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An AAV-SGCG Dose-Response Study in a γ-Sarcoglycanopathy Mouse Model in the Context of Mechanical Stress.

ABSTRACT: Sarcoglycanopathies are rare autosomic limb girdle muscular dystrophies caused by mutations in one of the genes coding for sarcoglycans. Sarcoglycans form a complex, which is an important part of the dystrophin-associated glycoprotein complex and which protects the sarcolemma against muscle contraction-induced damage. Absence of one of the sarcoglycans on the plasma membrane reduces the stability of the whole complex and perturbs muscle fiber membrane integrity. There is currently no curative treatment for any of the sarcoglycanopathies. A first clinical trial to evaluate the safety of a recombinant AAV2/1 vector expressing γ-sarcoglycan using an intramuscular route of administration showed limited expression of the transgene and good tolerance of the approach. In this report, we undertook a dose-effect study in mice to evaluate the efficiency of an AAV2/8-expressing γ-sarcoglycan controlled by a muscle-specific promoter with a systemic mode of administration. We observed a dose-related efficiency with a nearly complete restoration of gamma sarcoglycan (SGCG) expression, histological appearance, biomarker level, and whole-body strength at the highest dose tested. In addition, our data suggest that a high expression threshold level must be achieved for effective protection of the transduced muscle, while a suboptimal transgene expression level might be less protective in the context of mechanical stress.

SUBMITTER: Israeli D

PROVIDER: S-EPMC6545357 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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An AAV-SGCG Dose-Response Study in a γ-Sarcoglycanopathy Mouse Model in the Context of Mechanical Stress.

Israeli David D Cosette Jérémie J Corre Guillaume G Amor Fatima F Poupiot Jérôme J Stockholm Daniel D Montus Marie M Gjata Bernard B Richard Isabelle I

Molecular therapy. Methods & clinical development 20190510

Sarcoglycanopathies are rare autosomic limb girdle muscular dystrophies caused by mutations in one of the genes coding for sarcoglycans. Sarcoglycans form a complex, which is an important part of the dystrophin-associated glycoprotein complex and which protects the sarcolemma against muscle contraction-induced damage. Absence of one of the sarcoglycans on the plasma membrane reduces the stability of the whole complex and perturbs muscle fiber membrane integrity. There is currently no curative tr ...[more]

PMID: 31194043

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Project description:BackgroundThe molecular mechanisms of DNA repair following chronic medium-dose-rate (MDR) γ-ray-induced damage remain largely unknown.Methodology/principal findingsWe used a cell function imager to quantitatively measure the fluorescence intensity of γ-H2A.X foci in MDR (0.015 Gy/h and 0.06 Gy/h) or high-dose-rate (HDR) (54 Gy/h) γ-ray irradiated embryonic fibroblasts derived from DNA-dependent protein kinase mutated mice (scid/scid mouse embryonic fibroblasts (scid/scid MEFs)). The obtained results are as follows: (1) Automatic measurement of the intensity of radiation-induced γ-H2A.X foci by the cell function imager provides more accurate results compared to manual counting of γ-H2A.X foci. (2) In high-dose-rate (HDR) irradiation, γ-H2A.X foci with high fluorescence intensity were observed at 1 h after irradiation in both scid/scid and wild-type MEFs. These foci were gradually reduced through de-phosphorylation at 24 h or 72 h after irradiation. Furthermore, the fluorescence intensity at 24 h increased to a significantly greater extent in scid/scid MEFs than in wild-type MEFs in the G(1) phase, although no significant difference was observed in G(2)/M-phase MEFs, suggesting that DNA-PKcs might be associated with non-homologous-end-joining-dependent DNA repair in the G(1) phase following HDR γ-ray irradiation. (3) The intensity of γ-H2A.X foci for continuous MDR (0.06 Gy/h and 0.015 Gy/h) irradiation increased significantly and in a dose-dependent fashion. Furthermore, unlike HDR-irradiated scid/scid MEFs, the intensity of γ-H2A.X foci in MDR-irradiated scid/scid MEFs showed no significant increase in the G(1) phase at 24 h, indicating that DNA repair systems using proteins other than DNA-PKcs might induce cell functioning that are subjected to MDR γ-ray irradiation.ConclusionsOur results indicate that the mechanism of phosphorylation or de-phosphorylation of γ-H2A.X foci induced by chronic MDR γ-ray irradiation might be different from those induced by HDR γ-ray irradiation.

Project description:Congenital myasthenic syndromes are a group of inherited disorders characterized by defective neuromuscular transmission and fatigable muscle weakness. Causative mutations have been identified in over 30 genes, including DOK7, a gene encoding a post-synaptic protein crucial in the formation and stabilization of the neuromuscular junction. Mutations in this gene are one of the leading three most prevalent causes of congenital myasthenia in diverse populations across the globe. The majority of DOK7 congenital myasthenic patients experience varying degrees of disability despite receiving optimized treatment (usually salbutamol), necessitating the development of improved therapeutic approaches. Here, we executed a dose escalation pre-clinical trial using a DOK7 congenital myasthenic syndrome mouse model to assess the efficacy of AMP-101, an innovative recombinant adeno-associated viral gene replacement therapy. This mouse model harbours a duplication in the Dok7 gene that corresponds to the mutation most commonly found in DOK7 congenital myasthenia patients, c.1124-1127dupTGCC. The model has a much more severe phenotype than patients, and lives for only a few days. AMP-101 is based on AAVrh74 and contains human DOK7 cDNA under the control of a muscle-restricted promoter. Three doses of AMP-101 (2 × 1013 vg/kg, 6 × 1013 vg/kg or 1 × 1014 vg/kg) were administered intraperitoneally at 4 days of age. We show that the two higher doses of 6 × 1013 vg/kg and 1 × 1014 vg/kg generated enlarged neuromuscular junctions and rescued the very severe phenotype of the model. Treated mice became at least as strong as wild-type littermates, as demonstrated by using an inverted screen hang test, a rotarod test and a grip strength test. EMG showed that the treated model mice had decrement of compound muscle action potential on repetitive nerve stimulation, which indicates defective signalling at the neuromuscular junction. However, male models treated with 1 × 1014 vg/kg showed the least decrement that was not statistically different from wild-type littermates. Western blot analysis demonstrated robust expression of DOK7 in the diaphragm and tibialis anterior muscles. These data show that AMP-101 is an effective treatment in a mouse model for DOK7 congenital myasthenia, and suggests that AMP-101 is a promising candidate to move forward to clinic trials as a gene therapy for patients.

Dataset Information

An AAV-SGCG Dose-Response Study in a γ-Sarcoglycanopathy Mouse Model in the Context of Mechanical Stress.

Publications

An AAV-SGCG Dose-Response Study in a γ-Sarcoglycanopathy Mouse Model in the Context of Mechanical Stress.

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