Amelioration of Morphological Pathology in Cardiac, Respiratory, and Skeletal Muscles Following Intraosseous Administration of Human Dystrophin Expressing Chimeric (DEC) Cells in Duchenne Muscular Dystrophy Model.

Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutation in the dystrophin gene. Currently there is no cure for DMD. We introduced a novel human Dystrophin Expressing Chimeric (DEC) cell therapy of myoblast origin and confirmed the safety and efficacy of DEC in the mdx mouse models of DMD. In this study, we assessed histological and morphological changes in the cardiac, diaphragm, and gastrocnemius muscles of the mdx/scid mice after the transplantation of human DEC therapy via the systemic-intraosseous route. The efficacy of different DEC doses was evaluated at 90 days (0.5 × 106 and 1 × 106 DEC cells) and 180 days (1 × 106 and 5 × 106 DEC cells) after administration. The evaluation of Hematoxylin & Eosin (H&E)-stained sectional slices of cardiac, diaphragm, and gastrocnemius muscles included assessment of muscle fiber size by minimal Feret's diameter method using ImageJ software. The overall improvement in muscle morphology was observed in DMD-affected target muscles in both studies, as evidenced by a shift in fiber size distribution toward the wild type (WT) phenotype and by an increase in the mean Feret's diameter compared to the vehicle-injected controls. These findings confirm the long-term efficacy of human DEC therapy in the improvement of overall morphological pathology in the muscles affected by DMD and introduce DEC as a novel therapeutic approach for DMD patients.

Long-Term Biodistribution and Safety of Human Dystrophin Expressing Chimeric Cell Therapy After Systemic-Intraosseous Administration to Duchenne Muscular Dystrophy Model.

Duchenne muscular dystrophy (DMD) is a lethal disease caused by X-linked mutations in the dystrophin gene. Dystrophin deficiency results in progressive degeneration of cardiac, respiratory and skeletal muscles leading to premature death due to cardiopulmonary complications. Currently, no cure exists for DMD. Based on our previous reports confirming a protective effect of human dystrophin expressing chimeric (DEC) cell therapy on cardiac, respiratory, and skeletal muscle function after intraosseous administration, now we assessed long-term safety and biodistribution of human DEC therapy for potential clinical applications in DMD patients. Safety of different DEC doses (1 × 106 and 5 × 106) was assessed at 180 days after systemic-intraosseous administration to mdx/scid mice, a model of DMD. Assessments included: single cell gel electrophoresis assay (COMET assay) to confirm lack of genetic toxicology, magnetic resonance imaging (MRI) for tumorigenicity, and body, muscle and organ weights. Human DEC biodistribution to the target (heart, diaphragm, gastrocnemius muscle) and non-target (blood, bone marrow, lung, liver, spleen) organs was detected by flow cytometry assessment of HLA-ABC markers. Human origin of dystrophin was verified by co-localization of dystrophin and human spectrin by immunofluorescence. No complications were observed after intraosseous transplant of human DEC. COMET assay of donors and fused DEC cells confirmed lack of DNA damage. Biodistribution analysis of HLA-ABC expression revealed dose-dependent presence of human DEC cells in target organs, whereas negligible presence was detected in non-target organs. Human origin of dystrophin in the heart, diaphragm and gastrocnemius muscle was confirmed by co-localization of dystrophin expression with human spectrin. MRI revealed no evidence of tumor formation. Body mass and muscle and organ weights were stable and comparable to vehicle controls, further confirming DEC safety at 180 days post- transplant. This preclinical study confirmed long-term local and systemic safety of human DEC therapy at 180 days after intraosseous administration. Thus, DEC can be considered as a novel myoblast based advanced therapy medicinal product for DMD patients.

Long-Term Protective Effect of Human Dystrophin Expressing Chimeric (DEC) Cell Therapy on Amelioration of Function of Cardiac, Respiratory and Skeletal Muscles in Duchenne Muscular Dystrophy.

Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutations in dystrophin encoding gene, causing progressive degeneration of cardiac, respiratory, and skeletal muscles leading to premature death due to cardiac and respiratory failure. Currently, there is no cure for DMD. Therefore, novel therapeutic approaches are needed for DMD patients.We have previously reported functional improvements which correlated with increased dystrophin expression following administration of dystrophin expressing chimeric (DEC) cells of myoblast origin to the mdx mouse models of DMD.In the current study, we confirmed dose-dependent protective effect of human DEC therapy created from myoblasts of normal and DMD-affected donors, on restoration of dystrophin expression and amelioration of cardiac, respiratory, and skeletal muscle function at 180 days after systemic-intraosseous DEC administration to mdx/scid mouse model of DMD. Functional improvements included maintenance of ejection fraction and fractional shortening levels on echocardiography, reduced enhanced pause and expiration time on plethysmography and improved grip strength and maximum stretch induced contraction of skeletal muscles. Improved function was associated with amelioration of mdx muscle pathology revealed by reduced muscle fibrosis, reduced inflammation and improved muscle morphology confirmed by reduced number of centrally nucleated fibers and normalization of muscle fiber diameters. Our findings confirm the long-term systemic effect of DEC therapy in the most severely affected by DMD organs including heart, diaphragm, and long skeletal muscles.These encouraging preclinical data introduces human DEC as a novel therapeutic modality of Advanced Therapy Medicinal Product (ATMP) with the potential to improve or halt the progression of DMD and enhance quality of life of DMD patients. Human DEC as a novel therapeutic modality with the potential to improve or halt progression of the DMD disease and enhance quality of life of DMD patients. Graphical abstract represents manufacturing process of the human DEC therapy for the future clinical applications. 1. We report the long-term efficacy of human DEC therapy resulting in increased dystrophin expression and reduced mdx muscle pathology after systemic-intraosseous administration of human Dystrophin Expressing Chimeric (DEC) Cells to the mdx/scid mouse model of DMD. 2. Systemic administration of human DEC therapy resulted in amelioration of cardiac, respiratory and skeletal muscle function as confirmed by echocardiography, plethysmography and standard muscle strength tests respectively. 3. We introduce human DEC as a novel Advanced Therapy Medicinal Product (ATMP) for future clinical application in DMD patients.