Auswahl der wissenschaftlichen Literatur zum Thema „Microdystrophin“

Duchenne muscular dystrophy (DMD) is a rare neuromuscular disease affecting 1:5000 newborn males. No cure is currently available, but gene addition therapy, based on the adeno-associated viral (AAV) vector-mediated delivery of microdystrophin transgenes, is currently being tested in clinical trials. The muscles of DMD boys present significant fibrotic and adipogenic tissue deposition at the time the treatment starts. The presence of fibrosis not only worsens the disease pathology, but also diminishes the efficacy of gene therapy treatments. To gain an understanding of the efficacy of AAV-based microdystrophin gene addition in a relevant, fibrotic animal model of DMD, we conducted a systemic study in juvenile D2.mdx mice using the single intravenous administration of an AAV8 system expressing a sequence-optimized murine microdystrophin, named MD1 (AAV8-MD1). We mainly focused our study on the diaphragm, a respiratory muscle that is crucial for DMD pathology and that has never been analyzed after treatment with AAV-microdystrophin in this mouse model. We provide strong evidence here that the delivery of AAV8-MD1 provides significant improvement in body-wide muscle function. This is associated with the protection of the hindlimb muscle from contraction-induced damage and the prevention of fibrosis deposition in the diaphragm muscle. Our work corroborates the observation that the administration of gene therapy in DMD is beneficial in preventing muscle fibrosis.

Gene therapy is a good alternative for determined congenital disorders; however, there are numerous limitations for gene delivery in vivo including targeted cellular uptake, intracellular trafficking, and transport through the nuclear membrane. Here, a modified G5 polyamidoamine (G5 PAMAM) dendrimer–DNA complex was developed, which will allow cell-specific targeting to skeletal muscle cells and transport the DNA through the intracellular machinery and the nuclear membrane. The G5 PAMAM nanocarrier was modified with a skeletal muscle-targeting peptide (SMTP), a DLC8-binding peptide (DBP) for intracellular transport, and a nuclear localization signaling peptide (NLS) for nuclear uptake, and polyplexed with plasmid DNA containing the GFP-tagged microdystrophin (µDys) gene. The delivery of µDys has been considered as a therapeutic modality for patients suffering from a debilitating Duchenne muscular dystrophy (DMD) disorder. The nanocarrier–peptide–DNA polyplexes were prepared with different charge ratios and characterized for stability, size, surface charge, and cytotoxicity. Using the optimized nanocarrier polyplexes, the transfection efficiency in vitro was determined by demonstrating the expression of the GFP and the µDys protein using fluorescence and Western blotting studies, respectively. Protein expression in vivo was determined by injecting an optimal nanocarrier polyplex formulation to Duchenne model mice, mdx4Cv. Ultimately, these nanocarrier polyplexes will allow targeted delivery of the microdystrophin gene to skeletal muscle cells and result in improved muscle function in Duchenne muscular dystrophy patients.

In gene therapy for Duchenne muscular dystrophy there are two potential immunological obstacles. An individual with Duchenne muscular dystrophy has a genetic mutation in dystrophin, and therefore the wild-type protein is “foreign,” and thus potentially immunogenic. The adeno-associated virus serotype-6 (AAV6) vector for delivery of dystrophin is a viral-derived vector with its own inherent immunogenicity. We have developed a technology where an engineered plasmid DNA is delivered to reduce autoimmunity. We have taken this approach into humans, tolerizing to myelin proteins in multiple sclerosis and to proinsulin in type 1 diabetes. Here, we extend this technology to a model of gene therapy to reduce the immunogenicity of the AAV vector and of the wild-type protein product that is missing in the genetic disease. Following gene therapy with systemic administration of recombinant AAV6-microdystrophin to mdx/mTRG2 mice, we demonstrated the development of antibodies targeting dystrophin and AAV6 capsid in control mice. Treatment with the engineered DNA construct encoding microdystrophin markedly reduced antibody responses to dystrophin and to AAV6. Muscle force in the treated mice was also improved compared with control mice. These data highlight the potential benefits of administration of an engineered DNA plasmid encoding the delivered protein to overcome critical barriers in gene therapy to achieve optimal functional gene expression.

The cytotoxic T cell (CT) GalNAc transferase, or Galgt2, is a UDP-GalNAc:β1,4- N-acetylgalactosaminyltransferase that is localized to the neuromuscular synapse in adult skeletal muscle, where it creates the synaptic CT carbohydrate antigen {GalNAcβ1,4[NeuAc(orGc)α2, 3]Galβ1,4GlcNAcβ-}. Overexpression of Galgt2 in the skeletal muscles of transgenic mice inhibits the development of muscular dystrophy in mdx mice, a model for Duchenne muscular dystrophy. Here, we provide physiological evidence as to how Galgt2 may inhibit the development of muscle pathology in mdx animals. Both Galgt2 transgenic wild-type and mdx skeletal muscles showed a marked improvement in normalized isometric force during repetitive eccentric contractions relative to nontransgenic littermates, even using a paradigm where nontransgenic muscles had force reductions of 95% or more. Muscles from Galgt2 transgenic mice, however, showed a significant decrement in normalized specific force and in hindlimb and forelimb grip strength at some ages. Overexpression of Galgt2 in muscles of young adult mdx mice, where Galgt2 has no effect on muscle size, also caused a significant decrease in force drop during eccentric contractions and increased normalized specific force. A comparison of Galgt2 and microdystrophin overexpression using a therapeutically relevant intravascular gene delivery protocol showed Galgt2 was as effective as microdystrophin at preventing loss of force during eccentric contractions. These experiments provide a mechanism to explain why Galgt2 overexpression inhibits muscular dystrophy in mdx muscles. That overexpression also prevents loss of force in nondystrophic muscles suggests that Galgt2 is a therapeutic target with broad potential applications.

La dystrophie musculaire de Duchenne (DMD) est une maladie musculaire dégénérative touchant principalement les jeunes garçons, caractérisée par la perte de l'expression fonctionnelle de la dystrophine. Bien que la thérapie génique visant à restaurer une forme tronquée fonctionnelle de la dystrophine, appelée µ-dystrophine, ait montré des résultats prometteurs dans les études précliniques, son efficacité thérapeutique chez les patients DMD traités reste limitée, nécessitant des améliorations urgentes. Le travail présenté dans cette thèse vise d'abord à améliorer notre compréhension des perturbations métaboliques et cellulaires dans la DMD, puis à proposer de nouvelles approches thérapeutiques combinées, associant la thérapie génique aux traitements des dérégulations identifiées. Nous avons récemment identifié des dérégulations du métabolisme du cholestérol dans le muscle DMD, un phénomène souvent lié au dysfonctionnement lysosomal dans les troubles neurodégénératifs. Sur cette base, nous avons émis l'hypothèse d'une interaction potentielle entre l'accumulation de cholestérol et les perturbations lysosomales dans la pathogenèse de la DMD. Notre étude a identifié une augmentation de la Galectine-3 (LGALS3), un biomarqueur de la perméabilisation de la membrane des lysosomes (PML), dans les muscles dystrophiques des patients DMD et des modèles animaux, indiquant l'occurrence de la PML dans les myofibres dystrophiques. Ce phénomène a été corrélé à un stress lysosomal important, mis en évidence par des changements dans le nombre, la morphologie et le positionnement des lysosomes, ainsi que par l'activation des voies de biogenèse, de réparation et d'élimination des lysosomes. La PML a été exacerbée chez les souris nourries avec un régime riche en cholestérol et n'a pas été entièrement corrigée par la thérapie génique par µ-dystrophine. Pour remédier à cette limitation, nous avons sélectionné la tréhalose, un composé approuvé par la FDA et connu pour restaurer la fonction lysosomale, pour l'utiliser en combinaison avec une dose sous-optimale de thérapie génique par µ-dystrophine. Le traitement combiné a permis de corriger la PML et d'améliorer la correction des paramètres dystrophiques, y compris la fonction motrice, l'histologie musculaire et la signature transcriptomique, par rapport à la thérapie génique suboptimale de la µ-dystrophine seule. Les travaux de cette thèse soulignent l'importance des lésions lysosomales dans la physiopathologie de la DMD et suggèrent qu'une approche synergique combinant une supplémentation en tréhalose et une dose sous-optimale d'AAV µ-dystrophine est prometteuse pour améliorer les résultats thérapeutiques dans la DMD
Duchenne Muscular Dystrophy (DMD) is a muscle degenerative disease primarily affecting young boys, characterized by the loss of dystrophin expression. While gene therapy targeting the restoration of a functional truncated form of dystrophin, known as µ-dystrophin, has shown promise in preclinical studies, its therapeutic efficacy in treated DMD patients remains limited, necessitating urgent improvements. The aim of the work presented in this thesis is to first improve our understanding of the metabolic and cellular perturbations in DMD, and secondly to propose new combined therapy approaches, associating gene therapy to treatments of identified dysregulations. We have recently identified dysregulations of cholesterol metabolism in DMD muscle, a phenomenon frequently associated with lysosomal dysfunction in neurodegenerative disorders. Building upon this association, we hypothesized a potential interplay between cholesterol accumulation and lysosomal perturbations in DMD pathogenesis. Our study identified an upregulation of Galectin-3 (LGALS3), a known biomarker of lysosome membrane permeabilization (LMP), in the dystrophic muscle of DMD patients and animal models, indicating the occurrence of LMP within dystrophic myofibers. This correlated with significant lysosomal stress evidenced by changes in lysosome number, morphology, positioning and activation of lysosomal biogenesis, repair and removal pathways. Remarkably, LMP was exacerbated in mice fed a cholesterol-rich diet and was not fully corrected by µ-dystrophin gene therapy. Subsequently, we selected trehalose, an FDA-approved compound known to restore lysosomal function, for use in combination with a suboptimal dose of AAV-µ-dystrophin gene therapy. The combined treatment resulted in correction of lysosomal defects and improved correction of dystrophic parameters, including motor function, muscle histology, and transcriptome signature, compared to µ-dystrophin gene therapy alone. This work underscores the significance of lysosomal damage in DMD pathophysiology and suggests that a synergistic approach combining trehalose supplementation with a suboptimal dose of AAV µ-dystrophin holds promise for enhancing therapeutic outcomes in DMD