A gene therapy approach for the treatment of limb-girdle muscular dystrophy 2C/R5

Limb-girdle muscular dystrophy 2C/R5 (LGMD 2C/R5) is a progressive muscle disease caused by mutations in the γ-sarcoglycan gene that results in loss of γ-sarcoglycan protein. The γ-sarcoglycan protein is an integral component of the sarcoglycan subcomplex, which is part of the dystrophin glycoprotein complex (DGC) (Figure 1). The DGC serves as a transmembrane linkage system between the actin cytoskeleton inside the muscle cell and laminin in the extracellular matrix and serves as a molecular shock absorber to protect the sarcolemma during muscle contraction.1Wilson D.G.S. Tinker A. Iskratsch T. The role of the dystrophin glycoprotein complex in muscle cell mechanotransduction.Commun. Biol. 2022; 5: 1022Crossref PubMed Scopus (5) Google Scholar The absence of γ-sarcoglycan in LGMD 2C/R5 results in progressive muscle weakness with affected individuals experiencing loss of ambulation at a young age and, as the disease progresses, cardiac and respiratory dysfunction. Currently there is no cure for LGMD 2C/R5.2Pozsgai E. Griffin D. Potter R. Sahenk Z. Lehman K. Rodino-Klapac L.R. Mendell J.R. Unmet needs and evolving treatment for limb girdle muscular dystrophies.Neurodegener. Dis. Manag. 2021; 11: 411-429Crossref PubMed Scopus (16) Google Scholar In this issue of Molecular Therapy - Methods and Clinical Development, Rodino-Klapac and her team developed a recombinant adeno-associated virus (rAAV)-MCHK7-γ-sarcoglycan construct to deliver a normal copy of the γ-sarcoglycan to the muscle of a mouse model of LGMD 2C/R5.3Seo Y.E. Baine S.H. Kempton A.N. Rogers O.C. Lewis S. Adegboye K. Haile A. Griffin D.A. Peterson E.L. Pozsgai E.R. Potter R. Rodino-Klapac L.R. Systemic AAV-Mediated γ- Sarcoglycan Gene Transfer Therapy Results in Dose-dependent Correction of Muscle Deficits in LGMD 2C/R5 Mouse Model. Molecular therapy - Methods & clinical development, 2023Google Scholar The study design included a dose-ranging investigation to determine the efficacy and optimal dose of the gene replacement therapeutic in skeletal and cardiac muscle.3Seo Y.E. Baine S.H. Kempton A.N. Rogers O.C. Lewis S. Adegboye K. Haile A. Griffin D.A. Peterson E.L. Pozsgai E.R. Potter R. Rodino-Klapac L.R. Systemic AAV-Mediated γ- Sarcoglycan Gene Transfer Therapy Results in Dose-dependent Correction of Muscle Deficits in LGMD 2C/R5 Mouse Model. Molecular therapy - Methods & clinical development, 2023Google Scholar The study used an engineered version of the muscle creatine kinase (MHCK7) promoter4Salva M.Z. Himeda C.L. Tai P.W. Nishiuchi E. Gregorevic P. Allen J.M. Finn E.E. Nguyen Q.G. Blankinship M.J. Meuse L. et al.Design of tissue-specific regulatory cassettes for high-level rAAV- mediated expression in skeletal and cardiac muscle.Mol. Ther. 2007; 15: 320-329Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar to drive high-level expression of the normal copy of the γ-sarcoglycan gene in skeletal and cardiac muscle. The study showed γ-sarcoglycan expression at all dose ranges was associated with localization of γ-sarcoglycan to the skeletal muscle sarcolemma along with improvements in skeletal muscle pathology and function.3Seo Y.E. Baine S.H. Kempton A.N. Rogers O.C. Lewis S. Adegboye K. Haile A. Griffin D.A. Peterson E.L. Pozsgai E.R. Potter R. Rodino-Klapac L.R. Systemic AAV-Mediated γ- Sarcoglycan Gene Transfer Therapy Results in Dose-dependent Correction of Muscle Deficits in LGMD 2C/R5 Mouse Model. Molecular therapy - Methods & clinical development, 2023Google Scholar The authors report rAAV-MCHK7-γ-sarcoglycan treatment resulted in cytoplasmic localization of γ-sarcoglycan protein within cardiomyocytes of the heart.3Seo Y.E. Baine S.H. Kempton A.N. Rogers O.C. Lewis S. Adegboye K. Haile A. Griffin D.A. Peterson E.L. Pozsgai E.R. Potter R. Rodino-Klapac L.R. Systemic AAV-Mediated γ- Sarcoglycan Gene Transfer Therapy Results in Dose-dependent Correction of Muscle Deficits in LGMD 2C/R5 Mouse Model. Molecular therapy - Methods & clinical development, 2023Google Scholar The sarcoglycan subcomplex in skeletal and cardiac muscle is composed of at least four proteins: γ-sarcoglycan, α-sarcoglycan, β-sarcoglycan, and δ-sarcoglycan. Mutations in genes that encode each member of the sarcoglycan subcomplex cause distinct forms of LGMD.5Vainzof M. Souza L.S. Gurgel-Giannetti J. Zatz M. Sarcoglycanopathies: an update.Neuromuscul. Disord. 2021; 31: 1021-1027Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar Studies have shown the sarcoglycan subcomplex is assembled in a sequential fashion within the endoplasmic reticulum, and disruption of this assembly process can result in muscle disease (Figure 1).6Tarakci H. Berger J. The sarcoglycan complex in skeletal muscle.Front. Biosci. 2016; 21: 744-756Crossref PubMed Scopus (34) Google Scholar,7Allikian M.J. McNally E.M. Processing and assembly of the dystrophin glycoprotein complex.Traffic. 2007; 8: 177-183Crossref PubMed Scopus (62) Google Scholar In addition, studies indicate the stoichiometry of the sarcoglycans are critical to correct assembly of the sarcoglycan subcomplex.8Holt K.H. Campbell K.P. Assembly of the sarcoglycan complex. Insights for muscular dystrophy.J. Biol. Chem. 1998; 273: 34667-34670Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar,9Zhu X. Hadhazy M. Groh M.E. Wheeler M.T. Wollmann R. McNally E.M. Overexpression of gamma-sarcoglycan induces severe muscular dystrophy. Implications for the regulation of Sarcoglycan assembly.J. Biol. Chem. 2001; 276: 21785-21790Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar,10Shi W. Chen Z. Schottenfeld J. Stahl R.C. Kunkel L.M. Chan Y.M. Specific assembly pathway of sarcoglycans is dependent on beta- and delta-sarcoglycan.Muscle Nerve. 2004; 29: 409-419Crossref PubMed Scopus (50) Google Scholar The high level of expression of γ-sarcoglycan achieved using the MHCK7 promoter may have been a contributing factor to the cytoplasmic localization of γ-sarcoglycan protein in cardiac cells. This observation may suggest differences in tissue level expression of the sarcoglycans and how they are handled in forming the sarcoglycan subcomplex. The question is why is there an apparent difference in the localization of γ-sarcoglycan between cardiac and skeletal muscle? One possibility is skeletal muscle is composed of large multinucleated myofibers that may act to effectively dilute the dose of rAAV-γ-sarcoglycan allowing the correct assembly to the sarcoglycan complex with the DGC. In contrast, single cardiomyocytes transduced with the rAAV-γ-sarcoglycan may have relatively high levels of γ-sarcoglycan protein compared with skeletal muscle, which could impact the assembly of the sarcoglycan complex in the heart. Although the authors did not observe any adverse cardiac events in the LGMD 2C/R5 mouse model treated using rAAV-γ-sarcoglycan, cardiac function in these animals was not studied. This study suggests doses of rAAV-γ-sarcoglycan gene therapy used in this study could effectively ameliorate skeletal muscle disease progression in a mouse model of LGMD 2C/R5,3Seo Y.E. Baine S.H. Kempton A.N. Rogers O.C. Lewis S. Adegboye K. Haile A. Griffin D.A. Peterson E.L. Pozsgai E.R. Potter R. Rodino-Klapac L.R. Systemic AAV-Mediated γ- Sarcoglycan Gene Transfer Therapy Results in Dose-dependent Correction of Muscle Deficits in LGMD 2C/R5 Mouse Model. Molecular therapy - Methods & clinical development, 2023Google Scholar but further investigation will be needed to assess efficacy and impact of AAV-gene therapy on cardiac function in the mouse model. The cytoplasmic localization of γ-sarcoglycan highlights the need to conduct both short- and long-term therapeutic preclinical efficacy studies. These studies are often underperformed, as they are time consuming and expensive. It is possible that it takes longer than 12 weeks for the sarcoglycan complex to localize to the sarcolemma in cardiomyocytes. It is also possible the benefits observed in skeletal muscle with short-term treatments may be detrimental with long-term high-level expression of γ-sarcoglycan. This study also highlights the need to develop the next generation of gene therapy constructs to include elements that allow regulation of gene expression. A method to modulate gene expression employed in this study is a dose-ranging study using the AAV-γ-sarcoglycan construct to identify a dosage that works equally well in skeletal and cardiac muscle. However, it is possible that an overlapping optimal dose of rAAV-γ-sarcoglycan effective in both cardiac and skeletal muscle may not be identified. A second method is to develop promoters that allow transcriptional activity of the delivered γ-sarcoglycan to be regulated. The issue with this approach includes the current limits on the packaging capacity of the AAV delivery system could limit including regulatory sequences within the promoter. A third approach could be to embed small DNA sequences within the γ-sarcoglycan gene construct that will allow regulation of the transcript by endogenous cardiac-specific miRNA processing machinery. This approach could allow cardiac cells to regulate transcript levels of the exogenously delivered γ-sarcoglycan gene. This study demonstrates the clinical feasibility of a gene therapy approach for the treatment of LGMD 2C/R5. The development of the next generation of constructs that contain regulatory elements to modulate γ-sarcoglycan expression could allow a more effective treatment of LGMD 2C/R5-related dilated cardiomyopathy and could have wider application for the treatment of muscle disease. The authors have no competing interests to declare.