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Currently, available therapies for muscular dystrophy are not curative. Nevertheless, advances in gene delivery and editing tools are providing great opportunities for effective gene-replacement strategies. The majority of the preclinical and clinical strategies being studied rely on the use of AAV vectors. This gene delivery strategy successfully transduces critical target tissues, such as skeletal and cardiac muscles, affected in many types of muscular dystrophy.
What are muscular dystrophies?
Muscular dystrophy is a group of over thirty genetically heterogeneous disorders resulting in progressive muscle degeneration. Because various genes and mutations may be involved, disease presentation is widely diverse in onset, progression rate, severity, and the specific muscle groups involved (Patel et al. 2021). However, general hallmarks of disease progression include the loss of muscle fibers and replacement by fibrous tissue and fat. These changes result in progressive muscle weakness, which reduces mobility and ultimately leads to respiratory and cardiac malfunction.
Duchenne muscular dystrophy, the most common and severe form of these disorders, was first linked to loss of function mutations in the dystrophin gene in the 1980s. Similarly, during the same time, Becker muscular dystrophy, which has a milder presentation, was linked to mutations in the same gene. Nevertheless, dystrophin mutations in Becker muscular dystrophy didn’t entirely eliminate protein function; instead, truncated dystrophin with partial activity remained, explaining the milder phenotype (Guiraud et al. 2015).
Dystrophin is a large cytoskeletal protein (427 kDa) broadly expressed in different muscle tissues (e.g., skeletal and cardiac) but also found in the brain and other organs (Petkova et al. 2016). In the muscle, dystrophin is found in tight association with the cell membrane or sarcolemma and as part of a large protein complex known as the dystrophin-associated protein complex (DAPC) or dystrophin-glycoprotein complex.
It’s now well established that the dystrophin-glycoprotein complex plays a critical role in cytoskeletal organization, cell signaling, sarcolemmal stabilization, and force transmission in the skeletal muscle. Also, mutations in genes encoding various dystrophin-associated proteins or those critical for the function of the complex have been linked to several types of muscular dystrophies (e.g., Sarcoglycanopathy, Dysferlinopathy, Calpainopathy, and Dystroglycanopathy) (Balci-Hayta et al. 2018). Among these, Dystroglycanopathies result from gene mutations affecting the glycosylation status of alpha-Dystroglycan, a main component of the dystrophin-glycoprotein complex (Dowling et al. 2021, Yonekawa et al. 2022). Reduction or loss of alpha-Dystroglycan glycosylation impairs its function as a major extracellular matrix receptor in the skeletal muscle (Walimbe et al. 2020).
Dystrophin-Glycoprotein Complex. Dystroglycan is a main component of the Dystrophin Glycoprotein complex and consists of two main subunits, alpha, and beta-Dystroglycan. A single gene, DAG1, encodes both subunits, which are formed through post-translational processing, including extensive glycosylation and proteolytic cleavage. The transmembrane subunit, beta-Dystroglycan, interacts directly with Dystrophin, while the alpha subunit is localized to the cell surface where it interacts with beta-Dystroglycan and extracellular matrix proteins. Retrieved and modified to show only part of Figure 3 from Dowling et al. 2021.
Correcting Dystroglycan glycosylation
A new study has leveraged a mouse model of Dystroglycanopathy to evaluate the efficacy of a gene replacement strategy in restoring muscle function. The myd mouse is null for like-acetyl-glucosaminyltransferase-1 (LARGE1), an enzyme critical for the synthesis and extension of matriglycan onto alpha-Dystroglycan (Balci-Hayta et al. 2018, Dowling et al. 2021).
“The sequence encoding mouse Large1 was synthesized (GenScript, Piscataway, NJ) and cloned into the AAV backbone under the transcriptional control of the ubiquitous CMV promoter.” Yonekawa et al. 2022
Yonekawa and colleagues successfully restored LARGE1 by delivering the gene systemically with an AAV2/9 vector. The team chose to rescue LARGE1 expression in older mice to test the effectiveness of this approach in restoring muscle function under conditions of advanced muscle degeneration. To this end, they administered AAV-LARGE1 to ~34 weeks myd old mice via intraperitoneal/intravenous injection, which resulted in broad muscle overexpression of Large1. Evaluation of motor function and grip strength for over 28 weeks revealed restored locomotor and exploratory behaviors and significant improvements in forelimb grip strength. In agreement with the observed improved motor function, AAV-LARGE1 treatment improved muscle contractility. Moreover, Western blot and immunohistochemical analysis of muscle tissue derived from AAV- LARGE1 treated mice confirmed successful overexpression of LARGE1 and the presence of glycosylated and functional alpha- Dystroglycan.
Significantly, muscle pathology in AAV- LARGE1 treated mice was improved, evidenced by reduced fibrous tissue deposition and reduced atrophy. Lastly, AAV-LARGE1 treatment also led to improved respiratory pathology and function.
Overall, these preclinical findings by Yonekawa and colleagues support the potential effectiveness of gene replacement as a strategy in muscular dystrophies. Further, the results support that such strategies may benefit even when significant muscle atrophy has already occurred.
Balci-Hayta, B., Talim, B., Kale, G. & Dincer, P. LARGE expression in different types of muscular dystrophies other than dystroglycanopathy. BMC Neurol. (2018) doi:10.1186/s12883-018-1207-0.
Dowling, P. et al. The dystrophin node as integrator of cytoskeletal organization, lateral force transmission, fiber stability and cellular signaling in skeletal muscle. Proteomes (2021) doi:10.3390/proteomes9010009.
Guiraud, S. et al. The Pathogenesis and Therapy of Muscular Dystrophies. Annu. Rev. Genomics Hum. Genet. (2015) doi:10.1146/annurev-genom-090314-025003.
Patel, K. M. et al. Molecular Diagnosis of Muscular Dystrophy Patients in Western Indian Population: A Comprehensive Mutation Analysis Using Amplicon Sequencing. Front. Genet. (2021) doi:10.3389/fgene.2021.770350.
Petkova, M. V. et al. Characterization of a DmdEGFP reporter mouse as a tool to investigate dystrophin expression. Skelet. Muscle (2016) doi:10.1186/S13395-016-0095-5.
Walimbe, A. S. et al. Pomk regulates dystroglycan function via large1-mediated elongation of matriglycan. Elife (2020) doi:10.7554/ELIFE.61388.
Yonekawa, T. et al. Large1 gene transfer in older myd mice with severe muscular dystrophy restores muscle function and greatly improves survival. Science (2022) doi/10.1126/sciadv.abn0379.