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Sustained alpha-sarcoglycan gene expression after gene transfer in limb-girdle muscular dystrophy, type 2D.

ABSTRACT: The aim of this study was to attain long-lasting alpha-sarcoglycan gene expression in limb-girdle muscular dystrophy, type 2D (LGMD2D) subjects mediated by adeno-associated virus (AAV) gene transfer under control of a muscle specific promoter (tMCK).rAAV1.tMCK.hSGCA (3.25 × 10¹¹ vector genomes) was delivered to the extensor digitorum brevis muscle of 3 subjects with documented SGCA mutations via a double-blind, randomized, placebo controlled trial. Control sides received saline. The blind was not broken until the study was completed at 6 months and all results were reported to the oversight committee.Persistent alpha-sarcoglycan gene expression was achieved for 6 months in 2 of 3 LGMD2D subjects. Markers for muscle fiber transduction other than alpha-sarcoglycan included expression of major histocompatibility complex I, increase in muscle fiber size, and restoration of the full sarcoglycan complex. Mononuclear inflammatory cells recruited to the site of gene transfer appeared to undergo programmed cell death, demonstrated by terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick-end labeling and caspase-3 staining. A patient failing gene transfer demonstrated an early rise in neutralizing antibody titers and T-cell immunity to AAV, validated by enzyme-linked immunospot on the second day after gene injection. This was in clear distinction to other participants with satisfactory gene expression.The findings of this gene replacement study in LGMD2D subjects have important implications not previously demonstrated in muscular dystrophy. Long-term, sustainable gene expression of alpha-sarcoglycan was observed following gene transfer mediated by AAV. The merit of a muscle-specific tMCK promoter, not previously used in a clinical trial, was evident, and the potential for reversal of disease was displayed.

SUBMITTER: Mendell JR

PROVIDER: S-EPMC2970162 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Sustained alpha-sarcoglycan gene expression after gene transfer in limb-girdle muscular dystrophy, type 2D.

Mendell Jerry R JR Rodino-Klapac Louise R LR Rosales Xiomara Q XQ Coley Brian D BD Galloway Gloria G Lewis Sarah S Malik Vinod V Shilling Chris C Byrne Barry J BJ Conlon Thomas T Campbell Katherine J KJ Bremer William G WG Taylor Laura E LE Flanigan Kevin M KM Gastier-Foster Julie M JM Astbury Caroline C Kota Janaiah J Sahenk Zarife Z Walker Christopher M CM Clark K Reed KR

Annals of neurology 20101101 5

<h4>Objective</h4>The aim of this study was to attain long-lasting alpha-sarcoglycan gene expression in limb-girdle muscular dystrophy, type 2D (LGMD2D) subjects mediated by adeno-associated virus (AAV) gene transfer under control of a muscle specific promoter (tMCK).<h4>Methods</h4>rAAV1.tMCK.hSGCA (3.25 × 10¹¹ vector genomes) was delivered to the extensor digitorum brevis muscle of 3 subjects with documented SGCA mutations via a double-blind, randomized, placebo controlled trial. Control sides ...[more]

PMID: 21031578

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Project description:Limb-girdle muscular dystrophy type 2D/R3 (LGMD2D/R3) is a progressive muscular dystrophy that manifests with muscle weakness, respiratory abnormalities, and in rare cases cardiomyopathy. LGMD2D/R3 is caused by mutations in the SGCA gene resulting in loss of protein and concomitant loss of some or all components of the dystrophin-associated glycoprotein complex. The sgca-null (sgca-/-) mouse recapitulates the clinical phenotype of patients with LGMD2D/R3, including dystrophic features such as muscle necrosis and fibrosis, elevated serum creatine kinase (CK), and reduction in the generation of absolute muscle force and locomotor activity. Thus, sgca-/- mice provide a relevant model to test the safety and efficacy of gene transfer. We designed a self-complementary AAVrh74 vector containing a codon-optimized full-length human SGCA (hSGCA) transgene driven by a muscle-specific promoter, shortened muscle creatine kinase (tMCK). In this report, we test the efficacy and safety of scAAVrh74.tMCK.hSGCA in sgca-/- mice using a dose-escalation design to evaluate a single systemic injection of 1.0 × 1012, 3.0 × 1012, and 6.0 × 1012 vg total dose compared with vehicle-treatment and wild-type mice. In sgca-/- mice, treatment with scAAVrh74.tMCK.hSGCA resulted in robust expression of α-sarcoglycan protein at the sarcolemma membrane in skeletal muscle at all doses tested. In addition, scAAVrh74.tMCK.hSGCA was effective in improving the histopathology of limb and diaphragm muscle of sgca-/- mice, as indicated by reductions in fibrosis, central nucleation, and normalization of myofiber size. These molecular changes were concomitant with significant increases in specific force generation in the diaphragm and tibialis anterior muscle, protection against eccentric force loss, and reduction in serum CK. Locomotor activity was improved at all doses of vector-treated compared with vehicle-treated sgca-/- mice. Lastly, vector toxicity was not detected in a serum chemistry panel and by gross necropsy. Collectively, these findings provide support for a systemic delivery of scAAVrh74.tMCK.hSGCA in a clinical setting for the treatment of LGMD2D/R3.

Project description:In a previous limb-girdle muscular dystrophy type 2D (LGMD2D) clinical trial, robust alpha-sarcoglycan gene expression was confirmed following intramuscular gene (SGCA) transfer. This paved the way for first-in-human isolated limb infusion (ILI) gene transfer trial to the lower limbs. Delivery of scAAVrh74.tMCK.hSGCA via an intravascular route through the femoral artery predicted improved ambulation. This method was initially chosen to avoid safety concerns required for large systemic vascular delivery viral loads. ILI methods were adopted from the extensive chemotherapy experience for treatment of malignancies confined to the extremities. Six LGMD2D subjects were enrolled in a dose-ascending open-label clinical trial. Safety of the procedure was initially assessed in the single limb of a non-ambulant affected adult at a dose of 1 × 1012 vg/kg. Subsequently, ambulatory children (aged 8-13 years) were enrolled and dosed bilaterally with either 1 × 1012 vg/kg/limb or 3 × 1012 vg/kg/limb. The six-minute walk test (6MWT) served as the primary clinical outcome; secondary outcomes included muscle strength (maximum voluntary isometric force testing) and SGCA expression at 6 months. All ambulatory participants except one had pre- and post-treatment muscle biopsies. All four subjects biopsied had confirmed SGCA gene delivery by immunofluorescence, Western blot analysis (14-25% of normal), and vector genome copies (5.4 × 103-7.7 × 104 vg/μg). Muscle strength in the knee extensors (assessed by force generation in kilograms) showed improvement in two subjects that correlated with an increase in fiber diameter post gene delivery. Six-minute walk times decreased or remained the same. Vascular delivery of AAVrh74.tMCK.hSGCA was effective at producing SGCA protein at low doses that correlated with vector copies and local functional improvement restricted to targeted muscles. Future trials will focus on systemic administration to enable targeting of proximal muscles to maximize clinical benefit.

Project description:Limb-girdle muscular dystrophy types 2D and 2F (LGMD 2D and 2F) are autosomal recessive disorders caused by mutations in the alpha- and delta sarcoglycan genes, respectively, leading to severe muscle weakness and degeneration. The cause of the disease has been well characterized and a number of animal models are available for pre-clinical studies to test potential therapeutic interventions. To facilitate transition from drug discovery to clinical trials, standardized procedures and natural disease history data were collected for these mouse models. Implementing the TREAD-NMD standardized operating procedures, we here subjected LGMD2D (SGCA-null), LGMD2F (SGCD-null) and wild type (C57BL/6J) mice to five functional tests from the age of 4 to 32 weeks. To assess whether the functional test regime interfered with disease pathology, sedentary groups were taken along. Muscle physiology testing of tibialis anterior muscle was performed at the age of 34 weeks. Muscle histopathology and gene expression was analysed in skeletal muscles and heart. Muscle histopathology and gene expression was analysed in skeletal muscles and heart. Mice successfully accomplished the functional tests, which did not interfere with disease pathology. Muscle function of SGCA- and SGCD-null mice was impaired and declined over time. Interestingly, female SGCD-null mice outperformed males in the two and four limb hanging tests, which proved the most suitable non-invasive tests to assess muscle function. Muscle physiology testing of tibialis anterior muscle revealed lower specific force and higher susceptibility to eccentric-induced damage in LGMD mice. Analyzing muscle histopathology and gene expression, we identified the diaphragm as the most affected muscle in LGMD strains. Cardiac fibrosis was found in SGCD-null mice, being more severe in males than in females. Our study offers a comprehensive natural history dataset which will be useful to design standardized tests and future pre-clinical studies in LGMD2D and 2F mice.

Project description:ObjectiveTo identify novel biomarkers as an alternative diagnostic tool for limb girdle muscular dystrophy (LGMD).BackgroundLGMD encompasses a group of muscular dystrophies characterized by proximal muscles weakness, elevated CK levels and dystrophic findings on muscle biopsy. Heterozygous CAPN3 mutations are associated with autosomal dominant LGMD-4, while biallelic mutations can cause autosomal recessive LGMD-1. Diagnosis is currently often based on invasive methods requiring muscle biopsy or blood tests. In most cases Western blotting (WB) analysis from muscle biopsy is essential for a diagnosis, as muscle samples are currently the only known tissues to express the full-length CAPN3 isoform.MethodsWe analyzed CAPN3 in a cohort including 60 LGMD patients. Selected patients underwent a complete neurological examination, electromyography, muscle biopsy, and skin biopsies for primary fibroblasts isolation. The amount of CAPN3 was evaluated by WB analysis in muscle and skin tissues. The total RNA isolated from muscle, fibroblast and urine was processed, and cDNA was used for qualitative analysis. The expression of CAPN3 was investigated by qRT-PCR. The CAPN3 3D structure has been visualized and analyzed using PyMOL.ResultsAmong our patients, seven different CAPN3 mutations were detected, of which two were novel. After sequencing CAPN3 transcripts from fibroblast and urine, we detected different CAPN3 isoforms surprisingly including the full-length transcript. We found comparable protein levels from fibroblasts and muscle tissue; in particular, patients harboring a novel CAPN3 mutation showed a 30% reduction in protein compared to controls from both tissues.ConclusionsOur findings showed for the first time the presence of the CAPN3 full-length transcript in urine and skin samples. Moreover, we demonstrated surprisingly comparable CAPN3 protein levels between muscle and skin samples, thus allowing us to hypothesize the use of skin biopsy and probably of urine samples as an alternative less invasive method to assess the amount of CAPN3 when molecular diagnosis turns out to be inconclusive.

Dataset Information

Sustained alpha-sarcoglycan gene expression after gene transfer in limb-girdle muscular dystrophy, type 2D.

Publications

Sustained alpha-sarcoglycan gene expression after gene transfer in limb-girdle muscular dystrophy, type 2D.

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