Les dysferlinopathies sont des maladies musculaires dues à une déficience en protéine dysferline, codée par le gène DYSF. Dans ce travail de thèse, trois approches thérapeutiques ont été évaluées pour ces pathologies, sur des modèles cellulaires et murins. Un variant transcriptionnel court de la dysferline a été vectorisé dans un AAV8r et injecté dans le modèle murin Bla/J, déficient en dysferline. L’analyse des muscles des animaux traités montre une augmentation de la résistance des fibres musculaires au stress mécanique, mais n’apporte pas de correction histologique. Cette étude souligne également la toxicité de cette miniprotéine. L’anoctamine 5, impliquée dans des pathologies et des activités similaires à la dysferline, a été testée en tant que protéine compensatrice. L’anoctamine 5 surexprimée dans le modèle Bla/J ne permet pas la restauration d’un phénotype normal. La compensation de DYSF par ANO5 n’est donc pas une voie thérapeutique à exploiter pour les dysferlinopathies. Enfin, une thérapie génique par chirurgie de l’ARN dysferline a été évaluée en utilisant le trans-épissage médié par le splicéosome (SMaRT). La preuve de principe de la reprogrammation d’un minigène dysferline a été faite (ARN et protéine trans-épissée obtenus in vitro). L’efficacité du SMaRT dans un contexte endogène s’est en revanche révélée faible, et n’a pas permis la restauration d’une protéine dysferline fonctionnelle dans des myoblastes humains. De plus, l’observation de l’expression de protéines directement à partir du RTM (RNA-trans-splicing molecule) a fait apparaître des limites à l’utilisation du SMaRT pour la thérapie génique, et en particulier pour les dysferlinopathies. / Dysferlinopathies are muscular diseases due to mutations in DYSF gene, inducing dysferlin protein deficiency. In this thesis, three therapeutic approaches have been investigated for these pathologies, on cell or mice models. A short transcriptional dysferlin variant has been injected into Bla/J dysferlin deficient mouse model, using AAV8r vector. Muscle fibers of treated animals displayed an increased resistance to mechanical stress without therapeutic benefit. These experiments also pointed out the toxicity of this strategy. A protein compensation approach has been tested using anoctamin 5, known to be involved in pathologies and activities similar to dysferlin’s ones. AAVr mediated Anoctamin 5 overexpression in Bla/J model does not rescue their muscle phenotype. Overexpression of ANO5 does not seem to be a valuable therapeutic strategy for dysferlin deficiency. Dysferlin RNA surgery was evaluated as a possible genetic therapy using Spliceosome-Mediated RNA Trans-splicing (SMaRT). On a Minigene target, SMaRT is able to induce RNA reprogramming by trans-splicing, and produce the corresponding protein. But efficiency is by far decreased in endogenous context and not good enough to restore functional dysferlin in human myoblasts. Moreover, we described proteins resulting from RNA-trans-splicing molecule (RTM) self-expression, limiting the value of SMaRT as therapeutic strategy, especially for dysferlinopathies.

Les dysferlinopathies sont des dystrophies musculaires qui se manifestent par la dystrophie musculaire des ceintures de type 2B (LGMD2B) ou la myopathie de Miyoshi (MM). Elles sont causées par des mutations dans le gène dysferline. La dysferline est une protéine membranaire exprimée dans le muscle squelettique, responsable de la réparation des microlésions du sarcolemme. L’absence d’une telle réparation de la membrane entraîne une atrophie musculaire progressive. Ce travail de thèse explore le potentiel thérapeutique d'une stratégie de modulation d'épissage pour le traitement de la LGMD2B causée par la mutation faux-sens c4022T>C dans l'exon 38 du gène dysferline. Des oligonucléotides et des petits ARN U7 délivrés par un vecteur viral de type adéno-associé ont été utilisés comme outils antisens pour induire un saut d'exon in vitro et in vivo. Ce projet de thèse étudie également la capacité de la dysferline tronquée à se localiser de façon appropriée à la membrane et ainsi la réparer. / Dysferlinopathy is a muscular dystrophy that manifests as two major phenotypes: limb-girdle muscular dystrophy type 2B (LGMD2B) or Miyoshi myopathy (MM). It is caused by mutations in the dysferlin gene. Dysferlin is a membrane protein expressed in skeletal muscle. It is responsible for the repair of sarcolemma microlesions produced by muscle contractions. A compromised membrane repair leads to slowly progressing muscle wasting. This thesis explores the therapeutic potential of an antisense mediated splice switching strategy in LGMD2B caused by the missense mutation c4022T>C in the exon 38 of the dysferlin gene. Antisense oligonucleotides and U7 snRNAs delivered by an adeno-associated viral vector were used as antisense tools to trigger exon skipping in vitro and in vivo. The thesis investigates also if the truncated dysferlin maintainsa proper membrane localization and its membrane repair ability.

Muscular dystrophies are a heterogeneous group of genetic diseases that involve mutations in genes leading to progressive muscular weakness. Limb-Girdle Muscular Dystrophy 2B (LGMD2B) is a subset of muscular dystrophy caused by mutations in the DYSF gene, which encodes for dysferlin protein and has an incidence of 1/100,000-1/200,000 people, or 1/300 people of Libyan Jewish descent. Since there is no effective treatment that can cure or reverse effects of LGMD2B once diagnosed, our goal is to investigate and develop a protein therapy that mitigates effects of this disease in patients. Galectin-1 (Gal-1) is a small, soluble 14.5 kDa protein with a carbohydrate recognition domain capable of stabilizing the sarcolemma. The exact role that Gal-1 plays in myogenic cells is not fully understood, however, it is known that Gal-1 possesses anti-inflammatory properties and increases the terminal differentiation of committed myogenic cells. Our hypothesis is that Gal-1 treatment increases myogenic potential, improves membrane repair capability, and modulates the immune response in models of LGMD2B by stabilizing muscle integrity, leading to decreased disease manifestation. To test this hypothesis and assess the effect of Gal-1 treatment on myogenesis, anti-inflammatory modulation, and membrane repair, we designed, produced, and purified recombinant human galectin-1 (rHsGal-1) to be used in LGMD2B models. Our in vitro results indicate that after 2-3 days of treatment with 0.11μM rHsGal-1, A/J-/- myotubes enhance expression of myogenic late markers and increase in size and alignment. Additionally, after short-term treatment, rHsGal-1 improves membrane repair capability in a Ca2+ independent manner through an activated carbohydrate recognition domain (CRD) in in vitro and in vivo models of LGMD2B. We give evidence that rHsGal-1 upregulates anti-inflammatory cytokines, increases functional activity, and modulates the canonical NF-κB inflammatory pathway in dysferlin-deficient models by decreasing expression of TAK-1 and the p65 and p50 subunits in vitro and short-term in vivo treatment. Similar effects of the rHsGal-1 treatment were observed in patient-derived dysferlin-deficient human myotubes. Exploratory results show a potential decrease in muscle fat deposition in Bla/J mice. Furthermore, Gal-1 contributes to immune modulation by helping to initiate muscle regeneration by shifting M2 macrophage polarization. Together, our novel discoveries provide direct evidence that Gal-1 is a promising candidate to treat LGMD2B disease pathologies by improving expression of late-stage myogenic markers, improving membrane repair in vitro and short-term in vivo studies, promoting muscle regeneration through immune modulation, and reducing canonical NF-κB inflammation.

Limb-girdle muscular dystrophy type 2B (LGMD2B) is caused by mutations in the dysferlin gene, resulting in non-functional dysferlin, a key protein found in muscle membrane. Treatment options available for patients are chiefly palliative in nature and focus on maintaining ambulation. Our hypothesis is that galectin-1 (Gal-1), a soluble carbohydrate binding protein, increases membrane repair capacity, myogenic potential, M2 macrophage polarization and decreases NF-κB inflammation in dysferlin-deficient models. To test this hypothesis, we used recombinant human galectin-1 (rHsGal-1) to treat dysferlin-deficient models. We show that rHsGal-1 treatments of 48 h-72 h promotes myogenic maturation as indicated through improvements in size, myotube alignment, and myoblast migration in dysferlin-deficient myotubes. Furthermore, rHsGal-1 showed an increased membrane repair capacity of dysferlin-deficient myotubes. Improvements in membrane repair after only a 10 min rHsGal-1treatment suggests mechanical stabilization of the membrane due to interaction with glycosylated membrane bound, ECM or yet to be identified ligands through the CDR domain of Gal-1. rHsGal-l significantly reduces canonical NF-κB inflammation through TAK 1, P65, P50. Lastly we find 2.7 mg/kg in vivo rHsGal-1 treatment in BLA/J mice supports an M2 cyto-regenerative macrophage populations. Together our novel results reveal Gal-1 remediates disease pathologies in LGMD2B through changes in integral myogenic protein expression, mechanical membrane stabilization, immune modulation, and reducing canonical NF-κB inflammation.