

Luc Buée

Nicolas Sergeant
email: nicolas.sergeant@inserm.fr

Denis Furling
email: denis.furling@sorbonne-universite.fr

Decoy gene therapy reverses triplet repeat RNA toxicity in Myotonic Dystrophy

Keywords: onset Normal Juvenile Congenital Normal Affected Infantile CX 7 CX 6 CX dm1, cells, cultured, CTG repeats, models, biological, pathophysiology, human cells

Myotonic dystrophy (DM) is an autosomal neuromuscular disease encompassing two distinct forms, type 1 (DM1) and type 2 (DM2), which are caused by abnormal microsatellite expansions of C(C)TG repeats in the 3'UTR of the DMPK and first intron of ZNF9 genes, respectively. Mutant RNAs carrying expanded repeats are retained in the nucleus as riboprotein aggregates that abnormally sequester MBNL splicing factors leading to alternative splicing misregulations associated with clinical symptoms.

Although various therapeutic approaches for DM are under development, there is no effective therapy available so far. In this study, we designed a novel gene therapy strategy with the use of an engineered MBNL RNA-binding protein derivative that acts as a CUGexp-decoy to release sequestered endogenous MBNL factors and restore their proper functions. Expression of the decoy results in the correction of DM1-associated features in both in vitro and in vivo models of the disease. Subsequent optimization processes were applied to our engineered decoy and the most potent derivate that increases its functional capacity was selected for further therapeutic application. Additionally, we developed an autoregulatory system based on a splice-sensor strategy to control transgene product expression and provided a proof-ofconcept of its efficacy in both in vitro and in vivo systems.

In conclusion, my work establishes the potency of gene therapy treatment for DM and support the use of the decoy-based approach as an alternate or complementary therapeutic intervention for DM. Résumé XI Résumé Les Dystrophies Myotoniques de type 1 (DM1) et 2 (DM2) sont des maladies neuromusculaires autosomiques dominantes causées par l'expansion anormale de séquences microsatellites C(C)TG situées respectivement dans la région 3'UTR du gène DMPK et le premier intron du gène ZNF9. Les ARNs mutés contenant les expansions de répétitions sont retenus dans le noyau des cellules sous formes d'agrégats riboprotéiques et séquestrent les protéines de liaison à l'ARN de la famille MBNL conduisant à des dérégulations de l'épissage alternatif associés aux symptômes cliniques. Bien que différentes approches thérapeutiques pour la DM soient en développement, il n'existe à ce jour aucune thérapie. Dans ce travail de thèse, nous avons développé une stratégie innovante de thérapie génique basée sur une protéine MBNL1 modifiée. Ce dérivé MBNL∆ agit comme un leurre pour libérer les facteurs MBNL endogènes anormalement séquestrés par les ARN mutés, et restaurer leurs fonctions. Ainsi l'expression de MBNL∆ dans des modèles DM1 permet la correction des anomalies moléculaires et phénotypiques. Nous avons également réalisé différentes optimisations de cet outil dans le but d'accroître ces capacités fonctionnelles. Enfin, nous avons développé un système d'autorégulation basé sur un « senseur d'épissage » dans le but de contrôler l'expression protéique d'un transgène. La preuve de concept de ce système a été faite à l'aide de MBNL∆ et validée dans des modèles DM1. En conclusion, mon travail de thèse a permis d'établir le potentiel d'une approche de thérapie génique pour la DM de type « leurre » basée sur l'ingénierie de protéine de liaison à l'ARN. Preamble XII Preamble Myotonic dystrophy belongs to the group of muscle disorders characterized by progressive muscle weakness and wasting and affecting multiple organs. The central cause of the disease is the nuclear retention of transcripts containing expanded microsatellite expansions that sequester Muscleblind splicing factors leading to global missplicing changes associated with clinical symptoms. Although several therapies are under development, there is no curative treatment available so far. The work presented in this thesis focuses mostly on the development of a novel gene therapy strategy for myotonic dystrophy. Additionally, we bring fundamental knowledge about MBNL function in the progressive pathology of the disease.

The thesis is organized in the following order: Introduction comprises 4 chapters:

Description of microsatellite expansions in the human genome

This chapter gives an overview of the microsatellites' structure and functions and describe their causative role in microsatellite disorders including myotonic dystrophy.

Description of myotonic dystrophies

This chapter describes in details the two types of myotonic dystrophies, type 1 (DM1) and type 2 (DM2), and gives an overview of the clinical manifestation of the disease, the mechanisms underlying the physiopathology of the disease as well as models used to study myotonic dystrophy with a special focus on mouse models.

Characterization of Muscleblind family of proteins

This chapter presents the structure and functions of the three paralogs of Muscleblind proteins in mammals and indicates their role in the pathology of myotonic dystrophy.

Overview of therapeutic approaches for myotonic dystrophy

This chapter summarizes strategies for treating myotonic dystrophy, with a special interest in DM1, and highlights challenges of therapy development for multisystemic disease as DM1.

The Result chapter is divided into 4 parts: Each part comprises an introduction, the results, the discussion and materials and methods.

The last chapter will be the conclusions and perspectives:

This chapter summarizes all the results in a more general context and discusses future directions. 2012). To restart the fork, the one-ended DSB invades the sister chromatid to form a D-loop, however, does it imperfectly because of the presence of the repetitive sequence, thus leading to expansion. BIR has been extensively characterized in S. cerevisiae (

List of figures and tables

Introduction

Figure 2. Unusual DNA structures formed by expandable repeats a, An imperfect hairpin formed by (CNG)n repeats. b, A quadruplex like structure formed by the (CGG)n repeat. Brown rectangles indicate G quartets, and the yellow rectangle indicates an i motif. c, A slipped-stranded structure formed by the (CTG)n(CAG)n repeat. d, H-DNA and sticky DNA formed by the (GAA)n•(TTC)n repeat. e, A DNA-unwinding element formed by the (ATTCT)n(AGAAT)n repeat. The structure-prone strand of the repetitive run is shown in red, its complementary strand in green, and flanking DNA in beige (adapted from [START_REF] Mirkin | Replication fork stalling at natural impediments[END_REF].

Expanded (CNG)n repeats are able to fold into imperfect hairpin-like structures that consist of both Watson-Crick and non-Watson-Crick base pairs. The stability of such hairpins differs, depending on the sequence of the triplet repeats and decreases as a consequence of the energy contribution of the mismatched base pairs in the following order CGG>CTG>CAG=CCG [START_REF] Gacy | Trinucleotide repeats that expand in human disease form hairpin structures in vitro[END_REF]. In addition to expandable CNG sequences, individual strands of (CCTG)n/(CAGG)n repeats have also been shown to fold into hairpin-like structures [START_REF] Dere | Hairpin structure-forming propensity of the ({CCTG}.{CAGG}) tetranucleotide repeats contributes to the genetic instability associated with myotonic dystrophy type 2[END_REF]. Tetrahelical conformation stabilized by intertwined G quartets represents another peculiar conformation that is common for single-stranded stretches of (CGG)n, (CCG)n and (CGCG 4 CG 4 )n [START_REF] Fry | The fragile X syndrome d(CGG)n nucleotide repeats form a stable tetrahelical structure[END_REF][START_REF] Usdin | CGG repeats associated with DNA instability and chromosome fragility form structures that block DNA synthesis in vitro[END_REF]. However, single stranded DNA are not the only one that can adopt atypical structures. Such conformations have been observed as well for double stranded DNA (dsDNA) containing expanded repeats. Of special interest for dsDNA, is that denaturation and subsequent renaturation of the strands favors the folding into very stable so-called 'slipped-stranded' DNA conformation where the 'looped out' repeated sequences ultimately form a secondary hairpin-like structure [START_REF] Pearson | Alternative structures in duplex DNA formed within the trinucleotide repeats of the myotonic dystrophy and fragile X loci[END_REF]. Other characteristic unusual conformation for double stranded DNA is an intramolecular triplex (H-DNA) under the influence of negative supercoiling; however, such type of DNA conversion is most favorable for poly-purine/poly-pyrimidine sequences like (GAA) n /(TTC) n [START_REF] Gacy | GAA instability in Friedreich's Ataxia shares a common, DNA-directed and intraallelic mechanism with other trinucleotide diseases[END_REF]. Notably, the longer forms of these kind of repeats can adopt even more complicated composite triplex structure, called 'sticky DNA' [START_REF] Sakamoto | Sticky DNA: self-association properties of long GAA.TTC repeats in R.R.Y triplex structures from Friedreich's ataxia[END_REF]. AT-rich repeats can adopt other distinct conformation causing extensive unwinding of DNA that progresses with increasing negative superhelical stress [START_REF] Potaman | Unpaired structures in SCA10 (ATTCT)n.(AGAAT)n repeats[END_REF].

Interestingly, the formation of peculiar structures is restricted to repeats that are more prone to expand [START_REF] Gacy | Trinucleotide repeats that expand in human disease form hairpin structures in vitro[END_REF]. Closer investigation of the structure's alignment suggests that all expanded repeats contain in their conformations various mismatched base pairs and multiple junctions between substructural elements [START_REF] Mirkin | {DNA} structures, repeat expansions and human hereditary disorders[END_REF]. Therefore, it is plausible, that the structural imperfections may ensure the expanded tracts to remain stable.

Molecular mechanisms of repeat expansions

The mutational mechanisms of microsatellite repeats have been extensively studied. Several distinct models have been proposed that share a general common theme based on DNA metabolism associated with strand separation and reannealing at microsatellite sequences.

These events allow the formation of non-canonical structures and recruitment of DNA damage factors that subsequently lead to the expansion of the microsatellites. Within the various models proposed, three genetic processes (replication, repair and recombination) have been shown to be involved in either repeat expansion or contraction (Figure 3) [START_REF] Budworth | Bidirectional transcription of trinucleotide repeats: roles for excision repair[END_REF][START_REF] Lopez Castel | Repeat instability as the basis for human diseases and as a potential target for therapy[END_REF][START_REF] Mirkin | Replication fork stalling at natural impediments[END_REF][START_REF] Polleys | Role of recombination and replication fork restart in repeat instability[END_REF][START_REF] Polleys | Role of recombination and replication fork restart in repeat instability[END_REF].

Replication

In dividing cells, repeat expansion occurs mainly during DNA replication. Two main reasons support this hypothesis. Firstly -significant DNA synthesis is necessary to explain massive and rapid accumulation of repetitive sequences. Secondly -it is a main cellular process that involves separation of the DNA strands that can facilitate its folding into stable secondary structures. These can therefore evoke problems at the replication fork resulting in its stalling and restarting during strands synthesis that can increase the number of repetitions [START_REF] Follonier | Friedreich's ataxia-associated GAA repeats induce replication-fork reversal and unusual molecular junctions[END_REF][START_REF] Kerrest | SRS2 and SGS1 prevent chromosomal breaks and stabilize triplet repeats by restraining recombination[END_REF][START_REF] Kim | The balancing act of DNA repeat expansions[END_REF][START_REF] Mirkin | Replication fork stalling at natural impediments[END_REF]. Similarly, imperfect Okazaki fragment ligation may cause the adding of several repeats in the lagging DNA strand [START_REF] Freudenreich | Expansion and length-dependent fragility of CTG repeats in yeast[END_REF][START_REF] Gordenin | Repeat expansion--all in a flap?[END_REF][START_REF] Spiro | Inhibition of FEN-1 processing by DNA secondary structure at trinucleotide repeats[END_REF]. The misalignments of two strands of DNA force one of them to 'slip-out' leading to so-called replication slippage [START_REF] Hartenstine | Base stacking and even/odd behavior of hairpin loops in DNA triplet repeat slippage and expansion with DNA polymerase[END_REF][START_REF] Petruska | Analysis of Strand Slippage in DNA Polymerase Expansions of CAG/CTG Triplet Repeats Associated with Neurodegenerative Disease[END_REF]. This condition creates either expansion or contraction of the repeated track depending on the origin of the slippedout strand [START_REF] Kunkel | Nucleotide repeats. Slippery DNA and diseases[END_REF]. In fact, the pathway followed for repeats instability during replication processes has been shown to be highly influenced by several factors acting in cis, including expandable repeat sequence, tract length, distance from the replication origin and replication direction [START_REF] Cleary | Evidence of cis-acting factors in replication-mediated trinucleotide repeat instability in primate cells[END_REF]. Although replication has been proposed as a main cause of repeat instability in dividing cells, it is able to explain only short expansions rather than large-scale accumulations of repeated sequences. critical driver of repeat instability in non-dividing cells [START_REF] Foiry | Msh3 is a limiting factor in the formation of intergenerational {CTG} expansions in {DM}1 transgenic mice[END_REF][START_REF] Guo | DNA mismatch repair in trinucleotide repeat instability[END_REF][START_REF] Iyer | DNA triplet repeat expansion and mismatch repair[END_REF][START_REF] Schmidt | Disease-associated repeat instability and mismatch repair[END_REF]. MMR mechanism expands the pathogenic repeat tract via the recognition of unusual repetitive hairpins by the MutSβ enzyme (MSH2-MSH3 complex), which together with endonucleases remove DNA lesions. The subsequent repair processing by the polymerases may then inserts additional 1-3 repeats due to flawed priming or templating [START_REF] Chan | Coordinated processing of 3' slipped (CAG)n/(CTG)n hairpins by DNA polymerases β and δ preferentially induces repeat expansions[END_REF]. Such an incremental expansion generated through damage repair pathways might be further facilitated by transcription across the repeat. Although the precise mechanisms underlying transcription-induced repeat instability are not fully defined, it is possible that the separation of DNA strands during transcription results in DNA-RNA hybrids and secondary structures at the site of the repeats, followed by protein-induced stabilization of these structures [START_REF] Mcivor | New insights into repeat instability: role of RNA•DNA hybrids[END_REF]. Additionally, during bidirectional transcription, head-on collision between RNA polymerases may also occur causing activation of downstream DNA damage response pathways [START_REF] Lin | Transcription-induced DNA toxicity at trinucleotide repeats: double bubble is trouble[END_REF].

Recombination

Meiotic and mitotic crossing overs are two main recombination events taking place in cells. The simplest explanation for tracts expansion/contraction seem to be the unequal crossingover between the expanded repeats on homologous chromosomes during meiosis [START_REF] Warren | Polyalanine expansion in synpolydactyly might result from unequal crossing-over of HOXD13[END_REF]. Indeed, this phenomenon has been observed for expansions of (GCN)n repeat tracts [START_REF] Warren | Polyalanine expansion in synpolydactyly might result from unequal crossing-over of HOXD13[END_REF]. However, the vast majority of other types of repeats rather tend to stimulate recombination in mitotic cell division during single-end or double-strand breaks [START_REF] Meservy | Long {CTG} tracts from the myotonic dystrophy gene induce deletions and rearrangements during recombination at the {APRT} locus in {CHO} cells[END_REF][START_REF] Polleys | Role of recombination and replication fork restart in repeat instability[END_REF][START_REF] Warren | Polyalanine expansion in synpolydactyly might result from unequal crossing-over of HOXD13[END_REF]. One intriguing mechanism to explain single-step large expansions by homologous recombination is based on break-induced replication (BIR) [START_REF] Leffak | Break-induced replication links microsatellite expansion to complex genome rearrangements[END_REF]. BIR rescues collapsed or broken replication forks and is activated when a replisome collides with a broken single-end double strand break (DSB) (Malkova and Haber,

Functions of microsatellites 1.3.1 Biological functions of microsatellites

Given the extensive polymorphism rate of microsatellites, they quickly found the application in the basis for forensic DNA analyses and as markers for population genetics studies and gene mapping [START_REF] Brouwer | Cell recovery from {DM}1 transgenic mouse tissue to study ({CTG}) n instability and {DM}1 pathogenesis[END_REF][START_REF] Diegoli | Forensic typing of short tandem repeat markers on the X and Y chromosomes[END_REF][START_REF] Dumache | Molecular DNA Analysis in Forensic Identification[END_REF][START_REF] Payseur | A Genomic Portrait of Human Microsatellite Variation[END_REF].

However, for many years the proper biological function of microsatellite repeats remained vague. First line of evidence for their functional significance came from comparative studies showing that some microsatellites including noncoding repeats have remained conserved between species across hundreds of millions of years [START_REF] Fitzsimmons | Conservation and dynamics of microsatellite loci over 300 million years of marine turtle evolution[END_REF]. Multiple investigations of these regions gave rise to the observation that microsatellite polymorphism may modulate gene expression. One example is the binding of the tumor suppressor protein p53 to (TGYCC) 10-17 repeats located on the PIG3 gene. This direct interaction is necessary and sufficient for a stimulatory effect on transcription [START_REF] Contente | A polymorphic microsatellite that mediates induction of PIG3 by p53[END_REF]. Normal microsatellite polymorphism has also been linked to alternative splicing regulation. It has been reported that CA microsatellites in the introns could act as splicing enhancers or suppressors like for the eNOS gene for which pre-mRNA splicing efficiency depends on the length of the intronic CA repeats that varies between 19-38 (Hui et al., 2003). While the number of microsatellites with documented functions still remain very low relative to their overall abundance in the genome, it becomes evident that functional microsatellites are more prevalent than previously assumed, with some well described roles (Table 1). effect during neuronal development in the brain resulting in severe mental retardation.

Contrary, FRDA disease is caused by a reduced but not fully ablated expression of the frataxin protein due to the decreased level of FRDA mRNA by the expansion of GAA repeats in the first intron of the frataxin gene [START_REF] Wilson | Frataxin and frataxin deficiency in Friedreich's ataxia[END_REF]. As frataxin controls iron availability in mitochondria and has a role in biogenesis of iron-sulphur clusters and haem, its low levels decrease energy production and increase the release of free radicals [START_REF] Karthikeyan | Reduction in frataxin causes progressive accumulation of mitochondrial damage[END_REF][START_REF] Yoon | Iron-sulfur cluster biosynthesis. Characterization of frataxin as an iron donor for assembly of [2Fe-2S] clusters in ISU-type proteins[END_REF].

Gain-of-function diseases

Repeat expansions residing in the coding regions of the genes are found to result in gain-offunction mechanisms due to mutant protein expression. The aberrant proteins may therefore lead to abnormal interactions and affect a range of cellular processes including transcription and RNA metabolism [START_REF] Riley | Polyglutamine neurodegenerative diseases and regulation of transcription: assembling the puzzle[END_REF]. In most cases, the repeat causing disease is a (CAG)n that encodes an abnormal long polyglutamine stretches. So far, several different diseases have been recognized to be caused by the pathogenic polyQ tracts including Huntington's disease [START_REF] Li | Huntingtin-protein interactions and the pathogenesis of Huntington's disease[END_REF]), Kennedy's disease [START_REF] Spada | Androgen receptor gene mutations in {X}-linked spinal and bulbar muscular atrophy[END_REF] and at least six types of Spinocerebellar ataxias [START_REF] Orr | Trinucleotide repeat disorders[END_REF]. Despite the fact that expansion sizes, structures, cellular localization and functions of mutant proteins between the diseases substantially differ, all CAGn mediated disorders are found to be highly neurodegenerative.

Although polyglutamine diseases constitutes the largest group of gain-of-function diseases known so far, polyalanine stretches encoded by (GCG) n in the poly(A)-binding protein 2 in OPMD patients and polyalanine/polyleucine tracts of repeated (CTG) n in an alternatively spliced exon of the JPH3 gene in HD-like 2 (HDL2) syndrome found to be alike deleterious, yet not progressive or neurodegenerative [START_REF] Brais | Short GCG expansions in the PABP2 gene cause oculopharyngeal muscular dystrophy[END_REF][START_REF] Margolis | Huntington's disease like-2: review and update[END_REF]. Given the fact that expanded amino acids stretches have an inherent ability to aggregate, a recognizable hallmark of the gain-of-function protein diseases are cellular inclusions, which however, are not always the primary cause of cell toxicity [START_REF] Margolis | Expansion explosion: new clues to the pathogenesis of repeat expansion neurodegenerative diseases[END_REF].

Besides the obvious deleterious downstream pathogenesis of expansions located in the coding regions, microsatellites residing in non-coding sequences of introns or UTRs have also been linked to degenerative disorders, giving rise to the novel group of RNA-gain of function diseases. The acquisition of the deleterious secondary function of mutated RNA has been grounded in its unusual conformations stimulated by expanded sequences independently of its tract and composition. Trinucleotide (CUG)n motifs implicated in myotonic dystrophy type 1 (DM1), tetranucleotide (CCUG)n repetitions found in myotonic dystrophy type 2 (DM2) as well as pentanucleotide stretches of (AUUCU)n responsible for spinocerebellar ataxia 10 fold into stable hairpins structures that either inhibit or enhance the normal activities of important cellular factors [START_REF] Handa | The AUUCU repeats responsible for spinocerebellar ataxia type 10 form unusual RNA hairpins[END_REF][START_REF] Sobczak | RNA structure of trinucleotide repeats associated with human neurological diseases[END_REF][START_REF] Tapscott | Deconstructing myotonic dystrophy[END_REF]. Similar to expanded amino acids stretches, mutant RNAs can accumulate and form distinct aggregates and inclusions like those found in the nucleus of both myotonic dystrophies (type 1 and 2) and C9ORF72 ALS/FTD or in neurons and astrocytes in FXTAS [START_REF] Dejesus-Hernandez | Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS[END_REF][START_REF] Greco | Neuronal intranuclear inclusions in a new cerebellar tremor/ataxia syndrome among fragile X carriers[END_REF][START_REF] Taneja | Foci of trinucleotide repeat transcripts in nuclei of myotonic dystrophy cells and tissues[END_REF]. Those aggregates have been found to sequester and form complexes with several different proteins. Therefore, the agglomeration of expanded transcripts and the functional dysregulation of RNA-binding proteins is postulated to be a primary pathogenesis driven pathway of RNA toxic diseases [START_REF] Echeverria | {RNA}-binding proteins in microsatellite expansion disorders: mediators of {RNA} toxicity[END_REF].

However, this mechanism is not exclusive and RNA toxicity might contribute to the disease pathogenesis at several different levels. In the case of spinocerebellar ataxia type 8 (SCA8), bidirectional transcription on expanded RNAs produces both untranslated CUG repeats and CAG repeats recognized by repeat-associated non-ATG (RAN) translation and translated into toxic polyglutamine peptides [START_REF] Moseley | Bidirectional expression of {CUG} and {CAG} expansion transcripts and intranuclear polyglutamine inclusions in spinocerebellar ataxia type 8[END_REF]. SCA8 therefore appears as both an RNAand protein-gain of function disease [START_REF] Daughters | {RNA} gain-of-function in spinocerebellar ataxia type 8[END_REF][START_REF] Koob | An untranslated {CTG} expansion causes a novel form of spinocerebellar ataxia ({SCA}8)[END_REF]. Similarly, depending on the alternative splicing isoform, the HDL2 gene mRNA contains either expanded CUG repeats coding for polyalanines or polyleucines according to the selected ATG, or noncoding CUG repeats (Holmes et al., 2001a;[START_REF] Rudnicki | Huntington's disease-like 2 is associated with {CUG} repeat-containing {RNA} foci[END_REF]. Conversely, CAG repeats located in a coding region can form aggregates of RNA that sequester RNA-binding protein [START_REF] De Mezer | Mutant CAG repeats of Huntingtin transcript fold into hairpins, form nuclear foci and are targets for RNA interference[END_REF], therefore adding on the toxicity at the RNA level [START_REF] Hsu | Long tract of untranslated CAG repeats is deleterious in transgenic mice[END_REF].

So far, there are several diseases known to be primary mediated by RNA gain-of-function mechanism including fragile X tremor ataxia syndrome (FXTAS) [START_REF] Hagerman | Fragile X-associated tremor/ataxia syndrome (FXTAS)[END_REF], SCA type 8 (SCA 8) [START_REF] Daughters | {RNA} gain-of-function in spinocerebellar ataxia type 8[END_REF], SCA 10 (White et al., 2010), SCA 12 (Holmes et al., 2001b), SCA 31 (Sato et al., 2009), SCA 36 (Kobayashi et al., 2011), Huntington diseaselike 2 (HDL2) [START_REF] Rudnicki | Huntington's disease-like 2 is associated with {CUG} repeat-containing {RNA} foci[END_REF] and amyotrophic lateral sclerosis (ALS) [START_REF] Dejesus-Hernandez | Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS[END_REF]. However, the most studied examples so far are myotonic dystrophies type 1 and 2, which serve as a paradigm for the molecular mechanism of RNA-gain-of-function diseases. Both disorders will be discussed in details in the next chapters.

Myotonic dystrophy type 1 2.1.1 Mutation

The causative mutation of DM1 has been identified as an unstable expansion of a CTG trinucleotide repeat in the 3 ʹ -untranslated region (3'UTR) of the dystrophia myotonica protein kinase (DMPK) gene located on chromosome 19q13.3 (Brook et al., 1992;Mahadevan et al., 1992) (Figure 5a).

In DM1 patients, the number of CTG repeats within the mutant DMPK allele ranges from 50 to 5000 and globally, there is a correlation between the size of the expansion, the onset and severity of symptoms [START_REF] Harley | Size of the unstable CTG repeat sequence in relation to phenotype and parental transmission in myotonic dystrophy[END_REF]. Normal not mutated DMPK allele present in patients and healthy individuals carry from 5 to 37 CTG repeats at the same locus. A CTG expansion between 38 and 49 repetitions reflects a premutation state but affected individuals usually stay asymptomatic. In contrast, patients with the number of CTG repeats >50 develop clinical symptoms and disease-related pathology. In general, 5 different phenotypic groups of DM can be distinguished (Table 2): § late onset/asymptomatic § adult onset § juvenile § infantile § congenital Table 2. Clinical manifestations and disease stages in DM1 and DM2 (adapted from [START_REF] Taylor | MBNL splicing activity depends on RNA binding site structural context[END_REF].

CTG expansion

Anticipation

Although patients carrying expansion between 38-100 repeats present no or only few very mild symptoms, they are at great risk of having children with much longer expansions [START_REF] Thornton | Myotonic dystrophy with no trinucleotide repeat expansion[END_REF]. Thus, a common situation often observed in DM1 family would be a grandparent with almost no signs of the disease, a daughter with adult-onset disease that would be a mother of a child suffering from the severe congenital form of DM. The molecular basis of this phenomenon of anticipation (i.e. the severity of the disease increases while the age of onset decreases from one generation to another) is the size of the CTG expansion (CTGexp) that increases over successive generations due to its very high rate of instability in the germ-line cells [START_REF] Ashizawa | Myotonic dystrophy types 1 and 2[END_REF]. In most cases, the number of the repeats preferentially tends to increase; however, sporadic contractions of the CTG tract have been recorded in about 6,4% of transmissions, mostly when the mutated allele was coming from the father [START_REF] Lavedan | Myotonic dystrophy: size-and sex-dependent dynamics of CTG meiotic instability, and somatic mosaicism[END_REF].

Somatic mosaicism

Apart of an increasing number of expanded CTG repeats over successive generations, the size of the microsatellite expansion is also variable within the same individual. Variable number of CTGs in different tissues and cell types of the same patient results from repeat instability at the somatic level [START_REF] Higham | High levels of somatic DNA diversity at the myotonic dystrophy type 1 locus are driven by ultrafrequent expansion and contraction mutations[END_REF][START_REF] Morales | Somatic instability of the expanded CTG triplet repeat in myotonic dystrophy type 1 is a heritable quantitative trait and modifier of disease severity[END_REF]. As repeated tracks show big bias toward expansions, the number of repeats changes throughout whole life at various rate between tissues. For diagnosis and clinical evaluation, the correlation between the size of the repeats and the severity of the disease is done in the blood, and not in any other organ or tissue.

Interruptions within the repeats

In the majority of the cases, the repeated expansions within the DMPK gene consist of a pure and uninterrupted CTG tract. However, several patients containing interruptions of CCGn and/or GGCn within the overall CTG repeated stretch have been identified [START_REF] Braida | Variant CCG and GGC repeats within the CTG expansion dramatically modify mutational dynamics and likely contribute toward unusual symptoms in some myotonic dystrophy type 1 patients[END_REF][START_REF] Cumming | De novo repeat interruptions are associated with reduced somatic instability and mild or absent clinical features in myotonic dystrophy type 1[END_REF][START_REF] Leeflang | A novel repeat structure at the myotonic dystrophy locus in a 37 repeat allele with unexpectedly high stability[END_REF][START_REF] Musova | Highly unstable sequence interruptions of the CTG repeat in the myotonic dystrophy gene[END_REF][START_REF] Santoro | Expanded [CCTG]n repetitions are not associated with abnormal methylation at the CNBP locus in myotonic dystrophy type 2 (DM2) patients[END_REF][START_REF] Tomé | MSH3 polymorphisms and protein levels affect CAG repeat instability in Huntington's disease mice[END_REF]. The interruptions seem to affect the severity of the disease, as these patients show much milder phenotypes. It has been proposed that variant interspacing sequences exert stabilizing effect on the repeats, thereby reducing their expansion and consequently, slowing down the progression of the disease.

Myotonic dystrophy type 2

DM2 is caused by a CCTG tetranucleotide repeat expansion in intron 1 of the CNBP gene, also known as zinc finger protein 9 (ZNF9), on chromosome 3q21 (Liquori et al., 2001;[START_REF] Ranum | Genetic mapping of a second myotonic dystrophy locus[END_REF] (Figure 5b). The size of the CCTG repeats below 30 is commonly find in normal individuals while the range of the expansions in affected patients is huge and vary from 55 to 11000 with the average of 5000. Any relation between clinical phenotype and CCTG repeat length in DM2 has been found [START_REF] Day | Myotonic dystrophy type 2: molecular, diagnostic and clinical spectrum[END_REF]Liquori et al., 2001) and the size of the repeats in the blood seems to be rather associated with the age of the patient than with the severity of the disease. Interestingly, the interruptions of the CCTG tracts with one or more GCTG, TCTG or ACTG motifs have been observed, but in direct contrast to DM1, they reside in healthy range alleles rather than expanded ones, which typically stay uninterrupted (Bachinski et al.,

Muscle weakness

Muscle weakness is one of the most frequent symptom in DM, reported by more than half of the patients [START_REF] De Antonio | Unravelling the myotonic dystrophy type 1 clinical spectrum: A systematic registry-based study with implications for disease classification[END_REF][START_REF] Montagnese | Assessing the influence of age and gender on the phenotype of myotonic dystrophy type 2[END_REF]. Different groups of muscles are affected in DM1 and DM2, and muscle weakness is usually symmetrical in both diseases.

DM1 predominantly disturbs distal muscles, primarily involving finger flexors, wrist flexors and foot extensors [START_REF] Jiménez-Moreno | Falls and resulting fractures in Myotonic Dystrophy: Results from a multinational retrospective survey[END_REF]. During the course of the disease, muscle weakness regularly progresses, gradually involving proximal limb areas. Oropharyngeal and respiratory muscles are often involved causing difficulty in chewing, swallowing and breathing that commonly leads to fatal respiratory failure. Facial muscle wasting and atrophy are giving to the DM1 patients a characteristic 'hatchet' facial appearance, which is often seen as an early feature of the disease. This 'hatchet' phenotype is rarely seen in DM2, as facial and bulbar muscles are spared in the majority of DM2 patients. Contrary to DM1, type 2 myotonic dystrophy mainly involves proximal limb muscles having an early presentation in mild weakness of hip extensions, thigh flexions, and finger flexions [START_REF] Wenninger | Core Clinical Phenotypes in Myotonic Dystrophies[END_REF].

Histologically, skeletal muscles in myotonic dystrophy exhibit a number of characteristic abnormalities. In both types of the disease, affected muscles show significantly increased variability in the muscle fiber diameter, ring fibers, split fibers, as well as Z and I bands aberrations. In contrast to DM1 where fiber atrophy affects mainly type I fibers, a preferential atrophy of type II muscle fibers is observed in DM2. Pyknotic nuclear clumps that are a marker of late atrophic myofibers are also commonly observed in DM2 biopsies. Another histological feature observed in DM skeletal muscle fibers is a centronucleation. This phenomenon that is usually found in newly formed fibers after degeneration was not related to an active regenerative process in DM. Central nuclei could contribute to muscle dysfunction by disrupting sarcomere organization and affect myofibril contraction however, the mechanism of centralized nuclei in DM has not been investigated yet. While central nuclei are a hallmark of both DM1 and DM2, similar to muscle atrophy, they appear more frequently in type I fibers in DM1 and in fibers type II in DM2. Other less studied but histopathologically relevant characteristics of DM muscle include adipose depositions, sarcoplasmic masses late-stage fibrosis, nuclear chains, and large/irregularly shaped myonuclei (reviewed in Meola, 2015). [START_REF] Gourdon | Myotonic Dystrophies: State of the Art of New Therapeutic Developments for the CNS[END_REF].

Extra

A variety of histopathological changes have been observed in post mortem DM brain samples.

They include presenile neurofibrillary tangles [START_REF] Caillet-Boudin | Brain pathology in myotonic dystrophy: when tauopathy meets spliceopathy and RNAopathy[END_REF], neuronal loss [START_REF] Ono | Loss of serotonin-containing neurons in the raphe of patients with myotonic dystrophy: a quantitative immunohistochemical study and relation to hypersomnia[END_REF] and the presence of inclusion bodies and neurofibrils caused by intraneuronal aggregation of hyperphosphorylated Tau protein [START_REF] Vermersch | Specific tau variants in the brains of patients with myotonic dystrophy[END_REF].

Gastrointestinal track

Gastrointestinal manifestations are frequent in DM patients with dysphagia being one of the most common complaint. It is caused by reduced swallowing reflex and reduced esophageal motility [START_REF] Ercolin | Oral motor movements and swallowing in patients with myotonic dystrophy type 1[END_REF] causing the major clinical problem associated with a risk of aspiration. Other frequently reported symptoms include alternating constipation, bloating, and diarrhea accompanied by stomach cramps, reflux, and regurgitation, which are associated with smooth and striated muscle impairments and endocrine dysfunctions [START_REF] Bellini | Gastric emptying in myotonic dystrophic patients[END_REF][START_REF] Bellini | Gastrointestinal manifestations in myotonic muscular dystrophy[END_REF]. Liver problems have been also observed with elevated aspartate (AST) and alanine (ALT) transaminases. The exact causes of the gastrointestinal track pathogenesis remain to be ascertained, however, it is correlated with the timespan of disease and not necessarily the severity of skeletal muscle involvement [START_REF] Bellini | Gastrointestinal manifestations in myotonic muscular dystrophy[END_REF].

Other symptoms

A number of other features are common among patients with myotonic dystrophies. The most frequent extramuscular symptom is the occurrence of a characteristic "Christmas tree" earlyonset cataract, observed in about 50-60% of patients [START_REF] Day | Myotonic dystrophy type 2: molecular, diagnostic and clinical spectrum[END_REF][START_REF] Ekström | Visual function in congenital and childhood myotonic dystrophy type 1[END_REF][START_REF] Montagnese | Assessing the influence of age and gender on the phenotype of myotonic dystrophy type 2[END_REF]. However, the mechanisms underlying the pathophysiology of cataract in DMs are still largely unknown. Asymptomatic immunoglobulin M and immunoglobulin G hypogammaglobulinemia are also present in a majority of patients with both DM1 and DM2 but have not been associated with any important clinical implications [START_REF] Kaminsky | {IgG} deficiency and expansion of {CTG} repeats in myotonic dystrophy[END_REF][START_REF] Wochner | Accelerated breakdown of immunoglobulin G (IgG) in myotonic dystrophy: a hereditary error of immunoglobulin catabolism[END_REF]. In addition, a 2-fold increase in cancer susceptibility has been reported in myotonic dystrophy with endometrial, brain, ovarian, and colon cancers carrying the highest risk compared to the general population [START_REF] Gadalla | Cancer risk among patients with myotonic muscular dystrophy[END_REF]. Finally, frequent endocrine dysfunction occurrence increases with progression of the disease and may include hypogonadism, hyperinsulinism, likely related to insulin resistance, and frontal balding, apparent in at least half of DM patients [START_REF] Pisani | Early subclinical cochlear dysfunction in myotonic dystrophy type 1[END_REF]. Recently, a high incidence of venous thromboembolism has been described for DM1 patients [START_REF] Sochala | High Risk of Fatal and Nonfatal Venous Thromboembolism in Myotonic Dystrophy[END_REF].

Mechanism of the disease

Given the location of C(C)TG expansions ((C(C)TGexp) within non-coding regions and the fact that DM1 and DM2 are autosomal dominant inherited diseases sharing striking similarities, an overlapping pathogenic mechanism was suggested for both types of DM. Two mains hypothesis have been proposed. First, the haploinsufficiency of mutation harboring genes (DMPK, CNBP) and flanking gene (SIX homeobox 5 (SIX5)) for DM1. Second, an RNA-mediated toxicity caused by expanded microsatellite transcripts that affect downstream effectors.

Therefore, molecular mechanisms involved in DM could be considered at genomic, RNA and effector levels (Figure 11).

Genomic level

The haploinsufficiency model gained considerable interest with the original report of decreased DMPK mRNA and DMPK protein levels [START_REF] Fu | Decreased expression of myotoninprotein kinase messenger RNA and protein in adult form of myotonic dystrophy[END_REF] together with reduced expression of SIX5 neighboring gene in DM1 patient-derived cells and tissues [START_REF] Klesert | Trinucleotide repeat expansion at the myotonic dystrophy locus reduces expression of DMAHP[END_REF]. Transcriptional dysregulation, and subsequent decrease of gene expression, might result from alterations of nucleosome positioning by CTG expansions. Indeed, increased nucleosome occupancy on mutated DMPK alleles has been observed that further correlates with the number of the repeated tracts (H [START_REF] Wang | Preferential Nucleosome Assembly at DNA Triplet Repeats from the Myotonic Dystrophy Gene[END_REF]. Moreover, CpG island located upstream and downstream of the CTGexp, which remains unmethylated in healthy individuals and most of DM1 adult patients, are hypermethylated in congenital form of the disease suggesting a unique susceptibility of highly expanded DMPK alleles to certain epigenetic modifications (Barbé et al., 2017;[START_REF] Steinbach | The DMPK gene of severely affected myotonic dystrophy patients is hypermethylated proximal to the largely expanded CTG repeat[END_REF]. Even though initially suggested that DMPK sufficiency contributes to cardiac abnormalities and mild myopathy [START_REF] Berul | DMPK dosage alterations result in atrioventricular conduction abnormalities in a mouse myotonic dystrophy model[END_REF][START_REF] Berul | Progressive atrioventricular conduction block in a mouse myotonic dystrophy model[END_REF][START_REF] Saba | Localization of the sites of conduction abnormalities in a mouse model of myotonic dystrophy[END_REF], further studies have shown that neither homozygous nor heterozygous DMPK knock-out (KO) mice exhibit cardiac or skeletal muscle impairments, thereby excluding a major contribution of DMPK loss of function in DM1 muscle phenotypes [START_REF] Carrell | Dmpk gene deletion or antisense knockdown does not compromise cardiac or skeletal muscle function in mice[END_REF][START_REF] Mounsey | Phospholemman is a substrate for myotonic dystrophy protein kinase[END_REF]. Nevertheless, the chromatin structure surrounding the pathological repeat motif could contribute to the disease by affecting the expression of neighboring genes at the DM1 locus. The loss of the hypersensitive site-enhancer (HSE)

between the CTG repeats in DMPK's 3ʹUTR and the SIX5's promoter on the mutant DMPK allele has been suggested to be responsible for loss of SIX5 expression and contribution to DM1 phenotype [START_REF] Klesert | Trinucleotide repeat expansion at the myotonic dystrophy locus reduces expression of DMAHP[END_REF][START_REF] Otten | Triplet repeat expansion in myotonic dystrophy alters the adjacent chromatin structure[END_REF][START_REF] Thornton | Expansion of the myotonic dystrophy CTG repeat reduces expression of the flanking DMAHP gene[END_REF]. Studies from SIX5 KO mice have shown that homozygous animals develop cataracts and heterozygous mice subtle cardiac abnormalities. However, no evident link to DM pathology has been found [START_REF] Klesert | Mice deficient in Six5 develop cataracts: implications for myotonic dystrophy[END_REF][START_REF] Sarkar | Heterozygous loss of Six5 in mice is sufficient to cause ocular cataracts[END_REF]. All together, these compelling evidences fail to support a major contribution of DMPK or SIX5 haploinsufficiency to DM1 pathogenesis. It also excludes a common mechanism between the two types of DMs since the expression of these genes are not altered in DM2 caused by a CCTG expansion within the first intron of the CNBP gene located on a different chromosome. On the other hand, while DMPK knock-outs do not develop phenotype mimicking myotonic dystrophy, heterozygous CNBP KO is sufficient to produce multiorgan abnormalities resembling those found in DM patients including cataracts, cardiac defects and muscle wasting (Chen et al., 2007b). Although some discrepancies exist about the reduced level of CNBP in DM2 patients, it has been proposed that CNBP insufficiency could modulate DM2 pathophysiology and explain some differences between both types of the disease [START_REF] Raheem | Novel myosin heavy chain immunohistochemical double staining developed for the routine diagnostic separation of I, IIA and IIX fibers[END_REF].

A key distinction between DM1 and DM2-associated microsatellite expansions is the location of the repeats. In the DM1 context, mutant DMPK transcripts are expressed and undergo splicing, 5'capping and polyadenylation indicating that their nuclear retention is not the consequence of disrupted pre-mRNA processing [START_REF] Taneja | Foci of trinucleotide repeat transcripts in nuclei of myotonic dystrophy cells and tissues[END_REF]. In DM2, CCTGexp residing in intron are expected to be completely spliced out from the final mRNAs. The ribonuclear inclusions would be therefore enriched for the CCUG expansion alone, without flanking sequences. This expected processing pattern of mutant CNBP transcripts is supported by early studies [START_REF] Margolis | DM2 intronic expansions: evidence for CCUG accumulation without flanking sequence or effects on ZNF9 mRNA processing or protein expression[END_REF], however recent findings suggest that large microsatellite sequences might cause intron retention like in C9-ALS/FTD patient's lymphoblasts [START_REF] Niblock | Retention of hexanucleotide repeat-containing intron in C9orf72 mRNA: implications for the pathogenesis of ALS/FTD[END_REF]. Punctate nuclear localization of C(C)UGexp-RNAs and molecular crowding of electronegative A-type helix RNA with a U-U mispaired bulge provide a vast surface for interactions with RNA-binding proteins or other cellular factors that could significantly disturb their normal functions [START_REF] Mooers | The structural basis of myotonic dystrophy from the crystal structure of CUG repeats[END_REF][START_REF] Tian | Expanded CUG repeat RNAs form hairpins that activate the double-stranded RNA-dependent protein kinase PKR[END_REF]. Together, the diffusion rate of these ribonucleoprotein complexes is largely reduced as well as their ability to move out from the nucleus to the cytoplasm resulting in their nuclear retention [START_REF] Querido | Stochastic and reversible aggregation of mRNA with expanded CUG-triplet repeats[END_REF].

Effector level

The function of two family of RNA-binding proteins, Muscleblind-like (MBNL) and Elav-like (CELF), have been found to be markedly altered by the expression of C(C)UGexp-RNAs and the consequences of their disrupted activities are thought to play major roles in DM pathophysiology. Among their several functions, MBNL and CELF are known to be involved in the control of alternative splicing as key regulators of pre-mRNA splicing switch important for fetal-to-adult isoform transition during development. They play antagonistic roles in the regulation of splicing events that is not due to the direct competition for binding sites but rather via an independent recognition of distinct cis-acting RNA motifs and functional regulation of their downstream processing [START_REF] Kalsotra | A postnatal switch of CELF and MBNL proteins reprograms alternative splicing in the developing heart[END_REF]Wang et al., 2015b). The combined MBNL functional loss and CELF upregulation in DM results in an abnormal reexpression of fetal isoforms in adult tissues and their embryonic protein forms fail to meet the adult state requirements. This "spliceopathy" therefore directly contributes to the overall disease pathology.

CELF upregulation

CELF1 (previously known as CUG-BP1) has been identified as a protein having high affinity to UG-enriched RNAs in vitro; however, it does not colocalize with nuclear C(C)UGexp-RNA foci in DM patient derived cells (Fardaei et al., 2002;[START_REF] Faustino | Identification of putative new splicing targets for ETR-3 using sequences identified by systematic evolution of ligands by exponential enrichment[END_REF][START_REF] Teplova | Structural insights into RNA recognition by the alternate-splicing regulator CUG-binding protein 1[END_REF][START_REF] Timchenko | Myotonic dystrophy: the role of RNA CUG triplet repeats[END_REF]. Instead, CELF1 level is markedly elevated in DM1 skeletal and cardiac tissues [START_REF] Kalsotra | A postnatal switch of CELF and MBNL proteins reprograms alternative splicing in the developing heart[END_REF]Philips et al., 1998;Timchenko et al., 2001) and its overexpression in mice was sufficient to reproduce muscle wasting, myopathy and abnormal splicing of chloride channel gene (Clcn1) as observed in Mbnl1-deficient mice (Ward et al., 2010).

Contrary, protein upregulation in heart caused cardiac conduction defects with cardiomyopathy and early mortality [START_REF] Koshelev | Heart-specific overexpression of CUGBP1 reproduces functional and molecular abnormalities of myotonic dystrophy type 1[END_REF]. CELF1 upregulation in DM1 was associated with its hyperphosphorylation and stabilization due to an abnormal activation of the protein kinase C (PKC) pathway activation (Kuyumcu-Martinez et al., 2007). Transgenic DMPK CTG960 mice, which display increased CELF1 levels in severely affected muscles, show amelioration of symptoms and reduced CELF1 protein following PKC inhibition (Kuyumcu-Martinez et al., 2007;[START_REF] Morriss | Mechanisms of skeletal muscle wasting in a mouse model for myotonic dystrophy type 1[END_REF][START_REF] Wang | PKC inhibition ameliorates the cardiac phenotype in a mouse model of myotonic dystrophy type 1[END_REF]. However, other potential regulators of CELF1 steady-state levels include glycogen synthase kinase 3 beta (GSK3β), cyclin-dependent kinase 4 (CDK4)/cyclin D3, and calcineurin (Jones et al., 2012;[START_REF] Ravel-Chapuis | Pharmacological and physiological activation of AMPK improves the spliceopathy in DM1 mouse muscles[END_REF]. Unlike in DM1, conflicting reports showed either no change (Lin et al., 2006;Pelletier et al., 2009) or increased CEFL1 protein expression [START_REF] Salisbury | Expression of RNA CCUG repeats dysregulates translation and degradation of proteins in myotonic dystrophy 2 patients[END_REF] in DM2 patient tissues and cells.

Additional modulators/effectors

Beyond MBNL and CELF, it has been proposed that functions of other RNA binding proteins are mutually altered by the expression of expanded C(C)TG repeats. The level of the heterogeneous nuclear ribonucleoprotein H (hnRNP H) splice regulator was found elevated in DM1 muscle cells that is a part of an RNA-dependent repressor complex which coordinates alternative splicing regulation. Thus, its increased expression might affect the stoichiometry of the complexes and disrupt the downstream splicing processing (Kim et al., 2005;[START_REF] Paul | Expanded CUG repeats Dysregulate RNA splicing by altering the stoichiometry of the muscleblind 1 complex[END_REF]. Another protein, Staufen homolog 1 (STAUFEN1) was also found to be elevated in DM1 skeletal muscles and DM1 mouse models, but without direct evidence of localization in RNA foci in vivo. STAUFEN1 is involved in the transport and localization of mRNAs to different subcellular compartments and might have a role in the nuclear export of non-foci associated single CUGexp-DMPK RNA molecules (Ravel-chapuis et al., 2012). Its increased expression level can also contribute to the disease progression as STAUFEN1 modulates the splicing of several DM-relevant transcripts including insulin receptor and chloride channel [START_REF] Bondy-Chorney | Staufen1s role as a splicing factor and a disease modifier in Myotonic Dystrophy Type I[END_REF]. Two RNA-helicases, DEAD box protein 5 (DDX5) and DEAD box protein 6 (DDX6), have be identified to interact with expanded C(C)UG repeats that might modulate the hairpin structure by unwinding the RNA molecules making them more susceptible for turnover. Indeed, their overexpression in DM1 cells leads to a reduction of RNA foci and a partial correction of some missplicing events [START_REF] Jones | Reduction of toxic RNAs in myotonic dystrophies type 1 and type 2 by the RNA helicase p68/DDX5[END_REF][START_REF] Laurent | New function for the RNA helicase p68/DDX5 as a modifier of MBNL1 activity on expanded CUG repeats[END_REF][START_REF] Pettersson | DDX6 regulates sequestered nuclear CUG-expanded DMPK-mRNA in dystrophia myotonica type 1[END_REF]. More recently, the rbFOX splicing factor was found to specifically bind expanded CCUG repeats and colocalize with the RNA in DM2 but not DM1 muscle cells.

Importantly, rbFOX not only interact with repeated tracts but actively compete for the binding with MBNL proteins. As a consequence, the titration of MBNL is partially reduced that may result in an overall lower toxicity observed in DM2. Substantial role of Fox-1 holomolog A (rbFOX) has been additionally confirmed in DM2 fly model, where its overexpression significantly corrects splicing alterations and reduces muscle atrophy [START_REF] Sellier | rbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophy type 1/type 2 differences[END_REF].

Pathophysiologic mechanisms in DM

Splicing misregulation

Splicing is a well-known mechanism of RNA processing by which introns are removed from a pre-mRNA and exons are joined together to form a mature mRNA transcript [START_REF] Kornblihtt | Alternative splicing: a pivotal step between eukaryotic transcription and translation[END_REF]. Two types of RNA splicing exist, constitutive and alternative. The latter generates several unique mRNA transcripts depending on the inclusion or exclusion of particular exons, raising the biodiversity of proteins that can be encoded by the same genome. Although many human genetic disorders have been identified as the consequence of disruption of RNA splicing, cis-acting effects affecting the splicing of a single pre-mRNA represent the majority of the cases. Contrary, DM is caused by a trans-effect of alternative splicing regulators that affect a large number of transcripts regulated by these factors. This trans-dominant effect of C(C)UG expansions on alternative splicing is considered as the major mechanism responsible for the multisystemic phenotype of DM1 and DM2. Importantly, it selectively influences exons that are subjected to alternative splicing, and which additionally undergo a developmental regulation. In means that DM spliceopathy does not lead to the production of unusual splicing isoforms; instead, it is associated with the expression of fetal splicing products in adult tissues leading to the typical disease manifestations. To date, more than a hundred of RNA missplicing events have been identified in DM1 and some of them, based on animal model studies, were associated with symptoms observed in patients (Table 3).

MBNL loss-of-function [START_REF] Osborne | Transcriptional and post-transcriptional impact of toxic RNA in myotonic dystrophy[END_REF]. However, a direct effect of expanded repetitions on transcription factors has been proposed causing either their inappropriate distribution and lower availability as observed for specificity protein 1 (SP1) and retinoic acid receptor gamma (RARγ) in DM1 myocytes (Ebralidze et al., 2004), or upregulation like for NK2 homeobox 5 (NKX2-5) in DM1 heart and skeletal muscles, whose downstream consequences may contribute to defects in muscle regeneration and conduction abnormalities [START_REF] Yadava | RNA toxicity in myotonic muscular dystrophy induces NKX2-5 expression[END_REF]. Additionally, steady-state levels of some genes involved in muscle development were found to be altered in DM1 myoblasts due to the aberrant cytoplasmic localization of the SMART/HDAC1-associated repressor transcription factor (SHARP) (Dansithong et al., 2005).

mRNA translation, localization and stability

Besides well-established binding to intronic and exonic regions to regulate splicing, global cross-linking immunoprecipitation (CLIP) and RNA sequencing (RNAseq) analysis identified high abundance of MBNL potential binding sites in 3'UTR regions of messenger RNAs. Further analysis showed the MBNL proteins to be global regulators of RNA localization and membrane-associated translation as depletion of the proteins causes impaired mRNAs trafficking and their substantial relocalization away from the membrane compartments [START_REF] Adereth | RNA-dependent integrin alpha3 protein localization regulated by the Muscleblind-like protein MLP1[END_REF][START_REF] Taliaferro | Distal Alternative Last Exons Localize mRNAs to Neural Projections[END_REF][START_REF] Wang | Transcriptome-wide regulation of pre-mRNA splicing and mRNA localization by muscleblind proteins[END_REF]. In neurons, MBNL1 binding determines differential 3'UTR isoforms associated with the localization of mRNA at the soma or neurites of the neuron, respectively [START_REF] Taliaferro | Distal Alternative Last Exons Localize mRNAs to Neural Projections[END_REF]. Therefore, it demonstrates that the tight MBNL's regulatory effect on the composition of a transcript's 3ʹ-UTR and its RNA localization fate seem to be closely linked. Noteworthy, the regulation of the choice between alternative 3'UTR regions further affects the stability of the messenger RNAs (Wang et al., 2015a). MBNL's interactions with 3'UTR has been additionally implicated in the direct activation of the polyadenylation machinery and choice of the polyadenylation sites (PAS) (Batra et al., 2014). Accordingly, widespread alternative polyadenylation (APA) misregulation characterized by a shift from adult to fetal APA patterns has been found in both cell and animal DM models, corroborating that deregulation of pre-mRNA processing in myotonic dystrophy is affected at multiple levels. Therefore, it has been postulated that specific alterations in 3ʹ

UTR structures and consequent changes in RNA localization and/or protein isoforms and levels might be attributable to pathological changes present in DM [START_REF] Wang | Transcriptome-wide regulation of pre-mRNA splicing and mRNA localization by muscleblind proteins[END_REF].

Proteins from the CELF family have been found to equally bind 3ʹ-UTRs of selective mRNAs and regulate their expression levels (Wang et al., 2015a). CELF1 by interaction with GU-rich elements (GREs) localized in 3'UTR induces the decay of short-lived mRNAs [START_REF] Vlasova | Conserved GU-rich elements mediate mRNA decay by binding to CUG-binding protein 1[END_REF] and numerous of these CELF target transcripts encode proteins essential for muscle cell development and function [START_REF] Rattenbacher | Analysis of CUGBP1 targets identifies GU-repeat sequences that mediate rapid mRNA decay[END_REF][START_REF] Timchenko | Molecular mechanisms of muscle atrophy in myotonic dystrophies[END_REF]. CELF1 is additionally involved in regulation of mRNA translation and its affinity towards mRNA targets strongly depends on its phosphorylation status. For example, cyclin D3-CDK4-phosphorylated CELF1 interacts with eukaryotic initiation factor 2 (eIF2) initiation factor, leading to the recruitment of translational machinery to target mRNAs [START_REF] Dasgupta | The importance of CELF control: molecular and biological roles of the CUG-BP, Elav-like family of RNA-binding proteins[END_REF]. DM cells show decreased activity of cyclin D3-CDK4 that raises higher levels of unphosphorylated CELF1, which forms inactive complexes with eIF2 affecting translation of mRNAs required for myoblast differentiation. These CELF1-eIF2 complexes have been found to accumulate in cytoplasmic stress granules in both DM1 and DM2 myoblasts [START_REF] Barreau | Mammalian CELF/Bruno-like RNA-binding proteins: molecular characteristics and biological functions[END_REF][START_REF] Salisbury | Expression of RNA CCUG repeats dysregulates translation and degradation of proteins in myotonic dystrophy 2 patients[END_REF]. Perturbations of CELF1 cytoplasmic functions might also arise from its improper nucleus-cytoplasm distribution that is regulated by protein kinase B (AKT) signaling pathway and has been shown to be dysregulated in DM1 myoblasts (Huichalaf et al., 2010).

miRNA

miRNAs are small non-coding RNAs regulating gene expression at posttranscriptional level.

Their expression and intracellular distribution are often found to be considerably deregulated in human diseases including muscular dystrophies [START_REF] Eisenberg | miRNAS in normal and diseased skeletal muscle[END_REF][START_REF] Gambardella | Overexpression of microRNA-206 in the skeletal muscle from myotonic dystrophy type 1 patients[END_REF][START_REF] Perbellini | Dysregulation and cellular mislocalization of specific {miRNAs} in myotonic dystrophy type 1[END_REF]. Hence, some miRNAs in association with several downstream target RNAs were found to be significantly altered in DM patient-derived cells and muscle tissues [START_REF] Fernandez-Costa | Expanded CTG repeats trigger miRNA alterations in Drosophila that are conserved in myotonic dystrophy type 1 patients[END_REF]. In the heart tissues of DM1 patients and mouse model, a subset of developmentally regulated microRNAs associated with cardiac arrhythmias is largely downregulated [START_REF] Kalsotra | The Mef2 transcription network is disrupted in myotonic dystrophy heart tissue, dramatically altering miRNA and mRNA expression[END_REF]Rau et al., 2011). This is particularly the case for miR-1 family. Interestingly, MBNL1 itself is involved in miR-1 processing by binding to pre-miR-1 sequences and promoting their maturation. Therefore, the significant reduction of miR-1 and consequently, increased expression of their target transcripts including calcium voltagegated channel subunit alpha1 A (CACNA1C) and Gap junction alpha-1 protein (GJA1) in DM cardiac tissue, results from the depletion of functional MBNL1 (Rau et al., 2011). Potential MBNL1 binding sites have been also found in many other pre-miRNAs highlighting the possibility that MBNL1 is a global regulator of the maturation of miRNAs (Rau et al., 2011).

However, other study showed that the alteration in the expression of a subset of miRNAs in DM1 might be the consequence of dysregulation of myocyte enhancer factor-2 (MEF2) transcriptional network [START_REF] Kalsotra | The Mef2 transcription network is disrupted in myotonic dystrophy heart tissue, dramatically altering miRNA and mRNA expression[END_REF]. As myocyte enhancer factor-2 A (MEF2A)

protein levels are significantly reduced in DM1 heart tissue, it may have further implication in the steady state levels of particular miRNAs.

RNA interference

Hairpin structures formed by microsatellite containing RNAs have been suggested to be a target for RNA interference (RNAi) machinery to generate small interference RNAs (siRNAs) that negatively regulate genes expression [START_REF] Krol | Ribonuclease dicer cleaves triplet repeat hairpins into shorter repeats that silence specific targets[END_REF]. The stem structures would require processing by a Dicer protein, a ribonuclease involved in biogenesis of mi-and siRNA.

However, siRNA processing takes place in the cytoplasm, therefore due to the nuclear retention of the expanded transcripts, this event is highly unlikely. A small 21-nt RNA fragments has been suggested to be produced as a result of bidirectional transcription on the wild type allele at DM1 locus through an siRNA mechanism [START_REF] Osborne | Cell-free cloning of highly expanded CTG repeats by amplification of dimerized expanded repeats[END_REF].

However, no direct evidence has been found as these RNA fragments are generated by Dicer.

Thus far, siRNA pathway has been only demonstrated in C. elegans model of DM1 [START_REF] Qawasmi | Expanded CUG Repeats Trigger Disease Phenotype and Expression Changes through the RNAi Machinery in C. elegans[END_REF] with no proof of C(C)UG-containing small interference RNAs neither in DM patients nor in any other animal model of the disease.

Antisense transcription

Natural antisense transcripts are RNA molecules that are transcribed from the opposite DNA strand, compared with other transcripts, and overlap in part with sense RNA. Both sense and antisense RNAs can encode proteins or be non-protein-coding transcripts [START_REF] Katayama | Antisense transcription in the mammalian transcriptome[END_REF]. In accordance, it has been shown that bidirectionally transcription occurs also on microsatellite expansions including CTG repeats [START_REF] Batra | Partners in crime: bidirectional transcription in unstable microsatellite disease[END_REF][START_REF] Cho | Antisense transcription and heterochromatin at the DM1 CTG repeats are constrained by CTCF[END_REF]. The primary DM1 antisense transcript spans more than 6kb, contains alternative transcription start sites and undergo alternative splicing producing a mixture of antisense products with and without expanded CAG repeats [START_REF] Gudde | Trinucleotide-repeat expanded and normal DMPK transcripts contain unusually long poly(A) tails despite differential nuclear residence[END_REF]. The expression of these antisense RNAs appears upregulated in DM1 patients however their overall abundance remains very low in all tissues [START_REF] Gudde | Trinucleotide-repeat expanded and normal DMPK transcripts contain unusually long poly(A) tails despite differential nuclear residence[END_REF]. So far, both structure and biologic significance of these antisense genes are largely unknown and require further studies, but it has been suggested that they can contribute to the formation of toxic nuclear ribonucleoprotein (RNP) aggregates or generation RAN translation products in the cytoplasm [START_REF] Gudde | Trinucleotide-repeat expanded and normal DMPK transcripts contain unusually long poly(A) tails despite differential nuclear residence[END_REF]. Additionally, antisense transcription could stimulate repeat instability, leading to either expansion or contraction, by producing unstable alleles (Nakamori et al., 2011).

Non-ATG translation

Another not mutually exclusive mechanism for C(C)UG toxicity in DM is a non-canonical way of protein translation termed repeat associated non-ATG translation (RAN). It occurs independently of an ATG initiation codon in all reading frames producing homopolymeric peptides that aggregate both in the nucleus and cytoplasm [START_REF] Zu | Non-ATG-initiated translation directed by microsatellite expansions[END_REF]. DMPK and CNBP, as other genes with microsatellite expansions, are bidirectionally transcribed [START_REF] Batra | Partners in crime: bidirectional transcription in unstable microsatellite disease[END_REF]. Therefore, RAN translation across sense strand in DM1 can produce proteins of long polyleucine, polyalanine and polycysteine tracts while the antisense CAG strand would translate into polyglutamine, polyserine and polyalanine. Out of these potential products, so far only polyglutamine has been detected in human DM1 myoblasts and skeletal muscles [START_REF] Zu | Non-ATG-initiated translation directed by microsatellite expansions[END_REF]. On the other hand, RAN across CCUG tracts in DM2 produce tetrapeptide expansion proteins with Leu-Pro-Ala-Cys (LPAC) motif from the sense strand or Gln-Ala-Gly-Arg (QAGR) repeats from the antisense strand [START_REF] Zu | RAN Translation Regulated by Muscleblind Proteins in Myotonic Dystrophy Type 2[END_REF]. These products have not yet been identified in muscles, but they were readily spotted in the brain tissues of DM2 patients suggesting, that they might be responsible for some of the CNS features in DM [START_REF] Zu | RAN Translation Regulated by Muscleblind Proteins in Myotonic Dystrophy Type 2[END_REF].

It has been noted that cells with the RAN protein products are prone to apoptosis indicating their toxicity. However, specific roles of RAN products regarding their mechanism of action and regulation still remain to be determined. [START_REF] Garcia-Lopez | Genetic and chemical modifiers of a CUG toxicity model in Drosophila[END_REF][START_REF] Picchio | Novel Drosophila model of myotonic dystrophy type 1: phenotypic characterization and genome-wide view of altered gene expression[END_REF][START_REF] Yu | Modeling human trinucleotide repeat diseases in Drosophila[END_REF]. The flies additionally develop relevant muscle phenotypes, such as muscle wasting [START_REF] Garcia-Lopez | Genetic and chemical modifiers of a CUG toxicity model in Drosophila[END_REF] and hypercontraction [START_REF] Picchio | Novel Drosophila model of myotonic dystrophy type 1: phenotypic characterization and genome-wide view of altered gene expression[END_REF]. The expression of expanded CUG repeats also resulted in RNA foci and muscle phenotypes in zebrafish [START_REF] Delorimier | Modifications to toxic CUG RNAs induce structural stability, rescue mis-splicing in a myotonic dystrophy cell model and reduce toxicity in a myotonic dystrophy zebrafish model[END_REF][START_REF] Todd | Transcriptional changes and developmental abnormalities in a zebrafish model of myotonic dystrophy type 1[END_REF] and C. elegans (Chen et al., 2007a;[START_REF] Wang | Muscleblind participates in RNA toxicity of expanded CAG and CUG repeats in Caenorhabditis elegans[END_REF]. Although this great conservation of the core mechanism of RNA toxicity between species, the physiology of simple organisms substantially differs from humans. Therefore, to get the closest genetic, molecular and phenotypic characteristics of the disease, mice models have been the most extensively studied among other animal models to provide relevant insight into the mechanism of the disease. So far, a number of different mouse models have been developed providing suitable platform for investigation of particular disease features (Table 4). The most extensively used mouse models that reproduce specific characteristics of DM1 include the HSALR mice, the DMSXL mice and the MBNL1 deficient mice:

Models for DM

HSA LR : CTGexp in the human skeletal actin gene

The HSA long repeat (HSA LR ) mice express 250 CTG repeats in the 3' UTR of the human skeletal alpha actin (HSA) gene [START_REF] Mankodi | Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat[END_REF]. While the expanded CTG repeat tract is located in a non-DMPK gene and its expression restricted to skeletal muscles, HSA LR mice display CUGexp RNA foci with MBNL colocalization [START_REF] Mankodi | Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat[END_REF], abnormal muscle histopathology including centralized nuclei and aberrant fibers size [START_REF] Mankodi | Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat[END_REF], robust splicing abnormalities and myotonia as a functional outcome (Lin 2006). Although HSA LR mice do not recapitulate all the characteristic features of DM muscles like muscle wasting and weakness, and its phenotype is limited only to skeletal muscle, it constitutes a widely used model to study muscle pathology and to evaluate therapeutic approaches.

DMSXL: CUGexp repeats in human DMPK gene

The DMSXL mouse carries expanded CTG repeats within their natural context and was primarily generated through the insertion of 45 kb of human genomic sequence including the human DMPK gene with 300 CTG repeats (Seznec et al., 2001). Because of intergenerational instability of the expansion, the length of the CTG has increased to more than 1500 repeats (Gomes-Pereira et al., 2007). The human DMPK transgene that is under the control of its own promoter has an almost ubiquitous expression. However, in adult DMSXL mice, the level of expression of human DMPK is relatively low in skeletal muscles when compared to murine Dmpk but it is highly expressed in the brain. DMSXL mice display several DM1-associated phenotypes, including cognitive impairments growth retardation, muscle defects, nuclear foci and mild splicing abnormalities [START_REF] Fernandez-Costa | Expanded CTG repeats trigger miRNA alterations in Drosophila that are conserved in myotonic dystrophy type 1 patients[END_REF]Huguet et al., 2012;Vignaud et at., 2010). Due to the ubiquitous expression of the mutated transgene, DMSXL mice constitute a good model for studying the multisystemic disorder of DM1.

MBNL1 knockout mice

Given the central role of MBNL1 protein in the DM pathology, the MBNL1 deficient mouse was generated through the disruption of Mbnl constitutive exon 3 (Kanadia et al., 2003a).

Mbnl1 Δ3/Δ3 mice show robust missplicing changes mainly in the muscles and transcriptomic analysis has shown that functional depletion of MBNL1 protein is responsible for the vast majority of the splicing and expression defects observed in DM1 (Lin et al., 2006;Osbourne et al., 2009). MBNL1 deficient mice display other muscular symptoms including myotonia and presence of central nuclei (Kanadia et al., 2003a;Lin et al., 2006). However, atrophy and muscle weakness are not, or very poorly, reproduced in these mice. Loss of MBNL1 affects also other tissues, resulting in typical DM1 cataracts (Kanadia et al., 2003a), motivation deficits and apathy (Matynia et al., 2010) as well as conduction defects and missplicing in heart (Lee et al., 2013a).

In vitro cell models for myotonic dystrophy have been thoroughly described in two articles attached in the annex.

MBNL family of proteins

Being initially identified in Drosophila melanogaster as a protein required for terminal differentiation of muscles and photoreceptors [START_REF] Artero | The muscleblind gene participates in the organization of Z-bands and epidermal attachments of Drosophila muscles and is regulated by Dmef2[END_REF][START_REF] Begemann | muscleblind, a gene required for photoreceptor differentiation in Drosophila, encodes novel nuclear Cys3His-type zinc-finger-containing proteins[END_REF][START_REF] Pascual | The Muscleblind family of proteins : an emerging class of regulators of developmentally programmed alternative splicing[END_REF], MBNL proteins constitute a deeply conserved family of tissue specific RNA-binding factors. Having appeared about 800 million years ago, they represent a relatively modern group of proteins, with the expression being restricted to Metazoans as they have not been found in bacteria, fungi or plants [START_REF] Pascual | The Muscleblind family of proteins : an emerging class of regulators of developmentally programmed alternative splicing[END_REF]. Generally, all invertebrate genomes analyzed so far, possess only one muscleblind gene while most vertebrate species encode three muscleblind protein homologs. Deep conservation between MBNL paralogs suggest that they have arisen as a consequence of a modern gene duplication event from a common ancestor and over the years acquired similar, yet distinct functions.

Mammalian MBNLs

In mammals, MBNL1, MBNL2 and MBNL3 genes are located on chromosomes 3, 13 and X, respectively. Genomic organization analysis of MBNL paralogs revealed a high similarity of exons composition regarding their number and size (Fardaei et al., 2002). Accordingly, MBNL1 is composed of 10, MBNL2 of 8 and MBNL3 of 11 exons [START_REF] Pascual | The Muscleblind family of proteins : an emerging class of regulators of developmentally programmed alternative splicing[END_REF] and several exons undergo alternative splicing regulation giving rise to number of MBNL mRNA isoforms (Konieczny et al., 2014) (Figure 12). The similarity of amino acid sequences between MBNL paralogs reaches 65-73% with the highest found between MBNL1 and MBNL2. Sequences encoded by alternative exons modulate further the cellular localization, binding properties, multimerization capacity and overall general activity of the proteins [START_REF] Kino | Nuclear localization of MBNL1: Splicing-mediated autoregulation and repression of repeatderived aberrant proteins[END_REF][START_REF] Poulos | Progressive impairment of muscle regeneration in muscleblind-like 3 isoform knockout mice[END_REF][START_REF] Terenzi | Conserved developmental alternative splicing of muscleblind-like (MBNL) transcripts regulates MBNL localization and activity[END_REF][START_REF] Tran | Analysis of exonic regions involved in nuclear localization, splicing activity, and dimerization of {Muscleblind}like-1 isoforms[END_REF]. Besides their structural resemblance, the three MBNL proteins differ widely in their expression pattern (Figure 13). MBNL1 and MBNL2 are ubiquitously present but MBNL1 is the paralog that seems to serve primary roles in most tissues, with the highest expression reported in heart and skeletal muscles (Fardaei et al., 2002;[START_REF] Kanadia | Developmental expression of mouse muscleblind genes Mbnl1, Mbnl2 and Mbnl3[END_REF]. The exception remains the central nervous system where MBNL2 is predominantly expressed (Fardaei et al., 2002). Conversely, MBNL3 expression is more restricted to early stages of development but is also detectable in placenta, spleen and lung. In mouse, MBNL3 is also expressed in mouse muscle precursor cells where antagonizes muscle differentiation by suppressing myoblast determination protein 1 (MyoD) expression levels to prevent unwanted myogenic gene transcription. Additionally, MBNL3 is involved in the regenerative capacity of the murine skeletal muscle as its regeneration is impaired in aged MBNL3 deficient mice [START_REF] Poulos | Progressive impairment of muscle regeneration in muscleblind-like 3 isoform knockout mice[END_REF]. Likewise, the pattern of expression of MBNL proteins throughout muscle cell development and differentiation shows temporal changes that are species-specific (Miller et al., 2000). For instance, in human muscle cells only MBNL1 and MBNL2 are expressed while MBNL3 is not detectable. In contrast, in mouse all three paralogs are present in myoblasts, muscle precursor cells, and during the early stages of myogenesis but during differentiation only the level of MBNL1 and MBNL2 increases remarkably while MBNL3 is barely detectable in terminally differentiated myotubes [START_REF] Holt | Muscleblind-like proteins: similarities and differences in normal and myotonic dystrophy muscle[END_REF]. In addition, MBNL1 and MBNL2 could have some compensatory roles as the deletion of either MBNL1 or MBNL2 results in a significant increase of the other paralog that is associated with functional compensation (Lee et al., 2013a). The importance of this dependence is further emphasized by the fact, that double MBNL1:MBNL2 deficient mice are embryonic lethal. Moreover, the conditional deletion of MBNL2 on the MBNL1 KO background causes robust splicing deregulation, exceeding the ones seen in either of the single MBNL deficient mouse models and resembling the spliceopathy observed in DM patients (Lee et al., 2013a;[START_REF] Thomas | Disrupted prenatal RNA processing and myogenesis in congenital myotonic dystrophy[END_REF]. function: a Lev box that is localized immediately before the first zinc finger domain and consists of the consensus amino acid sequence WLXLEV [START_REF] Pascual | The Muscleblind family of proteins : an emerging class of regulators of developmentally programmed alternative splicing[END_REF], and a NGR motif builds of asparagine-glycine-arginine (NGR) core sequence followed by asparagine or valine residues. Together with two highly conserved apolar residues in front and after the NGR core, they constitute so-called NGR box. Two NGR boxes were reported to flank the second zinc finger of all studied Muscleblind proteins [START_REF] Pascual | The Muscleblind family of proteins : an emerging class of regulators of developmentally programmed alternative splicing[END_REF]. Additionally, MBNL paralogs contain unstructured (low complexity) regions rich in proline and alanine. Although the functional relevance of such domains is unclear, it has been suggested that they may be involved in protein-protein interactions (Kay et al.). Due to the high degree of similarity, it has been speculated that the two ZnF binding domains have equivalent RNA binding properties and may be functionally redundant. This hypothesis was supported by studies using Drosophila melanogaster and Caenorhabditis elegans's MBNL1, which contain a single ZnF domain orthologous to the human ZnF1/2, and efficiently regulate splicing of many MBNL1 target transcripts in mammalian cells [START_REF] Irion | Drosophila muscleblind codes for proteins with one and two tandem zinc finger motifs[END_REF][START_REF] Oddo | Conservation of context-dependent splicing activity in distant Muscleblind homologs[END_REF][START_REF] Vicente-Crespo | Drosophila muscleblind is involved in troponin {T} alternative splicing and apoptosis[END_REF]. However, combinatorial mutagenic analysis of the four ZnFs revealed that they are not functionally equivalent, but may regulate splicing in an exclusive or mutually compensating manner [START_REF] Purcell | Combinatorial Mutagenesis of MBNL1 Zinc Fingers Elucidates Distinct Classes of Regulatory Events[END_REF]. MBNL1 protein with a single functional ZnF1/2 retain high binding affinity to RNA substrates with approximately 80% of the normal splicing activity. On the other hand, MBNL1 depleted for the first pair of the domain with single functional ZnF3/4

shows drastic decline in binding to RNA target transcripts, with merely half of its splicing activity [START_REF] Purcell | Combinatorial Mutagenesis of MBNL1 Zinc Fingers Elucidates Distinct Classes of Regulatory Events[END_REF]. Interestingly, deletion of any of the binding domain alone markedly diminishes the interaction of MBNL1 with expanded CUG RNA in DM1 in vivo [START_REF] Kino | Muscleblind protein, {MBNL}1/{EXP}, binds specifically to {CHHG} repeats[END_REF][START_REF] Rehman | Development of an AP-FRET Based Analysis for Characterizing RNA-Protein Interactions in Myotonic Dystrophy (DM1)[END_REF]. Recent studies have confirmed that two pairs of ZnF act as independent units with remarkably different RNA binding specificities. In general, ZF1/2 specifically recognizes MBNL binding motifs and drives splicing regulation while ZF3/4 serves as a more general binding domain allowing the recognition of a broader range of targets [START_REF] Hale | An engineered RNA binding protein with improved splicing regulation[END_REF].

Exon 3-linker

The protein/RNA interaction is also determined by an 80 amino acids linker encoded by exon 3 that separates two ZnF tandems, as mentioned previously. Even if it does not contain any defined RNA binding domains, its deletion severely reduces MBNL binding affinity to target pre-mRNAs, indicating that its presence is required for optimal function of ZnF domains [START_REF] Kino | Muscleblind protein, {MBNL}1/{EXP}, binds specifically to {CHHG} repeats[END_REF][START_REF] Tran | Analysis of exonic regions involved in nuclear localization, splicing activity, and dimerization of {Muscleblind}like-1 isoforms[END_REF]. The possible explanation is that the linker could support protein flexibility facilitating the binding of separated RNA motifs in cis or in trans [START_REF] Cass | The four Zn fingers of MBNL1 provide a flexible platform for recognition of its RNA binding elements[END_REF][START_REF] Tran | Analysis of exonic regions involved in nuclear localization, splicing activity, and dimerization of {Muscleblind}like-1 isoforms[END_REF]. It has been also shown that the presence of regulatory regions within the sequence of the exon 3 are essential for the precise regulation of alternative exons of the insulin receptor (IR) and cardiac troponin T (cTNT) genes. Although the linker sequence greatly increases binding properties and its absence reducing capabilities to regulate splicing, it is not absolutely required for MBNL RNA binding activity as MBNL1 isoforms lacking exon 3 have been identified to colocalize in ribonuclear foci containing CUG repeats in DM1 [START_REF] Rehman | Development of an AP-FRET Based Analysis for Characterizing RNA-Protein Interactions in Myotonic Dystrophy (DM1)[END_REF][START_REF] Tran | Analysis of exonic regions involved in nuclear localization, splicing activity, and dimerization of {Muscleblind}like-1 isoforms[END_REF]. Additionally, two mouse isoforms lacking exon 3 are highly conserved among this organism implying their potentially important functions (Kanadia et al., 2003a). Surprisingly, despite its evident prominence, exon 3 shows only 50% similarity between all MBNLs, indicating its possible slightly different roles in each paralog [START_REF] Grammatikakis | Identification of MBNL1 and MBNL3 domains required for splicing activation and repression[END_REF]. [START_REF] Goers | MBNL1 binds GC motifs embedded in pyrimidines to regulate alternative splicing[END_REF]. Additional analysis of frequencies of nucleotides flanking core GpC dinucleotide revealed that the Y positions have a strong bias toward uridine and the UGCU motif, especially when repeated several times over a short sequence region, constitutes the most common binding element for all MBNL paralogs [START_REF] Goers | MBNL1 binds GC motifs embedded in pyrimidines to regulate alternative splicing[END_REF]) [START_REF] Masuda | CUGBP1 and MBNL1 preferentially bind to 3' UTRs and facilitate mRNA decay[END_REF]. The examination of crystal structure of ZnF3/4 from MBNL1 in conjugation with short (r(CGCUGU) sequence confirmed the recognition of the CpG containing motifs and showed that ZnF3 preferentially binds to GC whereas ZnF4 recognizes GCU sequences spatially separated in mRNA sequence [START_REF] Teplova | Structural insights into RNA recognition by the alternativesplicing regulator muscleblind-like MBNL1[END_REF]. Both zinc finger domains represent unprecedented overall fold that orients RNA binding surfaces away from each other. This leads to an antiparallel orientation of the two single-stranded RNA chains in a looped conformation with ZnFs lying between the complementary sequences. Interacting GC dinucleotides are buried into neighboring pockets containing conserved aromatic residues of lysine and arginine, with two zinc-coordinated cysteines lining the bottom. ZnF interactions have the character of intermolecular stacking of amino acid residues and nitrogen bases, bound by the hydrogen connection network [START_REF] Teplova | Structural insights into RNA recognition by the alternativesplicing regulator muscleblind-like MBNL1[END_REF]. Nuclear magnetic resonance spectroscopy (NMR) analysis of each of zinc fingers pairs showed that both of them fold into a compact globular tandem structure that participates in RNA binding however, the individual zinc fingers within ZnF1/2 or ZnF3/4 pairs are nonequivalent. In the complex with the short GUCUCGCUUUUCCCC sequence the primary RNA binding surface is formed in each pair by the second zinc finger (ZnF2 or ZnFf4), which interacts with the CGCU RNA sequence [START_REF] Park | Structural Basis for Interaction of the Tandem Zinc Finger Domains of Human Muscleblind with Cognate RNA from Human Cardiac Troponin T[END_REF]. Even though YGCY motif represents the core sequence crucial for MBNL recognition, additional 9-10 nucleotides flanking YGCY are required for high affinity binding [START_REF] Park | Structural Basis for Interaction of the Tandem Zinc Finger Domains of Human Muscleblind with Cognate RNA from Human Cardiac Troponin T[END_REF]. Besides, CLIP-seq analysis confirmed that independently of MBNL's interaction with introns, exons or 3'UTR, it recognizes the same consensus sequence, indicating the same mode of interplay with various RNA regions and in various cellular compartments [START_REF] Poulos | Progressive impairment of muscle regeneration in muscleblind-like 3 isoform knockout mice[END_REF][START_REF] Wang | Transcriptome-wide regulation of pre-mRNA splicing and mRNA localization by muscleblind proteins[END_REF].

Binding requirements

Considering the structural organization of MBNL RNA-binding motifs, it has been shown that periodic mispairings within the sequence are essential for MBNL recognition and their replacement with Watson-Crick base pairs completely abolishes the protein interaction (Warf and Berglund, 2007). While two CpG steps can remain either single or double-stranded, it seems that efficient binding might depend on the presence of single stranded pyrimidines neighboring YCGY [START_REF] Lambert | RNA Bind-n-Seq: quantitative assessment of the sequence and structural binding specificity of RNA binding proteins[END_REF] that destabilize RNA conformation. Several studies indicate that MBNL binding may influence the RNA structure equilibrium and shift the helical region toward partial unwinding, creating locally single-stranded conformation. Analogous conclusions can be drawn from the studies of the interaction of MBNL1 with expanded CUG, CCUG and CAG repetitions that create thermodynamically stable hairpin structures [START_REF] Ho | Colocalization of muscleblind with {RNA} foci is separable from mis-regulation of alternative splicing in myotonic dystrophy[END_REF][START_REF] Kino | Muscleblind protein, {MBNL}1/{EXP}, binds specifically to {CHHG} repeats[END_REF][START_REF] Krzyzosiak | Triplet repeat RNA structure and its role as pathogenic agent and therapeutic target[END_REF][START_REF] Mykowska | CAG repeats mimic CUG repeats in the misregulation of alternative splicing[END_REF][START_REF] Sobczak | Structural diversity of triplet repeat RNAs[END_REF]Warf and Berglund, 2007;[START_REF] Yuan | Muscleblind-like 1 interacts with RNA hairpins in splicing target and pathogenic RNAs[END_REF]. The core of the CUG and CAG repetitions forms an alpha helix with U-U and A-A mismatches. Weak pairing between U-U and A-A bases enables separation of both strands of RNA duplex and subsequent interaction of ZnF with GC steps [START_REF] Teplova | Structural insights into RNA recognition by the alternativesplicing regulator muscleblind-like MBNL1[END_REF]Warf and Berglund, 2007). Noteworthy, stabilization of CUG hairpins with pseudouridins effectively prevents MBNL binding similarly to CGG triplets that adopt stable hairpins structures with a strong two hydrogen bond between G-G pairs [START_REF] Delorimier | Modifications to toxic CUG RNAs induce structural stability, rescue mis-splicing in a myotonic dystrophy cell model and reduce toxicity in a myotonic dystrophy zebrafish model[END_REF][START_REF] Kiliszek | Crystal structures of CGG RNA repeats with implications for fragile X-associated tremor ataxia syndrome[END_REF][START_REF] Kino | Muscleblind protein, {MBNL}1/{EXP}, binds specifically to {CHHG} repeats[END_REF].

Structural context-consensus motif vs RNA structure

The activity of RNA binding proteins depends not only on their own requirement but also on the features of bounded target including sequence specificity, structural organization and distribution of RNA-binding motifs [START_REF] Lewis | RNA modifications and structures cooperate to guide RNA-protein interactions[END_REF][START_REF] Lunde | RNA-binding proteins: modular design for efficient function[END_REF][START_REF] Wang | Transcriptome-wide regulation of pre-mRNA splicing and mRNA localization by muscleblind proteins[END_REF].

For MBNLs, the number of UGCU positively correlates with binding affinity that is further associated with splicing activity [START_REF] Lewis | RNA modifications and structures cooperate to guide RNA-protein interactions[END_REF][START_REF] Lunde | RNA-binding proteins: modular design for efficient function[END_REF]Wang et al., 2015b). In general, two UGCU motifs are required for MBNLs interaction but their affinity to target RNAs rises 10 times when the amount of the motif is doubled [START_REF] Taylor | MBNL splicing activity depends on RNA binding site structural context[END_REF]. Indeed, for experimentally confirmed MBNLs targets the number of UGCU tandems is usually high and varies between 15 for LIM domain binding 3 LDB3 exon 10 (Cywoniuk et al., 2017), 12 for MBNL1 exon 1 (Konieczny et al., 2017) and 9 for insulin receptor (INSR) exon 11 [START_REF] Sen | Muscleblind-like 1 (Mbnl1) promotes insulin receptor exon 11 inclusion via binding to a downstream evolutionarily conserved intronic enhancer[END_REF]. Even though the number of the consensus motifs determines the impact of RNA binding, their structural organization can modulate proteins activity to a greater extent.

Hence, contiguous UGCU sequences (5'…UGCUUGCU…3') assures high binding affinity and splicing activity while overlapping UGCU motifs (5'…UGCUGCU…3') significantly lowers MBNLs' splicing regulation abilities without affecting their binding properties. In contrast, separation of individual motifs vastly perturbs both traits [START_REF] Taylor | MBNL splicing activity depends on RNA binding site structural context[END_REF]. Additionally, structural analysis of MBNLs' targets revealed high preferences of the proteins to UGCU located on one side of the stem of RNA hairpin and their distribution on the opposite side substantially weakens splicing activity [START_REF] Taylor | MBNL splicing activity depends on RNA binding site structural context[END_REF]. Even though all 3 paralogs regulate the same alternative events with different strengths, their activity are fine-tuned by the same RNA structural feature.

C-terminal part of the protein

Composed of six mostly alternatively spliced exons (exon 5 to 10), C-terminus part of the protein varies between MBNL paralogs. It does not contain any RNA binding motifs and its presence is not required for RNA recognition since MBNL1 protein ablated for this domain has similar affinity for its target RNAs as the entire protein [START_REF] Kino | Muscleblind protein, {MBNL}1/{EXP}, binds specifically to {CHHG} repeats[END_REF][START_REF] Terenzi | Conserved developmental alternative splicing of muscleblind-like (MBNL) transcripts regulates MBNL localization and activity[END_REF]. Instead, the ability of MBNLs to regulate splicing is lost by the absence of the C-terminal part [START_REF] Kino | Muscleblind protein, {MBNL}1/{EXP}, binds specifically to {CHHG} repeats[END_REF] suggesting an essential role of this domain in the protein activity either directly or by modulating proteins regions involved in its proper functions.

Exon 5 and 6-nuclear localization

Exon 5, localized just after the last ZnF domain, has been found to be crucial for determining the cellular localization of MBNL proteins. Its inclusion or exclusion from the pre-mRNA transcript switches the protein content from nucleo-cytoplasmic to prevalently nuclear localization (Fernandez-Costa et al., 2011;[START_REF] Kino | Nuclear localization of MBNL1: Splicing-mediated autoregulation and repression of repeatderived aberrant proteins[END_REF][START_REF] Terenzi | Conserved developmental alternative splicing of muscleblind-like (MBNL) transcripts regulates MBNL localization and activity[END_REF]Tran et al., 2011). Noteworthy, whereas the sequence of exon 5 present in Mbnl1 and Mbnl2 mRNA is highly conserved sharing 72% of similarity, no such sequence exists in Mbnl3 that causes MBNL3 protein to predominate in the cytoplasm [START_REF] Gates | Autoregulated Splicing of muscleblindlike 1[END_REF]. Studies from Drosophila melanogaster have shown that exon 5 constitutes only a part of a nuclear localization signal (NLS) present in MBNLs. A five amino-acids KRAEK NLS motif, conserved from C. elegans to vertebrates (Fernandez-Costa et al., 2011) has been identified in constitutive exon 6 in all MBNL paralogs suggesting that the nuclear retention of MBNL necessitates a bipartite signal with one part in the alternative exon 5 and the second in the constitutive exon 6. Noteworthy, NLS present in exon 6 is not functional upon absence of exon 5. Although MBNLs regulate alternative splicing, it is not clear whether its increasing concentration in the cell nucleus exerts a positive, negative or no effect on the splicing activity [START_REF] Kino | Nuclear localization of MBNL1: Splicing-mediated autoregulation and repression of repeatderived aberrant proteins[END_REF][START_REF] Terenzi | Conserved developmental alternative splicing of muscleblind-like (MBNL) transcripts regulates MBNL localization and activity[END_REF][START_REF] Tran | Analysis of exonic regions involved in nuclear localization, splicing activity, and dimerization of {Muscleblind}like-1 isoforms[END_REF]. On the other hand, even though exon 6 alone is unable to facilitate MBNL splicing, its presence has been shown to be essential for full MBNL1 splicing regulation activity. Exon 5 undergoes alternative splicing autoregulation and its repression is regulated by all MBNL paralogs by recognition of several binding sites in intron 4 [START_REF] Gates | Autoregulated Splicing of muscleblindlike 1[END_REF]. This autoregulatory loop is vastly disturbed in DM, where the sequestration of MBNL proteins causes increased inclusion of exon 5, growing accumulation of the protein in the nucleus and subsequently, its titration by expanded CUG-RNAs leading to disease progression and DM phenotype worsening.

Exon 7-multimerization

Encoding a very short 12 amino acids sequence, exon 7 shows high similarity (60-82%) between all three MBNL paralogs. Even though not a lot is known about its exact function, its presence induces formation of multimeric ring-like conformations [START_REF] Yuan | Muscleblind-like 1 interacts with RNA hairpins in splicing target and pathogenic RNAs[END_REF].

Additional studies showed exon 7-dependent MBNL1 self-dimerization, which has been suggested to facilitate protein binding to target sequences [START_REF] Tabaglio | MBNL1 alternative splicing isoforms play opposing roles in cancer[END_REF][START_REF] Tran | Analysis of exonic regions involved in nuclear localization, splicing activity, and dimerization of {Muscleblind}like-1 isoforms[END_REF]. Indeed, the presence of the exon 7 can either enhance protein affinity for mRNAs, or be important for the interaction with other trans-acting factors as its presence modulates alternative splicing activity of MBNL1 [START_REF] Sznajder | Mechanistic determinants of MBNL activity[END_REF]. Interestingly, MBNL isoforms containing exon 7 are elevated in DM suggesting that its improper inclusion might contribute to the disease pathogenesis likely by focal localization of MBNL proteins through its selfassociation and promotion of foci formation [START_REF] Tran | Analysis of exonic regions involved in nuclear localization, splicing activity, and dimerization of {Muscleblind}like-1 isoforms[END_REF]. Recently it has been reported that exon 7 might also play a crucial role in cancer progression. MBNL1 has a well described role as tumor suppressor and specifically, isoforms lacking exon 7 act as an antisurvival factor by inhibiting cell viability and migration [START_REF] Tabaglio | MBNL1 alternative splicing isoforms play opposing roles in cancer[END_REF].

Exon 8

Exon 8, composed of 95 nucleotides, shows great conservation between all three MBNL paralogs reaching nearly 87% of identity. Similar to exons 5 and 7, its expression undergoes significant changes during tissues and development, but also shows significant missplicing in DM [START_REF] Sznajder | Mechanistic determinants of MBNL activity[END_REF]. Interestingly, exon 8 inclusion is not controlled by MBNL itself but by another protein involved in DM pathology, CELF 1 [START_REF] Kalsotra | A postnatal switch of CELF and MBNL proteins reprograms alternative splicing in the developing heart[END_REF]Wang et al., 2015a).

Albeit not much is known about the function of exon 8, it has been suggested that its presence might be relevant for proper MBNLs splicing regulation by exerting positive effect on alternative exons exclusion [START_REF] Sznajder | Mechanistic determinants of MBNL activity[END_REF].

Additional MBNL regulatory elements

Recent investigations of MBNL paralogs, especially the most studied MBNL1, revealed a high complexity of the proteins regarding, in particular, their expression, regulation and localization.

Besides aforementioned loop based on autoregulatory splicing of exon 5 that determine protein localization, another feedback regulation has been depicted for exon 1. This exon mutually constitutes the 5 ʹ -untranslated region (5 ʹ UTR) and the N-terminus of the protein that encompasses first pair of ZnFs. Alternative splicing of exon 1 is regulated by its own protein product, as well as other MBNL paralogs. This autoregulation acts as an on/off switch of MBNL1 expression as the lack of exon 1 shifts the translation start to exon 2 that results in assembly of mature mRNA missing two of four ZnF domains. The exon 1-devoid transcript has substantially affected translational activity and its protein product displays high instability (Konieczny et al., 2017). Importantly, the regulation of exon 1 further depends on the transcription initiation site that vary between different tissues. In DM, activation of exon 1 inclusion and switch on/off autoregulatory feedback loop is vastly perturbed due to the lack of functional MBNL proteins [START_REF] Konieczny | Autoregulation of MBNL1 function by exon 1 exclusion from MBNL1 transcript[END_REF]. Interestingly, such an autoregulation seems to be virtually not active in two other MBNL paralogs.

Besides its autoregulatory function, exon 1 of Mbnl1 has been proposed to play an important role in other negative feedback loop mechanism. So far described in Drosophila melanogaster [START_REF] Ashwal-Fluss | circRNA Biogenesis Competes with Pre-mRNA Splicing[END_REF][START_REF] Pamudurti | Translation of CircRNAs[END_REF] but also found in human, it points out that MBNL1 binds to MBNL1 intron 1 pre-mRNA causing exon 1 removal and spliceosome-dependent exon 1 circularization. As the sequence of circular RNA (circRNA) contains potential MBNL1 binding sites, it can act as a sponge that grasp the excess of the protein [START_REF] Ashwal-Fluss | circRNA Biogenesis Competes with Pre-mRNA Splicing[END_REF]. Recently it has been also found that a subset of circular RNAs is translated in vivo including circRNA generated from the muscleblind locus however, the role of this polypeptide remains unknown. Specific downregulation of circMbl in Drosophila melanogaster led to particular defects that suggest its roles in muscle and neural tissues during development and in adult flies [START_REF] Pamudurti | Translation of CircRNAs[END_REF]. Although suggested for humans, the analysis of control and DM muscle samples showed similar levels of circMbnl RNA, despite significant increase in MBNL1 transcript levels containing exon 1 following MBNL sequestration in DM patients (Konieczny et al., 2017). Nonetheless, several other circRNAs have been found to be deregulated in DM1-skeletal muscle tissue indicating the potential role of RNA circularization in the disease progression [START_REF] Voellenkle | Dysregulation of Circular RNAs in Myotonic Dystrophy Type 1[END_REF].

Even though MBNL cellular compartment expression is mostly determined by the presence of nuclear localization signal, precise switch between exon 5 inclusion and exclusion is not the only mechanism influencing MBNL localization. In developing brain, lysin63-linked MBNL1 ubiquitination controls its cytoplasmic localization independent of the Mbnl1 exon 5 splicing pattern. This cytoplasmic localization has been shown to be crucial for proper neurite morphogenesis, as only cytoplasmic and not nuclear MBNL1 isoforms promoted neurite outgrowth. DM1 mouse model expressing toxic CTG repeats in the brain showed reduced level of MBNL1 ubiquitination suggesting that the expanded toxic repeats induce MBNL1 deubiquitylation, thereby resulting in nuclear translocation and diminished MBNL1 cytoplasmic content contributing to morphological impairment and DM1 neural pathogenesis [START_REF] Wang | Ubiquitination of MBNL1 Is Required for Its Cytoplasmic Localization and Function in Promoting Neurite Outgrowth[END_REF].

MBNLs' functions

Alternative splicing regulation

MBNL proteins regulate alternative splicing profile of hundreds of transcripts during development by either promoting exon inclusion or exclusion. This splicing function generally depends on the binding position relative to the regulated exon (Figure 15) [START_REF] Charizanis | Muscleblind-like 2-Mediated Alternative Splicing in the Developing Brain and Dysregulation in Myotonic Dystrophy[END_REF][START_REF] Goers | MBNL1 binds GC motifs embedded in pyrimidines to regulate alternative splicing[END_REF][START_REF] Kalsotra | A postnatal switch of CELF and MBNL proteins reprograms alternative splicing in the developing heart[END_REF][START_REF] Wang | Transcriptome-wide regulation of pre-mRNA splicing and mRNA localization by muscleblind proteins[END_REF].

However, there is no evidence for hairpin formation in these MBNLs' binding regions [START_REF] Grammatikakis | Identification of MBNL1 and MBNL3 domains required for splicing activation and repression[END_REF]. Splicing activation has been shown for several transcripts including very low density lipoprotein receptor (Vldlr) exon 16, sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (Atp2a1) exon 22 [START_REF] Hino | Molecular mechanisms responsible for aberrant splicing of SERCA1 in myotonic dystrophy type 1[END_REF]Kimura et al., 2005) and Insr exon 11 [START_REF] Echeverria | Muscleblind-like 1 activates insulin receptor exon 11 inclusion by enhancing {U}2AF65 binding and splicing of the upstream intron[END_REF]. In the latter, MBNL binds to the sequence of splicing enhancer in the downstream intron of the regulated exon that in turn, activates the interaction of splicing factors with upstream intronic regions potentially through MBNLinduced changes in the pre-mRNA conformation or assembly of other proteins involved in splicing regulation [START_REF] Echeverria | Muscleblind-like 1 activates insulin receptor exon 11 inclusion by enhancing {U}2AF65 binding and splicing of the upstream intron[END_REF]. Interestingly, it seems that although MBNLs regulate alternative splicing events, the proficiency of exon exclusion is significantly higher than exon inclusion [START_REF] Sznajder | Mechanistic determinants of MBNL activity[END_REF]. Moreover, MBNL paralogs can regulate the same alternative splicing events but with different efficiency, which might be a consequence of unlike affinity to RNA targets, probably due to previously described dissimilarities in a linker sequence or structure sensitivity [START_REF] Sznajder | Mechanistic determinants of MBNL activity[END_REF]. These differences might be also influenced by their relative level of expression and the distribution of their splicing isoforms that are expressed in both tissue and developmental dependent manner (Batra et al., 2014;[START_REF] Jog | RNA splicing is responsive to MBNL1 dose[END_REF][START_REF] Wang | Transcriptome-wide regulation of pre-mRNA splicing and mRNA localization by muscleblind proteins[END_REF].

3'UTR -mediated protein function

Binding in 3'untranslated region has been identified as the second major target site for the MBNL protein family [START_REF] Wang | Transcriptome-wide regulation of pre-mRNA splicing and mRNA localization by muscleblind proteins[END_REF]. This preferential interaction indicates MBNLs' involvement in the regulation of important cellular 3'UTR-mediated processes.

Alternative polyadenylation

A large number of transcripts are governed by alternative 3' end formation [START_REF] Danckwardt | 3' end mRNA processing: molecular mechanisms and implications for health and disease[END_REF]. Given the crucial role of these regions for interactions with trans-acting factors including RNA-binding proteins and microRNAs that lead to modulations of RNA localization, translation and turnover [START_REF] Tian | Alternative cleavage and polyadenylation: the long and short of it[END_REF], it highlights the necessity of proper 3' UTR assembly during the transition from embryonic to adult life. MBNL proteins have been shown to be important regulators of developmental switch of polyadenylation sites (Batra et al., 2014) that in turn generates alternative 3'UTRs.

RNA-binding proteins and myogenic differentiation factors, like paired like homeodomain 2 (PITX2) [START_REF] Masuda | CUGBP1 and MBNL1 preferentially bind to 3' UTRs and facilitate mRNA decay[END_REF]. However, other studies did not confirm these observations [START_REF] Wang | Transcriptome-wide regulation of pre-mRNA splicing and mRNA localization by muscleblind proteins[END_REF].

miRNA processing

MBNL1 has been shown to be directly implicated in the maturation of one of the miRNA family, namely miR-1, by binding a single UGC motif in the unpaired region of a terminal loop in the precursor miRNA (Rau et al., 2011). This interaction prevents miR-1 uridylation that will impede its further processing leading to the decreased level of mature miRNAs (Rau et al., 2011). Interestingly, miR-1 is so far the only reported MBNL1 target with a single consensus sequence.

Regulation of cells pluripotency

Two members of MBNL family, MBNL1 and MBNL2, have been identified as a central negative regulators of cells pluripotency. Together, they negatively control a large subset of alternatively spliced events that are differentially regulated between embryonic stem cells (ESCs) and other cell types. Among their specific targets in ESCs, the forkhead family transcription factor FOXP1 that plays a key role in pluripotency control has been identified. MBNL1 and 2 functional depletion significantly enhances the expression of the primary pluripotency genes and the formation of induced pluripotent stem cells (iPSCs) during somatic cell reprogramming [START_REF] Han | MBNL proteins repress ES-cell-specific alternative splicing and reprogramming[END_REF][START_REF] Venables | MBNL1 and RBFOX2 cooperate to establish a splicing programme involved in pluripotent stem cell differentiation[END_REF].

Therapy

Up to date, only symptoms management to alleviate pain and reduce myotonia to improve quality of life is offered to DM patients without any curative treatment. However, constant progress in understanding disease pathology revealed novel mechanisms and new potential therapeutic strategies. Preclinical attempts have proved that DM1 underlying pathology is responsive to therapeutic intervention and can be targeted at several different levels (Figure 18).

In a therapeutic context, two main points of DM biology need to be emphasized: i) mutant RNAs containing expanded repeats are retained in the nucleus as foci, a process that is substantially exacerbated by MBNL interactions, resulting in functional loss of MBNL proteins; ii) foci are not rigid structures but rather dynamic assemblies that constitute a reversible state [START_REF] Sznajder | Mechanistic determinants of MBNL activity[END_REF]. It is important to note that the extend of MBNLs sequestration depends on the length of the expanded repeats. Moreover, in the presence of short expansions, the fraction of trapped MBNLs undergoes dynamic exchange with free MBNL and partial activity of MBNL is retained. In contrast, most of MBNLs are sequestered by larger expansion and/or excess of C(C)UG repeats leading to the depletion of free MBNLs from the nucleoplasm and subsequent loss of MBNL functions [START_REF] Sznajder | Mechanistic determinants of MBNL activity[END_REF]. Both points might have important therapeutic implications. Firstly, it seems that therapeutics acting preferentially in the nucleus may carry a greater potential comparing to those acting in the cytoplasm. Secondly, early prevention of C(C)TG expansion may restrain the progression of the disease. And thirdly, foci complexes are accessible to drugs and other small agents that may evoke their dispersion.

Importantly, even a modest release of MBNL may have potentially an impact by mitigating the severity of the symptoms.

So far, therapeutic development focuses mostly on DM type 1 because the lack of DM2 mouse models substantially limits assessment of DM2-specific therapies (Day and Ranum, 2005;[START_REF] Margolis | DM2 intronic expansions: evidence for CCUG accumulation without flanking sequence or effects on ZNF9 mRNA processing or protein expression[END_REF]. Up to date, only some studies reported small molecules specific to CCUG expansions or examined post-transcriptional silencing of CNBP mutant allele [START_REF] Childs-Disney | Structure of the myotonic dystrophy type 2 RNA and designed small molecules that reduce toxicity[END_REF]Lee et al., 2009a;[START_REF] Nguyen | Small molecules that target the toxic RNA in myotonic dystrophy type 2[END_REF][START_REF] Wong | Selective inhibition of {MBNL}1-{CCUG} interaction by small molecules toward potential therapeutic agents for myotonic dystrophy type 2 ({DM}2)[END_REF]. Therefore, described below therapeutic strategies will concentrate on myotonic dystrophy type 1. [START_REF] Kramer | Spt4 selectively regulates the expression of C9orf72 sense and antisense mutant transcripts[END_REF][START_REF] Liu | Spt4 is selectively required for transcription of extended trinucleotide repeats[END_REF]. However, it is not clear yet whether SUPTH4H is required for transcription of expanded CTG/CCTG tracts in DM. The second approach targeting prevention of CTG expansions by direct binding to the repeated tracts utilized deactivated CRISPR associated protein 9 (dCas9) enzyme that proved to effectively impedes transcription across repeat-containing RNAs upon direct binding to the expanded sequence (Pinto et al., 2017).

Besides, several small molecules have also showed transcription inhibitory activity of long repeated tracts. For example, Actinomycin D (ActD), a DNA intercalator that preferentially binds GpC dinucleotides widely known as transcription inhibitor, can reduce the expression of CUGexp-RNA at doses below those generally needed for global impede of mRNA synthesis (Siboni et al., 2015). Likewise, pentamidine, an anti-parasitic drug traditionally used in pneumocystis pneumonia, and other related antibiotics have shown beneficial effects in DM1 models using a mechanism similar to ActD (Coonrod et al., 2013). DM1 cells treated with pentamidine showed a decrease expression of expanded CUG transcripts that is associated with the correction of splicing abnormalities in a dose dependent manner (Coonrod et al., 2013). Similarly, pentamidine delivery in vivo in HSA LR mice resulted in partial reversal of aberrant splicing but this effect did not last after the end of the treatment. Additionally, at the dose required to mediate meaningful phenotypic changes, pentamidine showed relative toxicity that further prevent the use of the drug in DM (Warf et al., 2009b). Subsequent development of pentamidine analogs showed that less toxic heptamidine corrects splicing defects and myotonia in HSA LR mice in a more potent manner (Coonrod et al., 2013). Taken together, the usage of pharmaceutical molecules already applied in human therapy can represent particularly attractive and rapid strategy for treatment of rare diseases.

Post-transcriptional silencing of expanded transcripts

So far, targeting the mutant RNA itself holds some of the greatest prospects for therapeutic intervention in DM and various nucleic acid-based technologies have been developed to achieve post-transcriptional knockdown of toxic repetitions.

Gapmer antisense oligonucleotides

One of the RNA-based strategies that has found the widest application is the use of antisense oligonucleotides (ASOs). These short single-stranded stretches of nucleic acids hybridize to complementary sequence in mRNA via Watson-Crick base-pairing and inhibit gene expression or modulate alternative splicing or degrade targeted RNAs. This latest strategy using ribonuclease H (RNase-H)-active ASOs have been already assessed in a DM1 clinical trial. These ASOs called 'gapmers' commonly consist of 8-10 DNA nucleotides flanked by chemically modified base pairs. The presence of non-modified nucleotides represents a "gap" in the formed ASO/RNA heteroduplex, which allows the RNase-H to recognize the DNA/RNA hybrid and cleave the target RNA before exonuclease degradation of the RNA fragments. The targeting sequence could be located upstream or downstream of the repeat tract, taking advantage of the sequence specificity of the antisense technology. This strategy has been successfully used by intramuscular injection in DM1 HSA LR transgenic mice leading to efficient reduction of CUGexp-RNA expression and nearly complete rescue of molecular and functional DM1-associated phenotype including alternative splicing, myotonia and general muscle architecture (Wheeler et al., 2012). Importantly, this beneficial effect sustained for a long period of time, up to one year after the end of the treatment. Aside local activity, systemic application of gapmers also reduced the level of CUGexp-DMPK-mRNAs in skeletal muscles as well as in heart of DMSXL mice, improving body weight mass and muscles strength (Jauvin et al., 2017). However, in the DM1 patient context, this approach will target both mutated and non-mutated DMPK transcripts.

RNA endonuclease

Even though gapmer-ASOs show high potency, conceptually similar engineered artificial sitespecific RNA endonucleases (ASREs) can be used to cleave CUG-containing transcripts. These ASREs possesses two domains, one specific to RNA tracts and the second comprising endonuclease activity to cut the target sequence [START_REF] Zhang | Treatment of type 1 myotonic dystrophy by engineering site-specific RNA endonucleases that target (CUG)(n) repeats[END_REF]. ASREs have been tested in DM1-derived myoblasts where they reduced the number of nuclear foci and restored normal alternative splicing pattern [START_REF] Zhang | Treatment of type 1 myotonic dystrophy by engineering site-specific RNA endonucleases that target (CUG)(n) repeats[END_REF].

RNAi

RNA interference has also been tested to tackle CUGexp-DMPK transcripts since the particular hairpin structure formed by the expanded repeats have been shown to be substrates for Dicer, a ribonuclease crucial in RNA interference pathway that recognize double-stranded RNA sequences and induce their cleavage into shorter, 21-nt duplexes [START_REF] Krol | Ribonuclease dicer cleaves triplet repeat hairpins into shorter repeats that silence specific targets[END_REF]. These duplexes are then incorporated into the RNA-induced silencing complex and, as such, function to guide substrate selection for degradation. Known to be active in the cytoplasm, RNAi has proved to occur also in the nucleus as observed in DM1 patient fibroblasts treated with synthetic oligoribonucleotide (CUG) 7 that resulted in a selective reduction of transcripts containing expanded CUG repeats [START_REF] Krol | Ribonuclease dicer cleaves triplet repeat hairpins into shorter repeats that silence specific targets[END_REF]. This effect supported the hypothesis in which the use of siRNAs targeting mutant transcript would tag this RNA for degradation via Dicer pathway. Therefore, expression of a lentivirus-delivered short hairpin RNA (shRNA) complementary to DMPK mRNA, which is processed in the cell to 21-nt siRNAs, in DM1 myoblasts led to the degradation of mutant nuclear mRNA but also cytoplasmic wild-type transcripts (Langlois et al., 2005). The in vivo use of CAG/CUG siRNA duplex designed directly against the expanded repeats, resulted in a decrease of CUGexp-RNA following electroporation into skeletal muscle of HSA LR mice. Several splicing defects were rescued, including CLCN1 missplicing that was associated with myotonia correction [START_REF] Sobczak | RNA structure of trinucleotide repeats associated with human neurological diseases[END_REF]. Similar therapeutic effect of RNAi machinery has been observed after systemic delivery of AAV vector containing shRNA sequence designed against the sequence of 3'UTR of HSA mRNA and not CUG repeats themselves [START_REF] Bisset | Therapeutic impact of systemic AAV-mediated RNA interference in a mouse model of myotonic dystrophy[END_REF]. Additionally, modulating the activity of the RNAi machinery by knocking down of their main components in C. elegans DM1 model, led to significant improvement of the disease phenotypes [START_REF] Qawasmi | Expanded CUG Repeats Trigger Disease Phenotype and Expression Changes through the RNAi Machinery in C. elegans[END_REF].

Ribozymes

Ribozymes are RNA molecules containing enzymatic functions that are able to either directly cleave the target RNA or replace it with the ribozyme-embedded sequences [START_REF] Sioud | Ribozyme-and siRNA-mediated mRNA degradation: a general introduction[END_REF].

Nuclear-retained hammerhead ribozyme designed to target and induce endonucleolytic cleavage of the mutant DMPK have been tested in DM1 myoblasts. Unfortunately, similar to previously described gapmer antisense oligonucleotides, the enzyme affected also the wild type allele, causing 50% reduction in its expression (Langlois et al., 2003). Nevertheless, the treatment triggered a reduction in the foci number, a decrease of CUGexp-RNA expression and a partial restoration of insulin receptor pre-mRNA missplicing. Other type of ribozyme, a group 1 intron ribozyme, was used to target 12 CUG repeats in the 3' UTR of human DMPK transcripts and replace them with a shorter, containing 5 CUG repeats, sequence. The approach was successful in both in vitro assay and in cultured human fibroblasts [START_REF] Phylactou | Ribozyme-mediated trans-splicing of a trinucleotide repeat[END_REF].

Blocking RNA-proteins interactions

Considering very high affinity of MBNL proteins to expanded C(C)UG repeats, a reasonable therapeutic approach would be to disrupt deleterious RNA-MBNL binding in order to release sequestered MBNL proteins and restore their native functions. For this purpose, various tools have been developed.

ASOs without RNase-H activity

In contrast to gapmer-ASOs used for RNA degradation, these ASOs comprise of fully modified CAG repeats of about 15-30 base pairs long designed to directly target repeated tracks and specifically mask the nucleotide sequence. Two CAG25-ASOs of different chemistry showed high potency to bind to CUG expansions and displace sequestered MBNL from nuclear RNA foci leading to MBNL relocalization and restoration of its functional activity (González-Barriga et al., 2013;Mulders et al., 2009;Wheeler et al., 2009). Although not intended to evoke RNase-H cleavage, ASOs caused substantial reduction of toxic CUGexp-RNA levels in DM1 cells (González-Barriga et al., 2013;Mulders et al., 2009) and transgenic mouse models (Mulders et al., 2009a;Wheeler et al., 2009). Similar results have been obtained with the U7-small nuclear RNA carrying an antisense (CAG)15 sequence. Lentiviral transduction of DM1 cells, to ensure efficient and long-lasting effect, resulted in an 80% reduction of mutated DMPK transcript expression followed by improvement of myoblast fusion and adult splicing pattern restoration (Francois et al., 2011). It suggests that once released from the abnormal MBNL binding, the mutant transcripts undergo degradation by another unknown pathway.

Small molecules

A parallel approach to ASOs strategy encompasses high-throughput screens to identify small molecules that either bind to the C(C)UG repeats to inhibit MBNL1:C(C)UG interaction, disperse or perturb the formation of RNA foci, or improve a specific deleterious phenotype in DM1 models [START_REF] Arambula | A simple ligand that selectively targets {CUG} trinucleotide repeats and inhibits {MBNL} protein binding[END_REF][START_REF] García-López | In vivo discovery of a peptide that prevents CUG-RNA hairpin formation and reverses RNA toxicity in myotonic dystrophy models[END_REF][START_REF] Gareiss | Dynamic combinatorial selection of molecules capable of inhibiting the (CUG) repeat RNA-MBNL1 interaction in vitro: discovery of lead compounds targeting myotonic dystrophy (DM1)[END_REF]Ketley et al., 2014;[START_REF] Luu | A Potent Inhibitor of Protein Sequestration by Expanded Triplet (CUG) Repeats that Shows Phenotypic Improvements in a Drosophila Model of Myotonic Dystrophy[END_REF]Nakamori et al., 2016;[START_REF] Ofori | From dynamic combinatorial "hit" to lead: in vitro and in vivo activity of compounds targeting the pathogenic {RNAs} that cause myotonic dystrophy[END_REF][START_REF] Rzuczek | Studying a Drug-like, RNA-Focused Small Molecule Library Identifies Compounds That Inhibit RNA Toxicity in Myotonic Dystrophy[END_REF]Warf et al., 2009b). One of such an example is an ABP1 peptide that reduces CUG-induced toxicity by binding to expanded tracts and inducing a conformational shift in CUG secondary structure. ABP1 showed to be effective in fly and mouse model of DM1 [START_REF] García-López | In vivo discovery of a peptide that prevents CUG-RNA hairpin formation and reverses RNA toxicity in myotonic dystrophy models[END_REF].

Besides, some natural compounds have been found to pose an attractive alternative in DM treatment. These alkaloids intercalate between the bases in double-stranded RNAs and disturb the binding of other proteins to these structures. Even if substantially potent in correcting DM1 phenotypes in vitro and in vivo, the studies highlighted specificity issues and potential toxicity [START_REF] Herrendorff | Identification of Plant-derived Alkaloids with Therapeutic Potential for Myotonic Dystrophy Type I[END_REF].

Additionally, considering the biophysical properties of expanded C(C)UG tracts, compounds with higher affinity and specificity could be developed. For example, several CUG-binding monomers have been identified and designed to form multimers using precisely arranged linker of defined length so that the inter-subunit distance is tuned to the periodicity of the CUG-CCUG-repeats. The efficacy of these molecules to displace MBNLs from CUG expansions has been shown in DM1-cell models (Childs-Disney et al., 2012;[START_REF] Jahromi | Developing bivalent ligands to target {CUG} triplet repeats, the causative agent of myotonic dystrophy type 1[END_REF]Lee et al., 2009b). Also, small ligands may tend to drive strong interaction with reiterated binding motif in expanded RNAs offering an interesting alternative for therapy approach [START_REF] Childs-Disney | A small molecule microarray platform to select RNA internal loop-ligand interactions[END_REF]Lee et al., 2009b;[START_REF] Maury | Pluripotent Stem Cell-Based Drug Screening Reveals Cardiac Glycosides as Modulators of Myotonic Dystrophy Type 1[END_REF].

Targeting proteins altered in the disease

MBNLs overexpression

Another therapeutic strategy is to restore functional levels of MBNL1 either by its direct overexpression or modulating MBNL abundance via gene expression modifiers. Intramuscular injection of AAV-MBNL1 vectors in HSA LR mice resulted in a 2-fold increase of MBNL1 protein level that led to the rescue of some of the DM features including correction of myotonia and restoration of splicing deficits but without any sign of muscle histopathology improvement [START_REF] Chamberlain | Mouse model of muscleblind-like 1 overexpression: skeletal muscle effects and therapeutic promise[END_REF][START_REF] Kanadia | Reversal of RNA missplicing and myotonia after muscleblind overexpression in a mouse poly(CUG) model for myotonic dystrophy[END_REF]. In contrast, overexpression of MBNL1 in an inducible mouse model of RNA toxicity was not sufficient to reverse DM1 phenotypes such as myotonia and cardiac conduction abnormalities and show only minor rescue of aberrant alternative splicing pattern. Moreover, exogenous MBNL1 expression even worsen muscle pathology [START_REF] Yadava | MBNL1 overexpression is not sufficient to rescue the phenotypes in a mouse model of RNA toxicity[END_REF].

Recently, another approach aims to increase endogenous MBNL level through targeting miRNAs that regulate MBNL gene expression. Silencing of miR-23b and miR-218 with antagomiRs significantly increased the level of MBNL proteins in DM1 patients-derived myoblasts and HSA LR mice. Subsequently, a partial correction of alternative splicing, myotonia and muscle histology was found in treated HSA LR mice [START_REF] Cerro-Herreros | Derepressing muscleblind expression by miRNA sponges ameliorates myotonic dystrophy-like phenotypes in Drosophila[END_REF]. Therefore, identification of miRNA repressors of all MBNL paralogs might hold a great promise for future therapeutic targets to restore adequate protein levels.

CELF downregulation

Abnormal upregulation of CELF protein in DM1 physiopathology raised the question of modulating protein expression as a potential therapeutic strategy. Thus, approaches aimed to target signaling pathway activated downstream of C(C)UGexp expression such as PKC and GSK3b, which are considered to be responsible for CELF upregulation through its phosphorylation and stabilization. Inhibition of PKC by small molecules in EpA960 mice leads to normalization of CELF protein levels followed by the improvement of cardiac phenotype and rescue of muscle wasting [START_REF] Wang | PKC inhibition ameliorates the cardiac phenotype in a mouse model of myotonic dystrophy type 1[END_REF]. Similarly, inhibition of GSK3b cascade balances CELF protein level and recovers muscle wasting in a transgenic mouse model (Jones et al., 2012b). Interestingly, GSK3b inhibitors are already in clinical trials for diabetes, cancer and Alzheimer's disease (Zhang Y 2018, James A. McCubrey 2014). Other approached using miRNA, likewise for MBNL, showed that overexpression of miR-206 helped rescue myogenic deficiency by inhibiting CELF1 expression in DM cell models [START_REF] Dong | Mir-206 partially rescues myogenesis deficiency by inhibiting CUGBP1 accumulation in the cell models of myotonic dystrophy[END_REF].

Targeting downstream effects

Curing the effect and not the cause of the disease is an alternative therapy for DM. Considering myotonic dystrophy as a severe spliceopathy with some misspliced transcripts directly responsible for some signs of the disease, targeting crucial DM-associated splicing deficiencies might have a potential to improve pivotal, but individual features of the disease. As a proof of concept, antisense oligonucleotides targeting alternatively spliced exon 7a of Clcn1 that contains a stop codon and is included in the presence of CUGexp-RNA, has been used in HSA LR mice [START_REF] Wheeler | Correction of {ClC}-1 splicing eliminates chloride channelopathy and myotonia in mouse models of myotonic dystrophy[END_REF]. ASOs efficiently blocked the access of the splicing machinery to 3' splice site of exon 7a pre-mRNA, resulting in effective and persistent exon exclusion from the mature transcript. As a consequence, functional CLCN1 protein was produces leading to alleviation of myotonia, one of the main hallmarks of the disease [START_REF] Wheeler | Correction of {ClC}-1 splicing eliminates chloride channelopathy and myotonia in mouse models of myotonic dystrophy[END_REF].

Genome editing

Emerging genome engineering technologies including targeted gene editing and gene regulation have become widely explored for several genetic neuromuscular disorders including myotonic dystrophy. Seemingly the most straightforward strategy in DM, direct removal of expanded repeats in the genomic sequence, has been attempted by CRISPR-Cas technology. Cas9 nuclease with a pair of guides RNA targeting a specific locus upstream and downstream the CTG repeats in DM1 cells, resulted in the deletion of the expansions, diminution of RNA foci and correction of missplicing defects (van Agtmaal et al., 2017;[START_REF] Dastidar | Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophy patient-derived iPS and myogenic cells[END_REF]Provenzano et al., 2017). Expanded repeats have been equally targeted at the RNA level by a modified CRISPR nuclease-dead Cas9 conjugated to PIN endonuclease domain to degrade CUGexp-RNAs. Expression of this complex in DM1 cells lowered the amount of toxic RNA and rescued the alternative splicing pattern (Batra et al., 2017). Another approach to eliminate expanded repeats utilized Transcription Activator Like Effector Nucleases (TALEN). Precise induction of a DSB inside the repeat tract induced an efficient contraction of the repeats to a non-pathogenic length in 99% of cases in yeast cells [START_REF] Mosbach | TALEN-Induced Double-Strand Break Repair of CTG Trinucleotide Repeats[END_REF][START_REF] Richard | Highly specific contractions of a single CAG/CTG trinucleotide repeat by TALEN in yeast[END_REF]. TALEN-and CRISPR-Cas9-based approach has been also successfully used in genome modification by placement of exogenous polyadenylation signals upstream of the DMPK CTG sequence. The insertion of PASs leads to premature arrest of transcription machinery and therefore, prevention of production of toxic mutant mRNA. The lack of expanded tracks expression reversed the phenotypes in the wide array of cells including DM1 iPS cells and their progeny, differentiated neural stem cells, forebrain neurons, cardiomyocytes, and skeletal muscle myofibers (Gao et al., 2016;Xia et al., 2015).

DM therapy challenges

Given that DM is a highly multisystemic disease, the delivery and tissue distribution represent one of the main challenges for efficient therapy development. Penetration of ASOs and siRNA into skeletal muscles, heart and CNS is minimal and need further development. Improvement in tissue delivery has been already made by incorporation of wide range of modifications into the chemical structures of antisense oligonucleotides to increase their stability and longevity or by combining them with lipids or peptides that ensure enhanced cellular uptake [START_REF] Grijalvo | Oligonucleotide delivery: a patent review (2010 -2013)[END_REF][START_REF] Khvorova | Functional siRNAs and miRNAs exhibit strand bias[END_REF]. siRNAs are more challenging as they are quickly degraded by cellular RNsases and tolerate only a limited number of modifications at the risk of losing their effectiveness [START_REF] Garber | Worth the RISC?[END_REF]. Using siRNA also raises the question of specificity and potential side effects/toxicity similarly to other tested drugs and small molecules like Actinomycin D, pentamidine and engineered or natural compounds. The need of chemical modifications might evoke potential immunogenicity or toxicity, which might be also a question for the genome editing components or delivery particles. Presently, increasing number of therapies utilize viral-vector mediated delivery system with some genetic treatments using AAVs (adeno-associated vector) in clinical trials [START_REF] Duan | Duchenne muscular dystrophy gene therapy: {Lost} in translation?[END_REF][START_REF] Kotterman | Engineering adeno-associated viruses for clinical gene therapy[END_REF][START_REF] Mendell | Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy[END_REF]. AAV-based approach proved to be relatively safe and effective, therefore constituting an emerging alternative for whole-body gene therapies.

Additionally, viral-mediated delivery ensures long-lasting effect in contrast to ASOs, siRNA and other small molecules that need repetitive administration. In the light of sustained effect, the development of autologous stem cell therapy by genome editing of induced pluripotent stem cells hold an attractive promise that however takes considerable limitations for a multisystemic diseases (Gao et al., 2016).

So far, a substantial amount of therapies for DM1 focuses on the reduction of the level of toxic RNA. However, it remains unclear how it may affect the function of skeletal muscles and heart, two tissues with the highest DMPK expression. The therapeutic outcome suggests that collateral knockdown of wild-type and mutant DMPK may be well tolerated, as shown in mice and monkeys but only for a short period of time (Pandey et al., 2015). It allows one of the ASOs (DMPK-Rx) to enter first clinical trials for DM1 (ClinicalTrials.gov identifier: NCT02312011). Designed to promote RNase-H-mediated degradation of mutant DMPK transcript, DMPK-Rx did not reach a sufficient concentration in muscle tissues due to the inadequate biodistribution and thus, the trial was not pursuing in Phase III. As therapeutic advances to human trials, it will be necessary to consider the possibility that addressing only one mechanism may be not sufficient and a combination of several strategies simultaneously targeted several disease steps will be required for additive or synergistic effects.

Aims

Substantial progress in understanding the genetics and mechanism underlying the pathology of myotonic dystrophy gave rise to development of several distinct therapeutic approaches.

However, besides numerous routes to reverse the disease-associated toxicity, there is no curative treatment available so far. Due to that unmet need for DM treatment, the main aim of my thesis was to develop and assess a novel therapeutic strategy for myotonic dystrophy.

Specifically, the objectives were:

Design and validation of a gene therapy approach based on engineered RNA-binding protein

We aimed to engineer and determine both mechanism of action and therapeutic potential of an RNA-binding protein derivative using DM1 muscle cells in vitro, and further assess its ability to rescue DM-associated phenotypes via viral vector delivery in a disease mouse model.

Optimization of the therapeutic RNA-binding protein

With the view to improve the therapeutic capacity of our engineered protein tool, we intended to apply numerous modifications to its nucleotide sequence while maintaining its favorable properties. For that reason, several different protein derivatives have been developed and their potential were thoroughly revised in both, in vitro and in vivo systems in order to select the most potent candidate.

Development of an auto-regulatory system that controls transgene product expression

Precise control of the transgene product abundance is of high importance in all gene therapy approaches. For that reason, we designed an innovative strategy that assures spatiotemporal changes in the transgene product expression depending on the current demand of the cell.

Bringing a fundamental knowledge about MBNL1 contribution in DM pathogenesis

A central role of the MBNL1 protein's functional depletion in myotonic dystrophy is well established. However, whether and how the protein contributes to the disease progression was not studied in details yet. Therefore, we aimed to decipher the potential implication of MBNL1 in pathologic process with a special focus on its alternatively spliced exons. This knowledge might be useful for a better understanding of the molecular basis of myotonic dystrophy and gain new insight into development of therapeutic strategies for DM.

Results

Part 1

Design and validation of a gene therapy approach based on engineered RNA-binding protein Introduction RNA-binding proteins (RBPs) play a key role in the regulation of RNA processing, and the disruption of their activities is responsible for a growing number of diseases (1). Among them, Muscleblind-like 1 (MBNL1) regulates developmental alternative splicing events during the transition from fetal to adult isoforms, and its functional loss represents a central pathophysiologic mechanism of myotonic dystrophy type 1 (DM1) disease (2) (3). DM1, one of the most common form of muscular dystrophy in adults, is caused by expanded CTGn repeats (n>50) located in the 3'UTR of the DMPK gene (4) (5). Mutant DMPK transcripts containing expanded CUG repeats (CUGexp) are retained in the nucleus of DM1 cells and sequester MBNL1 in discrete ribonucleoprotein foci (6) (7) (8). Due to their high affinity for the YGCY RNA motif, MBNL1 proteins are titrated by long pathogenic tracts of CUG repeats resulting in their functional loss and ultimately, in muscle impairment. Thus, a number of alternative splicing events controlled by MBNL1 are misregulated in striated muscles of DM1 patients (9), and splicing alterations of CLCN1, INSR, BIN1, DMD and SCN5A pre-mRNAs are associated with myotonia, insulin resistance, muscle weakness, dystrophic process and cardiac conduction defects, respectively, all symptoms of DM1 (10) (11) (12) (13) (14).

Therapeutic strategies for DM1 aim to target CUGexp-transcripts either by inducing their degradation or by blocking the deleterious MBNL1:CUGexp interaction (15)( 16) (17) (18) (19) (20) (21). Several approaches that have shown promising beneficial effects in disease models including antisense oligonucleotides, small compounds or more recently RNAtargeting Cas9, share a common feature: the release of sequestered MBNL1 from CUGexp-RNA foci that is associated with the recovery of MBNL1 activity and ultimately, the correction of DM1-associated phenotypes including splicing defects and myotonia. Furthermore, overexpression of MBNL1 in skeletal muscles of DM1 mice using recombinant adeno-associated viral (rAAV) vectors reverses DM1-associated phenotypes, confirming that CUGexp-RNA toxicity is largely mediated by a MBNL1 loss-of-function mechanism in muscles (22). Consistently, myotonia and several missplicing events detected in affected skeletal muscles of DM1 patients were also found in Mbnl1 deficient mice (3). Although MBNL1 is the major member of the MBNL family expressed in striated muscles, Mbnl1 deficient mice do not recapitulate the entire spectrum of DM1 symptoms due to a compensatory increase of MBNL2 protein levels and associated splicing activity. Indeed, Mbnl1-/-:Mbnl2+/-mice display major splicing changes characteristic for advanced DM1 disease and cardinal features including myotonia, skeletal muscle weakness, heart defects and reduced lifespan (23).

Restoration of functional MBNL1 in DM1 muscle cells represents a valuable strategy for therapeutic intervention. However MBNL1 is part of a RBP family composed of 3 paralogs (MBNL1, MBNL2 and MBNL3) with more than ten isoforms per paralog, and all MBNL isoforms are sequestered by CUGexp-RNA foci resulting in altered function of specific MBNL proteins according to DM1 tissues (24) (25). For instance, disruption of MBNL2, which is mainly expressed in the brain leads to specific missplicing events in mice that are similarly misregulated in DM1 patients'brain supporting a prominent role of MBNL2 loss of function in pathological changes observed in DM1 central nervous system (26). Given that the expression of MBNL paralog isoforms is highly regulated in a cell/tissue-specific manner, the overexpression of a single MBNL isoform appears as a challenging approach for restoring specific functions of all MBNL paralog isoforms (27). Thus, we propose a decoy-based gene therapy in which engineered CUGexp-binding protein decoys interact with CUGexp to release functional endogenous MBNL proteins in DM1 cells. To evaluate this strategy, we engineered a modified RBP (called MBNL∆) that competes with endogenous MBNL1 proteins for CUGexp binding. We show that MBNL∆ expression rescues most of the splicing misregulations in DM1 muscle cells and demonstrate that the action of MBNL∆ is triggered by the release and the recovery of endogenous MBNL1 functionality in DM1 muscle cells. Moreover, rAAV-mediated delivery of MBNL∆ in skeletal muscles of DM1 mice leads to an efficient and long-term correction of DM1-associated phenotypes including splicing defects, myopathy and myotonia. These results support the concept of a decoy gene therapy using engineered RNA-binding proteins with high CUGexp-binding affinities as a therapeutic avenue for DM1.

Results

MBNL∆ CUGexp-binding protein reverses splicing defects in DM1 muscle cells

Taking advantage of the high binding affinity of MBNL1 for CUGexp that is provided by two tandem zing finger (ZnF) domains located in the N-terminal part of the protein, we engineered a modified MBNL1 protein (referred here as MBNL∆) lacking its C-terminal domain (Fig. 1A). We showed previously that the MBNL∆ protein maintained a binding affinity for YGCY RNA motif similar to MBNL1 since the C-terminal domain of MBNL1 is rather implicated in splicing activity, cellular localization and oligomerization (28). Indeed, co-transfection experiments with plasmids containing MBNL∆ or MBNL1 and splicing reporter minigenes derived from DM1 misspliced events in HeLa cells confirmed that MBNL∆ had a reduced splicing activity when compared to MBNL1 (Fig. S1A). To assess whether MBNL∆ was able to target CUGexp-RNA foci, we performed co-transfection experiments in Hela cells expressing a 960 CTG construct. We found that MBNL∆ did colocalize with nuclear CUGexp-RNA foci like MBNL1 itself (Fig. 1B). Moreover, UV-cross-linking experiments demonstrated that MBNL∆ binds to CUGexp and is able to displace MBNL1 from CUGexp-RNA in a dose dependent manner (Fig. S1B). Based on these results, we hypothesized that MBNL∆ could act as a CUGexp-decoy to promote the release of endogenous MBNL1 from CUGexp allowing the recovery of a functional pool of endogenous MBNL1 in DM1 cells.

To determine whether the expression of MBNL∆ could restore endogenous MBNL1 activity in DM1 muscle cells, the modulation of misspliced events that are mainly regulated by MBNL1 in DM1 striated muscles was used as a biomarker of functional MBNL1 recovery (2)( 9) (29). For this purpose, human immortalized DM1 muscle cells (30) displaying DM1-associated phenotypes including nuclear CUGexp-RNA foci and alternative splicing misregulations were transduced with lentiviral vectors expressing a conditional Tet-on GFP-tagged MBNL∆. Integration of this transgene that contains a hygromycin selection cassette allowed the generation of a DM1 muscle cell line in which MBNL∆ expression is inducible by doxycycline. We showed that the induction of MBNL∆ in differentiated DM1 muscle cells normalized splicing defects of MBNL1 exon 5, DMD exon 78, NFIX exon 7, MBNL2 exon 5 and ATP2A1 exon 22 pre-mRNAs suggesting a recovery of functional MBNL1 in MBNL∆-treated DM1 cells (Fig. 1C and Fig. S1C). Moreover, transcriptomic analyses following global RNA sequencing confirmed that the vast majority (91%) of misregulated splicing events measured in differentiated DM1 muscles cells were normalized or partially corrected by MBNL∆, with a median level of correction of 83% for excluded events and 75% for included events (Fig. 1D and Table S1). At the gene expression level, 87% of the transcripts that are deregulated in differentiated DM1 muscle cells were also corrected by MBNL∆, with a mean level of restoration of 61% (Fig. 1E and Table S2). These results confirm that the overall transcriptome of differentiated DM1 muscle cells is significantly improved by the expression of MBNL∆.

MBNL∆ acts as a CUGexp-decoy to restore functional endogenous MBNL in DM1 cells

To examine whether MBNL∆ could compete with endogenous MBNL1 for the binding to CUGexp in DM1 muscle cells and release sequestered MBNL1 from RNA foci, we performed combined FISH-immunofluorescence to analyze the distribution of GFP-tagged MBNL∆ and endogenous MBNL1 within the nuclear CUGexp-RNA foci (Fig. 2A andS2A). In untreated DM1 muscle cells, MBNL1 colocalized with almost all nuclear foci as previously reported. Following MBNL∆ induction, all nuclear CUGexp-RNA foci colocalized with MBNL∆ whereas the mean intensity of endogenous MBNL1 per CUGexp-RNA foci was significantly reduced by 66% in MBNL∆-expressing DM1 cells compared to untreated DM1 cells, suggesting that MBNL∆ displaces MBNL1 from CUGexp-RNA foci (Fig. 2B). In addition, the MBNL∆:CUGexp interaction also modified the behavior of the nuclear CUGexp-RNA foci with a significant 2.5-fold reduction in the mean number of foci per nucleus in MBNL∆-expressing DM1 cells compared to untreated DM1 cells (Fig. 2C). The reduction in the mean number of CUGexp-RNA foci per nucleus, which was largely associated with a decrease in the number of small or medium foci and an increase of large RNA foci took place rapidly, within 4 hours following MBNL∆ induction (Fig. S2B). Interestingly, this change in the RNA foci fate occurred prior to the correction of splicing defects, which mostly became significant 6 hours following MBNL∆ induction (Fig. S2C). Altogether, these results support a CUGexp-decoy mechanism through which MBNL∆ favors the release of free and functional endogenous MBNL1 from CUGexp-RNA foci.

To demonstrate that the action of MBNL∆ requires free endogenous MBNL1 proteins for the recovery of MBNL activity, both MBNL1 and MBNL2 mRNAs were silenced in differentiated WT or DM1 muscle cells expressing conditional MBNL∆. Transfection of specific siRNA directed against MBNL1 and MBNL2 mRNAs but not MBNL∆ transcripts showed a significant 50% and 70% reduction in the level of MBNL1 and MBNL2 transcripts, respectively (Fig. S3A). As expected, dual MBNL1/MBNL2 mRNA silencing had no effect on misspliced events in DM1 cells whereas in WT cells, it led to splicing changes of MBNL1 exon 5, NFIX exon 7 and DMD exon 78 pre-mRNAs similar to those found in DM1 cells (Fig. 2D andS3B). In striking contrast, corrections of splicing misregulations of MBNL1, NFIX and DMD pre-mRNAs in MBNL∆expressing DM1 muscle cells were abolished by the dual MBNL1/MBNL2 silencing demonstrating that the mechanism of action of MBNL∆ requires endogenous functional MBNL proteins (Fig. 2D andS3B). Interestingly, splicing changes in MBNL1/MBNL2-siRNA WT cells were not corrected by the expression of MBNL∆ indicating that the residual splicing activity of MBNL∆ did not compensate for MBNL1/MBNL2 deficiency. As well, the expression of MBNL∆ in WT muscle cells did not change the alternative splicing profiles of pre-mRNAs, suggesting that MBNL∆ had no effect in WT cells. Overall, these data support the finding that MBNL∆ acts through the release of endogenous MBNL1 from CUGexp to restore endogenous functional MBNL1 in DM1 cells.

Efficient and long-term correction of DM1-associated phenotypes in rAAV-MBNL∆-treated DM1 mice

To test the efficacy of the modified MBNL∆ CUGexp-binding protein in vivo, we used the wellestablished HSA LR mouse model of DM1 (31). HSA LR mice express 250 CTG repeats in the 3'UTR of a human skeletal actin (ACTA1) transgene and recapitulate characteristic molecular and phenotypic features of DM1, including splicing abnormalities and myotonia in skeletal muscles. GFP-tagged MBNL∆ was packaged into a rAAV9 vector since this serotype ensures an efficient transduction and transgene-delivery into skeletal muscles (32). Next, Gastrocnemius muscles of HSA LR mice were injected with rAAV-MBNL∆ vectors (1x10 11 vector genomes) whereas contralateral muscles received saline vehicle. Seven weeks following injection, myotonia that is revealed by delayed muscle force relaxation in HSA LR mice compared to WT mice was measured in situ (Fig. 3A). As shown in Fig. 3B, myotonia was completely abolished in rAAV-MBNL∆-injected HSA LR muscles. Consistent with this result, the splicing alteration of Clcn1 exon 7a that is responsible for myotonia (33) was normalized by MBNL∆ (Fig. 3C). Moreover, additional characteristic DM1 splicing defects that are MBNL1-dependent such as Atp2a1 exon 22 and Mbnl1 exon 5 pre-mRNAs were also significantly corrected by MBNL∆ in HSA LR muscles (Fig. 3C). Furthermore, combined FISH-immunofluorescence staining performed on muscle sections showed that MBNL∆ colocalized with almost all CUGexp-RNA foci in myonuclei of rAAV-MBNL∆-injected HSA LR muscles (Fig. 3D and Fig. S4), supporting a model in which MBNL∆ acts through a CUGexp-decoy mechanism.

To assess the amount of MBNL∆ expressed in skeletal muscles of HSA LR mice and needed to reverse DM1-associated phenotypes, MBNL∆ and MBNL1 levels were determined in rAAV-MBNL∆-injected muscles and saline-injected contralateral muscles. Slot-blot analysis using a monoclonal antibody recognizing both MBNL∆ and MBNL1 proteins showed that the amount of MBNL∆ following rAAV-MBNL∆ transduction was four-time higher than the amount of endogenous MBNL1 (Fig. 3E). This result correlated with the corresponding mRNA level measured by RT-qPCR analysis that showed a six-fold increase of MBNL∆ mRNA compared to Mbnl1 mRNA levels (Fig. S5). At the histological level, no changes were observed in rAAV-MBNL∆-injected HSA LR muscles (data not shown). To confirm that the expression of MBNL∆ had no adverse effects on muscle, wild-type FVB mice were injected intramuscularly with the same dose of rAAV-MBNL∆. Three, four or six weeks post-injection, muscles showed no signs of degeneration/regeneration as revealed by the absence of central nuclei and regenerating fibers expressing embryonic myosin heavy chain, nor histological changes in hematoxylin and eosin (H&E)-stained cryosections (Fig. S6A). Moreover, the splicing profile of Clcn1 exon 7a, Atp2a1 exon 22 and Mbnl1 exon 5 pre-mRNAs that are abnormally spliced in DM1 was not altered by MBNL∆ in wild-type FVB muscles (Fig. S6B).

To further establish that indeed MBNL∆ acts through a decoy mechanism regarding the correction of MBNL1-dependent missplicing events, we engineered another CUGexp-decoy from MBNL2 rather than MBNL1. Based on the same rational, a MBNL∆2 construct that contains only the two tandem ZnF domains required for RNA binding activity and without the Cterminal domain was cloned into a rAAV9 backbone. Then, Gastrocnemius muscles of HSA LR mice were injected with rAAV-V5-MBNL∆2 vectors (1x10 11 vector genomes) whereas contralateral muscles received saline vehicle as control. Seven weeks following injection, MBNL1-dependent splicing events misregulated in DM1 were used as biomarkers of functional MBNL1 recovery. As observed in Fig. 3F, MBNL∆2 rescued abnormal splicing of Clcn1 exon 7a, Atp2a1 exon 22 and Mbnl1 exon 5 pre-mRNAs that are regulated by MBNL1 in skeletal muscle to a similar extent than MBNL∆ derived from MBNL1. These results suggest that engineered RBP proteins with high CUGexp-binding affinities represent attractive decoys to displace endogenous MBNL1 from CUGexp-RNA and reverse MBNL1-dependent defects.

Finally, to evaluate the duration of MBNL∆ action, three-months-old HSA LR mice that have undergone a single intramuscular injection of rAAV-MBNL∆ were examined at fifteen months of age, one year later. As observed for short-term experiments performed seven weeks postinjection, myotonia and splicing defects of Clcn1 exon 7a, Atp2a1 exon 22 and Mbnl1 exon 5 pre-mRNAs were still normalized after one year in HSA LR -injected muscles demonstrating a long-lasting and efficient effect of the rAAV-MBNL∆ treatment (Fig. 4A andB). At the histological level, age-dependent myopathic changes have been described in ageing HSA LR mice (over twelve months of age) with an increased number of internalized nuclei in skeletal muscles fibers (20). One-year post-injection, a significant 30 % reduction in the number of fibers with internal nuclei was observed in MBNL∆-treated muscles compared to contralateral salineinjected muscles of fifteen-months-old HSA LR mice indicating that the expression of MBNL∆ partially prevents the progressive muscle pathology (Fig. 4C andD)

Discussion

In this study, we propose a novel gene therapy approach to neutralize the RNA dominant mechanism responsible for DM1 pathogenesis. We demonstrated that an engineered RBP protein with a high binding affinity for pathogenic CUG expanded tracts acts as a decoy to release functional MBNL1 from CUGexpRNA foci and reverse disease-associated defects in both DM1 patient muscle cells and skeletal muscles of DM1 mice. As a proof of concept and because MBNL proteins have a high binding affinity for CUGexp, we engineered a modified MBNL∆ CUGexp-binding protein derived from a C-terminal truncated MBNL1 protein since this region is not required for RNA binding affinity. MBNL∆ contains the two tandem ZnF domains of MBNL1 necessary for high binding RNA affinity and preserves its ability to bind CUGexp like MBNL1 itself. In contrast, the regulatory splicing activity of MBNL∆ is reduced compared to MBNL1 and more importantly, its residual activity is not sufficient to compensate splicing changes following dual MBNL1 and MBNL2 silencing in human muscle cells. We showed that MBNL∆ promotes the competition for MBNL-binding sites on CUGexp-RNA leading to the dissociation of endogenous sequestered MBNL1 from these structures. By competing for the binding to CUGexp, the four-fold excess of MBNL∆ compared to endogenous MBNL1 saturates CUGexp-RNA foci and improves the mobility of endogenous sequestered MBNL1 proteins. In our model, MBNL∆ modifies the dynamic of endogenous MBNL1 from a toxic unsaturated state in which MBNL1 proteins are sequestered within CUGexp foci, to a non-toxic saturated state in which the formation of MBNL∆:CUGexp foci promotes MBNL1 mobility and subsequently, the recovery of a pool of free and functional MBNL1 as confirmed by the correction of DM1misspliced events. This model is in accordance with previous results from Sznajder et al. (34), showing that the dynamic of sequestration of MBNL proteins in CUGexp-RNA foci is related to the number of MBNL-binding sites on CUGexp. In our condition, the switch towards a mobile state of MBNL1 in DM1 cells is probably also favored by the reduced mobility of MBNL∆ within CUGexp-foci compared to MBNL1 isoforms, as determined previously for a comparable C-terminal truncated MBNL1 protein (34). In addition, the release of MBNL1 in the presence of MBNL∆ is also accompanied by a reduction in the mean number of foci per nucleus in DM1 muscle cells. This behavioral modification of the CUGexp-foci occurs rapidly after MBNL∆ expression and precedes the correction of MBNL1-dependent splicing defects, supporting the proposed mechanism of CUGexp-decoy for MBNL∆ action. Nevertheless, CUGexp-RNA foci are still present in both MBNL∆-treated DM1 muscle cells and HSA LR muscle fibers despite the reversal of DM1-associated phenotypes. These results strongly suggest that MBNL∆decoys:CUGexp foci are not or less pathogenic in contrast to MBNL:CUGexp foci associated with a titration of MBNL proteins, supporting the idea that nuclear CUGexp-RNA foci per se are not necessarily toxic, depending on their composition.

Correction of splicing abnormalities that are mainly regulated by MBNL1 in skeletal muscles confirms the recovery of functional MBNL1 in MBNL∆-treated DM1 patient muscle cells and DM1 mice. MBNL∆ treatment leads to the reversal of splicing defects associated with DM1 symptoms such as myotonia, muscle weakness or dystrophic process but also to the normalization of the aberrant splicing pattern of MBNL1 exon 5, which promotes almost exclusive localization of MBNL proteins in the nucleus of DM1 cells (28). Hence, the recovery of MBNL isoforms prone to shuttle from nucleus to cytoplasm compartments in MBNL∆-treated DM1 cells will also compensate for the loss of cytoplasmic MBNL functions involved in the regulation of mRNA stability and localization, which has been proposed to contribute to DM1 pathogenesis (35). In addition to the correction of the vast majority of abnormal splicing events, MBNL∆ expression also significantly improves the overall transcriptome of DM1 patient derived muscle cells. Finally, it seems likely that MBNL∆ CUGexp-binding proteins delocalize all sequestered MBNL paralogs from CUGexp-RNA foci. Indeed, splicing misregulations that are MBNL1-dependent in skeletal muscles of DM1 mice were also normalized by the expression of MBNL∆2 compounds derived from MBNL2 rather than MBNL1. This result supports the concept of an engineered RBP-decoy aims to release all sequestered MBNL proteins from nuclear CUGexp-foci and consequently, restore appropriate MBNL paralog isoforms and activities in a cell/tissue specific manner. Because MBNL1 functions are essential for striated muscle homeostasis and MBNL2 is a key factor disrupted in the central nervous system of DM1 patients, a decoy approach based on engineered RBPs having the ability to compete with all MBNL paralogs for CUGexp-binding represents an attractive therapeutic strategy in contrast to a gene therapy based on the overexpression of one single MBNL paralog isoform. This work paves the way for the design, optimization and development of innovative engineered or synthetic RBP-decoys (36) that have higher affinity for CUGexp in order to neutralize CUGexp-RNA pathogenicity and restore endogenous functional MBNL paralog isoforms in a cell/tissue-specific manner. Moreover, such a decoy-strategy could be extended to myotonic dystrophy type 2 (DM2) caused by the sequestration of MBNL by CCUGexp-RNAs but also adapted to other expanded repeat RNA-dominant disorders (37) (38).

Materials and Methods

Study design

The objective of our study was to establish the proof of concept of a decoy gene therapy for the RNA-dominant myotonic dystrophy (DM1) disease. A modified RNA-binding protein (MBNL∆) with a high affinity for expanded CUG repeats was engineered to act as a decoy to release sequestered MBNL1 proteins from nuclear CUGexp-RNA foci and reverse DM1associated features. Both relevance and mechanism of action of engineered MBNL∆ proteins were determined in human DM1 muscle cells displaying nuclear CUGexp-RNA foci that sequester MBNL1 splicing factors and splicing defects. Then, in vivo efficacy of this decoy gene therapy approach was evaluated in a DM1 mouse model. rAAV vectors were used to ensure efficient delivery of MBNL∆ in Gastrocnemius muscles of DM1 mice. Molecular and physiologic readouts including splicing defects, myopathy and myotonia were measured to assess the therapeutic effect of rAAV-MBNL∆ treatment.

Constructions and cell cultures

The cloning of GFP-tagged MBNL1 full length (isoform 43) or MBNL∆ (previously named MBNL∆CT3) that contains amino acids 1 to 269 of MBNL1 was performed as previously described (28). GFP-MBNL∆ was cloned in an all-in-one Tet-On lentiviral construct containing a hygromycin selection cassette (39) and the production of lentiviral vector was done as previously described (19). V5-tagged MBNL∆2 containing amino acids 1 to 271 of MBNL2 and GFP-MBNL∆ constructs were cloned in a pSMD2 plasmid allowing the production of rAAV9 vectors as described elsewhere (40). Human immortalized myoblasts derived from a healthy individual and a DM1 patient with 2600 CTG were grown and transduced with Tet-On GFP-MBNL∆ lentiviral vectors as described previously (30), and then selected in the presence of hygromycin (1 μg/ml) for one week. Induction of the conditional Tet-On GFP-tagged MBNLΔ construct was achieved by adding doxycycline (4 μg/ml) into the medium (Sigma-Aldrich). For gene silencing experiments, differentiated muscle cells were transfected by a mix of siRNA (Eurogentec) directed against MBNL1 and MBNL2 mRNAs (5′-CAGACAGACUUGAGGUAUGdTdT-3′; 5′-GAAGAGUAAUUGCCUGCUUUUdTdT-3′, respectively) using Lipofectamine RNAiMAX reagent (Life Technologies) as described previously (41). HeLa cells (ECACC 93021013) were grown to 70% confluence in Dulbecco's modified essential medium supplemented with 10% FBS (Life Technologies) and transfected with 1 µg of DMPK 3' UTR plasmid including interrupted 960 CTG (CTG 960 ) and either 1 µg of GFP-tagged MBNL1 or MBNL∆ plasmid, using FuGENE HD transfection reagent (Roche Diagnostics) according to the manufacturer's instructions.

Fluorescent in situ hybridization and immunofluorescence

Fluorescent in situ hybridization (FISH) and combined FISH-Immunofluorescence experiments were performed as described previously (19). Briefly, a Cy3-labeled 2′OMe (CAG)7 probe was used to detect CUGexp-RNA foci. An Alexa 488 conjugated polyclonal anti-GFP antibody (1:200, Life Technologies, A-21311) and a polyclonal anti-MBNL1 antibody (gift from C. Thornton 1:1000, A2764) followed by a secondary Alexa Fluor 647-conjugated goat anti-rabbit (1:500, Life technologies, A-11029) antibody were used to detect GFP-tagged MBNL∆ or MBNL1, respectively. Nuclei were revealed by Hoechst coloration. Pictures were captured using an Olympus BX60 microscope and Metamorph software (Molecular Devices), and processed with Adobe Photoshop software. Counting of nuclei with foci, number of foci per nuclei or intensity of MBNL1 signal was performed using Fiji software and custom scripts.

RNA isolation, RT-PCR and RT-qPCR analysis

Total RNAs were isolated using TRI-Reagent according to the manufacturer's protocol. Then, one microgram of RNA was reverse transcribed using M-MLV first-strand synthesis system (Life technologies) in a total of 20 µl. One microliter of cDNA preparation was subsequently used in a semi-quantitative PCR analysis according to standard protocol (ReddyMix, Thermo Scientific). PCR amplification was carried out for 18 cycles for GFP and 30-35 cycles for splicing of pre-mRNA within the linear range of amplification for each gene. PCR products were quantified with ImageJ software. The ratio of exon inclusion was quantified as a percentage of inclusion relative to total intensity of isoform signals. To quantify the mRNA expression, realtime PCR was performed using a Lightcycler 480 (Roche Diagnostics). Reactions were performed with SYBR Green kit (Roche) according to the manufacturer's instructions. PCR cycles were a 15 min denaturation step followed by 50 cycles with a 94 °C denaturation for 15 s, 58 °C annealing for 20 s and 72 °C extension for 20 s. Mouse Rrlp0 mRNA were used as standard. Data were analyzed with the Lightcycler 480 analysis software. All primers are described in Table S3.

RNA sequencing and analysis

Total stranded RNA-seq was performed by the Centre National de Génotypage (Institut de Génomique, CEA). After complete RNA quality control on each sample (quantification in duplicate and RNA6000 Nano LabChip analysis on Bioanalyzer from Agilent), libraries were prepared using the TruSeq Stranded Total RNA with Ribo-Zero Kit (Illumina), with an input of 1 μg. Library quality was checked by Bioanalyzer analysis, and sample libraries were pooled before sequencing to reach the expected sequencing depth. Sequencing was performed on an Illumina HiSeq2000 as paired-end 100 bp reads, using Illumina TruSeq V3 reagents. RNA-seq reads were trimmed using Trimmomatic (42) and aligned to human hg19 reference genome using Tophat2 (43). Raw count tables were calculated with HT-seq (44). Differentially expressed genes and differential splicing events between two groups of samples were respectively identified using DESeq2 (45) and DEXSeq (44) [FDR≤0.1, abs(log2 fold change)≥0.58]. To detect differential splicing events, a minimum read coverage of 30 was required in at least one condition to perform analysis at exon level. Each splicing event was annotated using BioMart (46) and exon numbering was obtained based on FasterDB annotation (47). After analysis, a manual clean out was performed to remove remaining technical artifacts, and precisely annotate alternative splicing events.

In vivo gene transfer and muscle histology

All mouse procedures were done according to the protocol n°01204.02 approved by the Ethical Committee on Animal Resources at the Centre Experimentation Fonctionnelle of Pitie-Salpetriere animal facility and under appropriate biological containment. Three month-old adult control FVB or HSA LR transgenic mice (gift from C. Thornton) were intramuscularly injected with PBS, rAAV9-MBNLΔ or rAAV9-MBNL∆2 vectors. At sacrifice, muscles were snapfrozen in isopentane chilled with liquid nitrogen and then sliced at 10 μm with a cryostat. Hematoxylin and eosin (H&E) staining was done according to the standard procedures. Images were captured on a Leica DMR microscope using a NikonDS-Ri1 camera and NIS-Element software. Fibers with internal nuclei were counted on muscle sections with more than 1200 fibers/5-6 fields per muscle.

In situ muscle force relaxation measurement

The isometric contractile properties of gastrocnemius muscle were studied in situ as previously described (48). Mice were anesthetized with a solution of Ketamine/Xylasine (80 mg.kg-1 and 15 mg.kg-1, respectively). The knee and foot were fixed with clamps and pins. The distal tendon of the GA muscle was attached to a lever arm of a servomoteur system (305B, Dual-Mode Lever). Data were recorded and analyzed using PowerLab system (4SP, ADInstruments) and software (Chart 4, ADInstruments). The sciatic nerve (proximally crushed) was stimulated by a bipolar silver electrode using a supramaximal (10-V) square wave pulse of 0.1 ms duration. Absolute maximal isometric tetanic force (P0) was measured during isometric contractions in response to electrical stimulation (frequency of 25 to 150 Hz, train of stimulation of 500 ms). Myotonia is measured as the increase in the area under the relaxation force/time curve.

Slot-Blot

Tissue samples were lysed in a RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.0, 0.1 mM ethylenediamine tetraacetic acid (EDTA), 1% NP-40, 0.5% sodium dodecyl sulfate (SDS)) supplemented with Complete Protease Inhibitor Cocktail (Roche). Lysates were sonicated at 4•C and centrifuged at 14 000 × g for 10 min at 4•C. Protein concentration was determined with the BCA protein assay kit (Thermo scientific). Slot-blot was performed using a 48-well Bio-Dot SF apparatus. Two fold dilution of total protein lysates were prepared in tris-buffered saline (TBS) with 0.5% SDS and loaded onto nitrocellulose membrane in the well of the slot blot apparatus for 45 min. Then, the membrane was washed with TBS-Tween (TBST) solution twice, blocked with 5% milk in TBST for 1h, incubated with a antibody against MBNL1 (1:1000, MB1a) for 1h, washed 3 times with TBST and incubated with a anti-mouse Horseradish Peroxidase antibody (1:5000; Jackson Immunoresearch) for 45 min. Immunoreactivity was detected using the Immobilon Western Chemiluminescent HRP Substrate system (EMD Millipore).

Statistical analysis

All statistical analyses were performed using the GraphPad Prism software (Version 6, GraphPad Software Inc.). Data distribution was assessed by the Kolmogorov-Smirnov test prior to the use of either Student t-test or one-way ANOVA analyses of variance as appropriate.

Supplementary materials

Materials and Methods

Figures

Supplementary materials

Materials and Methods

Minigene experiments and RT-PCR

HeLa cells grown to 70% confluence were transfected with 1 µg of minigene plasmid DNA, 1µg of CTG 960 plasmid and 3 µg of MBNL plasmid DNA, using FuGENE HD transfection reagent (Roche Diagnostics) according to the manufacturer's instructions. Minigenes expressing cTNT exon 5 and INSR exon 11 were previously described (49) (11). Next, total RNA was isolated 48 hrs post-transfection using a total RNA extraction kit (Nucleospin® RNA II kit, Macherey Nagel). One µg of RNA was reverse transcribed using random hexamers and M-MLV reverse transcriptase (Life technologies) according to standard protocols. PCR was performed as previously described (28) and PCR products were visualized on polyacrylamide gels using a FluoroImager scanner (Claravision).

Immunohistochemistry

Muscle cryo-sections were stained using Mouse on Mouse (M.O.M) kit (Vector Labs). Primary laminin (1:300, rabbit polyclonal, Chemicon) and embryonic Myosin Heavy Chain (1:100, mouse monoclonal, Developmental Studies Hybridoma Bank) antibodies were incubated overnight at 4°C followed by 3 washes with PBS-Tween (0.1%) and then, incubated with goat anti-rabbit secondary antibodies and goat anti-mouse (Life Technologies; 1:400) conjugated to Alexa 555 and Alexa 488 respectively.

Purification of recombinant proteins and UV cross-linking experiments

Plasmid pSP72 containing 95 CTG repeats was constructed as described (41). Plasmid pGEX-6P1-MBNL1/40 kDa isoform and pGEXMBNL∆ were described previously (28). Recombinant protein GST-MBNL1 (isoform 40) and GST-MBNL1∆ were expressed in E. coli BL21 (DE3) and purified using Glutathione Uniflow resin (BD biosciences Clontech) according to standard procedure. GST-MBNL∆ was treated by PreScission protease (GE healthcare) to eliminate the GST tag according to standard procedure. In brief 100µg of recombinant GST-MBNL∆ was treated with 2U of PreScission protease for 16 hrs at 4°C in 10 mM Tris buffer pH7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT. The protein was then mixed with glutathionne sepharose preequilibrated with the digestion buffer for 4 hrs at 4°C. The cleaved MBNL∆ protein was recovered after centrifugation and dialyzed against buffer D (20 mM HEPES pH7.9, 20% glycerol, 100mM KCl, 0.2 mM EDTA, 0.5 mM PMSF and 0.5mM DTT). RNAs for UV-crosslinking experiments were transcribed using a cap analogue and SP6 RNA polymerase in the presence of [α-32 P]UTP. Uniformly labeled RNA (10 fmol) were incubated for 15 min at 30°C, in 10 µl with a fixed concentration of recombinant GST-MBNL1 and increasing concentration of MBNL∆ as indicated in the figure under splicing conditions with 0.5 mM ATP, 1 mM MgCl2, 20 mM creatine-phosphate. Samples were UV irradiated for 15 min and treated with 0.3 µg RNase A and 10 U of RNase T1 at 37°C for 30min. The cross-linked proteins were resolved on a 12% SDS poly-acrylamide gel and visualized by a PhosphorImager.

Figures

Part 2

Optimization of the therapeutic RNA-binding protein Introduction Myotonic dystrophy (DM), the most common form of adult onset muscular dystrophy, is a disease characterized by myotonia, muscle wasting, insulin resistance, cardiac and cognitive dysfunctions [START_REF] Meola | Myotonic dystrophies: An update on clinical aspects, genetic, pathology, and molecular pathomechanisms[END_REF]. DM has two clinical manifestations: type 1 and type 2 (DM1 and DM2) caused by the inherited expansions of CUG repeats in the 3' UTR of the DMPK gene and CCUG repeats in the intron 1 of CUGBP gene, respectively (Brook et al., 1992;Liquori et al., 2001;Mahadevan et al., 1992). When transcribed into RNA, the C(C)UG repeats serve as binding sites for RNA-binding proteins, including MBNL splicing factors [START_REF] Miller | Recruitment of human muscleblind proteins to (CUG)n expansions associated with myotonic dystrophy[END_REF].

Consistent with this model, combined immunofluorescent (IF) and fluorescent in vivo hybridization (FISH) experiments demonstrated that expanded CUG repeats form nuclear aggregates, or foci, containing MBNL proteins (Fardaei et al., 2002;[START_REF] Ho | Colocalization of muscleblind with {RNA} foci is separable from mis-regulation of alternative splicing in myotonic dystrophy[END_REF]. All MBNL paralogs (MBNL1, MBNL2, MBNL3) share a similar arrangement of four Zinc finger RNAbinding domains, an overlapping set of target binding sites and high affinity for C(C)UG repeats (Fardaei et al., 2002;Mankodi et al., 2001;[START_REF] Pascual | The Muscleblind family of proteins : an emerging class of regulators of developmentally programmed alternative splicing[END_REF][START_REF] Sznajder | Mechanistic determinants of MBNL activity[END_REF]. By binding to and aggregating with mutant C(C)UGexp-RNAs, MBNL proteins are sequestered leading to their loss of function that affect alternative splicing, polyadenylation and expression level or localization of hundreds of genes (Batra et al., 2014;Du et al., 2010;[START_REF] Wang | Transcriptome-wide regulation of pre-mRNA splicing and mRNA localization by muscleblind proteins[END_REF].

Distinct routes to increase functional pool of MBNL proteins in DM tissues have been developed so far: i) releasing MBNL molecules from pathogenic C(C)UG expansions using antisense oligonucleotides (ASO) or small compounds; ii) boosting the content of MBNL to saturate C(C)UGexp-RNAs in order that the excess protein could fulfill its normal functions and ultimately corrects DM1-associated phenotypes. However, the expression of MBNL paralogs and their isoforms are highly regulated in a cell/tissue-specific manner therefore, the overexpression of a single MBNL isoform appears as a challenging approach for restoring specific functions of all MBNL paralog isoforms. To overcome these limitations, we recently developed an engineered protein acting as a decoy to release all endogenous MBNLs sequestered on expanded CUG tracks. The decoy, referred as MBNL1∆, is a MBNL1-derivative truncated for its C-terminal part. Encompassing four first exons of MBNL1, the engineered protein displays high binding affinity due to two pairs of zinc fingers domains therefore preserving its intrinsic capabilities to specifically interact with CUG repeats. We reported in both, in vitro and in vivo models that the decoy efficiently competes with endogenous MBNL proteins for binding to expanded CUG tracts, subsequently leading to release of MBNL and correction of DM1 phenotypes (Arandel et al., in preparation). Here, we aimed to assess the optimization of the decoy-protein to further improve its target specificity and general efficacy while maintaining its favorable properties. For that reason, we engineered several MBNL∆ protein derivatives (Figure 1) and evaluated their therapeutic effect in vivo in well-established HSA LR DM1 mouse model that expresses 250 repeats in the 3'UTR of the human skeletal actin gene and recapitulates DM1 splicing abnormalities. Splicing rescue of MBNL-dependent events including MBNL1 ex. 5, Atp2a1 ex. 22 and CLCN1 ex. 7a have been used as a biomarker of endogenous functional recovery of MBNL proteins.

Results

Codon optimization

The potential to increase protein expression by altering codon assignments has led to the broad use of codon-optimized mRNAs for gene therapies that profit from enhancing the effectiveness of the treatment with lower dose of the transgene [START_REF] Mauro | A critical analysis of codon optimization in human therapeutics[END_REF].

Taking advantage of this well-established approach, the mRNA sequence of MBNL1∆ decoy was optimized for codon usage bias. As codon optimization should substantially increase protein production, we firstly evaluated the protein expression level of sequence-optimized MBNL1 transgene in transfected Hek293T cells. Surprisingly, we did not notice a more efficient protein translation with codon opt-MBNL1∆ when compared to control MBNL1∆ (Figure 2a), which could be due to the mRNA secondary structure or other variables that may inadvertently affect protein expression like for example adaptation of codons to the tRNA pool. Next, both constructs were cloned in AAV9 vectors and injected intramuscularly in Gastrocnemius muscles of HSA LR mice at the dose of 3*10^11 vg. Five weeks post-injection, the level of MBNL1∆ was determined by Western blot. Remarkably, we noticed much lower protein expression level of opt-MBNL1∆ when compared to MBNL1∆ constructs (Figure 2b).

Accordingly, splicing analysis of MBNL1 ex.5, Atp2a1 ex. 22 and CLCN1 ex. 7a pre-mRNAs showed that only treatment with AAV-MBNL1∆ led to significant alternative splicing correction whereas codon optimized construct had no effect on splicing rescue suggesting, that the amount of the protein expressed in injected muscles was not sufficient to displace endogenous MBNL proteins from expanded repeats (Figure 2c,d). These results indicate that the optimized MBNL1∆ construct, in contrast to what has been expected, has no decoy activity.

Zinc fingers and linker domains

MBNL proteins binds to RNA via two pairs of highly-conserved tandem ZnF (ZnF1-2 and ZnF3-4) domains. However, despite their high similarity, they are not functionally equivalent.

Recent findings using chimeric recombinant MBNL1 proteins containing either duplicated

ZnF1-2 or Zn3-4 domains gave additional insight into the function of each ZnF pair [START_REF] Hale | An engineered RNA binding protein with improved splicing regulation[END_REF]. Both bind to YGCY RNA consensus motifs however, ZnF1-2 specifically recognizes YGCY sequences while ZnF3-4 acts like a more general RNA binding domain. Potentially due to that specific recognition, ZnF1-2 conveys very strong splicing activity whereas ZnF3-4 shows drastic decrease in splicing regulation. To gain a complete insight into the behavior of these chimeric proteins in vivo, we engineered MBNL1∆ with either duplicated ZnFs1-2 or ZnFs3-4 domains.

To determine whether the ZnF domains swapping in MBNL1∆ affects relative protein expression, we determined protein level in transfected Hek293T cells. We found that MBNL1∆ with two ZnF1-2 domains was expressed at an approximately one-fold higher level as compared to native MBNL1∆ without binding domains rearrangement. In contrast, the presence of two ZnF3-4 motives caused a 6-fold increase in protein expression when normalized to native MBNL1∆ (Figure 3a), confirming that ZnF3-4 domain might confer additional stability to the protein as previously described [START_REF] Hale | An engineered RNA binding protein with improved splicing regulation[END_REF]. To further examine how variations of ZnFs would affect the MBNL1∆ decoy functionality, MBNL1∆ 1-2/1-2 and MBNL1∆ 3-4/3-4 recombinant proteins were evaluated in vivo following intramuscular injection of corresponding AAV9 vectors in HSA LR mice. Correction of splicing changes induced by MBNL1∆ 1-2/1-2 was not significantly different from that noticed with MBNL1∆ (Figure 3b,c). However, MBNL1∆ 3-4/3-4 did not exert any effect on missplicing events found in HSA LR mice (Figure 3b,c). RT-qPCR analysis showed that both transgenes mRNA, MBNL1∆ 1-2/1-2 and MBNL1∆ 3-4/3-4, was expressed at similar level (Figure 3d). Next, we assessed the protein level by immunoblot analysis and showed that MBNL1∆ 1-2/1-2 was expressed approximately 0.5-fold higher than MBNL1∆ in agreement with in vitro results (Figure 3e). In contrast, we did not detect MBNL1∆ expression with duplicated ZnF3-4 domains (Figure 3e). To test whether the lack of protein observed with the MBNL1∆ 3-4/3-4 construct was due to the disease model and/or specific post-transcriptional regulation of the transgene, we injected muscles of WT mice with the same AAV-MBNL1∆ 3-4/3-4 and AAV-MBNL1∆ constructs used in HSA LR mice.

As previously observed, we did not detect the expression of MBNL1∆ 3-4/3-4 in WT injected mice (Supplementary Figure 1a). In parallel, the AAV constructs were validated by transducing We previously demonstrated that MBNL1∆ has a reduced splicing activity when compared to full length MBNL1 and does not regulate splicing events in WT and DM1 muscle cells upon depletion of endogenous MBNL1 and MBNL2 proteins by RNA interference (Arandel et al. in preparation). To determine whether the constitutive exon 3 of MBNL1 that is localized between the two pairs of zinc fingers and acts as a flexible linker could modulate splicing properties of the decoy, we engineered an MBNL1∆ construct lacking its exon 3 (MBNL1∆ -ex.

3). Indeed, it has been previously shown that its absence in MBNL1 protein leads to very inefficient splicing regulation [START_REF] Tran | Analysis of exonic regions involved in nuclear localization, splicing activity, and dimerization of {Muscleblind}like-1 isoforms[END_REF]. To test firstly whether MBNL1∆ -ex. 3 preserved its binding properties, we co-transfected Hek293T cells with plasmids expressing 115 CTG repeats and MBNL1∆ -ex. 3 sequence. As visualized in the Figure 4a by FISH-IF analysis, MBNL1∆ -ex. 3 colocalizes with CUGexp-RNA foci in the nucleus suggesting that the linker between ZnF domains is not crucial to assure binding to expanded CUG repeats as previously reported [START_REF] Rehman | Development of an AP-FRET Based Analysis for Characterizing RNA-Protein Interactions in Myotonic Dystrophy (DM1)[END_REF][START_REF] Tran | Analysis of exonic regions involved in nuclear localization, splicing activity, and dimerization of {Muscleblind}like-1 isoforms[END_REF]. Next, MBNL1∆ -ex. 3 sequence was cloned into an AAV9 backbone and vectors were injected intramuscularly in HSA LR mice.

While similar level of protein expression was detected in HSA LR muscles transduced with the same dose of either AAV-MBNL1∆ -ex. 3 or AAV-MBNL1∆ vectors (Figure 4b), MBNL1∆ -ex. 3 did not exert any effect on the splicing abnormalities when compared to MBNL1∆ (Figure 4c,d). Thus, the presence of exon 3 appears to have a pivotal role in MBNL1∆ to drive its role as a decoy as its removal markedly abrogates MBNL1∆ decoy activity.

Changes in splicing are represented as difference in percent exon inclusion (PSI, ψ) when compared to saline-injected muscles. d, Combined analysis of splicing changes in injected muscles with both proteins (expressed as ∆ψ). The represented mean and standard deviation came from three biological replicas. Statistical significance was determined by one-way ANOVA followed by Tukey's test (ns: no significant, *p < 0.1, **p < 0.01, ***p < 0.001).

Protein localization

Microscopy analysis of cellular distribution of MBNL1∆ fused to GFP showed that the protein preferentially localizes in both cytoplasm and nucleus compartments (Supplementary Figure 2). Since MBNL1∆ lacks the C-terminal domains including exon 5, which is part of a bipartite nuclear localization signal (NLS) located in exons 5 and 6, we added an eight amino acid SV40 NLS at the end of its sequence and assessed whether increased nuclear localization influences protein's decoy-activity. Considering that cytoplasmic MBNL1∆ does not serve as a decoy since pathogenic repeats are retained within the nuclear compartment, we expected that MBNL1∆-NLS would lead to higher concentration of the protein in the nucleus or similar nuclear levels as MBNL1∆ but at lower therapeutic doses. Hence, less vectors, and subsequently, less decoy would need to be delivered to convey a therapeutic impact. As expected, conjugation of an NLS to MBNL1∆ shifted the protein almost exclusively to the nucleoplasm (Figure 5a).

Immunoblot analysis showed that the presence of the NLS did not change MBNL1∆ protein expression level in injected HSA LR muscles (Figure 5b). However, nuclear retention of MBNL1∆ induced by the NLS enhances its efficiency when compared to MBNL∆ alone as observed by improved correction of missplicing events (Figure 5c,d). Thus, direct localization of MBNL1∆ to the nucleus via addition of nuclear localization signal to the protein sequence exerts favorable and robust effect on its decoy function. percent spliced in (PSI, ψ) (i.e. percent exon inclusion) after treatment with MBNL1∆ NLS protein and compared to saline-injected HSA muscles. c, Combined analysis of splicing changes in injected muscles after treatment with MBNL1∆ and MBNL1∆ NLS proteins (expressed as ∆ψ). The represented mean and standard deviation come from three biological replicas. Statistical significance was determined by Student's t-test (ns: no significant, *p < 0.1, **p < 0.01, ***p < 0.001).

MBNL paralogs

As mentioned previously, Muscleblind family consists of three highly conserved members namely MBNL1, MBNL2 and MBNL3. A direct comparison of the proteins' activity showed that they bind the same RNA motifs with different affinities and in general, MBNL1 demonstrates the highest splicing activity while MBNL3 the lowest. Contrary, all three paralogs show exceptionally alike binding affinity for expanded CUG and CCUG repeats [START_REF] Sznajder | Mechanistic determinants of MBNL activity[END_REF]; however, FRAP analysis demonstrated that MBNL1 and MBNL2 are less densely packed with higher rates of diffusion and greater mobility between the foci than MBNL3 that is rather static and more prone to be tightly packed on toxic repeats. To determine whether these characteristics would impact the effectiveness of the decoy approach, we directly compared the derivatives of the three MBNL paralogs with truncated C-terminal part. MBNL1∆, MBNL2∆, and MBNL3∆ evaluation in HSA LR mice revealed that all decoys exerted strong effect on alternative splicing rescue of MBNL-dependent transcripts when compared to saline-injected matched controls (Figure 6a). Nevertheless, comparison of the splicing switch percentage rate between the three proteins showed that MBNL3∆ is the paralog with the highest efficiency (Figure 6b), whereas MBNL1∆ and MBNL2∆ have comparable efficacy (except exon 5 of MBNL1 that showed a better rescue after treatment with MBNL1∆). Importantly, immunoblot analyses showed no differences in the expression of all three MBNL∆ paralogs (Figure 6c).

Therefore, the greater extent of the therapeutic effect of MBNL3∆ was neither driven by its higher levels in injected muscles nor by changes in HSA transgene levels in the treated mice as all three proteins led to comparable CUGexp-RNA reduction (Supplementary Figure 3).

Since MBNL1∆ still possesses a residual splicing activity when tested in minigene assays (Arandel et al., in preparation;[START_REF] Hale | An engineered RNA binding protein with improved splicing regulation[END_REF], we examined whether differences observed between MBNL3∆ and other paralogs are not due to the direct splicing promotion. Thus, we co-transfected Hek293T cells with splicing reporter minigene plasmids (either the mouse ATP2A1 or the human IR constructs) containing number of MBNL binding sites required for exon regulation together with plasmids expressing each MBNL∆. In this in vitro assay we observed that all three proteins promote the inclusion of ATP2A1 ex. 22 or IR ex. 11 (Figure 7a); however, splicing changes showed the same percent exon inclusion values for each MBNL∆ paralogs indicating that none of the truncated protein exhibits higher splicing activity.

To test whether MBNL∆ have the same effect on endogenous targets in Hek293T cells, we analyzed two endogenous MBNL-dependent alternatively spliced events, Mbnl ex. 5 and Opa1 ex. 5. None of the MBNL∆ paralogs significantly influenced exon inclusion, thereby demonstrating that MBNL∆ decoys do not possess splicing activity for intrinsic endogenous targets (Figure 7b). To examine MBNL∆ derivates in a DM1 muscle context, human immortalized DM1 muscle cells carrying 2600 CTG and displaying nuclear CUGexp-RNA foci and alternative splicing misregulations were transduced with lentiviral vectors expressing a conditional Dox-dependent Tet-on GFP-tagged MBNL2∆ or MBNL3∆ as previously described for GFP-tagged MBNL1∆, which was used as a control. Because the presence of nuclear localization signal had supplementary effect on decoy activity, we additionally transduced DM1 cells with the lentivirus containing the sequence of GFP-MBNL3∆ fused to NLS signal. First, we evaluated the level of expression of all four MBNL∆ constructs tested. No significant changes in the expression between the three MBNL∆ derivates have been found neither at the mRNA nor protein levels. In contrast, expression of MBNL3∆-NLS was reduced by 0.5-fold at both mRNA and protein levels (Figure 7c,d). Whether this result is due to the integration of the transgene(s) in less active genome region(s) or to reduced number of integrated transgenes should be assessed. To further examine the effect of the four MBNL∆ decoys, we analyzed the correction of alternative splicing events that were used as biomarkers of functional MBNL1 recovery after dox-induced expression of each decoy in DM1 differentiated myoblasts. The induction of each MBNL∆ normalized splicing defects of Mbnl1 exon 5, Atp2a1 exon 22, Nfix exon 7, pre-mRNAs to various degrees. MBNL1∆ and MBNL3∆ induced similar missplicing correction; however, MBNL3∆ did not lead to increased missplicing recovery for any of the transcripts tested (Figure 7e). In contrast, MBNL2∆ had the weakest activity between all four decoys despite their similar expression levels. Surprisingly, NLS-conjugated MBNL3∆ exerted the same effect as MBNL1∆ with only half amount of the protein expressed when compared to other MBNL∆ decoys. Observed discrepancies between alternative splicing recovery among MBNL1∆, MBNL2∆ and MBNL3∆ are not due to the cellular localization of the proteins as their expression pattern showed similar distribution in GFP-MBNL∆-transfected control myoblasts. All paralogs localized in both cytoplasmic and nuclear compartments with approximately 50%-50% ratio while combination of MBNL3∆ with an NLS resulted in 90% nuclear localization of the protein (Figure 7f and Supplementary Figure 4). Moreover, we previously showed that MBNL1∆ expression modifies the behavior of the nuclear CUGexp-RNA foci causing a significant 2.5-fold reduction in the mean number of foci per nucleus when compared to untreated DM1 cells. The reduction was further associated with a decrease or almost complete disappearance of small and medium size foci (Arandel et al., in preparation).

Foci analysis in differentiated DM1 muscle cells after treatment with MBNL2∆, MBNL3∆ or MBNL3∆ NLS showed that all decoys lead to decrease in the quantity of foci per nucleus but only MBNL3∆ and MBNL3∆ NLS reduce their number significantly (Figure 7g,h). This can be explained by considerably lower amount of MBNL2∆ found on CUGexp RNA when compared to other decoys (Figure 7i). The shortage in alternative splicing rescue in cells expressing MBNL2∆ might be then due to the insufficient binding to expanded tracts or excessive protein mobility between the foci and nucleoplasm. Besides its low efficiency, in accordance to previous observations (Arandel et al., in preparation) we noticed decreased number or almost complete disappearance of small foci in cells expressing all MBNL∆ paralogs further confirming their decoy activity.

Discussion

In the previous study (part 1), we described a new gene therapy to neutralize the RNA dominant mechanism underlying the pathology of myotonic dystrophies. MBNL1 derivative lacking its C-terminal region (MBNL1∆) showed robust reversal of DM1-associated features in vitro and in vivo by acting as a decoy to displace endogenous MBNL proteins from expanded repeats due to its high binding affinity to CUG tracks. As a consequence, the pool of free and functional endogenous MBNL proteins is restored in the nucleoplasm resulting in decreased number of foci, long-lasting global correction of abnormal splicing events and myotonia normalization in HSA LR treated mice. To further improve its decoy activity, we evaluated several engineered MBNL∆ derivatives in vivo aiming to influence its cellular distribution, binding properties and behavior on expanded CUG RNA tracks.

Our results showed that MBNL∆ decoy competes with endogenous MBNL1 for the binding to expanded CUG repeats rather than endogenous splicing sites. In fact, MBNL1∆ lacks the protein region that has been shown to be crucial for splicing activity [START_REF] Tran | Analysis of exonic regions involved in nuclear localization, splicing activity, and dimerization of {Muscleblind}like-1 isoforms[END_REF]. Since the linker encodes by the exon 3 joins the second and third zinc finger domains, it has been shown to be essential for MBNL1 efficient binding to its pre-mRNA targets substantially influencing general MBNLs splicing activity, we assessed its removal from our MBNL1∆ construct. We noticed that MBNL∆ lacking exon 3 colocalizes with expanded tracts in vitro, however, it does not display decoy activity in injected HSA LR mice. It has been suggested that the linker between two ZnF pairs is necessary for their decent spatial positioning and structural integrity of MBNL to assure binding to the target sequence [START_REF] Kino | Muscleblind protein, {MBNL}1/{EXP}, binds specifically to {CHHG} repeats[END_REF]. Therefore, besides its binding capacity, its affinity to CUGexp might be weaker as previously observed [START_REF] Kino | Muscleblind protein, {MBNL}1/{EXP}, binds specifically to {CHHG} repeats[END_REF][START_REF] Tran | Analysis of exonic regions involved in nuclear localization, splicing activity, and dimerization of {Muscleblind}like-1 isoforms[END_REF] and might result in faster dissociation from expanded tracts when compared to endogenous MBNL proteins; thus, MBNL∆ lacking exon 3 might not compete efficiently with MBNL that remains trapped on CUGexp-RNA.

MBNL proteins bind to their RNA targets via two tandem of highly conserved zinc finger motifs.

Recently Hale et al. attempted to determine the RNA binding specificities and activity of ZnF1-2 and ZnF3-4 domains [START_REF] Hale | An engineered RNA binding protein with improved splicing regulation[END_REF]. MBNL1 proteins containing only the first (ZnF1-2) or the second (ZnF3-4) pair of ZnF domain show very weak binding to substrate RNAs suggesting, that the cooperative work of both tandems is necessary to assure high binding affinity despite acting as the independent domains [START_REF] Fu | {MBNL}1-{RNA} recognition: contributions of {MBNL}1 sequence and {RNA} conformation[END_REF]. Synthetic MBNL with either duplicated ZnF1-2 or ZnF3-4 demonstrated that the first pair drives very strong splicing activity while ZnF3-4 acts like a more general binding domain recognizing YGCY motifs. While more of the protein would be needed for efficient splicing regulation, MBNL1 with duplicated ZnF3-4 showed nearly identical binding affinities to CUG repeats as WT MBNL. Therefore, it seems that observed dissimilarities in splicing activity are rather driven by RNA binding specificities than general binding affinity. It has been noted that ZnF domains rearrangement additionally modify protein steady state levels. In our study, duplication of the first pair of ZnF caused approximately 1-fold increase in protein expression while MBNL1∆ with two ZnF3-4 motifs was expressed at 6-fold higher level than MBNL1∆ protein with canonical ZnFs organization.

These results are slightly different from published data [START_REF] Hale | An engineered RNA binding protein with improved splicing regulation[END_REF] showing 0.5-fold lower expression of MBNL with duplicated 1-2 domain and 2.5-fold higher expression of MBNL with duplicated 3-4 motifs. However, it has to be noted that protein sequences were not identical between both studies with the lack of NLS signal at the C-terminal part of the protein in our study and various N-terminal tags (HA vs V5). Low splicing activity together with high affinity to CUG repeats and elevated protein stability would confer substantial advantages to MBNL∆ decoy. Curiously, MBNL∆ 3-4/3-4 was efficiently expressed in muscle cells in vitro but there was no protein translation in muscles in vivo. This event was not species-dependent as we observed considerable protein expression in human muscle cells as well as in murine C2C12 myoblasts. It has been previously shown that higher protein expression with duplicated ZnF3-4 domains was not due to the raised level of mRNA in transfected Hek293T and Hela cells [START_REF] Hale | An engineered RNA binding protein with improved splicing regulation[END_REF]. This is in accordance with our in vivo observations following AAVs injections, indicating that MBNL∆ 3-4/3-4 mRNA was produced but an unknown mechanism could impede its translation or increase it degradation. Nevertheless, it is worth to mention that both MBNL∆ with duplicated Zinc Finger domains are synthetic proteins that are not expressed in living organisms; therefore, their usage as a therapeutic approach might be questionable due to potential immune response. Thus, they will need further diligent examination before in vivo application.

Composed wholly of four first MBNL1's exons, MBNL1∆ lacks protein's intrinsic bipartite NLS located in exon 5 and 6. However, in accordance with a previous report [START_REF] Tran | Analysis of exonic regions involved in nuclear localization, splicing activity, and dimerization of {Muscleblind}like-1 isoforms[END_REF], MBNL1∆ devoid of this region show dual nucleocytoplasmic localization presumably due to the putative weak NLS localized in exon 3. Nevertheless, it has been demonstrated that MBNL1 isoforms lacking exon 5 and 6 freely shuttle between both cellular compartments [START_REF] Tran | Analysis of exonic regions involved in nuclear localization, splicing activity, and dimerization of {Muscleblind}like-1 isoforms[END_REF]. Although MBNL1∆ partially localizes in the nucleus and drives the therapeutic effect, adding SV40 NLS signal to the protein markedly increased its decoy activity most likely due to higher occupancy on the CUG tracts. We assume that higher crowding of the decoy in the nucleus leads to more active competition with endogenous MBNLs for binding to expanded CUG repeats, resulting in more effective release of functional endogenous MBNL1 proteins as reflected by the complete missplicing correction in treated DM1-mice. Additionally, localization of the decoy to the nucleus shifts it away from potential cytoplasmic off-target binding. While it has been shown that MBNL1 and MBNL2 are implicated in cellular mRNAs localization and local translation in neuronal cells, not much is known about MBNLs' cytoplasmic functions in muscle cells. While we thoroughly demonstrated that MBNL∆ does not carry splicing activity, a well-defined MBNL nuclear function, one cannot totally exclude that its cytoplasmic action might be perturbed. Further evaluation of potential cytoplasmic events should be performed but biomarkers of MBNL cytoplasmic activity in muscles are lacking yet.

In spite of the discovery of three MBNL paralogs, much attention has been focused on MBNL1 and MBNL2 in DM due to their high expression in the muscles. Nevertheless, all three proteins have been found to be sequestered on RNA-foci (Fardaei et al., 2002;[START_REF] Ho | Colocalization of muscleblind with {RNA} foci is separable from mis-regulation of alternative splicing in myotonic dystrophy[END_REF] with almost the same binding affinities to expanded CUG tract [START_REF] Sznajder | Mechanistic determinants of MBNL activity[END_REF]. Recent findings directly comparing all three proteins revealed that despite their strong sequence similarity of RNA binding modules, they do not behave equally when sequestered within CUGexp-RNA foci [START_REF] Sznajder | Mechanistic determinants of MBNL activity[END_REF]. We found that MBNL∆-decoys derived from three different MBNL paralogs differ in their activity. Thus, MBNL3∆ drives the highest splicing recovery after treatment in HSA LR mice, which may be due to differences of paralogs mobility between the foci. In fact, MBNL3 isoform shows the lowest mobility rate when compared to two other paralogs [START_REF] Sznajder | Mechanistic determinants of MBNL activity[END_REF]. Even though these dissimilarities have not been evaluated for C-terminal truncated MBNL proteins, it seems that MBNL3∆ might be prone to be more stably stacked on expanded repeats making these sites less accessible for MBNL proteins present in the nucleoplasm. The higher therapeutic effect observed with V5-MBNL3∆ in vivo was however not found in vitro in DM1 cells expressing GFP-fused MBNL∆ derivatives. MBNL3∆ did not leads to better splicing rescue of any of the pre-mRNAs tested when compared to MBNL1∆. Surprisingly, MBNL2∆ exerted very weak effect on DM1 phenotype despite quite the same protein expression between three paralog derivatives. Noteworthy, three bands have been visualized in immunoblots of each MBNL∆ that might correspond to several ATG start codons present in the sequence of GFP as we did not observe such events in Hek293T-transfected MBNL∆ decoys fused to V5 tag (Supplementary Figure 5). However, we cannot exclude that different proteins' sizes somehow contribute to decoys stability. In line with our previous findings, nuclear localization signal substantially increased MBNL3∆ decoy capacity: similar correction of splicing defects as MBNL1∆ but with lower amount of the protein. It implies that nuclear localization added to the decoy sequence will allow to significantly decrease the dose of the treatment keeping simultaneously the same therapeutic outcome.

Although all three decoy MBNL paralogs have proven to drive their elementary function, from our results we conclude, that MBNL3∆, especially in fusion with nuclear localization signal, represents the most favorable decoy derivative. As MBNL3 is mostly expressed only during embryogenesis but also in muscle stem cells that are activated during the regenerative process in adult murine damaged muscles, some of its specific endogenous targets could be absent in mature muscles and adult tissues that would help to avoid unlike side-effects.

Therefore, expression of MBNL3∆ fused with a nuclear localization signal could add an additional benefit in further decoy gene therapy development.

Materials and methods

Constructs preparation

An N-terminal v5-tag (5'-GGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACG-3'), together with a Kozac sequence with a dedicated Start codon were inserted during the PCR-amplification via the forward cloning primer. MBNL∆ protein paralogs correspond to the amino-acids ranging from 1 to 269 for MBNL1∆, from 1 to 268 for MBNL2∆ and from 1 to 257 for MBNL3∆ (accession number Q9NR56, Q5VZF2 and Q9NUK0, respectively). MBNL1∆ isoform lacking exon 3 is devoid of amino-acids 116 to 183. MBNL1∆ contains two duplex of zinc finger domains (ZnF): ZnF1/2 followed by ZnF3/4. To build the ZnF chimeric constructs, we duplicated either the ZnF1/2 duplex or the ZnF3/4 duplex of MBNL1∆.

The obtained MBNL1∆ ZnF1/2-1/2 protein correspond to the amino-acids 1 to 178 fused with amino-acids 13 to 97. The ZnF3/4-3/4 starts with amino-acids 1 to 11 followed by amino-acids 179 to 241 fused with amino-acids 74 to 263. All constructs were cloned into a single-stranded AAV-expression vector (psmd2) with a CMV promotor. For in vitro transduction, MBNL1∆ constructs were N-terminally fused with GFP and cloned into pSin vector.

Cell culture

Hek293T cells were cultured as a monolayer in Dulbecco's modified Eagle's medium (DMEM)-Glutamax (Gibco) supplemented with 10% fetal bovine serum at 37 °C under 5% CO2. Human immortalized myoblasts were cultivated in a growth medium consisting of a mix of 199: DMEM (1:4 ratio; Life Technologies) supplemented with 20% FBS (Life Technologies), 25 μg/ml fetuin, 0.5 ng/ml bFGF, 5 ng/ml EGF and 0.2 μg/ml dexamethasone (Sigma-Aldrich) t 37 °C under 5% CO2.

Transfection and transduction

Prior to transfection, Hek293T cells were plated in twelve-well plates allowed to reach up to 70% confluency. For minigenes transfection, 100 ng ng of splicing minigene and 250 ng of MBNL∆ constructs were co-transfected into each well with Lipofectamine LTX (Invitrogen) according to the manufacturer's protocol and harvested after 24 h for RNA extraction. For MBNL∆ colocalization with CUG repeats, cells were co-transfected with 100 ng of plasmid expressing 115 CTG repeats and 250 ng of MBNL∆ constructs with Lipofectamine LTX (Invitrogen) according to the manufacturer's protocol. 24h later cells were fixed in 4% paraformaldehyde in PBS for 10 minutes. For MBNL∆ cellular localization, human myoblasts or HeLa cells were plated in 6-wells plate and transfected with 1 µg of the GFP-tagged MBNL∆ construct. Cells were analyzed 24h post-transfection.

Human immortalized myoblasts derived from DM1 patient with 2600 CTG were grown and transduced with lentiviral vectors as described previously [START_REF] Arandel | Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds[END_REF]. The use of a Tet-On lentiviral vector allowed the expression a conditional GFP-tagged MBNL∆ constructs by adding 4 μg/ml of doxycycline into the medium (Sigma-Aldrich).

Fluorescent in situ hybridization and immunofluorescence

Fluorescent in situ hybridization (FISH) and combined FISH-Immunofluorescence experiments were performed as described previously (Francois et al., 2011). Briefly, a Cy3-labeled 2ʹOMe (CAG)7 probe was used to detect CUGexp-RNA foci. An Alexa 488 conjugated polyclonal anti-GFP antibody (1:200, Life Technologies, A-21311) and a polyclonal anti-MBNL1 antibody (gift from C. Thornton 1:1000, A2764) followed by a secondary Alexa Fluor 647-conjugated goat anti-rabbit (1:500, Life technologies, A-11029) antibody was used to detect GFP-tagged MBNL∆ or MBNL1, respectively. Nuclei were revealed by Hoechst coloration. Images were acquired with a Nikon Ti2 microscope equipped with a motorized stage and a Yokogawa CSU-W1 spinning disk head coupled with a Prime 95 sCMOS camera (Photometrics). Counting of nuclei with foci, number of foci per nuclei or intensity of MBNL1 signal was performed using Fiji software and custom scripts.

In vivo gene transfer

All mouse procedures were done according to the protocol 01204.02 approved by the Ethical Committee on Animal Resources at the Centre Experimentation Fonctionnelle of Pitie-Salpetriere animal facility and under appropriate biological containment. WT FVB or HSA LR transgenic mice (gift from C. Thornton) were intramuscularly injected with PBS or rAAV9-MBNLΔ vectors.

RNA isolation, RT-PCR and RT-qPCR analysis

Prior to RNA extraction, muscle tissues were lyzed in TRI-Reagent (Sigma-Aldrich) using Fastprep system and Lysing Matrix D tubes (MP biomedicals. Then, total RNAs were isolated using TRI-Reagent according to the manufacturer's protocol. Total RNA from Hek293T cells and immortalized myoblasts were isolated using TRI-Reagent (Sigma-Aldrich). One microgram of RNA was reverse transcribed using M-MLV first-strand synthesis system (Life technologies) in a total of 20 µl. One microliter of cDNA preparation was subsequently used in a semi-quantitative PCR analysis according to standard protocol (ReddyMix, Thermo Scientific).

PCR amplification was carried out for 30 cycles for splicing of pre-mRNA within the linear range of amplification for each gene. PCR products were quantified with ImageJ software. The ratio of exon inclusion was quantified as a percentage of inclusion relative to total intensity of isoform signals. To quantify the mRNA expression, real-time PCR was performed using a Lightcycler 480 (Roche). Reactions were performed with SYBR Green kit (Roche) according to the manufacturer's instructions. PCR cycles were a 15 min denaturation step followed by 50 cycles with a 94 °C denaturation for 15 s, 58 °C annealing for 20 s and 72 °C extension for 20 s. Mouse Rrlp0 mRNA were used as standard. Data were analyzed with the Lightcycler 480 analysis software.

Statistical analysis

For statistical analysis, either Student's t-test or one-way analyses of variance were used as appropriate using GraphPad Prism software (Version 6, GraphPad Software Inc.).

Introduction

In the recent years, interest in gene therapy has raised substantially due to its high versatility [START_REF] Keeler | Gene Therapy 2017: Progress and Future Directions[END_REF]. However, targeting a therapeutic product to the disease-affected cells or tissues is crucial for the success of any gene therapy treatment since the expression of a transgene in the non-affected tissues or cells would be highly ineffective, or could lead to potential side effects. In the case of multisystemic disorders, efficient delivery of the transgene holds the greatest promise and simultaneously, the greatest challenge in treatment design. A layer of complexity is added in progressive diseases associated with constant changes in their pathogenic conditions. One of such an example is myotonic dystrophy type 1 (DM1) due to the unstable nature of the CTG expansion within the non-coding region of the DMPK gene (Brook et al., 1992;Mahadevan et al., 1992). The long repetitions within the mutant transcript sequester MBNL RNA-binding splicing factors, which are crucial regulators of fetal-to-adult developmental alternative splicing switch [START_REF] Kalsotra | A postnatal switch of CELF and MBNL proteins reprograms alternative splicing in the developing heart[END_REF]. The stoichiometry between functional and sequestered MBNL proteins and subsequent severity of the symptoms, depends on the size of the microsatellite that is modulated by somatic instability and increases during the lifespan of DM1 patients [START_REF] Higham | High levels of somatic DNA diversity at the myotonic dystrophy type 1 locus are driven by ultrafrequent expansion and contraction mutations[END_REF]Morales et al., 2012). It implies that the amount of the therapeutic compound required for effective treatment might likely increase over time in DM1 patients. Additionally, expression of the DMPK gene as well as the repeat's size varied between different tissues [START_REF] Higham | High levels of somatic DNA diversity at the myotonic dystrophy type 1 locus are driven by ultrafrequent expansion and contraction mutations[END_REF][START_REF] Morales | Somatic instability of the expanded CTG triplet repeat in myotonic dystrophy type 1 is a heritable quantitative trait and modifier of disease severity[END_REF]. Therefore, fine tuning of desirable concentration of the therapeutic product needed in particular tissues would be of great advantage to avoid unwanted or adverse effects. To address this issue, we developed an innovative tool called 'splice-sensor' that ensures an auto-regulatory expression of a transgene product according to the activity of a specific RNA binding protein (RBP) within the host cell or tissue. To assess the functionality of the splice-sensor, we took advantage of DM1 paradigm, in which functional loss of MBNL proteins results in splicing misregulation (Du et al., 2010;[START_REF] Wang | Transcriptome-wide regulation of pre-mRNA splicing and mRNA localization by muscleblind proteins[END_REF]. We combined this approach with our recently developed MBNL∆ gene therapy that acts like a decoy to release sequestered proteins from expanded CUG tracts (Arandel et al., in preparation). constructs as reported before in WT mice (Figure 5a). Immunoblot analysis showed MBNL1∆ protein expression in injected muscles with both AAV constructs independently of the presence of the splice-sensor sequence (Figure 5b). It confirms that in the DM1 condition with reduced functional MBNL1, the splice sensor is fully active allowing the production of MBNL1∆. However, we noticed slightly lower MBNL∆ protein amount in muscles injected with AAV-splice-sensor-MBNL∆, which is in line with the analysis of splice-sensor Atp2a1 exon 22 transcript that was not totally excluded (Figure 5a). Nonetheless, these results showed that the sensor is fine-tuned by MBNL1 activity. We assume that sufficient amount of MBNL∆decoy has been produced to release functional endogenous MBNL1 proteins and therefore, the excess of the decoy was shut down. Consistent with this hypothesis splicing defects were corrected to similar extend by either AAV9-V5-MBNL1∆ or AAV9-splice-sensor-V5-MBNL1∆ (Figure 5c). In a scenario of renewed decrease of MBNL1 activity, due to possible expansion of the CUG repeat size, the splice-sensor translation regulatory sequence will autoregulate the required level of the decoy (Figure 5d).

Conclusions

Here we describe an innovative approach to control the expression of a transgene product using a splice-sensor that is regulated by the activity of specific RNA binding proteins in host cells or tissues. We proved the functionality of the splice-sensor by taking advantage of the recently developed gene therapy for DM1 (Arandel et al., in preparation) that works like a decoy on expanded CUG repeats to release sequestered MBNL1 splicing factors. The splicesensor being under the control of the activity of MBNL1 protein regulates the expression of the decoy due to stop codons comprised in the sequence of the alternatively spliced ATP2A1 exon 22 within the sensor. Normal level of functional MBNL in cell or muscle tissue leads to positive regulation of the exon 22 and subsequent translation termination of the transgene decoy product. In contrast, when the MBNL1 activity is reduced like in DM1 mice, MBNLdependent exon 22 is excluded from the mature splice-sensor mRNA permitting the expression of the transgene decoy product. Based on that proof-of-concept, we propose the splice-sensor as a tool to control any transgene products that need transient or tissue-specific expression. Splice-sensor consists of a natural and/or a synthetic intron-exon-intron sequence associated with motifs required for alternative exon splicing. The exon that contains at least one stop codon and/or any sequence aimed to block translation or perturb mRNA stability, would be alternatively spliced according to the activity of a specific RNA binding proteins. The transgene is a chimeric construct containing a splice-sensor sequence and any defined cDNA.

Thus, the expression of the transgene at the protein level would be under the constant control of the splice-sensor's alternative exon exclusion or inclusion depending on the current demand in the cells.

Material and methods

Constructs preparation

The splice-sensor described here was derived from the human ATP2A1 gene and contains partial exon 21 (34 nucleotides from the 3'-end of the exon), intron 21, exon22, intron 22 and partial exon 23 (22 nucleotides from the 5'-end of the exon). This splice-sensor ATP2A1 exon22 was inserted in front of either an EGFP or a V5-MBNL∆ sequence into a pcDNA3.1(+) expression vector. Both constructs were verified by sequencing. MBNL∆ containing amino acids from 1 to 269 has been derived from MBNL1, tagged to V5 protein and cloned into pcDNA3.1(+) or pSMD2 vectors with a CMV promotor. The MBNL1 isoform 43 was cloned into a pCDNA5/ His/Protein C tag / TEV binding and cleavage site vector.

Cell culture and transfection

A Flp-In TM T-Rex TM 293 cells (Invitrogen) stable cell line containing tetracycline-inducible full length MBNL1 43 isoform protein was generated. Cells were routinely cultured as a monolayer in Dulbecco's modified Eagle's medium (DMEM)-GlutaMax (Invitrogen) supplemented with 10% fetal bovine serum (Gibco) at 37° under 5% CO 2 . Prior to transfection, cells were seeded into twenty-four-well plate at density 1,6 x 10 5 cells/well. Cells were transfected 24h later at approximately 80% of confluence. Plasmid (20 ng/well) was transfected into each well using Lipofectamine LTX Reagent (Invitrogen) following the manufacturer's protocol. After 4h the medium was changed to DMEM supplemented with 5% fetal bovine serum and containing doxycycline at the concentration 1 μg/ml (Sigma-Aldrich). Cells were harvested 48h post-transfection.

In vivo gene transfer

All mouse procedures were done according to the protocol n°01204.02 approved by the Ethical Committee on Animal Resources at the Centre Experimentation Fonctionnelle of Pitie-Salpetriere animal facility and under appropriate biological containment. Adult control WT (FVB) or DM1 (HSA LR ) transgenic mice were intramuscularly injected with saline or AAV9 vectors and sacrificed seven weeks after. AAV9-V5-MBNL∆ or AAV9-splice-sensor-V5-MBNL∆ were produced by the AAV core facility of the Centre de Recherche en Myologie.

RNA isolation and RT-PCR

Total RNA was isolated from cells or muscles using TRI Reagent (Sigma) and 1ug of total RNA was reverse transcribed using M-MLV first-strand synthesis system (Life technologies) in a total of 20 µl. Samples were treated with RQ1 DNase (Promega) and 1ul of cDNA preparation was used in a semi-quantitative PCR analysis according to standard protocol (ReddyMix, Thermo Scientific). PCR amplification was carried out for 30 cycles at 60°.

Western blotting

HEK cell pellets or muscle tissues were lysed in RIPA buffer ( 150 Anti-rabbit (1:20 000, Invitrogen) and anti-mouse (1:2000, Invitrogen) secondary antibodies were conjugated with horseradish peroxidase and detected using the Immobilon Western Chemiluminescent HRP Substrate system (EMD Millipore).

Part 4

Bringing a fundamental knowledge about MBNL1 contribution in DM pathogenesis Introduction Among various processes required for tissue development, the maturation of the transcriptome is finely orchestrated by a multitude of RNA-binding proteins (RBPs) that cooperatively regulate localization, translation, processing and turnover of mRNAs to ensure proteome complexity in a cell-and time-specific manner. Hence, defective functions of some RBPs have been directly linked to a number of human diseases [START_REF] Scotti | RNA mis-splicing in disease[END_REF].

For example, functional loss of MBNL RBPs play a pivotal role in the pathophysiology of myotonic dystrophy disease (DM). In DM1 muscle cells, MBNL1 proteins are sequestered by expanded CUG transcripts leading to the formation of ribonuclear aggregates, so-called foci, and splicing misregulations. Interestingly, MBNL1 it-self also undergoes alternative splicing regulation and various exons combinations give rise to several MBNL1 protein variants with distinct functional characteristics (Konieczny et al., 2014;[START_REF] Pascual | The Muscleblind family of proteins : an emerging class of regulators of developmentally programmed alternative splicing[END_REF]. More recently, it has been suggested that the presence of toxic microsatellite repeats dysregulates the intracellular concentration and partitioning of MBNL1 proteins during early stages of myogenesis in DM1 [START_REF] André | CTG)n repeatmediated dysregulation of MBNL1 and MBNL2 expression during myogenesis in DM1 occurs already at the myoblast stage[END_REF]. Since the regulation of MBNL1 exons 5 and 7 is perturbed in DM1, we investigated their role in muscle cells in relation to the presence of CUGexp-RNA foci. First of all, we confirmed that reduced level of MBNL1 in differentiated DM1 myoblasts is a posttranscriptional event that occurs within the first day of the myogenic differentiation process. Our results suggested that the regulation of the exon 5 is a key determinant for MBNL1 expression, which is further related to the number of the expanded repeats in the toxic RNAs. Additionally, we suggested that the aberrant inclusion of alternatively spliced MBNL1 exon 7 promotes nuclear foci retention and MBNL1 sequestration on expanded RNAs. Altogether we demonstrated that aberrant splicing of MBNL1 transcripts found in DM1 might contribute to the pathophysiologic process of the disease.

Results

The level of MBNL protein decreases during the differentiation process of DM1 myoblasts

Since the expression of DMPK transcripts is regulated during early stages of myogenic differentiation, which is associated with an increase of both mutant and normal DMPK mRNA in DM1 myoblasts (Furling et al., 2001), we assessed whether this change has an impact on MBNL1 protein expression. For this purpose, whole cell protein lysates were prepared from immortalized human control and DM1 myoblasts derived from a healthy individual and a DM1-patient carrying 2600 CTG repeat expansion. In proliferating myoblasts (50% of confluency), the total level of MBNL1 is unchanged between control and DM1 cells. This pattern was sustained in cells that reached 100% of confluency, just before starting the differentiation process (Figure 1a). Noteworthy, protein expression pattern was the same among two cells populations with the highest expression of most abundant low molecular MBNL1 isoforms (40 kDa and 41 kDa). After 4 days of differentiation, the level and expression pattern of MBNL1 in control muscle cells did not differ from the one observed previously in proliferating myoblasts. However, a drastic 70% decrease in MBNL1 expression was observed in DM1 differentiated myotubes that was associated with a switch of MBNL1 isoform and a prominent expression of high molecular protein isoforms (42 kDa and 43 kDa) (Figure 1b).

These isoforms are encoded by MBNL mRNA transcripts containing alternatively spliced exon 5 and exons 5 and 7, respectively. Both exons 5 and 7 of MBNL1 are found to be aberrantly included in muscle tissues from DM1 patients (Botta et al., 2013). Noteworthy, similar observations were noticed in another clone of immortalized DM1 cell line (Figure 1c). Next, we examined whether MBNL decrease is correlated with the time of myogenic differentiation.

We differentiated control and DM1 cells for 24, 48, 72 and 96 hours and assessed the level of the MBNL1 protein at each time point. Surprisingly, we noticed that in DM1 muscle cells the decline in MBNL1 protein expression occurs rapidly, 24h after differentiation and is sustained in differentiated myotubes over time (Figure 1d). This change is concomitant to increased levels of mutant DMPK transcript that occurs during the first 24h of differentiation (Furling et al., 2001). To confirm that altered MBN1 protein composition and expression is not driven by the immortalization process, we assessed the level of MBNL1 in primary human control and DM1 muscle cells carrying the same number of repeats (DM1 parental cells used for immortalization). As shown previously, we did not notice changes in MBNL1 protein level or expression pattern at the proliferating stage. However, myoblast-to-myotube differentiation resulted in similar reduction in MBNL1 expression in DM1 cells as found in immortalized DM1 myoblasts (Figure 1e). These results indicate that MBNL1 expression is perturbed during myogenic differentiation in muscle cells carrying large CTG expansion.

RT-PCR analysis of MBNL1 mRNA splice pattern confirmed an overt inclusion of both exons in differentiated immortalized DM1 muscle cells when compared to controls muscle cells not expressing expanded CUG tracts (Figure 2b). These results suggest that altered MBNL1 protein expression in differentiated DM1 myoblasts is related to a posttranslational process that might rather dependent on mRNA isoforms composition since the steady state level of MBNL1 transcripts remains unchanged between proliferation and differentiation stages.

MBNL1 level depends on the presence of exon 5

Since western blots showed a prominent expression of MBNL1 protein isoforms containing exon 5 (isoform MBNL42) or exons 5 and 7 (isoform MBNL43) in differentiated DM1 myoblasts, we wondered whether altered total MBNL1 protein level could be associated with the abnormal inclusion of exons 5 or 7. Therefore, we decided to selectively reverse exon 5 and 7 inclusion in differentiated DM1 myoblasts. For this purpose, we rationally designed steric hindrance 2'-O-Me antisense oligonucleotides (AONs) that bind to the target pre-mRNA sequence to mask splicing regulatory cis-elements. The consequence is an alternative splicing "switch" from one splicing isoform to another, without direct degradation of the target transcript. The use of selective AONs in differentiated DM1 cells resulted in effective skipping (up to 90%) of both exons, restoring the splicing pattern characteristic for differentiated control muscle cells (Figure 3a). Subsequent immunoblot analysis showed that exon 5 exclusion using the AON ex. 5 results in the re-expression of low molecular weight MBNL isoforms (Figure 3b). Notably, the change in the MBNL1 protein composition in differentiated DM1 myoblasts led to the restoration of the total amount of MBNL1 similar to the level detected in differentiated control myoblasts (Figure 3b). In contrast, skipping of MBNL1 exon 7 did not change the total MBNL1 protein level neither caused detectable switch in the MBNL1 isoforms pattern (Figure 3c). Remarkably, exon 5 skipping does not evoke elevation of total MBNL1 mRNA in DM1-treated cells (Figure 3d) suggesting that the recovery of MBNL1 protein level induced by the skipping of MBNL1 exon 5 is related to the expression of a specific isoform rather than a higher abundance of the MBNL1 transcript per se.

Normalized MBNL1 protein level is not sufficient to reverse DM1-associated phenotypes

We then wondered whether normalized MBNL1 protein level leads to the correction of DM-associated phenotypes in DM1 muscle cells. As differentiated DM1 myoblasts display missplicing abnormalities, we performed RT-PCR on RNA isolated from both not-treated and AON ex. 5-treated DM1 cells. Surprisingly, analysis of MBNL1-dependent pre-mRNA showed no changes in the splicing pattern of any of the transcripts tested (Figure 4a). Since the cells are expressing high level of CUGexp-RNA, we speculated that MBNL1 protein remains sequestered on expanded microsatellites making its functionally inactive. Combined FISH-immunostaining analysis demonstrated that indeed, RNA foci are still present in the AON-treated cells that colocalize with MBNL1 proteins. However, the phenotype of the foci was more variable in AON ex. 5-treated when compared to not-treated DM1 cells. We noticed several individual cytoplasmic foci and general less foci number in the nuclei (Figure 4b); however, these observations were not quantified yet and therefore need further examination.

Because MBNL1 exon 5 is part of a bipartite NLS signal and it has been shown that MBNL isoforms devoid of exon 5 localize in both, nucleus and cytoplasm compartments, we wondered what is the effect of exon 5 removal on cellular localization of MBNL1. Subcellular fractionation followed by western blotting showed that in differentiated DM1 treated myoblasts, MBNL142/MBNL143 variants were more abundant in the nuclear fraction with barely detectable MBNL140/41 cytoplasmic isoforms. In contrast, selective MBNL1 exon 5 skipping greatly increased protein level, causing its predominant localization in the cytoplasmic fraction (Figure 4c). We assumed that the amount of MBNL1 detected in the nuclear fraction constitutes the pool of the protein sequestered on expanded tracks and observed previously in our immunostaining experiment. The predominant MBNL1 protein localization in the cytoplasm together with its functional depletion in the nucleus due to the RNA sequestration, could therefore explain the insufficiency in alternative splicing rescue in AON ex. 5 DM1-treated cells.

MBNL exon 7 promotes nuclear foci retention

While AON-induced skipping of MBNL1 exon 7 did not modulate the level of MBNL1, it remains aberrantly included in DM1 muscle cells. Thus, one of the most prominent MBNL1 variants present in DM1 muscle cells is the isoform MBNL143, containing both exons 5 and 7.

Interestingly, it has been previously suggested that the sequence encoded by exon 7 significantly intensifies interactions between MBNL1 proteins leading to their multimerization [START_REF] Tabaglio | MBNL1 alternative splicing isoforms play opposing roles in cancer[END_REF][START_REF] Tran | Analysis of exonic regions involved in nuclear localization, splicing activity, and dimerization of {Muscleblind}like-1 isoforms[END_REF][START_REF] Yuan | Muscleblind-like 1 interacts with RNA hairpins in splicing target and pathogenic RNAs[END_REF]. This process might have specific implication in protein sequestration on expanded tracts where exon 7 would facilitate the recruitment of other molecules to create homodimers and increase local MBNL concentration on CUG repeats. To assess whether presence of exon 7 contributes to any DM1-specific features, we specifically forced the skipping of exon 7 in DM1 differentiated myoblasts and examine the ribonuclear foci behavior. By FISH analysis, we detected an increase number of foci in the cytoplasm of DM1 myotubes skipped for MBNL1 exon 7 when compared to not treated DM1 cells (Figure 6a). In an attempt to correlate cytoplasmic foci relocation with MBNL1 exon 7 removal, we performed a time course experiment after AON-exon7 treatment (Figure 6b). At the RNA level, we observed that the skipping of the exon starts as soon as 4-6h post-transfection and reaches almost 100% after 16h. Up to 6h post-transfection we did not notice any foci in the cytoplasmic compartment. After 8h, some cytoplasmic foci were detected with the more pronounce phenotype after 16h when the skipping efficiency reached over 80%. Quantification of CUGexp-RNA foci in the treated and not-treated DM1 myotubes carrying 2600 CTG showed that exon 7 removal significantly reduces total number of the foci by 30% (Fig 6c). Subsequently, there was less detectable foci in the nucleus with 65% more foci in the cytoplasmic compartment when compared to not-treated cells. Subsequent immunostaining analysis showed that while MBNL1 protein colocalizes with CUGexp-RNA in the nucleus, cytoplasmic foci that are smaller than those found in the nucleus were almost MBNL1-negative (Figure 6d). RT-PCR analysis of MBNL1-dependent alternatively spliced events did not show missplicing restoration, which is in line with considerable sequestration of the MBNL1 protein in nuclear CUGexp-RNAs (Figure 6d). Altogether our results indicate that the appearance of cytoplasmic foci correlates with the level of exon 7 exclusion and above all, the presence of MBNL1 exon 7 favors the nuclear retention of CUGexp-RNA foci. This finding was reproduced in two other DM1-cellular models, myo-convertible DM1 human fibroblasts carrying 1300 CTG repeats and control myoblast expressing inducible 115 CTG myoblasts undergoing myogenic differentiation. Concomitantly, a 5-fold increment in DMPK mRNA level occurs within the first day of myogenic differentiation in vitro (Furling et al., 2001) suggesting a causal effect of CTG expansion expression on MBNL1 protein level. In differentiated DM1 muscle cells carrying 2600 CTG repeats, the ratio between the DMPK transcript and MBNL1 protein level does not change over the time of differentiation indicating that elevated mutant mRNA expression assures lower MBNL1 protein quantity. However, the steady-state level of MBNL1 mRNA did not change during myoblasts-to-myotubes transition therefore, the reduced level of MBNL1 occurring during this time-frame should be mediated by the post-transcriptional events such as alternative splicing regulation. Consistent with this hypothesis, we observed a considerable change in MBNL1 expression pattern that switch from low to high-molecular weight isoforms during myogenic differentiation of DM1 myoblasts. This result is in accordance with the disproportionate inclusion of alternatively spliced MBNL1 exon 5 and exon 7 found in differentiated DM1 myoblasts underlying the correlation between MBNL1 mRNA and protein splice variants composition. It has been demonstrated that MBNL1 controls its own exon 5 exclusion however the regulation of its exon 7 is not a MBNL1dependent event. We found that forced exon 5 skipping shifts MBNL1 to the cytoplasm and restores its proper level in differentiated DM1 myoblasts. This result indicates that the differences in MBNL1 abundance might be the consequence of distinct stability or translation efficiency of the various MBNL1 isoforms, which can be additionally dependent on subcellular localization. Therefore, further analysis needs to be performed to determine the stability and half-life of the various MBNL1 isoforms. It has been also suggested that depending on the composition of mature mRNA, the efficiency of its nuclear export substantially differs [START_REF] Macmorris | An exon that prevents transport of a mature mRNA[END_REF]. Although the total level of MBNL1 mRNA does not change in the presence of expanded CUG tracts, it would be interesting to quantify the level of particular isoforms in both cellular compartments since it is not excluded that some MBNL1 splice variants might be more prone to degradation. Also, we can hypothesize that excessive abundance of MBNL142 and MBNL143 isoforms could mediate the proteins to the proteasome degradation pathways. Interestingly, the predominant MBNL140/41 isoforms found in control muscle cells are devoid of exon 5 and preferentially localize in the cytoplasm. Nevertheless, they freely shuttle between both compartments allowing effective alternative splicing regulation. In DM1 background, specific MBNL1 exon 5 skipping normalizes the level of MBNL1 protein but is not sufficient to correct aberrant alternative splicing events. However, it cannot be excluded that forced exon 5 exclusion perturb additional regulatory mechanism necessary to assure MBNL1 activity. Recently Wang et al. reported that the ubiquitination of the cytoplasmic MBNL1 protein participates in the control of its function in neuronal cells [START_REF] Wang | Ubiquitination of MBNL1 Is Required for Its Cytoplasmic Localization and Function in Promoting Neurite Outgrowth[END_REF]; however, we were not able to detect such a post-translational modification in our human myoblast cell model in a preliminary experiment (data not shown). Nevertheless, it has been shown that down-regulation of MBNL proteins remarkably impairs foci formation [START_REF] Kino | Nuclear localization of MBNL1: Splicing-mediated autoregulation and repression of repeatderived aberrant proteins[END_REF][START_REF] Querido | Stochastic and reversible aggregation of mRNA with expanded CUG-triplet repeats[END_REF]. We also noticed that shifting the protein out of the nucleus by removing MBNL1 exon 5 affects the foci physiology by decreasing their number and intensity; however further analyses are needed to quantify and confirm these observations. In differentiated DM1 myoblasts carrying 2600 CTG, missplicing of MBNL1 exon 5 leads to a reduce level of MBNL1 protein however, the corresponding MBNL1 isoforms are mainly nuclear due to the NLS signals present within the exon 5.

Next, we were wondering whether missplicing of MBNL1 it-self could contribute to the DM1 disease state? DM1 patients carry from 38 to several thousand repeats units and the size of the repeat tract correlates with the degree of alternative splicing defects and subsequently, severity of the clinical symptoms [START_REF] Yum | Myotonic Dystrophy: Disease Repeat Range, Penetrance, Age of Onset, and Relationship Between Repeat Size and Phenotypes[END_REF]. In our cell models, while 2600 CTG repeats causes 90% inclusion of MBNL1 exon 5, expression of 115 CTG repetitions leads only to half of this change. This nearly 40% inclusion was insufficient to modify the MBNL1 protein expression pattern that seem to be critical in the shift of the protein abundance. Noteworthy, in the well characterized DM1 HSA LR mouse model that carries 250 CTG repeats in the human actin skeletal gene, the number of repetitions provokes a similar 40-50% inclusion of MBNL1 exon 5, which does not reflect in the modifications of MBNL1 isoforms composition and consequently, total MBNL1 protein expression does not differ from the one observed in WT mice. Therefore, it would be interesting to determine whether there is a cut-off in the number of CTG repeats that provokes persistent alterations in the protein expression pattern and detrimental contribution to the disease.

Besides exon 5 involvement, we found that MBNL1 transcripts containing exon 7 are also overrepresented MBNL1 variants in DM1 muscle cells (2600 CTG). Its abnormal inclusion has been previously reported in DM1-patients brain and muscle tissues [START_REF] Dhaenens | Overexpression of {MBNL}1 fetal isoforms and modified splicing of {Tau} in the {DM}1 brain: two individual consequences of {CUG} trinucleotide repeats[END_REF][START_REF] Sznajder | Mechanistic determinants of MBNL activity[END_REF] and has been shown to be responsible for protein homodimerization [START_REF] Tabaglio | MBNL1 alternative splicing isoforms play opposing roles in cancer[END_REF][START_REF] Tran | Analysis of exonic regions involved in nuclear localization, splicing activity, and dimerization of {Muscleblind}like-1 isoforms[END_REF][START_REF] Yuan | Muscleblind-like 1 interacts with RNA hairpins in splicing target and pathogenic RNAs[END_REF]. However, so far, its contribution to the disease has not been investigated. We found that its efficient skipping in DM1 muscle cells broadly affects foci subcellular localization independently of the size of the CTG tract. We therefore assume that its excessive inclusion promotes foci nuclear retention probably due to the formation of stable protein-protein complexes on expanded tracts. Indeed, exon 7 exclusion in DM1 myotubes does not change the level of MBNL1 but led to significant decrease in foci volume and number. It has been shown that RNA-protein aggregations in DM1 have a decreased diffusion rate that might explain the failure of the majority of these mutated transcripts to reach nuclear pores and be exported to the cytoplasm [START_REF] Querido | Stochastic and reversible aggregation of mRNA with expanded CUG-triplet repeats[END_REF]. Noteworthy, we consistently observed in MBNL1-exon 7-skipped-DM1 myotubes that cytoplasmic foci were almost MBNL1-negative. It is plausible, that these RNA-foci were therefore more mobile with an increased diffusion and velocity rates that allowed their transportation from the nucleus. However, it cannot be also excluded that mutated DMPK transcripts detected in the cytoplasmic compartment constitute short stretches of already partially degraded mRNAs that do not sequester MBNL protein. Therefore, further investigation of exon 7 absence on the behavior of the RNA-foci should be pursued.

Materials and methods

Cell culture

Human immortalized myoblasts were cultivated in a growth medium consisting of a mix of 199:DMEM (1:4 ratio; Life Technologies) supplemented with 20% FBS (Life Technologies), 25 μg/ml fetuin, 0.5 ng/ml bFGF, 5 ng/ml EGF and 0.2 μg/ml dexamethasone (Sigma-Aldrich) at 37 °C under 5% CO2. Fibroblasts were cultivated in a DMEM growth medium supplemented with 15% FBS. Myogenic differentiation was induced by switching confluent cell cultures to DMEM for myoblasts, and additional 4 μg/ml of doxycycline (Sigma-Aldrich) for immortalized MYOD1-inducible fibroblasts.

AON design and transfection

AON were designed to block the 3ʹ exon splice sites of either MBNL1 exon 5 or exon 7.

For transfection, differentiated human immortalized myoblasts (2 days) or myotubesconverted DM1 fibroblasts (4 days of differentiation) were treated with AONs at the final concentration of 50 nM using Lipofectamine RNAiMAX transfection reagent (Life Technologies) following the manufacturer's instructions. Cells were harvested for analysis 24h after transfection, if not otherwise stated.

Western blotting

Cell pellets were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 8, 0.1 mM ethylenediamine tetra-acetic acid (EDTA), 1% NP-40, 0.5% sodium dodecyl sulfate (SDS)) supplemented with Complete Protease Inhibitor Cocktail (Roche)). Lysates were sonicated at 4•C and centrifuged at 14 000 × g for 10 min at 4•C. Protein concentration was determined with the BCA protein assay kit (Thermo scientific). Samples were diluted in Laemmli Reducing Sample Buffer supplemented with 50mM DTT, heated to 70 • C for 5 min, separated on 4-12% Bis-Tris polyacrylamide gels (Thermo scientific) and transferred to nitrocellulose membrane (Porablot NCP, Macherey-Nagel) using a wet transfer apparatus (1 h, 100 V, 4 • C). Membrane were blocked for 1 h in 5% milk in PBST buffer (phosphate buffered saline (PBS), 0.1% Tween-20) and incubated with a primary antibody against MBNL1 (MB1a 1:1000), V5 (1:1000, Invitrogen) GFP (1:1000, Clontech) or GAPDH (1:1000, Santa Cruz). Anti-rabbit (1:5000, Invitrogen) and anti-mouse (1:5000, Invitrogen) secondary antibodies were conjugated with horseradish peroxidase and detected using the Immobilon Western Chemiluminescent HRP Substrate system (EMD Millipore).

Subcellular fractionation

Cells were harvested by trypsin-EDTA, collected by centrifugation and washed two times in ice-cold PBS. Remaining cell pellets were resuspended in NC buffer (0,5%NP-40, 150mM NaCl, 1,5mM MgCl2, 10mM Tris pH8) supplemented with cOmplete TM protease inhibitor cocktail (Roche) and incubated for 10min on ice. Nuclei were pelleted by centrifugation at 3500 rpm for 2 min at 4°C and supernatant containing cytoplasmic protein were collected. Remaining nuclei were wash with Lysis buffer and lysed with RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 8, 0.1 mM ethylenediamine tetra-acetic acid (EDTA), 1% NP-40, 0.5% sodium dodecyl sulfate (SDS)) supplemented with cOmplete TM protease inhibitor cocktail (Roche) using the same volume used of NC buffer. Incubated for 10 min on ice, sonicated and centrifuged for 10min at 13000 rpm at 4°C. Supernatant containing nuclei proteins were collected. For Western blot, equal volumes of cytoplasmic and nuclear fractions were analyzed to allow the comparison of a protein's cytoplasmic versus nuclear distribution within the cell.

RNA isolation, RT-PCR and RT-qPCR analysis

Prior to RNA extraction, muscle tissues were lyzed in TRI-Reagent (Sigma-Aldrich) using

Fastprep system and Lysing Matrix D tubes (MP biomedicals. Then, total RNAs were isolated using TRI-Reagent according to the manufacturer's protocol. Total RNA from Hek293T cells and immortalized myoblasts were isolated using TRI-Reagent (Sigma-Aldrich). One microgram of RNA was reverse transcribed using M-MLV first-strand synthesis system (Life technologies) in a total of 20 µl. One microliter of cDNA preparation was subsequently used in a semiquantitative PCR analysis according to standard protocol (ReddyMix, Thermo Scientific). PCR amplification was carried out for 30 cycles for splicing of pre-mRNA within the linear range of amplification for each gene. PCR products were quantified with ImageJ software. The ratio of exon inclusion was quantified as a percentage of inclusion relative to total intensity of isoform signals. To quantify the mRNA expression, real-time PCR was performed using a Lightcycler 480 (Roche). Reactions were performed with SYBR Green kit (Roche) according to the manufacturer's instructions. PCR cycles were a 15 min denaturation step followed by 50 cycles with a 94 °C denaturation for 15 s, 58 °C annealing for 20 s and 72 °C extension for 20 s. Mouse Rrlp0 mRNA were used as standard. Data were analyzed with the Lightcycler 480 analysis software.

Fluorescent in situ hybridization and immunofluorescence

Fluorescent in situ hybridization (FISH) and combined FISH-Immunofluorescence experiments were performed as described previously (Francois et al., 2011). Briefly, a Cy3-labeled 2ʹOMe (CAG)7 probe was used to detect CUGexp-RNA foci. An Alexa 488 conjugated polyclonal anti-GFP antibody (1:200, Life Technologies, A-21311) and a polyclonal anti-MBNL1 antibody (gift from C. Thornton 1:1000, A2764) followed by a secondary Alexa Fluor 647-conjugated goat anti-rabbit (1:500, Life technologies, A-11029) antibody was used to detect GFP-tagged MBNL∆ or MBNL1, respectively. Nuclei were revealed by Hoechst coloration. Images were acquired with a Nikon Ti2 microscope equipped with a motorized stage and a Yokogawa CSU-W1 spinning disk head coupled with a Prime 95 sCMOS camera (Photometrics). Counting of nuclei with foci, number of foci per nuclei or intensity of MBNL1 signal was performed using Fiji software and custom scripts.

Statistical analysis

For statistical analysis, either Student's t-test or one-way analyses of variance were used as appropriate using GraphPad Prism software (Version 6, GraphPad Software Inc.).

General conclusions

Present work aimed to develop a new gene therapy strategy for myotonic dystrophy and more specifically, to neutralize RNA toxicity associated with expanded CUG repeats, which is the central pathophysiologic mechanism of the disease. We showed that a modified derivative of MBNL RNA-binding protein (MBNLD), due to its prominent binding properties to expanded CUG tracts via two tandem Zinc finger domains, acts as a decoy to counteract the CUGexp-RNA gain-of-function effect by displacing sequestered endogenous MBNL proteins and restoring their cellular functions. Thus, expression of MBNLD leads to the reversal of DM1-associated phenotypes in both in vitro and in vivo models. In vitro, global transcriptome analysis by RNA-sequencing showed that the vast majority of missplicing events are no more significantly altered in DM1-treated muscle cells, indicating an almost complete recovery of MBNL1 activity. In vivo, intramuscular injection of AAV vectors carrying the decoy construct assures sustained and robust therapeutic effect including correction of alternative missplicing and myotonia in HSA LR mice. Moreover, comprehensive investigation of several MBNLD derivates allowed us to discriminate the most potent candidate for future preclinical investigations. We noted that besides striking nucleotide similarities between the three MBNL paralogs, MBNL3 N-terminal protein derivative in fusion with a nuclear localization signal displays the highest decoy activity. Additionally, by using our decoy approach and the paradigm of DM1 in which MBNL splicing factors are sequestered by expanded CUG RNA tracts and therefore functionally inactive, we designed a splice-sensor autoregulatory system to control the expression of the decoy transgene product that is govern by MBNL proteins activity. The splice-sensor proved its functionality in both in vitro and in vivo proof-of-concept assays, further underlining its promising therapeutic application.

Altogether, this work supports the development of engineered RNA-binding proteins with improved potency and specificity to expanded microsatellite tracts for therapeutic application in myotonic dystrophy and potentially, other expanded repeat RNA-dominant disorders.

Besides, we suggest that the MBNL1 protein can contribute to the severity of myotonic dystrophy not only through its nuclear sequestration but also through the secondary effect of the expression of long expansions on cellular MBNL1 abundance. We showed that specific isoforms composition of Mbnl mRNA has a direct effect on steady-state level of MBNL1 protein during myogenic process. More specifically, the excessive inclusion of aberrantly spliced alternative exon 5, that is regulated by MBNL1 protein itself, evoked by large microsatellite expansions, directly affects protein expression level that might contribute to the progressive severity of DM. On the other hand, presence of alternatively spliced exon 7 seems to play crucial role in nuclear foci retention. Given the central role of MBNL1 in the pathology of the disease, these fundamental results underline the importance of better understanding of the molecular basis of myotonic dystrophy.

Perspectives

MBNL functions

DM1 very often refers as a 'spliceopathy' due to pronounced alternative splicing misregulations associated with the disease. However, as described in the introduction, MBNL RNA binding protein family is involved in the regulation of other RNA metabolic steps [START_REF] Wang | Transcriptome-wide regulation of pre-mRNA splicing and mRNA localization by muscleblind proteins[END_REF][START_REF] Masuda | CUGBP1 and MBNL1 preferentially bind to 3' UTRs and facilitate mRNA decay[END_REF][START_REF] Taliaferro | Distal Alternative Last Exons Localize mRNAs to Neural Projections[END_REF]Rau et al., 2011;Batra et al., 2014).

Since MBNLD releases endogenous MBNL proteins in DM1 cells, it would be of great importance to investigate whether functional protein restoration by MBNL∆ as well as other therapeutic approaches also improve MBNL functions that are not related to alternative splicing regulation. Unfortunately, no well-established cytoplasmic targets and functions associated with MBNL1 activity have been described in muscle cells yet, in contrast to neuronal cells where cytoplasmic MBNL is implicated in the localization of mRNA transcripts [START_REF] Taliaferro | Distal Alternative Last Exons Localize mRNAs to Neural Projections[END_REF]. Therefore, we could consider testing our decoy approach in neuronal DM1-cellular models with characterized MBNL cytoplasmic activity as currently, we cannot conclude whether MBNLD restores or interferes with cytoplasmic MBNL functions.

Nevertheless, by fusing MBNLD with an NLS signal, we were able to reduce its cytoplasmic localization while maintaining or even improving its decoy ability. In addition to splicing corrections, other nuclear processes regulated by MBNL like alternative polyadenylation (Batra et al. 2014) should be evaluated. Finally, MBNL-dependent or -independent mechanisms could be investigated by more global approaches including transcriptomic and proteomic analyses.

RNA foci dynamic

Although we prove that MBNLD triggers the correction of DM1-associated features through the interference with expanded microsatellite tracts and the release of sequestered endogenous MBNL proteins, the consequence of its binding to nuclear foci, i.e. a decrease in the total number of the foci, was unexpected. To fully understand why the binding of the truncated versions of MBNL proteins perturbs the behavior of the CUGexp-RNA foci would require further investigations. Interestingly, our AON-exon skipping analysis showed that removal of either exon 5 or exon 7, both absent in the sequence of MBNLD, also affects nuclear foci behavior. It might suggest that the C-terminal part of the MBNL protein is crucial for the formation and/or maintenance of the nuclear CUGexp-RNA foci as observed in DM1cells. Live-imagining together with Fluoresence Recovery After Fotobleaching (FRAP) experiments would be helpful to visualize and quantify the immobilization of MBNLD in nuclear RNA aggregates as it has been previously shown that foci are dynamic structures, which freely dissolve and re-agglomerate over time [START_REF] Querido | Stochastic and reversible aggregation of mRNA with expanded CUG-triplet repeats[END_REF][START_REF] Sznajder | Mechanistic determinants of MBNL activity[END_REF]. Moreover, their formation depends on the ribonuclear aggregate composition and different MBNL isoforms exhibit various mobility rates between the expanded tracts [START_REF] Sznajder | Mechanistic determinants of MBNL activity[END_REF]. One plausible explanation for the disappearance of small foci is that larger ribonuclear inclusions are less prone to dissolve when stably bound to MBNLD decoy that are consequently visualized as 'big foci' in our FISH analysis using fluorescent-CAG probes.

Noteworthy, it has been reported that treatment with CAG25 morpholino oligonucleotides targeting CUGexp tracts leads also to the release of sequestered endogenous MBNL and substantial reduction of nuclear foci associated with their cytoplasmic export (Wheeleof et al. 2009). We did not observe cytoplasmic relocation or export of nuclear CUGexp-RNA foci, however it cannot be excluded that MBNLD decoy directly impacts CUGexp-RNA turnover.

Additional experiments to assess and quantify the level and subcellular localization of mutant DMPK mRNA following decoy treatment would be helpful to propose and explore plausible hypothesis to better understand the mechanism involved in mutant DMPK transcript degradation.

DM1 mouse model

So far, in vivo functional validation of the decoy was performed in the DM1 HSA LR mouse model that exhibits only muscle-related disease phenotypes. Systemic AAV9-MBNL∆ administration is currently ongoing in HSA LR mice, however to further determine whether our decoy can reverse other DM1 characteristic features, it is crucial to evaluate its action in another DM1 mouse model such as DMSXL transgenic mice that ubiquitously express more than 1000 CTG repetitions in the human DMPK transgene. These mice display reduced motor performance, decreased muscle strength and DM-characteristic skeletal muscle abnormalities, which are lacking in the HSA LR model, probably due to the small CTG expansion size. Also, DMSXL mice exhibit cardiac features including conduction abnormalities (Algalarrondo et al., 2015), which are the second most common cause of death in DM1 patients. In addition, this mouse model show disease-related changes in the central nervous system similar to the one observed in patients (Seznec et al., 2001;Panaite et al., 2008;Guiraud-Dogan et al., 2007). Investigation of behavioral abnormalities would be very informative since MBNL2 constitutes the paralog the most relevant in the brain and MBNL2-deficiency was associated with DM1 CNS features. Hence, DMSXL mice represent a good model to evaluate the multisystemic effect of either systemic or intrathecal AAV-decoy administration considering the use of either ubiquitous or tissue-specific promoters.

Concluding remarks 1. Therapeutic development for myotonic dystrophy

Since the discovery of DM1-causing mutation in 1992, much progress has been made in identifying molecular targets and developing therapeutic strategies. So far, the RNA toxicity through sequestration of MBNL1 splicing factors that is the best characterized mechanism for DM1 pathology, constitutes the most attractive target for advanced therapies. To date, the most promising results have been achieved with antisense oligonucleotides approaches that either cause CUGexp RNA degradation or bind to expanded tracts to sterically block their interaction with MBNL proteins. The usage of AONs is already under clinical development in other diseases and have been successfully used in spinal muscular atrophy (SMA) (Hua et al. 2010). A great progress in antisense strategy and encouraging results obtained with RNase-H active ASO causing degradation of CUGexp-RNAs, allowed to progress to a phase 1/2a clinical trial. However, the study was not pursued due to the lack of therapeutic benefit after a short period of time, probably due to the inefficient tissue distribution, highlighting bio-distribution and long-lasting effect as critical points to long-term therapy outcome. It needs to be underlined that administration of other therapeutics like small molecules would have to tackle similar obstacles. Additionally, drugs, small molecules and engineered compounds rise the question of potential side effects or toxicity.

More recently, AAV-based gene therapies hold a great promise for therapeutic interventions with potential high efficacy and safety [START_REF] Kotterman | Engineering adeno-associated viruses for clinical gene therapy[END_REF]. So far, few attempts using AAV-mediated gene therapy approach have been proposed for myotonic dystrophy that is not solely a muscular dystrophy but rather a multisystemic disease.

Targeting the muscles

The first described endeavor using AAV-based therapy for myotonic dystrophy has been carried out by the group of Swanson in 2006 [START_REF] Kanadia | Reversal of RNA missplicing and myotonia after muscleblind overexpression in a mouse poly(CUG) model for myotonic dystrophy[END_REF]). The study aimed to overexpress MBNL1 protein in HSA LR mice by intramuscular injection of AAV vectors carrying the sequence of MBNL1-40kDa isoform. While the outcome was rather promising, with significant elevation of MBNL1 expression, reversal of muscle-related missplicing events and long lasting effect, the treatment failed to counteract some disease features including aberrant muscle histology [START_REF] Kanadia | Reversal of RNA missplicing and myotonia after muscleblind overexpression in a mouse poly(CUG) model for myotonic dystrophy[END_REF]. It therefore raises the concern whether the overexpression of a singular MBNL1 isoform would be sufficient enough to assure reversal of all DM-associated phenotypes. Taking into consideration three MBNL paralogs, each containing alternatively spliced exons giving rise to several different isoforms with distinct spatial distribution throughout the body organs and tissues, which MBNL paralog/isoform candidate should be overexpressed? Additionally, all three MBNL paralogs, except for alternative slicing regulation, may vary in their cellular functions that cannot always be compensated by the presence of another paralog. To overcome such concerns, our MBNL∆ approach represents an interesting answer. MBNLD acting as a decoy would allow the release of any MBNL paralogue members and their different isoforms in a tissue-specific manner, independently of the MBNL derivative used as all three paralogs show high affinity for CUGrepeats [START_REF] Sznajder | Mechanistic determinants of MBNL activity[END_REF][START_REF] Yuan | Muscleblind-like 1 interacts with RNA hairpins in splicing target and pathogenic RNAs[END_REF]Haghighat Jahromi et al. 2013;M. Bryan Warf et Berglund 2007). Accordingly, single-intramuscular AAV-MBNL∆ injection in HSA LR mice resulted in long-lasting effect without any signs of toxicity further underlying the efficacy of the treatment and presumable lack of immune response against MBNL∆. Likewise, in contrast to full-length protein overexpression approach, MBNLD provides thorough restoration of DMassociated characteristic features found in HSA LR mice including missplicing abnormalities, myotonia abolition and muscle histology improvement.

Targeting whole body

Successful intramuscular administration of AAV vectors hold a great promise for sustained therapeutic treatment; however, considering myotonic dystrophy being a multisystemic disease, the route of AAV-administration stands the main point that need to be addressed.

Besides latest improvement that has been done in the field of AAV engineering, its systemic use still raises some concerns. AAV tropism, as any other virus, is determined by the specific interaction between distinct viral capsid proteins and their cognate cellular receptors.

Therefore, targeting wide range of organs and tissues might constitute a substantial impediment that need further consideration in AAV-whole-body disease treatment. As an example, AAV vectors generally do not cross blood-brain barrier to reach brain tissues, which might be relevant for myotonic mystrophy patients due to broad central nervous systems impairments. However, the development of new capsids that efficiently transduce the central nervous systems following intravenous administration together with cell-type-specific promoters opens new avenues for AAV-based treatment of multisystemic diseases (Chan et al. 2017). So far, the only attempt for systemic administration of therapeutic agents in DM using AAV vectors was pursued by Pinto et al (Pinto et al. 2017) using a dCas9 approach, however the study was performed solely in newborn HSA LR mice. Although this approach did not address the problem of immune response against the transgene because of the timing of administration, it paved the way for systemic targeting of the disease. Nevertheless, considering that immune reactions are usually mediated against foreign viral particles or proteins, the use of derivates from naturally occurring cell-components might open new avenues. This could be an advantage for the MBNLD decoy used in our study since it is a part of already existing protein that is ubiquitously expressed. However, its immune reactivity has not been tested yet and needs further examination.

The use of RNA-binding proteins for therapeutic application As mentioned previously, RNA binding proteins have essential functions in several aspects of RNA biology and are expressed in a fine-tuned and specific spatiotemporal manner. Given their widespread role, it calls into question the reasonability of RBPs usage for therapeutic application. Recently, Yadava et al. reported that long-term constant overexpression of fulllength MBNL1 protein in mouse model of DM evokes a panel of adverse effects [START_REF] Yadava | MBNL1 overexpression is not sufficient to rescue the phenotypes in a mouse model of RNA toxicity[END_REF]. However, considering the number of diseases associated with altered RNA binding protein functions, including Myotonic Dystrophies, spinocerebellar ataxia type 8 (SCA8) and Fuchs Endothelial Corneal Dystrophy (FECD), rationally designed engineered RBP could constitute an interesting target for therapeutic application. In our study we provide a proofof-concept of the use of RNA-binding proteins derivates for DM1. However, the precise design and expression control stands as primary issues that need to be addressed to prevent off-target effects. We show that the expression of the transgene could be fine-tuned according to current needs by using a chimeric splice-sensor system. Therefore, when rationally designed with improved specificity and rigorous spatiotemporal control of its expression, engineered RBP might hold therapeutic relevance for future interventions.

Treatment of multisystemic diseases

Given the complexity of multisystemic diseases, their treatment constitutes particularly critical challenge. While targeting all affected tissues would be of great benefit, such a therapy does not exist so far. Recently widely studied genome editing holds a great promise for sustained effect, however considerable limitations effectively slower its therapeutic application. Whereas a burden of therapeutic approaches has been proposed for DM, none exert beneficial effect on all disease-associated symptoms. Additionally, it needs to be underline that the disease scope will differ between divers' body tissues. Altogether, it puts emphasis on the development of complementary therapies for multisystemic diseases like DM to ensure highly potent and synergistic therapeutic effect. In the current study, we extensively proved the functionality of decoy-approach in skeletal muscles. However, our knowledge of its therapeutic impact in other tissues like for example heart or brain remains to be determined. Considering limitations of all therapeutic approaches being currently under investigation including tissue distribution, delivery, efficacy and toxicity, we propose our decoy-based gene therapy approach to be considered as a complementary treatment for DM.

The role of MBNL1 in mechanism of myotonic dystrophy

MBNL sequestration and its subsequent functional depletion is considered a central cause of myotonic dystrophy, and the interplay between the CUGexp-RNA and MBNL protein ratio has been proposed to be the major modifier of disease severity [START_REF] Meola | Myotonic dystrophies: An update on clinical aspects, genetic, pathology, and molecular pathomechanisms[END_REF]. Thus, under the condition of small number of CUG repetitions, MBNL proteins are in considerable excess, therefore merely a fraction of MBNLs is titrated on expanded tracts resulting in only partial loss of their activity. However, in the situation of expansion grow and therefore, increase number of CUG binding sites, MBNL depletion becomes more strike leading to its functional loss-of-function and worsening of the disease state. In addition to this disease scenario of CUGexp/MBNL interplay, we found that the level of MBNL1 protein could be reduced in differentiated DM1 muscle cells expressing large CUG expansions. In the results described in the part 4, we showed that alternatively regulated MBNL1 exon 5, which is aberrantly misspliced in DM1, affects the level of MBNL1 protein expression. In the situation of relatively low number of expanded CUG repetitions, MBNL is partially sequestered and causes only minor changes in missplicing events. While the number of CUG repeats increases, alternative splicing misregulations are dramatically exacerbated including marked inclusion of MBNL1 exon 5 containing an NLS signal. This splicing switch causes not only considerable changes in the expression pattern of the protein, but also drastic decrease in the total protein level that remains in the nucleus. Therefore, we propose that the level of MBNL1 it-self being with adequate phenotype-causing mechanism that can add additional layer for therapeutic interventions. Altogether our results put emphasis on the importance to increase our knowledge and understanding of the molecular basis of myotonic dystrophy.

Annexes

INTRODUCTION

Myotonic dystrophy (DM) including both type 1 and type 2 forms is one of the most prevalent muscular dystrophies in adults. Both DM forms are autosomal dominant disorders characterized by myotonia, progressive muscle weakness and wasting, cardiac conduction defects as well as cognitive impairments and endocrine dysfunctions (Harper, 2001). The most common type 1 form (DM1, also called Steinert disease) is caused by the abnormal expansion of a repeated CTG trinucleotide in the 3′UTR of the DMPK gene (Brook et al., 1992;Fu et al., 1992;Mahadevan et al., 1992). The size of the expansion can reach more than 4000 CTG repeats in DM1 patients compared with 5-37 CTG repeats in nonaffected individuals. The unstable CTG expansion increases over successive generations [START_REF] Lavedan | Myotonic dystrophy: size-and sex-dependent dynamics of CTG meiotic instability, and somatic mosaicism[END_REF] and the size of the expanded repeats globally correlates with disease severity [START_REF] Groh | Survival and CTG repeat expansion in adults with myotonic dystrophy type 1[END_REF]. The type 2 form (DM2) is due to a large CCTG expansion that can reach up to 11,000 repeats in the first intron of the CNBP gene (Liquori et al., 2001). Both DM forms share similar clinical features; however, differences exist such as age of onset or pattern of muscle wasting that affects predominantly distal muscles in DM1 and proximal muscles in DM2 (Day and Ranum, 2005). In addition, clinical symptoms are milder in DM2 than in DM1 and, in contrast to DM1, there is no congenital form in DM2.

Myotonic dystrophy is part of a new family of RNA gain-offunction diseases [START_REF] Klein | Gain of RNA function in pathological cases: focus on myotonic dystrophy[END_REF] and both DM forms share a common pathophysiological feature: expression of mutant RNAs containing expanded C/CUG repeats (C/CUGexp-RNA) that are retained in the nucleus as discrete aggregates (Davis et al., 1997;Liquori et al., 2001;Taneja et al., 1995). In skeletal muscles, nuclear aggregates of C/CUGexp-RNAs sequester the regulatory splicing factor MBNL1 leading to its functional loss and subsequently, to alternative splicing misregulation (Fardaei et al., 2002;Lin et al., 2006;Liquori et al., 2001;Mankodi et al., 2001;Miller et al., 2000). More than 40 mis-splicing events have been confirmed in affected skeletal muscles of DM1 patients (Nakamori et al., 2013); in particular, altered splicing of CLCN1, INSR, BIN1 and DMD pre-mRNAs [START_REF] Savkur | Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing[END_REF]Savkur et al., 2001;Fugier et al., 2011;Rau et al., 2015) have been associated with myotonia, insulin resistance, muscle weakness and muscle fiber disorganization, respectively, which are all typical symptoms of myotonic dystrophy.

Although animal models including mouse, fly, zebrafish or worm have been developed during the last 15 years to investigate pathophysiologic mechanisms involved in DM1, and despite the fact that several therapeutic strategies are under development [START_REF] Klein | Therapeutic approaches for dominant muscle diseases: highlight on myotonic dystrophy[END_REF], there is still no cure for DM1 to date. However, it is worth noting that an antisense oligonucleotide (ASO) approach (IONIS-DMPK-2.5Rx) is currently being tested in a Phase 1/2a clinical trial (clinicaltrials.gov, NCT02312011) (Pandey et al., 2015;Wheeler et al., 2012). Mouse models expressing expanded CTG repeats in skeletal muscles have also been used to evaluate the efficacy of therapeutic approaches, including antisense oligonucleotides (ASOs), gene therapies and small molecules, on DM1-associated molecular features such as the presence of nuclear CUGexp-RNA aggregates and alternative splicing misregulation, or muscle dysfunction, such as myotonia [START_REF] Gomes-Pereira | Myotonic dystrophy mouse models: towards rational therapy development[END_REF]. Nevertheless, there remains a need for cellular models to evaluate compounds in vitro to allow middle-or high-throughput screenings, before in vivo validation. For this purpose, primary muscle cell cultures derived from muscle biopsies of DM1 patients represent a valuable model since the CTG expansion is expressed within its natural genomic context. Moreover, DM1 muscle cells show assessable DM1-associated molecular features including CUGexp-RNA nuclear aggregates that sequester MBNL1 and subsequent alternative splicing defects (Botta et al., 2013;Dansithong et al., 2005;Francois et al., 2011;Furling et al., 2001;Holt et al., 2007;Loro et al., 2010). However, several difficulties may limit the use of primary muscle cell cultures, especially considering high-throughput screening approaches that require a large number of cells with a reliable and robust phenotype. A major concern is the accessibility and availability of muscle biopsies from DM patients. In addition, the limited proliferative capacity of adult human myoblasts that is inversely correlated to the age of the dystrophic patients constitutes another constraint [START_REF] Hayflick | The limited in vitro lifetime of human diploid cell strains[END_REF]Renault et al., 2000). Furthermore, proliferation of DM primary myoblasts is reduced when compared with age-matched control myoblasts because of the triggering of premature replicative senescence (Bigot et al., 2009;[START_REF] Renna | Premature senescence in primary muscle cultures of myotonic dystrophy type 2 is not associated with p16 induction[END_REF][START_REF] Thornell | Satellite cell dysfunction contributes to the progressive muscle atrophy in myotonic dystrophy type 1[END_REF]. All these limitations support the development of immortalized human DM muscle cell lines in which replicative senescence is bypassed but that display robust and reliable disease-associated features.

Here, we established and characterized novel DM cellular models of human immortalized muscle cell lines. Proof-of-concept using antisense approaches and transcriptomic analyses was performed to validate these immortalized DM muscle cell lines as dependable tools to assess the in vitro effects of therapeutic compounds on RNA toxicity associated with DM mutations.

RESULTS

Generation of immortalized human DM muscle cell lines

To establish DM myoblast cell lines, primary muscle cell cultures obtained from muscle biopsies of DM patients carrying either a DM1 mutation with 2600 CTG or a DM2 mutation with 4000 CCTG were selected. Characterization of these primary cell cultures showed that DM1 myoblasts form smaller myotubes under differentiation conditions and presented a reduced fusion capacity when compared with non-DM control (Ctrl) myoblasts (Fig. 1A,B). In contrast, the fusion index of differentiated DM2 myoblast cultures was not significantly different from differentiated Ctrl myoblast cultures and the size of the multinucleated DM2 myotubes was comparable to that of Ctrl myotubes, as reported previously for these primary muscle cells (Pelletier et al., 2009). At the molecular level, nuclear RNA aggregates of expanded CUG or CCUG repeats were detected by fluorescent in situ hybridization (FISH) in DM1 and DM2 muscle cells, respectively (data not shown), in agreement with previous studies (Furling et al., 2001;[START_REF] Giagnacovo | Nuclear ribonucleoprotein-containing foci increase in size in non-dividing cells from patients with myotonic dystrophy type 2[END_REF]. Alternative splicing changes affecting BIN1 exon 11, DMD exon 78 and LDB3 exon 11 were also detected in differentiated DM1 muscle cell cultures but not in differentiated DM2 or Ctrl muscle cell cultures (Fig. 1C), in accordance with a previous report in affected muscles of a large cohort of DM1 patients (Nakamori et al., 2013).

For immortalization, primary DM1, DM2 and Ctrl myoblasts were co-transduced with two lentiviral vectors expressing the catalytic subunit of the human telomerase (TERT) and the natural p16 (CDKN2A) ligand, CDK4. Co-transduced cells were then selected as previously described (Zhu et al., 2007;Mamchaoui et al., 2011) and several immortalized myogenic clones were isolated from each population. After testing their ability to differentiate into myotubes, we selected clones showing the highest fusion efficiency, and one clone of each cell line (DM1-cl5, DM2-cl26 and Ctrl-cl4) was further characterized. Under differentiation conditions, immortalized myoblasts behave similarly to the primary myoblast cultures from which they were derived. Thus, DM1-cl5 myoblasts form smaller myotubes than Ctrl-cl4 myoblasts (Fig. 2A) and the fusion index of differentiated DM1-cl5 myoblast cultures was reduced when compared with Ctrl-cl4 myoblast cultures (Fig. 2B). In contrast, DM2-cl26 myotubes were similar to Ctrl-cl4 myotubes and there was no difference between the fusion capacity of differentiated DM2-cl26 and Ctrl-cl4 muscle cell cultures. Then, we investigated whether the molecular hallmarks of DM such as nuclear RNA aggregates and alternative splicing misregulation were found in these muscle cell lines. As expected, FISH and immunofluorescence analyses allowed the detection of nuclear aggregates of CUGexp-and CCUGexp-RNA in immortalized DM1 and DM2 cells, respectively, and the colocalization of MBNL1 with CUGexp-and CCUGexp-RNA foci (Fig. 2C). However the level of CELF1 RNA binding protein that is altered in cells expressing CUGexp-RNA was not significantly changed in either of the immortalized DM1 clones compared with the control (Fig. S1). Furthermore, RT-PCR analysis showed that the splicing pattern of BIN1 exon 11, DMD exon 78 or LDB3 exon 11 pre-mRNAs was significantly deregulated in differentiated DM1-cl5 or DM1-cl6 muscle cells compared with Ctrl-cl4 or Ctrl-cl48 and DM2-cl26 or DM2-cl33 muscle cells (Fig. 2D). Finally, Southern blot analysis indicated that the size of the expanded CTG repeat (∼2600 CTG), was similar between immortalized DM1 myoblasts (either cl5 or cl6) and the parental primary population of DM1 myoblasts used to establish these cell lines (Fig. 2E).

An alternative method to produce human muscle cell lines, when muscle biopsies are not available, is to convert immortalized skin fibroblasts into multinucleated myotubes by forced expression of the myogenic regulator factor MYOD1 (Chaouch et al., 2009;[START_REF] Cooper | Dystrophinopathy carrier determination and detection of protein deficiencies in muscular dystrophy using lentiviral MyoDforced myogenesis[END_REF][START_REF] Lattanzi | High efficiency myogenic conversion of human fibroblasts by adenoviral vectormediated MyoD gene transfer. An alternative strategy for ex vivo gene therapy of primary myopathies[END_REF]. Briefly, primary fibroblasts isolated from a skin biopsy are immortalized by the sole re-expression of TERT [START_REF] Bodnar | Extension of life-span by introduction of telomerase into normal human cells[END_REF] before a subsequent transduction with lentiviral vectors containing an inducible Tet-on MYOD1 construct. Under non-permissive conditions, immortalized cells can proliferate indefinitely, but when supplemented with doxycycline, cells express MYOD1 that activates the myogenic program and their fusion into myotubes. Here, human primary fibroblasts isolated from skin biopsies obtained from a control individual (Ctrl) and from a DM1 patient, were used to establish immortalized myo-converted or transdifferentiated cell lines (hereafter referred to as transdifferentiated myotubes). Of note, both muscle and skin biopsies were obtained from the same DM1 patient. We showed that forced expression of MYOD1 following doxycycline supplementation leads to the formation of large multinucleated DM1 transdifferentiated myotubes comparable to Ctrl transdifferentiated myotubes (Fig. 3A). FISH and immunofluorescence experiments revealed that DM1 myotubes contain nuclear CUGexp-RNA aggregates that colocalized with MBNL1 splicing factor (Fig. 3B). Interestingly, DM1 transdifferentiated myotubes displayed splicing changes of many transcripts including ATP2A1, BIN1, INSR, LDB3,

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RESOURCE ARTICLE

Disease Models & Mechanisms (2017) 10, 487-497 doi:10.1242/dmm.027367

Disease Models & Mechanisms

MBNL1 and TNNT2 (Fig. 3C,D) that are also misregulated in skeletal muscles of DM1 patients (Nakamori et al., 2013). Collectively, these results show that immortalized DM1 cell lines exhibit the characteristic features of DM1 pathology.

DM1 muscle cell lines to assess therapeutic compounds

To determine whether these novel immortalized DM muscle cell lines represent relevant tools for in vitro screening or evaluation of therapeutic approaches, we used as a proof-of-concept, compounds known to neutralize the CUGexp-RNA toxicity in our model of DM1 muscle cell lines. We analyzed the effects of ASOs targeting the expanded CUG tract of mutant DMPK transcripts that inhibit the deleterious sequestration of MBNL1 splicing factor and reduce the number of nuclear CUGexp-RNA aggregates, thereby correcting DM1 splicing defects in DM1 mouse models (Mulders et al., 2009;Wheeler et al., 2009). In accordance with previous results obtained in DM1 myoblasts (Mulders et al., 2009;Pantic et al., 2016), 2′OMe-PT-(CAG)7 ASO treatment of DM1-cl5 myoblasts significantly decreased the number of nuclei with nuclear RNA aggregates (Fig. 4A), which was associated with a redistribution of sequestered MBNL1 proteins into the nucleoplasm (Fig. 4B). DM1 transdifferentiated myotubes were also treated with 2′OMe-PT-(CAG)7 ASO and a similar reduction in the number of nuclear CUGexp-RNA aggregates was observed after treatment (Fig. 4C) as previously shown by Larsen et al. (2011) in DM1 transdifferentiated myoblasts. Next, we tested the effects of 2′OMe-PT-(CAG)7 ASO treatment on splicing misregulation measured in DM1 transdifferentiated myotubes since only mild effects, if any, were described for this ASO both in vivo and in vitro (Mulders et al., 2009;Pantic et al., 2016). In accordance with these studies, splicing changes in BIN1 exon 11, ATP2A1 exon 22, TNNT2 exon 5 and INSR exon 11 (Fig. 4D and data not shown) were not reversed. However, missplicing of MBNL1 exon 5 and LDB3 exon 11 were significantly corrected upon treatment with 2′OMe-PT-(CAG)7 ASO (Fig. 4D).

A second antisense approach using a modified hU7-snRNA conjugated to a (CAG)15 sequence [hU7-(CAG)15] was then assessed in the immortalized DM1-cl5 myoblast cell line.

Previously, we demonstrated the ability of hU7-(CAG)15 to silence CUGexp-RNA and correct splicing as well as differentiation defects in DM1 primary muscle cells (Francois et al., 2011). DM1-cl5 myoblasts were transduced with lentiviral vectors expressing the hU7-(CAG)15 construct and cultures were then switched to differentiation conditions. Myotubes formed by treated DM1-cl5 cells were similar to those observed in Ctrl-cl4 cells (Fig. 5A). The fusion index of differentiated DM1-cl5 myoblast cultures expressing hU7-CAG15 was increased compared with untreated differentiated DM1-cl5 myoblast cultures, and comparable to differentiated Ctrl-cl4 myoblast cultures (Fig. 5B). Moreover, expression of hU7-(CAG)15 antisense transcripts reduced the number of nuclear CUGexp-RNA aggregates in DM1-cl5 muscle cells (Fig. 5C) and partially corrected DM1-associated splicing changes for BIN1 exon 11, DMD exon 78 and LDB3 exon 11 (Fig. 5D).

To evaluate the overall effect of hU7-(CAG)15-antisense on DM1-splicing changes, we performed paired-end RNA sequencing (RNA-seq) on total RNA extracted from Ctrl-cl4, DM1-cl5 and hU7-(CAG)15-treated DM1-cl5 differentiated myoblasts. Unfortunately, the RNA preparation method used in this experiment did not allow an efficient extraction of mutant DMPK transcripts carrying large CUG expansions. Bioinformatics analyses using DEX-seq software showed that 349 alternative splicing events (or exon_bin, listed in Table S1) were differentially regulated (fold change ≥1.5, adjusted P-value for multiple testing ≤0.1) in differentiated DM1-cl5 muscle cells when compared with Ctrl-cl4 differentiated muscle cells. Several mis-spliced events previously validated in skeletal muscles of DM1 patients such as BIN1 exon 11, CAPZB exon 8, DMD exons 71 and 78, GFPT1 exon 9, KIF13A exon 32, ANK2 exon 21, UBE2D3 exon 10 and LDB3 exon 5 were also detected in the differentiated DM1-cl5 myoblast cell line. As shown in Fig. 6A, 53% of abnormal splicing events affect cassette exons and the vast majority of them (84%) are skipped single exon. Additional splicing events, including retention of alternative first or last exons and introns, were also found to be altered at 15%, 20% and 8%, respectively. Interestingly, the hU7-(CAG)15 antisense treatment affected 90% of mis-spliced events in differentiated

Disease Models & Mechanisms

DM1-cl5 myoblasts. The median level of rescue in treated DM1 cells was 41% for excluded events and 37% for included events (Fig. 6B). However, the level of correction in treated DM1 cells when compared with Ctrl cells was different for each mis-spliced event, as shown in Fig. 6C and listed in Table S1, with alternative splicing changes that were fully normalized (58% for excluded events and 51% for included events; red lines), partially corrected (28% for excluded or included events; blue lines) and not corrected or with less than 10% correction (14% for excluded events and 21% for included events; green lines). Globally, more than half (56%) of DM1-misregulated splicing events were no longer significantly altered in treated DM1 muscle cells when compared with Ctrl-cl4

DISCUSSION

Development of relevant and robust cellular models for myotonic dystrophy in which the mutation is expressed within its natural context is essential to facilitate efforts that are pushed forward to identify new therapeutic compounds for this neuromuscular disorder. Among them, DM1-hES human embryonic stem cells and DM1 induced pluripotent stem (iPS) cells have been developed and provide powerful models as they have the ability to differentiate into many different cell types (Gao et al., 2016;Laustriat et al., 2015;Marteyn et al., 2011;Yanovsky-Dagan et al., 2015). Recently, protocols to differentiate human iPS cells in skeletal muscle cells have also been established; however, it remains a time-consuming procedure with variable efficiencies [START_REF] Caron | A human pluripotent stem cell model of facioscapulohumeral muscular dystrophy-affected skeletal muscles[END_REF][START_REF] Chal | Differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy[END_REF]. In addition, primary muscle cell cultures derived from muscle biopsies of DM1 patients that reproduce characteristic molecular disease-associated features such as nuclear CUGexp-RNA aggregates have been used in many studies to either investigate molecular mechanisms or assess therapeutic compounds (Botta et al., 2013;Dansithong et al., 2005;Francois et al., 2011;Furling et al., 2001;Holt et al., 2007;Loro et al., 2010). However, one major limitation of this model, apart from the availability and accessibility of muscle biopsies from DM patients, is the limited proliferative capacity of human primary muscle cells due to replicative senescence (Bigot et al., 2009). To bypass these limitations and generate DM skeletal muscle cell lines, we immortalized human DM1 and DM2 primary myoblasts and selected clones from both cell lines that recapitulate phenotypic and molecular features similar to those found in primary DM muscle cell cultures.

Immortalization of DM muscle cells

To prevent primary human skeletal muscle cells from entering replicative cellular senescence induced by telomere shortening and/or an active p16 pathway, it has been shown that re-expression of the catalytic subunit of the telomerase TERT to maintain telomere lengths associated with overexpression of CDK4, the natural ligand of p16 that inhibits its activity, are sufficient to extend the proliferative capacity and immortalize human primary myoblasts isolated from either healthy donors or muscular dystrophic patients (Mamchaoui et al., 2011). We previously demonstrated that the dual ectopic expression of TERT and CDK4 enables DM1 primary myoblasts to overcome pre-

Disease Models & Mechanisms

senescence and significantly extend their proliferative lifespan (Bigot et al., 2009). Immortalization of DM1 primary myoblast populations can also be achieved by combined ectopic expression of TERT, mutated CDK4(R24C) and cyclin D1 (CCND1), which increases the CDK4 kinase activity for efficient Rb hyperphosphorylation (Pantic et al., 2016;Shiomi et al., 2011). In the present study, transduction of primary myoblasts using lentiviral vectors allows integration of TERT and CDK4 constructs that contain selection markers and subsequently, establishment of stable cell lines. Based on their ability to differentiate and form myotubes, clones of immortalized Ctrl and DM myoblasts were isolated and the fusion index indicated that selected DM clones behave as parental primary muscle cell cultures.

DM-associated molecular features in DM1 and DM2 muscle cell lines

At the molecular level, the presence of nuclear aggregates of either CUGexp-or CCUGexp-RNAs in immortalized DM1 and DM2 myoblasts, respectively, confirmed that both DM mutations were expressed in the corresponding muscle cell lines. Because the nuclear accumulation of mutant DM transcripts alters the function of RNA splicing factors such as MBNL1, misregulation of alternative splicing was assessed in immortalized DM muscle cells. Several abnormal splicing events that were validated in skeletal muscle of DM patients were indeed detected in differentiated DM1 cl5/cl6 myoblasts but not in differentiated DM2 clones, raising several questions. Independent of the nature of the different DM mutations, the size of the expanded repeat tract (2600 CTG in DM1 cells versus 4000 CCTG in DM2 cells) could also impact the pathophysiological mechanisms and ultimately explain the difference in splicing misregulation. To address this question, it would be interesting to have access to DM2 myoblasts with larger CCTG expansions, which can reach up to 11,000 repeats compared with the 5000 repeats in DM1. However, additional parameters may contribute to this difference, as DM2 pathology that affects mostly adults is usually milder than DM1, which affects adults and infants, and displays a severe congenital form (Liquori et al., 2001). Thus, the level of expression of C/CUG-expanded transcripts and the composition and/or the structure of the expanded tract itself (CUG versus CCUG) should also influence the behavior of the ribonucleoprotein complexes and sequestered/associated proteins. Although MBNL1 colocalized with both C/CUGexp-RNA aggregates, the extent of sequestered MBNL1 and subsequently, the levels of remaining free and functional protein may be different in DM1 and DM2 cells, particularly if the dynamics of accumulation of MBNL1 into

Disease Models & Mechanisms

CUGexp-and CCUGexp-RNA aggregates is not the same. Moreover, additional molecular alterations and/or modifiers that have been described in DM1 muscles could also be involved in this difference. Finally, altered myogenic differentiation and fusion of DM1 myoblasts might also interfere with the level of alternative splicing changes. However, this seems unlikely because similar splicing defects were detected in DM1 transdifferentiated myotubes overexpressing MYOD1 that display similar myogenic differentiation to Ctrl transdifferentiated myotubes, as previously described (Larsen et al., 2011). Furthermore, MYOD1 overexpression has also been shown to rescue the differentiation defects observed in myoblasts expressing CUGexp-RNA and associated with a compromised MYOD1 level [START_REF] Amack | Mutant DMPK 3′-UTR transcripts disrupt C2C12 myogenic differentiation by compromising MyoD[END_REF]. Overall, our data demonstrate that the molecular hallmarks associated with the toxic RNA gain-of-function mechanism, such as nuclear RNA aggregates and splicing defects are retained in immortalized DM1 muscle cells, and represent valuable readouts to assess the efficacy of therapeutic compounds.

Therapeutic approach assessment

To confirm that our new immortalized DM1 muscle cell lines are reliable tools for therapeutic compound screenings, we tested antisense approaches that have shown their efficacy to reverse CUGexp-RNA toxicity (Francois et al., 2011;Gonzalez-Barriga et al., 2013;Mulders et al., 2009;Wheeler et al., 2009). We found that CAG antisense treatments that target the expanded CUG tract such as hU7-(CAG)15 or ASO-(CAG)7, decrease the number of nuclear CUGexp-RNA aggregates, which has previously been shown to correlate reduced levels of CUGexp-DMPK mRNA, and correct splicing defects in our immortalized DM1 muscle cells. Of note, the normalization of DM splicing changes following CAG-antisense treatment in immortalized DM1 muscle cell models was not reported before (Pantic et al., 2016;Larsen et al., 2011). Our results are in accordance with an increasing body of evidence, indicating that functional loss of MBNL1 due to its sequestration into nuclear RNA aggregates results in misregulation of alternative splicing [START_REF] Cardani | Muscleblind-like protein 1 nuclear sequestration is a molecular pathology marker of DM1 and DM2[END_REF]Lin et al., 2006;Mankodi et al., 2001;Miller et al., 2000).

As the number of nuclear CUGexp-RNA aggregates sequestering MBNL1 in immortalized DM1 muscle cells is greatly reduced by antisense treatment, we hypothesize that the pool of free and functional MBNL1 is increased leading ultimately to the rescue of abnormal splicing events in DM1 cells. Accordingly, 90% of the 349 mis-spliced events identified by RNA-seq analysis in DM1-cl5 differentiated myoblasts were affected by antisense treatment, and In conclusion, we describe the development and characterization of innovative new immortalized DM muscle cell lines, as well as their potential use to screen therapeutic strategies and compounds. We show that selected clones of immortalized DM1 and DM2 myoblasts behave like parental primary myoblasts isolated from patient muscle biopsies, and contain nuclear aggregates of RNA containing expanded repeat tract, which are typical molecular features associated with the expression of DM mutations. In addition, RNA-seq analysis showed that more than 300 splicing events are altered in immortalized DM1 muscle cells compared with immortalized Ctrl muscle cells. Lastly, as a proof-of-concept, we were able to reverse CUGexp-RNA toxicity associated with nuclear RNA aggregates and abnormal splicing changes in immortalized DM1 muscle cells using CAG-antisense treatments. Therefore, immortalized DM muscle cell lines represent new valuable models for the assessment of potential therapeutic compounds.

MATERIALS AND METHODS

Cell culture

Muscle and skin biopsies were obtained from the MyoBank-AFM bank of tissues for research, a partner in the EU network EuroBioBank, in accordance with European recommendations and French legislation. Muscle

Disease Models & Mechanisms

biopsies were obtained from a semitendinosus muscle of a 25-year-old unaffected male individual (Ctl), a quadriceps muscle of a 53-year-old female patient suffering from DM2 (4000 CCTG in the muscle) and a gastrocnemius muscle of an 11-year-old female patient suffering from an infantile DM1 form (1300 CTG in the blood). Primary cells were isolated from skeletal muscle or skin biopsies as described previously (Chaouch et al., 2009;[START_REF] Edom | Clones of human satellite cells can express in vitro both fast and slow myosin heavy chains[END_REF]. Myoblasts are cultivated in a growth medium consisting of a mix of 199:DMEM (1:4 ratio; Life Technologies) supplemented with 20% FBS (Life Technologies), 50 μg/ml gentamycin (Life technologies), 25 µg/ml fetuin, 0.5 ng/ml bFGF, 5 ng/ml EGF and 0.2 µg/ml dexamethasone (Sigma-Aldrich). Fibroblasts are cultivated in a DMEM growth medium supplemented with 15% FBS and 50 μg/ml gentamycin. Myogenic differentiation is induced by switching confluent cell cultures to DMEM supplemented with 5 µg/ml insulin for myoblasts, and a additional 4 µg/ml of doxycycline (Sigma-Aldrich) for immortalized MYOD1-inducible fibroblasts.

Immortalization of primary myoblast and fibroblast

Immortalization and selection processes were done as previously described (Chaouch et al., 2009;Mamchaoui et al., 2011). Briefly, TERT and Cdk4 cDNAs were cloned into different lentiviral vectors containing puromycin and neomycin selection markers, respectively. Myoblasts were transduced with both TERT and Cdk4 lentiviral vectors while fibroblasts were transduced with TERT vectors only, with a MOI of 3-5 in the presence of 4 µg/ml of polybrene (Sigma-Aldrich). Transduced cell cultures were selected with puromycin (0.2 μg/ml) and/or neomycin (0.3 mg/ml) for 8 days. Transduced cells were seeded at clonal density and selected individual clones were isolated from each population. For conditional expression of MYOD1, immortalized fibroblasts were transduced with lentiviral vectors expressing murine Myod1 cDNA under the control of a Tet-on inducible construct [START_REF] Barde | Efficient control of gene expression in the hematopoietic system using a single Tet-on inducible lentiviral vector[END_REF].

Fluorescent in situ hybridization and immunofluorescence FISH experiments were done as previously described (Francois et al., 2011) using a Cy3-labeled 2′OMe (CAG) 7 or (CAGG) 5 probe. Immunofluorescence (IF) experiments were done as previously described [START_REF] Klein | PABPN1 polyalanine tract deletion and long expansions modify its aggregation pattern and expression[END_REF]

RNA isolation and RT-PCR

Total RNAs were isolated using TRIzol reagent (Life Technologies) according to the manufacturer's protocol. One µg of RNA was reverse transcribed using M-MLV first-strand synthesis system according to the manufacturer's instructions (Life Technologies) in a total volume of 20 µl. One microliter of cDNA preparation was subsequently used for PCR according to standard procedures (ReddyMix, Thermo Scientific). Primers used to analyze the splicing profiles of the different transcripts are indicated in Table 1.

Antisense treatments

For hU7-(CAG)15 expression, 1.5×10 5 immortalized DM1 myoblasts were transduced with 1×10 7 vg/ml of hU7-(CAG)15 lentiviral vectors. Transduction was performed twice for 4 h in the presence of 4 µg/ml of polybrene (Sigma-Aldrich) and the transduced cells were grown at least 1 week before differentiation and/or analyses. For ASO treatment, proliferating immortalized DM1 myoblasts or myotubes-converted DM1 fibroblast cell lines (after 4 days of differentiation) were transfected with 2 µg/ml of 2-OMe-PT-(CAG)7 ASOs using RNAi Max transfection reagent (Life Technologies) according to the manufacturer's protocol. Cells were harvested for analysis 24 h after transfection.

Southern blot analysis

Ten micrograms of genomic DNA were digested by HindIII and SacI and subjected to 0.7% agarose gel electrophoresis for 48 h with 1 kb Ladder (New England Biolabs) as molecular size markers. After denaturation (1 M NaOH) and neutralization 3 M NaCl,pH 8.3), DNA was transferred to a Hybond-N membrane in 6× SSC. Hybridization was carried out using a pM10M6 32 P-labeled probe, a 1.4 kb BamHI fragment containing the CTG repeat tract (Brook et al., 1992;Furling et al., 2001).

The membrane was then washed and analyzed by autoradiography. The size of the CTG repeat was measured using ImageJ software and the value was determined from the middle of the smear. DNA isolated from control cells were used as a negative control.

RNA sequencing and analysis

Total strand RNA-seq was performed by the Centre National de Génotypage (Institut de Génomique, CEA). After complete RNA quality control on each sample (quantification in duplicate and RNA6000 Nano LabChip analysis on Bioanalyzer from Agilent), libraries were prepared using the TruSeq Stranded Total RNA with Ribo-Zero Kit (Illumina), with an input of 1 µg. Library quality was checked by Bioanalyzer analysis, and sample libraries were pooled before sequencing to reach the expected sequencing depth.

Sequencing was performed on an Illumina HiSeq2000 as paired-end 100 bp reads, using Illumina TruSeq V3 reagents RNA-seq reads were trimmed using Trimmomatic [START_REF] Bolger | Trimmomatic: a flexible trimmer for Illumina sequence data[END_REF]) and aligned to human transcriptome using Tophat2 (Kim et al., 2013). Raw count tables were calculated using HT-seq [START_REF] Anders | Detecting differential usage of exons from RNA-seq data[END_REF] and differential splicing events between two groups of samples were identified using DEX-seq [FDR≤0.1, abs(log2 fold change)≥0.58] ( [START_REF] Anders | Detecting differential usage of exons from RNA-seq data[END_REF]. A minimum read coverage of 30 was required in at least one condition to performed analysis at exon level. After analysis, a manual curation was performed to remove remaining technical artefacts and precisely annotate alternative splicing events.

Statistical analysis

All group data are expressed as mean±s.e.m. Between groups, the comparison was performed by Mann-Whitney test or one-way ANOVA with Newman-Keuls post-test using Prism 5 software (GraphPad). Differences between groups were considered significant when P<0.05 (*P<0.05; **P<0.01; ***P<0.001).

INTRODUCTION

Myotonic dystrophies (DM) are a group of dominant disorders that are among the most prevalent neuromuscular diseases in adults (1). he main characteristics of these multisystemic diseases are myotonia, progressive muscle weakness and wasting, cardiac-conduction defects, cognitive impairments together with other endocrine dysfunctions (1). Two DM forms have been identiied so far: type 1 (DM1 also called Steinert Disease) and type 2 (DM2, previously known as PROMM), which is generally less severe than DM1. Both share a similar molecular mechanism in which deleterious expansion of microsatellite repeats in non-coding regions, (CTG)n in 3′UTR of dystrophia myotonica protein kinase (DMPK) gene in DM1 (2)(3)(4) and (CCTG)n in intron 1 of CNBP gene in DM2 ( 5) are transcribed into expanded C/CUG-RNA that are retained in the nucleus as discrete foci. hese ribonuclear foci sequester muscleblind-like (MBNL) RNA-binding proteins, resulting in their functional loss and consequently, RNA metabolism alterations (6)(7)(8)(9)(10)(11). hus, misregulation of alternative splicing events within downstream efector genes were found in striated muscles of DM1 patients and associated with clinical symptoms, such as insulin resistance, myotonia, muscle weakness, and cardiac defects (12)(13)(14)(15)(16)(17)(18).

During the past 20 years, several animal models, including mouse, ly, zebraish, and worm have been developed to investigate DM1 pathophysiologic mechanisms. hey largely contributed to the current state of the art on myotonic dystrophies, which also beneited from research performed on cell cultures. At present, more than 100 years from the irst descriptions of Steinert disease, there is still a need for cellular models to decipher disease-related molecular mechanisms and evaluate therapeutic approaches before in vivo validation. Because DM is a multisystemic disease afecting many tissues and cell types, various cell models are required to cover all DM-associated defects. hanks to technological progresses, we have access to new cellular models allowing more comprehensive and adequate studies. Herein, we will discuss the use of in vitro cell models through the advances in myotonic dystrophy research and describe the available cellular models, from exogenous expression of CTG repeats to patient's derived cells, which were developed for the study of DM1.

CELLULAR MODELS IN DM1 RESEARCH HISTORY

In the early 1900s, Dr. Hans Steinert provided for the irst time a detailed description of a neuromuscular disorder characterized by dystrophic progression with myotonia and degeneration of skeletal muscle (1). Since then, Steinert's disease that was renamed as myotonic dystrophy type 1 or DM1 by the International Myotonic Dystrophy Consortium has been extensively investigated at both clinical and pathophysiologic level. Even before the discovery of the mutation responsible for DM1, primary cells derived from DM1 patients have been used to uncover diferences in behavior or cytochemistry (19)(20)(21)(22) to study metabolism (23)(24)(25)(26) or to understand mechanisms leading to symptoms described in patients, like widely observed insulin resistance (27)(28)(29)(30). However, besides learning about the clinical, physiological, and cellular manifestations of DM1, it was essential to deine the molecular bases of the disease. he irst breakthrough came in 1992, when the mutation responsible for DM1 was identiied as an unstable CTG expansion within the 3′ non-coding region of the DMPK gene (2-4, 27, 31, 32). he next challenge was to understand how this expansion leads to molecular and cellular defects observed in DM1 cells. As it became striking that mutant DMPK mRNA was altered in DM1, the use of diferent cellular models provided initially confusing conclusions about its expression in the disease (33,34). Nevertheless, the observation that the level of mutant DMPK mRNA decreased when the size of the repeats increased, led to the hypothesis that expanded repeats were rather impairing post-transcriptional processing of the mutant DM1 allele (35). he compelling evidence for this postulate came shortly ater, when discrete ribonuclear foci were reported for the irst time in DM1 ibroblasts (36). Additionally, experiments performed with patient-derived myoblasts and ibroblasts determined that mutant DMPK transcripts, while correctly spliced and polyadenylated, were not exported to the cytoplasm but retained in the nucleus (37,38), causing approximately 50% reduction of the DMPK protein levels in DM1 myoblasts (39). hese indings obtained from patient-derived cells gave rise to the idea of a RNA gain-of-function mechanism in DM1. his concept was proposed following the identiication of a RNA-binding protein, CELF1 (also called CUG-BP) that could bind to single-stranded UG motifs and is aberrantly accumulated in the nucleus of cells derived from DM1 patients (40)(41)(42)(43). Upregulation of CELF1 and its splicing regulatory activity have been associated with abnormal splicing of downstream targets, suggesting a trans-dominant efect of CUG repeats on RNA processing in DM1 (18,44) which was further conirmed in cell models overexpressing exogenous CUG expanded tracts with increasing lengths (16). Finally, at the beginning of the 2000s, a second breakthrough has been reached with the identiication of RNA-binding proteins that bind speciically to CUG repeats proportionally to the size of the expansion (45). hese proteins belong to the MBNL family, which includes three paralogs (MBNL1, MBNL2, and MBNL3), and all of them are sequestered within the nuclear RNA foci in DM1 patient cells (46). Among their functions, MBNL proteins are splicing regulatory factors that control developmental switch between fetal and adult isoforms of many transcripts (47). hus, titration of MBNL proteins by nuclear CUGexp-RNA results in alternative splicing misregulations of several pre-mRNAs in DM1, and some of them are associated to DM1 phenotypic features, establishing the deiciency of functional MBNLs as a central cause of the disease (12)(13)(14)(15)(16)(17)(18)(48)(49)[START_REF] Sergeant | Dysregulation of human brain microtubule-associated tau mRNA maturation in myotonic dystrophy type 1[END_REF][START_REF] Tang | Muscle weakness in myotonic dystrophy associated with misregulated splicing and altered gating of CaV1.1 calcium channel[END_REF].

CELL MODELS EXPRESSING EXOGENOUS CTG REPEATS

Several years ater the identiication of the mutation, the expression of exogenously expressed CTG tracts in cellular models was widely used as a tool to conirm the direct role of the repeats in the pathologic mechanisms of DM1. he repeats, usually inserted in the 3′UTR of a truncated DMPK gene commonly under the control of a CMV promoter, are transiently or stably expressed in well-characterized human or murine cell lines, such as HeLa, HEK, or C2 cells. Even if they are lacking the entire genomic context of the CTG expansion and its own speciic promoter regulation, they still recapitulate several DM1-associated features like the formation of ribonuclear foci that colocalizes with MBNL proteins and the splicing defect (16,[START_REF] Ho | Muscleblind proteins regulate alternative splicing[END_REF][START_REF] Warf | MBNL binds similar RNA structures in the CUG repeats of myotonic dystrophy and its pre-mRNA substrate cardiac troponin T[END_REF]. hus, they provide fast and reproducible tools for informative screening readout. Up to date, several constructs containing expanded CTG repeats have been described. A construct expressing interrupted 960 CTG repeats has been used in a wide range of studies including molecular mechanism investigations and validation of therapeutic approaches [START_REF] Konieczny | Myotonic dystrophy: candidate small molecule therapeutics[END_REF]. To ensure the stability of the expansion, the CTG tracts are interrupted with TCGA sequences every 20 repeats (16). Several similar constructs with diferent lengths of CTG repeats and/or promoters were developed (16,[START_REF] Ho | Muscleblind proteins regulate alternative splicing[END_REF][START_REF] Lee | RNase H-mediated degradation of toxic RNA in myotonic dystrophy type 1[END_REF][START_REF] Rolfsmeier | Stabilizing efects of interruptions on trinucleotide repeat expansions in Saccharomyces cerevisiae[END_REF], however, the potential impact of these interruptions is not well deined yet. Additionally, constructs expressing short but pure repeats were also developed. Stable muscle cell lines expressing 200 CTG showed nuclear aggregates of mutated RNA that may cause disruptions in myogenic diferentiation according to the 3′UTR-DMPK environments of the CTG repeats [START_REF] Amack | he myotonic dystrophy expanded CUG repeat tract is necessary but not suicient to disrupt C2C12 myoblast differentiation[END_REF][START_REF] Amack | Cis and trans efects of the myotonic dystrophy (DM) mutation in a cell culture model[END_REF]. Overexpression of large pure repeats is more challenging due to their instability and technical issues associated with the cloning of long tracts of CTG repeats that substantially restricts their length [START_REF] Kang | Expansion and deletion of CTG repeats from human disease genes are determined by the direction of replication in E. coli[END_REF][START_REF] Kim | Generation of long tracts of disease-associated DNA repeats[END_REF]. However, some works partially overcame this constraint by expressing plasmids reaching 800 and 914 uninterrupted CTG repeats in the 3′UTR context of the DMPK gene or in inducible construct expressing GFP, respectively (10,[START_REF] Nakamori | Bidirectional transcription stimulates expansion and contraction of expanded (CTG)*(CAG) repeats[END_REF][START_REF] Nakamori | Oral administration of erythromycin decreases RNA toxicity in myotonic dystrophy[END_REF][START_REF] Sobczak | RNA interference targeting CUG repeats in a mouse model of myotonic dystrophy[END_REF].

In vitro cell models expressing exogenous CTG repeats have been widely used for small molecules screenings, therapeutic approaches, or molecular investigations (16,[START_REF] Konieczny | Myotonic dystrophy: candidate small molecule therapeutics[END_REF][START_REF] Lee | RNase H-mediated degradation of toxic RNA in myotonic dystrophy type 1[END_REF][START_REF] Rolfsmeier | Stabilizing efects of interruptions on trinucleotide repeat expansions in Saccharomyces cerevisiae[END_REF]. However, these models may encounter some limitations associated with the level of CTG overexpression that is not under the control of the endogenous DMPK promoter and the absence of the complete DMPK genomic context that could limit their use in speciic tissue or molecular mechanism studies.

DM1 PATIENTS' DERIVED CELL MODELS

Cells obtained directly from patients are of great utility in modeling human genetic disorders if they reproduce molecular hallmarks of the disease. Regarding DM1 patients' derived cells, they express the whole range of mutation lengths observed in afected individuals within their natural genomic context and reproduce other canonical features of the disease (Figure 1) such as CUGexp-RNA foci that colocalize with the MBNL family members (45,46,[START_REF] Denis | mTOR-dependent proliferation defect in human ES-derived neural stem cells afected by myotonic dystrophy type 1[END_REF][START_REF] Gao | Genome therapy of myotonic dystrophy type 1 iPS cells for development of autologous stem cell therapy[END_REF][START_REF] Holt | Defective mRNA in myotonic dystrophy accumulates at the periphery of nuclear splicing speckles[END_REF][START_REF] Marteyn | Mutant human embryonic stem cells reveal neurite and synapse formation defects in type 1 myotonic dystrophy[END_REF][START_REF] Xia | Dynamic changes of nuclear RNA foci in proliferating DM1 cells[END_REF][START_REF] Xia | Generation of neural cells from DM1 induced pluripotent stem cells as cellular model for the study of central nervous system neuropathogenesis[END_REF][START_REF] Xia | Genome modiication leads to phenotype reversal in human myotonic dystrophy type 1 iPS-cell derived neural stem cells[END_REF], alternative splicing misregulations (48,[START_REF] Gao | Genome therapy of myotonic dystrophy type 1 iPS cells for development of autologous stem cell therapy[END_REF][START_REF] Marteyn | Mutant human embryonic stem cells reveal neurite and synapse formation defects in type 1 myotonic dystrophy[END_REF][START_REF] Xia | Genome modiication leads to phenotype reversal in human myotonic dystrophy type 1 iPS-cell derived neural stem cells[END_REF][START_REF] Arandel | Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds[END_REF][START_REF] Laustriat | In vitro and in vivo modulation of alternative splicing by the biguanide metformin[END_REF][START_REF] Ueki | Myotonic dystrophy type 1 patient-derived iPSCs for the investigation of CTG repeat instability[END_REF], and alterations of metabolic pathways [START_REF] Botta | MBNL142 and MBNL143 gene isoforms, overexpressed in DM1-patient muscle, encode for nuclear proteins interacting with Src family kinases[END_REF][START_REF] Renna | Receptor and post-receptor abnormalities contribute to insulin resistance in myotonic dystrophy type 1 and type 2 skeletal muscle[END_REF]. However, considering variable parameters, like for instance samples from patients with diferent forms of the disease (from congenital to adult), culture conditions, or replicative senescence of primary cells, one should be aware of experimental variability between them. he generation of patient's cell lines or stem cells with their vast reprogramming abilities represent additional tools for deciphering molecular DM1 pathogenesis but also for translational research including drugs screening [START_REF] Laustriat | In vitro and in vivo modulation of alternative splicing by the biguanide metformin[END_REF][START_REF] O'leary | HTS-compatible patient-derived cell-based assay to identify small molecule modulators of aberrant splicing in myotonic dystrophy type 1[END_REF] and therapeutics development [START_REF] Gao | Genome therapy of myotonic dystrophy type 1 iPS cells for development of autologous stem cell therapy[END_REF][START_REF] Xia | Genome modiication leads to phenotype reversal in human myotonic dystrophy type 1 iPS-cell derived neural stem cells[END_REF][START_REF] Francois | Selective silencing of mutated mRNAs in DM1 by using modiied hU7-snRNAs[END_REF][START_REF] Furling | Viral vector producing antisense RNA restores myotonic dystrophy myoblast functions[END_REF][START_REF] Langlois | Hammerhead ribozymemediated destruction of nuclear foci in myotonic dystrophy myoblasts[END_REF].

Human Primary Cells

Currently in the DM1 ield, primary cell cultures mainly consist of dermal ibroblasts and skeletal muscle cells, also called myoblasts. Isolated directly from patient's tissue by either an enzymatic digestion of the extracellular matrix or an explant technique (small sliced pieces of biopsy from which cells grow out), primary cells potentially maintain physiological characteristics of their origin tissue environment. Primary ibroblasts are oten used because of their relative accessibility from patients and their ease of manipulation in culture. On the other hand, primary myoblasts have the advantage to initiate a myogenic diferentiation that results in their fusion into multinucleated cells or myotubes (Figure 1). Concomitantly, the expression of the DMPK gene is upregulated during the myogenic diferentiation process (39) and diferentiated DM1 muscle cells conserve some features found in DM1 muscles such as alternative splicing misregulation of muscle-speciic transcripts (49,[START_REF] Furling | Viral vector producing antisense RNA restores myotonic dystrophy myoblast functions[END_REF][START_REF] Langlois | Hammerhead ribozymemediated destruction of nuclear foci in myotonic dystrophy myoblasts[END_REF][START_REF] Beaulieu | Abnormal prostaglandin E2 production blocks myogenic diferentiation in myotonic dystrophy[END_REF][START_REF] Bigot | Large CTG repeats trigger p16-dependent premature senescence in myotonic dystrophy type 1 muscle precursor cells[END_REF][START_REF] Cardani | Ribonuclear inclusions as biomarker of myotonic dystrophy type 2, even in improperly frozen or defrozen skeletal muscle biopsies[END_REF][START_REF] Furling | Defective satellite cells in congenital myotonic dystrophy[END_REF][START_REF] Furling | Changes in myotonic dystrophy protein kinase levels and muscle development in congenital myotonic dystrophy[END_REF][START_REF] Loro | Normal myogenesis and increased apoptosis in myotonic dystrophy type-1 muscle cells[END_REF][START_REF] Nakamori | Splicing biomarkers of disease severity in myotonic dystrophy[END_REF][START_REF] Perdoni | RNA/MBNL1-containing foci in myoblast nuclei from patients afected by myotonic dystrophy type 2: an immunocytochemical study[END_REF][START_REF] Nakamori | Aberrantly spliced alpha-dystrobrevin alters alpha-syntrophin binding in myotonic dystrophy type 1[END_REF]. Given the relative diiculty to have access to muscle biopsies of dystrophic patients, an alternative source of muscle-like cells was designed. hus, primary ibroblasts were transduced with a viral construct expressing the key myogenic factor MYOD1 to force the expression of the myogenic program. his trans-diferentiation leads to the formation of diferentiated muscle-like cells expressing muscle-speciic transcripts presenting similar splicing abnormalities that those found in DM1 primary myoblasts and patients' muscles (18,[START_REF] Kuyumcu-Martinez | Misregulation of alternative splicing causes pathogenesis in myotonic dystrophy[END_REF][START_REF] Ravel-Chapuis | he RNA-binding protein Staufen1 is increased in DM1 skeletal muscle and promotes alternative pre-mRNA splicing[END_REF]. However, working with primary cells has also some constraints. Asides from the limited accessibility and availability of biopsies from patients, all somatic cells enter into replicative senescence ater a deine number of divisions that is inversely correlated with the age of the donor [START_REF] Haylick | he limited in vitro lifetime of human diploid cell strains[END_REF][START_REF] Renault | Skeletal muscle regeneration and the mitotic clock[END_REF]. his phenomenon is even more pronounced in DM cells as their proliferative capacity is reduced when compared with age-matched control due to a premature entry into replicative senescence [START_REF] Bigot | Large CTG repeats trigger p16-dependent premature senescence in myotonic dystrophy type 1 muscle precursor cells[END_REF]. Another diiculty, except the lengths of the repeats itself, is that primary cells could relect the variability of the individual they are isolated from. Indeed, the age of the donor (fetal vs. adult), the tissue origin (distal vs. proximal muscle), and impairment, and the severity of the patient symptoms could inluence cells behavior when grown in culture. In addition, various optimizations of cell cultures and medias or manipulation conditions may potentially lead to discrepancies between results.

Immortalized Human Fibroblast and Myoblasts

To circumvent the limitation of replicative senescence and keep the cells in a proliferative state, immortalized cell lines from DM1 primary ibroblasts, trans-diferentiated ibroblasts, and myoblasts have been established (Figure 2) [START_REF] Arandel | Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds[END_REF][START_REF] O'leary | HTS-compatible patient-derived cell-based assay to identify small molecule modulators of aberrant splicing in myotonic dystrophy type 1[END_REF][START_REF] Larsen | Myoblasts generated by lentiviral mediated MyoD transduction of myotonic dystrophy type 1 (DM1) ibroblasts can be used for assays of therapeutic molecules[END_REF][START_REF] Pantic | Reliable and versatile immortal muscle cell models from healthy and myotonic dystrophy type 1 primary human myoblasts[END_REF][START_REF] Provenzano | CRISPR/Cas9-mediated deletion of CTG expansions recovers normal phenotype in myogenic cells derived from myotonic dystrophy 1 patients[END_REF]. he immortalization process of human ibroblasts requires the stable re-expression of the human telomerase (hTERT) to prevent the excessive shortening of telomeres that triggers the entry in replicative senescence. Additional inhibition of the dominant p16 pathway by overexpressing CDK4 (the natural ligand of p16) in association, or not, with CCND1 is needed for the immortalization of human myoblasts [START_REF] Mamchaoui | Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders[END_REF][START_REF] Shiomi | CDK4 and cyclin D1 allow human myogenic cells to recapture growth property without compromising diferentiation potential[END_REF][START_REF] Zhu | Cellular senescence in human myoblasts is overcome by human telomerase reverse transcriptase and cyclin-dependent kinase 4: consequences in aging muscle and therapeutic strategies for muscular dystrophies[END_REF]. As a result, the immortalized DM1 cell lines display potentially unlimited number of divisions while keeping most of the tissue-and disease-speciic characteristics. Furthermore, clonal selection leads to homogeneous cell cultures, which allow to provide more consistent and reproducible results. Immortalized cells due to their unlimited lifespan are of special interest when considerably large amounts of cellular material is needed, e.g., for high-throughput screenings [START_REF] Konieczny | Myotonic dystrophy: candidate small molecule therapeutics[END_REF][START_REF] O'leary | HTS-compatible patient-derived cell-based assay to identify small molecule modulators of aberrant splicing in myotonic dystrophy type 1[END_REF][START_REF] Ketley | High-content screening identiies small molecules that remove nuclear foci, afect MBNL distribution and CELF1 protein levels via a PKC-independent pathway in myotonic dystrophy cell lines[END_REF].

Although the immortalized cell lines present a high value, it is not fully determined yet whether the immortalization process that requires viral transduction for genomic integration and stable expression of hTERT and CDK4 transgenes has any consequences on cellular behavior. Further validations and the use of diferent cell lines might also be needed to determine whether increasing number of divisions may alter disease-or tissues-characteristics of immortalized cells.

Human Pluripotent Stem Cells (hPSCs)

Cultures of some primary cells types like human neuronal cells constitute a major challenge due to limited biopsies availability and delay of tissue harvesting. he diiculty to obtain material relecting early stages of the disease process also represents considerable limitations for disease investigations (100). Fortunately, the ability to generate deined cell types from hPSCs ofers a unique opportunity to study disease mechanism in a cell-speciic manner. hPSCs comprising both embryonic and induced pluripotent stem cells (iPSCs), carry the potential to diferentiate them into a wide spectrum of cell types in vitro, including the recapitulation of early human embryo development states (101-103). herefore, they provide an attractive prospect for modeling celltype-speciic disorders.

Embryonic stem cells (hESCs) are isolated from the inner mass of the blastocyst, and can be distinguished by their remarkable long-term proliferative potential along with the ability to diferentiate into practically any cell type (Figure 2) (102). Although hESCs allow the generation of diverse cell types, up to date most of the work in the DM1 ield has been performed in neural stem cells. Lately, they proved to be additionally useful in identiication of new pathways misregulated in the context of DM1 mutationlike disruptions in mTor signaling or defective neuritogenesis [START_REF] Denis | mTOR-dependent proliferation defect in human ES-derived neural stem cells afected by myotonic dystrophy type 1[END_REF][START_REF] Marteyn | Mutant human embryonic stem cells reveal neurite and synapse formation defects in type 1 myotonic dystrophy[END_REF]. However, hESC research raises several ethical issues and has been a subject of controversy over the past 15 years. To obviate the concerns of hESCs, a more recent method of generating patient-speciic cells has arisen. It is built on the discovery that somatic cell nuclei can be "reprogrammed" to an embryonic-like state. he whole process begins for obtaining somatic cells, e.g., ibroblasts, from the afected individuals, which are then subjected to delivery of reprogramming factor cocktails (Figure 2). Such modiied cells are referred to as iPSCs (101). Most importantly, this approach can be applied to human somatic cells, ofering a unique opportunity to derive patient-speciic cell lines from readily available material (100, 101). Several hiPSc derived from primary ibroblasts of DM patients have already been described [START_REF] Gao | Genome therapy of myotonic dystrophy type 1 iPS cells for development of autologous stem cell therapy[END_REF][START_REF] Ueki | Myotonic dystrophy type 1 patient-derived iPSCs for the investigation of CTG repeat instability[END_REF](104)(105)(106)(107). Interestingly, the ability to reprogram DM1 immortalized lymphoblastic cells carrying 200 CTG repeats into hiPSC also opens the possibility to obtain, in the near future, hiPSC directly from patient's blood samples rather than skin biopsies (106).

Intriguingly, regarding the pluripotent state of the cells, CTG repeats are highly unstable during both reprogramming and subsequent passages, with a more rapid expansion when the initial CTG tract is longer (108). Contradictory to what is observed in vivo, several reports have shown CTG repeats instability during culture of undiferentiated DM1-hPSCs but not in diferentiated cells, which might be related to some epigenetic diferences in these cells [START_REF] Ueki | Myotonic dystrophy type 1 patient-derived iPSCs for the investigation of CTG repeat instability[END_REF]108,109). In DM1 maternally derived hESC lines, hypermethylation occurs upstream of CTG repeats when repeats number exceeded 300, however, the hypermethylation observed during reprogramming of patients ibroblasts into hiPSCs is not associated with the expansion of CTG repeats (110,111).

In the past years, a substantial progress in the culture and differentiation technologies associated with hiPSC has been done. Comparing to tissue harvesting, hiPSCs have the advantage of a nearly endless supply. hey might be expanded to large quantities and stored for a future expansion or manipulation. Nevertheless, the equivalence of iPS-derived cells to mature in vivo cells might vary because they oten do not capture the entire mature phenotype. Also, other issues such as homogenous culture of diferentiated cells, chromosomal rearrangement during reprogramming and relatively high cost of hPSC maintenance, constitute additional challenges. However, this technology ofers a unique opportunity to investigate speciic human disease cell types such as neuronal cells or cardiomyocytes for which there are none other or highly limited biological resources.

DM1 CELL MODELS AS A TOOL FOR THE DEVELOPMENT OF THERAPEUTIC APPROACHES

In vitro studies using DM1 cell models contribute also to the development of therapeutic approaches for myotonic dystrophies.

Comprehensive studies have been performed in various DM1 cell models to determine and support translational potential of new strategies. hus, diferent approaches aim to degrade mutant DMPK mRNAs have been tested in DM1 derived cells including gapmer antisense oligonucleotides (ASOs) directed against the CUGexp repeats [START_REF] Lee | RNase H-mediated degradation of toxic RNA in myotonic dystrophy type 1[END_REF]112) or the DMPK transcript it-self (113) as well as shRNA (114), which have showed signiicant eicacy in decreasing the level of CUGexp-transcripts. In another hand, CUGexp-steric blocking approaches by using fully modiied ASOs or viral-derived antisense RNA proved also to be efective in reversal disease molecular features when tested in DM1 cell ular models [START_REF] Francois | Selective silencing of mutated mRNAs in DM1 by using modiied hU7-snRNAs[END_REF][START_REF] Furling | Viral vector producing antisense RNA restores myotonic dystrophy myoblast functions[END_REF]112,115,116). As a matter of fact, DM1 cells are not only used for therapeutic compounds validation. Indeed this tool is also utilized in screening assays allowing the identiication of molecules that either interfere with the abnormal MBNL1:CUGexp interaction such as pentamidine (117,118) or lomofungin (119), reduce the expression of mutant DMPK mRNAs like actinomycin D, modulate splicing changes [START_REF] Laustriat | In vitro and in vivo modulation of alternative splicing by the biguanide metformin[END_REF] or afect the behavior of nuclear foci (120-122). Besides, reliable DM1 cell models are essential in the perspective of the recent progress made in genome engineering. TALEN and CRISPR-Cas approaches are being successfully applied in diferent disease cell ular models giving rise to a wide range of possibilities for future therapeutic interventions [START_REF] Gao | Genome therapy of myotonic dystrophy type 1 iPS cells for development of autologous stem cell therapy[END_REF][START_REF] Xia | Genome modiication leads to phenotype reversal in human myotonic dystrophy type 1 iPS-cell derived neural stem cells[END_REF][START_REF] Provenzano | CRISPR/Cas9-mediated deletion of CTG expansions recovers normal phenotype in myogenic cells derived from myotonic dystrophy 1 patients[END_REF](123)(124)(125). DM1 cell models constitute, therefore, an inescapable source and a lexible platform for thorough studies and validation of disease therapeutics.

CONCLUSION

hrough the years, cultured cells showed to be an essential model for both fundamental and translational research on myotonic dystrophy. Despite the fact that cells do not relect the complexity of a whole organ or body, each cellular model, from the patients' derived cells to more artiicial models overexpressing CTG expanded tracts, is suitable for diferent investigations. hey were and are used in many studies addressing various questions related to myotonic dystrophy diseases like mutation lengths, instability, polymorphisms or tissue-speciic mechanisms, molecular alterations, and efect of therapeutic approaches. Understanding all of those features paved the way to decipher molecular basics of DM1 and DM2, as both forms share common features, i.e., abnormal expansion of repeated sequences, formation of RNA-positive foci and trans-dominant efect on alternative splicing. Even though DM1 cells served as archetype for DM research, it is noteworthy that some cellular models have been also established for myotonic dystrophy type 2. Further investigations in those cells may emphasize the diferences between both DM forms and promote better understanding of their pathological mechanisms. Besides, recent advances in cellavailability and -engineering have given rise to unprecedented experimental opportunities to study disease mechanisms and therapeutic strategies. Late genome engineering tools, with the particular use of emerging development of TALENs and the CRISPR-Cas9 systems, facilitate the next generation of therapeutic interventions and hold a great promise for permanent

Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .

 123456 Figure 1. Nucleotide repeat lengths within the human genome ............................................. Figure 2. Unusual DNA structures formed by expandable repeats .......................................... Figure 3. Molecular mechanism of microsatellite expansions ................................................. Figure 4. Location of expandable repeats responsible for human diseases ........................... Figure 5. Genomic localization of nucleotide expansions responsible for myotonic dystrophies .......................................................................................................................... Figure 6. Multisystem effect of myotonic dystrophy.............................................................Figure 7. Presence of myotonia in DM patients .................................................................... Figure 8. Histological features of muscles in DM1 and DM2 ................................................. Figure 9. RNA foci in myotonic dystrophy ............................................................................. Figure 10. MBNL sequestration ............................................................................................ Figure 11. Molecular mechanism underlying DM pathology ................................................. Figure 12. MBNL1, MBNL2 and MBNL3 genomic composition .............................................. Figure 13. The level of mRNA expression of MBNL paralogs in various organs and tissues ... Figure 14. ZnF domains architecture of MBNL proteins ........................................................ Figure 15. Schematic representation of MBNL-dependent alternative exons regulation ...... Figure 16. Schematic representation of alternative polyadenylation regulation ................... Figure 17. Schematic representation of the MBNL-dependent mRNA localization ................ Figure 18. Therapeutic interventions for myotonic dystrophy type 1 ................................... Figure 19. Potential additional mechanism of RNA toxicity in DM1 ....................................

Figure 7 .Figure 8 .Figure 9 .Figure 10 .Figure 11 .Figure 13 .Figure 14 .Figure 15 .Figure 16 .Figure 17 .Figure 18 .Figure 19 .Table 1 . 2 . 3 .

 789101113141516171819123 Figure 1. Nucleotide repeat lengths within the human genome ............................................. Figure 2. Unusual DNA structures formed by expandable repeats .......................................... Figure 3. Molecular mechanism of microsatellite expansions ................................................. Figure 4. Location of expandable repeats responsible for human diseases ........................... Figure 5. Genomic localization of nucleotide expansions responsible for myotonic dystrophies .......................................................................................................................... Figure 6. Multisystem effect of myotonic dystrophy.............................................................Figure 7. Presence of myotonia in DM patients .................................................................... Figure 8. Histological features of muscles in DM1 and DM2 ................................................. Figure 9. RNA foci in myotonic dystrophy ............................................................................. Figure 10. MBNL sequestration ............................................................................................ Figure 11. Molecular mechanism underlying DM pathology ................................................. Figure 12. MBNL1, MBNL2 and MBNL3 genomic composition .............................................. Figure 13. The level of mRNA expression of MBNL paralogs in various organs and tissues ... Figure 14. ZnF domains architecture of MBNL proteins ........................................................ Figure 15. Schematic representation of MBNL-dependent alternative exons regulation ...... Figure 16. Schematic representation of alternative polyadenylation regulation ................... Figure 17. Schematic representation of the MBNL-dependent mRNA localization ................ Figure 18. Therapeutic interventions for myotonic dystrophy type 1 ................................... Figure 19. Potential additional mechanism of RNA toxicity in DM1 ....................................

Figure 12 .

 12 Figure 12. MBNL1, MBNL2 and MBNL3 genomic compositionSchematic representation of MBNL1, MBNL2 and MBNL3 genes. Localized on chromosomes 3, 13 and X, respectively, each paralog contains several alternatively spliced exons giving rise to different isoform (based on[START_REF] Pascual | The Muscleblind family of proteins : an emerging class of regulators of developmentally programmed alternative splicing[END_REF].

Figure 14 .

 14 Figure 14. ZnF domains architecture of MBNL proteins a, schematic representation of four ZnF domains in MBNL proteins with indicated cysteine and histidine spacing. Structures of free MBNL1 ZnF1-2 b, and ZnF3-4 c, and in complex with RNA sequence (d and e) (adapted from Teplova and Patel, 2008).

  Several consequential studies identified minimal consensus motif for MBNL binding as YGCY (where Y indicate U or C)

Fig. S1 .

 S1 Fig. S1. Function of MBNL∆.

Fig. S2 .

 S2 Fig. S2. CUGexp-RNA foci fate is changed by MBNL∆ prior to missplicing correction Fig. S3. Silencing of MBNL proteins abolished MBNL∆ effects. Fig. S4. In vivo colocalization of MBNL∆ with CUGexp-RNA foci in myonuclei Fig. S5. Level of MBNL∆ overexpression in HSA LR muscles Fig. S6. Expression of MBNL∆ in muscles of WT mice has no effect.

Figure 1 .

 1 Figure 1. MBNL∆ derivatives. Schematic representation of eight MBNL∆ expression constructs fused to V5-tag used in the study.

  mM NaCl, 50 mM Tris-HCl pH 8, 0.1 mM ethylenediamine tetra-acetic acid (EDTA), 1% NP-40, 0.5% sodium dodecyl sulfate (SDS) supplemented with Complete Protease Inhibitor Cocktail (Roche). Lysates were sonicated at 4 • C and centrifuged at 14 000 × g for 10 min at 4 • C. Protein concentration was determined with the BCA protein assay kit (Thermo scientific). Samples were diluted in Laemmli Reducing Sample Buffer supplemented with 50mM DTT, heated to 70 • C for 5 min, separated on 4-12% Bis-Tris polyacrylamide gels (Thermo scientific) and transferred to nitrocellulose membrane (Porablot NCP, Macherey-Nagel) using a wet transfer apparatus (1 h, 100 V, 4 • C). Membrane were blocked for 1 h in 5% milk in PBST buffer (phosphate buffered saline (PBS), 0.1% Tween-20) and incubated with a primary antibody against MBNL1 (MB1a 1:1000), V5 (1:1000, Invitrogen) GFP (1:1000, Clontech) or GAPDH (1:1000, Santa Cruz).

Annex 1 :

 1 Article 1 Arandel L, Polay-Espinoza M, Matloka M, Bazinet A, De Dea Diniz D, Rau F, Jollet A, Edom-Vovard F, Mamchaoui K,Tarnopolsky M, Puymirat J, Battail C, Boland A, Deleuze JF, Mouly V, Klein AF, Furling D, Immortalized human Myotonic Dystrophy muscle cell lines to assess therapeutic compounds, Disease models&mechanisms, 2016 Annex 2: Article 2 Matloka M, Klein AF, Rau F, Furling D, Cells of matter-in vitro models for myotonic dystrophy, Frontiers Neurology, 2018

Fig. 1 .

 1 Fig. 1. Features of primary DM muscle cells. (A) Control (Ctrl), DM1 and DM2 primary myoblasts were differentiated for 4 days and then fixed for immunofluorescence analysis using desmin antibody (green) and nuclei staining (blue) with Hoechst. (B) The fusion index of differentiated primary muscle cell cultures determined by the percentage of the number of nuclei in myotubes (>2 myonuclei) to the total number of nuclei in desmin-positive cells (n=4 for Ctrl, n=3 for DM1 and DM2, >1000 nuclei counted per experiment). (C) RT-PCR analysis of alternative splicing profile of BIN1 exon 11, DMD exon 78 and LDB3 exon 11 for differentiated cultures of Ctrl, DM1 and DM2 primary muscle cells (n=3 in duplicate for each condition). Data are expressed as mean±s.e.m. Comparison with one-way ANOVA with Newman-Keuls post-test; **P<0.01.

Fig. 2 .

 2 Fig. 2. Characterization of DM myoblast cell lines. (A) Representative cultures of Ctrl (cl4), DM1 (cl5) and DM2 (cl26) immortalized myoblasts differentiated for 4 days and then fixed for immunofluorescence analysis using desmin antibody (green) and Hoechst nuclear staining (blue). (B) The fusion index of differentiated immortal myoblast cultures was determined by the percentage of the number of nuclei in myotubes (>2 myonuclei) to the total number of nuclei in desmin-positive cells (n=3 for each condition, >1000 nuclei counted per experiment). (C) Nuclear RNA aggregates of expanded-CUG or -CCUG repeats in immortalized DM1 and DM2 myoblasts were detected by FISH using a Cy3-(CAG)7 or Cy3-(CCAG)5 probe (red), respectively. (D) Quantification of alternative splicing profile of BIN1 exon 11, DMD exon 78 and LDB3 exon 11 by RT-PCR analysis in differentiated cultures of Ctrl, DM1 and DM2 immortal myoblast cell lines (n=3 for each condition). (E) Southern blot analysis using a DMPK probe to determine the size of the CTG expansion in primary DM1 muscle cells (Pr) and two clones of immortalized DM1 myoblasts (Cl5 and Cl6) derived from the same primary muscle cell culture. Data are expressed as mean±s.e.m. Comparison with one-way ANOVA with Newman-Keuls post-test; **P<0.01, ***P<0.001.

Fig. 3 .

 3 Fig. 3. Characterization of DM1 and Ctrl myotube cultures derived from conditional MyoD-converted fibroblast cell lines. (A) Desmin (Desm) immunofluorescence staining (green) of myotube cultures derived from conditional MYOD1 converted Ctrl (left) and DM1 (right) fibroblast cell lines under permissive conditions to express MYOD1. (B) FISH using a Cy3-CAG7 probe (red) and MBNL1 immunostaining (green) showing the colocalization of MBNL1 with nuclear CUGexp-RNA aggregates in muscle converted DM1 immortalized fibroblasts. Hoe, Hoechst staining. (C,D) RT-PCR analysis and quantification of alternative splicing changes in ATP2A1, BIN1, IR, LDB3, MBNL1 and TNNT2 transcripts extracted from myotube cultures derived from converted DM1 and Ctrl fibroblast cell lines (n=4 for each condition). Data are expressed as mean±s.e.m. Comparison with Mann-Whitney test; **P<0.01.

Fig. 4 .

 4 Fig. 4. (CAG)7-ASO reverses CUGexp-RNA toxicity in immortalized muscle DM1 cells. (A) Percentage of immortalized DM1 myoblasts containing nuclear CUGexp-RNA aggregates (foci) following 2′OMe-(CAG)7 treatment (n=4). Data are expressed as mean±s.e.m. Comparison with Mann-Whitney test; ***P<0.001. (B) FISH immunofluorescence staining using a Cy3-(CAG)7 probe and an anti-MBNL1 antibody to detect nuclear foci (red) and MBNL1 (green), respectively, in immortalized DM1-cl5 myoblasts treated with 2′OMe-(CAG)7 ASOs compared with non-treated (NT) cells. (C) FISH experiment to detect CUGexp-RNA foci (red) in the nucleus (blue) of myotubes derived from converted DM1 fibroblast cell lines treated with 2′OMe-(CAG)7 ASOs. (D) Quantification of MBNL1 exon 7, LDB3 exon 11 and BIN1 exon 11 splicing profiles determined by RT-PCR in myotubes cultures of converted Ctrl and DM1 fibroblast cell lines treated or not with 2′OMe-(CAG)7 ASOs (n=8, except for BIN1 where n=6). Data are expressed as mean±s.e.m. Comparison with one-way ANOVA with Newman-Keuls post-test; **P<0.01, ***P<0.001; ns, not significant.

Fig. 5 .

 5 Fig. 5. hU7-(CAG)15 reverses CUGexp-RNA toxicity in DM1 myoblast cell lines. (A) Differentiation of DM1-cl5 myoblast cell line expressing hU7-(CAG)15 antisense transcripts was assessed by desmin (green) and Hoechst (blue) immunostaining and compared with differentiated DM1-cl5 and Ctrl cells. (B) Fusion index of differentiated cultures of immortalized DM1 myoblasts transduced or not with hU7-(CAG)15 lentiviral vectors compared with Ctrl-cl4 (n=4 for each condition, >1000 nuclei counted per experiment). Data are expressed as mean±s.e.m. Comparison with Mann-Whitney test; *P<0.05. (C) Nuclear CUGexp-RNA aggregates in differentiated DM1-cl5 myoblast cell line expressing hU7-(CAG)15. (D) RT-PCR analysis of BIN1 exon 11, DMD exon 78 and LDB3 exon 11 splicing profiles in differentiated cultures of immortalized Ctrl-Cl4 and DM1-cl5 myoblasts transduced or not with hU7-(CAG)15 lentiviral vectors (n=6 for each condition). Data are expressed as mean±s.e.m. Comparison with oneway ANOVA with Newman-Keuls post-test; ***P<0.001.

Fig. 6 .

 6 Fig. 6. Effect of hU7-(CAG)15 antisense oligonucleotide on global DM1-splicing changes. Total RNA extracted from Ctrl-cl4, DM1-cl5 and hU7-(CAG)15-treated DM1-cl5 differentiated myoblasts analyzed with a paired-end RNA sequencing (RNA-seq). (A) Repartition of 349 mis-splicing events in DM1-cl5 compared with Ctrl-cl4 cells (number of events; percentage compared with total number of events). (B) Box plot representing splicing inclusion or EUC (exon usage coefficient, determined with DEX-seq software) of the 349 splicing events (exon_bin) regrouped in excluded exons (n=245, left panel) and included exons (n=104, right panel) in Ctrl-cl4, DM1-cl5 and DM1-cl5 treated with hU7-(CAG)15. In box plot, lower whisker represents smallest observation greater than or equal to lower hinge-1.5x interquartile range (IQR); lower hinge is 25% quantile, middle line is median; upper hinge is 75% quantile; upper whisker is largest observation less than or equal to upper hinge+1.5xIQR. (C) Analysis of individual exon_bin exclusion (left panel) or inclusion (right panel) event in DM1-cl5 cells compared with Ctrl-cl4 cells. More than 50% of events are fully normalized (red), 28% are partially restored (blue) and around 20% are not significantly modulated (green) by treatment with hU7-(CAG)15.

FIGURE 1 |

 1 FIGURE 1 | Features of DM1 cells in vitro.

3

  Matloka et al. Cellular Models for DM1 Frontiers in Neurology | www.frontiersin.org May 2018 | Volume 9 | Article 361

FIGURE 2 |

 2 FIGURE 2 | Models of DM1 patients' derived cells.

-muscular symptoms 2.3.2.1 Central nervous system

	Brain involvement in both myotonic dystrophies show similar cognitive and behavioral
	dysfunctions, with slightly milder manifestation in DM2. Nevertheless, the most common
	reported CNS-related symptoms include fatigue, apathy, and cognitive impairment with low
	IQ scores, altered visuo-spatial and executive functions, reduced attention, avoidant
	behavioral trait, and depression. In DM1, cognitive problems are broadly associated with CTG
	repeat size as congenital form of the disease is characterized by very severe cognitive delay
	and mental retardation. Besides cognitive and behavioral features, DM patients suffer from
	excessive daytime sleepiness and sleep apneas. MRI studies revealed diffuse brain atrophy
	and white matter lesions which have been found to correlate with cognitive impairment in a
	group of DM1 patients (reviewed in

Table 4 . Mouse models for myotonic dystrophy Mouse model Rationale Features Limitations Contribution Reference Knock-out and overexpression models

 4

	Dmpk knockout	DMPK haploinsufficiency	Overall normal; older mice	Lack of symptomps; conflicting	Established that DM1 is not primarily	Reddy et al., 1996
		in DM1	develop calcium homeostasis	reports on impacts of Dmpk ablation	caused by DMPK haploinsufficiency	Carrell et al., 2016
			problems	in mice
	Six5 knockout	Six5 haploinsufficiency	Cataracts, reduced male fertility,	Animals develop nuclear cataracts	Established that DM1 is not caused by	Klesert et al., 2000
		in DM1	heart problems	different from the subcapsular	DMPK haploinsufficiency	Sarkar et al., 2000
				cataracts in DM1		Wakimoto et al., 2002
						Sarkar et al., 2004
	Cnbp knockout	CNBP haploinsufficiency	Heterozygous mice present	Conflicting reports of CNBP	haploinsufficiency of CNBP causes	Chen et al. 2003
		in DM2	myotonia, cardiac conduction	downregulation in DM2; lack of	several major symptoms of DM2	Margolis et al., 2006
			defects, cataracts, and myopathic	strong alternative splicig alterations		Chen et al., 2007
			features			Raheem et al., 2010
	Mbnl1	Mbnl1 loss-of-function	Myotonia, aberrant splicing,	No muscle weakness, normal CELF1	The loss of MBNL1 leads to the major	Kanadia et al., 2003
	knockout		centralized myonuclei, subcapsular	expression level; not a CCTGexp	features observed in DM	Lee et al., 2013
			cataracts, muscular and cardiac	model so lacking some repeat-
			myopathy	associated mechanisms like RAN
				translation
	Mbnl2	Mbnl2 loss-of-function	Myotonia, myopathy, aberrant	Alterations mainlyin the brain;	The loss of MBNL2 is responsible for	Charizanis et al., 2012
	knockout		splicing, DM-relevant learning	not a CCTG exp model so lacking	several specific aspects of DM, mainly
			deficits and REM sleep	some repeat-associated mechanisms	in the brain
			disturbances	like RAN transmation
	Mbnl3	Mbnl3 loss-of-function	Impairment at later stages of	Absence of nuclear isoform of the	The loss of MBNL3 could participate to	Poulos et al., 2013
	knockout		muscle regeneration	protein, low expression level of	the progressive muscle weakness and
				MBNL3 in adult tissues	wasting observed in DM1
	Mbnl1 /Mbnl2	Constitutive deletion of Mbnl1	Growth retardation, myotonia,	High mortality, reproductive	Loss of both MBNL1 and MBNL2 leads	Lee et al., 2013
	double-knockout	and conditional deletion of	muscular atrophy, locomotion	problems, phenotype limited to	to severe phenotypes, characteristic of	Chou et al., 2017
		Mbnl2 in skeletal muscles	difficultiess, strong deregulation of	skeletal muscles	congenital DM1 patients;
			alternative splicing, heart		constitutive deletion of MBNL1 and
			conduction block, clinical myofibril		MBNL2 leads to an early embryonic
			ultrastructural abnormality and		death
			cardiac arrhythmias
	Mbnl1	Constitutive deletion of Mbnl1	Post-natal growth retardation,	Only muscle-specific knockout	Provides a model to study congenital	Thomas et al., 2017
	/Mbnl2/Mbnl3	and conditional deletion of	muscle wasting and weakness,	model	form of DM1
	triple-knockout	Mbnl2 and Mbnl3	muscle histopathology, respiratory
			distress, missplicing
	CELF	Overexpression of CELF in	Muscular and cardiac myopathy,	Premature lethality, only partial	Overexpression of CELF1 recapitulates	Timchenko et al., 2004
	overexpression	skeletal muscle or heart	heart conduction defects,	reproduction of symptoms, limited	some features of DM1	Ward et al., 2010
			missplicing of CELF1 targets	applications for therapeutic		Koshelev et al., 2010
				development
	MBNL2 deficient	MBNL2 gene trap-insertion of	Myotonia, lardosis, decreased	Lack of study of brain function,	Independent role of MBNL2 in DM1	Hao et al., 2008
		mutation in intron 2	fiber CSA, cental nuclei, fibrosis,	conflicing reports with other studies	pathogenesis
			Clcn1 splicing alteration
		MBNL2 gene trap -insertion of	No myotonia, normal muscle	Conflicing reports with other studies No direct role of MBNL2 in DM1	Lin et al., 2006
		GT in Mbnl2 intron 4	histology, no muscle splicing		skeletal muscle pathogenesis
			abnormalities
	Nestin-Cre	MBNL1 constitutive nulls with	DKOs only survived until ∼23	Phenotype of the double-knockout	Compound loss of MBNL1 and MBNL2	Goddwin et al., 2015
	double-knockout	MBNL2 expression selectively	weeks of age, early-onset severe	limited only to the nervous system,	is required for loss of adult exon
		ablated in the nervous system	motor and grip deficits	mice do not survive longer than a	splicing in the mammalian brain,
				couple of months	MBNL depletion in the mouse brain
					recapitulates the aberrant CNS splicing
					patterns of DM1 and DM2

To xic RNA expresison models

	Dmt	First DM1 transgenic model.	Contractions and expansions of	Absence of promoter elements	Occurrence of somatic and	Monckton et al., 1997
		Transgene comprises ∼162 CTG	the repeats (-7 to +7), parent-of-	prevents transgene expression,	intergenerational instability	Fortune et al., 2000
		repeats and ∼750 bp of flanking	origin effects, ), age dependent	limiting the applicability of this
		DNA from the DMPK 3' UTR	somatic instability in various	model to studies on repeat
		without any promoter element	tissues; highest in kidney (-20 to	instability
		or coding sequence	+540), age-dependent
	HSA LR	Transgenic overexpression of	Shows RNA foci that sequester	Transgene expression only in	Established the RNA gain-of-function	Mankodi et al., 2000
		250 CTG within the human	MBNL proteins, splicing	postdevelopmental skeletal muscle	mechanism underlying DM1.
		skeletal actin (HSA) 3' UTR	alterations, myotonia, myopathy,	tissues at levels much higher than	Widely used for therapeutic
			muscle loss	the endogenous Dmpk gene;	development.
				absence of muscle weakness or
				wasting; no effect on CELF levels
	DM300	Transgenic line carrying a 45 kb	Occurrence of somatic and	Variable transgene expression levels;	Allows the study of the ubiquitous	Seznec et al., 2000
		region from the DM1 locus	intergenerational repeat instability.	overall mild phenotype	CUGexp RNA within its natural context	Seznec et al., 2001
		including a mutant DMPK gene	Myotonia, myopathy, altered Tau
		with ∼300 CTG repeats	splice forms
	DMSXL	Transgenic line derived from the	Splicing changes in the central	Derived from a single founder; low	Allows the study of the ubiquitous	Gomes-Pereira et al., 2007
		DM300 model. Contains > 1000	nervous system and skeletal	DMPK expression; requires	CUGexp RNA within its natural context	Huguet et al., 2012
		CTG repeats	muscle of homozygous mice,	homozygosity to model a dominant
			myotonia, myopathy, aggregation	disease; time-consuming breeding
			of Tau in the brain.	strategies
	EpA960	Tissue-specific overexpression of	Myotonia, myopathy, muscle loss,	Interrupted repeat tract; heart-	Expression of 960 CUG repeats leads to	Wang et al., 2007
		960 interrupted CTG repeats	cardiac conduction defects, CUG	specific model displayed premature	major features of DM; suggests that	Orengo et al., 2008
		within exon 15 of DMPK	RNA foci that sequester MBNL1	death	CELF1 upregulation is dependent on
			and stabilize CELF1, splicing		the DMPK genomic context
			alterations
	TRE-EGFP-CTG5	Transgenic line with inducible	Myotonia, myopathy, cardiac	No muscle weakness and RNA foci,	Dox-induced CUG-containing transgene	Mahadevan et al., 2006
	and CTG200	expression of DMPK 3' UTR	conduction defects, alterations of	short repeat sequence, high	activation leads to myotonia and
		carrying 5 or 200 CTG repeats	splicing	mortality; overexpression of a	cardiac defects. Dox withdrawal reverts
				nonpathogenic repeat, CTG5, results	these phenotypes.
				in DM1 features
	EpA960/CaMKII-	Mouse model expressing 960	Nuclear RNA foci, misregulation of	Interrupted repeat tract	Brain-specific mouse model for DM	Wang et al., 2017
	Cre	CUG repeats in the postnatal	alternative splicing, brain atrophy-
		brain	associated morphological defects
			in axons and dendrites, reduced
			cytoplasmic MBNL1 expression in
			dendrites at 6months, followed by
			reduced axon and dendrite
			integrity at 9 months and reduced
			MBNL2 expression, learning
			disability, neurodegeneration,

Table 1 .

 1 Primers used in the PCR analysis of the splicing profile of DM specific misregulated transcripts

	Transcript	Forward (5′-3′)	Reverse (5′-3′)	Source
	ATP2A1 exon 22	ATCTTCAAGCTCCGGGCCCT	CAGCTCTGCCTGAAGATGTG	Kimura et al., 2005
	BIN exon 11	AGAACCTCAATGATGTGCTGG	TCGTGTTGACTCTGATCTCGG	Fugier et al., 2011
	DMD exon 78	TTAGAGGAGGTGATGGAGCA	GATACTAAGGACTCCATCGC	Rau et al., 2015
	IR exon 11	CCAAAGACAGACTCTCAGAT	AACATCGCCAAGGGACCTGC	Savkur et al., 2001
	LDB3 exon 11	GCAAGACCCTGATGAAGAAGCTC	GACAGAAGGCCGGATGCTG	Francois et al., 2011
	MBNL1 exon 7	GCTGCCCAATACCAGGTCAAC	TGGTGGGAGAAATGCTGTATGC	Present study
	TNNT2 exon 5	ATAGAAGAGGTGGTGGAAGAGTAC	GTCTCAGCCTCTGCTTCAGCATCC	Philips et al., 1998

Acknowledgments

We would like to thank Cadot B., Jollet A., Lemaitre M. and Neuillet C. from the CRM for their help, and Battail C, Boland A and Deleuze J-F from the CEA/IG/CNG for RNA sequencing. Funding: This work was supported by AFM (Association Francaise contre les Myopathies), France Génomique infrastructure and ANR (Agence National de la Recherche). Author contributions: L.A. and M.M. conducted most of the experiments. F.R. made the constructs and A.K., J.M., T.H. and L.B. supported some experiments. C.M. and B.S. produced lentiviral vectors, A.F. measured the muscle force, and P-E.M. and N.N. performed RNAseq analyses. N.S. and D.F. supervised the project and wrote the manuscript. Competing interests:

The method described in this paper is the subject of a patent application (inventors N.S., L.A. and D.F.). Data and materials availability: We thank I. Holt and G. Morris (CIND, RJAH Orthopeadic Hospital, UK) as well as "The Muscular Dystrophy Association Monoclonal Antibody Resource" for the MBNL1 (MB1a) antibody, T. Cooper for the 960 CTG construct, C. Thornton for the MBNL1 polyclonal antibody and the HSA LR mouse model, the AAV platform of the Centre de Recherche en Myologie (CRM), the Penn Vector Core, Gene Therapy Program, University of Pennsylvania (Philadelphia) for providing the pAAV2/9 plasmid (p5E18-VD29).

Acknowledgements

We thank the MyoBank-AFM and the platform for immortalization of human cells from the Institut de Myologie (Paris, France) as well as J. Marie and V. Allamand for helpful discussions.

Funding

This research was supported by the Association Francaise contre les Myopathies (AFM), UniversitéPierre et Marie Curie (UPMC), Institut National de la Santéet de la Recherche Medicale (INSERM), Centre National de la Recherche Scientifique (CNRS) and the France Geńomique infrastructure, managed by the Agence Nationale de la Recherche (ANR-10-INBS-09), part of the Investment for the Future program.

FUNDING

his work was supported by Sorbonne Université, Inserm, CNRS and Association Institut de Myologie.

Abbreviations

ALS Amyotrophic lateral sclerosis

Table S1. Effect of MBNL∆ treatment on misregulated splicing events in DM1 muscle cells Table S2. Effect of MBNL∆ treatment on altered gene expression in DM1 muscle cells Table S3. List of primers

Part 3

Development of an auto-regulatory system that controls transgene product expression

Conclusions and perspectives

RESOURCE ARTICLE

Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded repeats, a hallmark of myotonic dystrophy. Selected clones of DM1 and DM2 immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of immortalized DM1 myoblasts when compared with control and DM2 cells. Alternative splicing defects were observed in differentiated DM1 muscle cell lines, but not in DM2 lines. Splicing alterations did not result from differentiation delay because similar changes were found in immortalized DM1 transdifferentiated fibroblasts in which myogenic differentiation has been forced by overexpression of MYOD1. As a proof-of-concept, we show that antisense approaches alleviate disease-associated defects, and an RNA-seq analysis confirmed that the vast majority of mis-spliced events in immortalized DM1 muscle cells were affected by antisense treatment, with half of them significantly rescued in treated DM1 cells. Immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA aggregates and splicing defects can be used as robust readouts for the screening of therapeutic compounds. Therefore, immortalized DM1 and DM2 muscle cell lines represent new models and tools to investigate molecular pathophysiological mechanisms and evaluate the in vitro effects of compounds on RNA toxicity associated with myotonic dystrophy mutations.

KEY WORDS: Myotonic dystrophy, Expanded repeats, Muscle cell line, Nuclear aggregates, Alternative splicing, Therapeutic compounds

Competing interests

The authors declare no competing or financial interests.

Author contributions

Experiments were performed by L.A.

Supplementary information

Supplementary genetic corrections [START_REF] Gao | Genome therapy of myotonic dystrophy type 1 iPS cells for development of autologous stem cell therapy[END_REF][START_REF] Xia | Genome modiication leads to phenotype reversal in human myotonic dystrophy type 1 iPS-cell derived neural stem cells[END_REF][START_REF] Provenzano | CRISPR/Cas9-mediated deletion of CTG expansions recovers normal phenotype in myogenic cells derived from myotonic dystrophy 1 patients[END_REF](123)(124)(125). Furthermore, it opens the door for the development of isogenic cell lines providing a genetically matched "control cells. " Alternate possibilities brought by genome editing tools combined with hiPSC technologies promise the generation of novel tissue-speciic cell lines opening new horizons for the development of more reined wide-ranging myotonic dystrophy cell models, which will push forward future disease investigations.

AUTHOR CONTRIBUTIONS

MM, AK, FR and DF wrote the review.