Chimeric Cell Therapies as a Novel Approach for Duchenne Muscular Dystrophy (DMD) and Muscle Regeneration.

Chimerism-based strategies represent a pioneering concept which has led to groundbreaking advancements in regenerative medicine and transplantation. This new approach offers therapeutic potential for the treatment of various diseases, including inherited disorders. The ongoing studies on chimeric cells prompted the development of Dystrophin-Expressing Chimeric (DEC) cells which were introduced as a potential therapy for Duchenne Muscular Dystrophy (DMD). DMD is a genetic condition that leads to premature death in adolescent boys and remains incurable with current methods. DEC therapy, created via the fusion of human myoblasts derived from normal and DMD-affected donors, has proven to be safe and efficacious when tested in experimental models of DMD after systemic-intraosseous administration. These studies confirmed increased dystrophin expression, which correlated with functional and morphological improvements in DMD-affected muscles, including cardiac, respiratory, and skeletal muscles. Furthermore, the application of DEC therapy in a clinical study confirmed its long-term safety and efficacy in DMD patients. This review summarizes the development of chimeric cell technology tested in preclinical models and clinical studies, highlighting the potential of DEC therapy in muscle regeneration and repair, and introduces chimeric cell-based therapies as a promising, novel approach for muscle regeneration and the treatment of DMD and other neuromuscular disorders.

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.

Intraosseous transplant of dystrophin expressing chimeric (DEC) cells improves skeletal muscle function in mdx mouse model of Duchenne muscular dystrophy.

Introduction: We previously reported that systemic delivery of dystrophin expressing chimeric (DEC) cells of normal (wt) and dystrophin-deficient (mdx) myoblast (MB) or mesenchymal stem cell (MSC) origin restored dystrophin expression and improved cardiac function in the mdx mouse model of Duchenne muscular dystrophy (DMD). Aim: This study evaluated the effect of intraosseous delivery of murine DEC lines of MB (MB wt /MB mdx ) and MSC (MB wt /MSC mdx ) origin on function of gastrocnemius muscle (GM). Material and methods: DEC lines created by ex vivo fusion were tested in the mdx mouse model of DMD: Group 1 - vehicle (control), Group 2 - non-fused 0.25 × 106 MB wt and 0.25 × 106 MSC mdx (control), Group 3 - fused 0.5 × 106 MB wt /MB mdx DEC and Group 4 - fused 0.5 × 106 MB wt /MSCmdx DEC. In situ and in vitro muscle force tests assessed GM function at 90 days post-transplant. Results: Application of MB wt /MSC mdx and MB wt /MB mdx DEC significantly improved the fatigue ratio of GM compared to vehicle-injected controls detected by in vivo muscle force tests (0.567 ±0.116, p = 0.045 and 0.489 ±0.087, p < 0.05, respectively). MB wt /MSCmdx DEC recipients presented enhanced maximum force at tetanus (0.145 ±0.040 g/mg, p < 0.05); furthermore, recipients of MB wt /MBmdx DEC showed a significant increase in the maximum force generation rate compared to vehicle controls (4.447 ±1.090 g/s/mg, p < 0.05). The ex vivo GM force testing in MB wt /MSCmdx DEC recipients detected increased average GM force compared to vehicle and non-fused controls. Conclusions: Systemic-intraosseous administration of MB wt /MBmdx and MB wt /MSCmdx DEC therapy combining the myogenic and immunomodulatory properties of MB and MSC significantly improved skeletal muscle (GM) function of force and resistance to fatigue in an mdx mouse model of DMD.

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.

Transplantation of Dystrophin Expressing Chimeric Human Cells of Myoblast/Mesenchymal Stem Cell Origin Improves Function in Duchenne Muscular Dystrophy Model.

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder caused by mutations in dystrophin gene. Currently, there is no cure for DMD. Cell therapies are challenged by limited engraftment and rejection. Thus, more effective and safer therapeutic approaches are needed for DMD. We previously reported increased dystrophin expression correlating with improved function after transplantation of dystrophin expressing chimeric (DEC) cells of myoblast origin in the mdx mouse models of DMD. This study established new DEC cell line of myoblasts and mesenchymal stem cells (MSC) origin and tested its efficacy and therapeutic potential in mdx/scid mouse model of DMD. Fifteen ex vivo cell fusions of allogenic human myoblast [normal myoblasts (MBN)] and normal human bone marrow-derived MSC (MSCN) from normal donors were performed using polyethylene glycol. Flow cytometry, confocal microscopy, polymerase chain reaction (PCR)-short tandem repeats, polymerase chain reaction-reverse sequence-specific oligonucleotide probe assessed chimeric state of fused MBN/MSCN DEC cells, whereas Comet assay assessed fusion procedure safety testing genotoxicity. Immunofluorescence and real-time PCR assessed dystrophin expression and myogenic differentiation. Mixed lymphocyte reaction (MLR) evaluated DEC's immunogenicity. To test MBN/MSCN DEC efficacy in vivo, gastrocnemius muscle of mdx/scid mice were injected with vehicle (n = 12), nonfused MBN and MSCN (n = 9, 0.25 × 106/each) or MBN/MSCN DEC (n = 9, 0.5 × 106). Animals were evaluated for 90 days using ex vivo and in vivo muscle strength tests. Histology and immunofluorescence staining assessed dystrophin expression, centrally nucleated fibers and scar tissue formation. Post-fusion, MBN/MSCN DEC chimeric state, myogenic differentiation, and dystrophin expression were confirmed. MLR reveled reduced DEC's immune response compared with controls (P < 0.05). At 90 days post-DEC transplant, increase in dystrophin expression (20.26% ± 2.5%, P < 0.05) correlated with improved muscle strength and function in mdx/scid mice. The created human MBN/MSCN DEC cell line introduces novel therapeutic approach combining myogenic and immunomodulatory properties of MB and MSC, and as such may open a universal approach for muscle regeneration in DMD.

Human dystrophin expressing chimeric (DEC) cell therapy ameliorates cardiac, respiratory, and skeletal muscle's function in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive and lethal disease, caused by X-linked mutations of the dystrophin encoding gene. The lack of dystrophin leads to muscle weakness, degeneration, fibrosis, and progressive loss of skeletal, cardiac, and respiratory muscle function resulting in premature death due to the cardiac and respiratory failure. There is no cure for DMD and current therapies neither cure nor arrest disease progression. Thus, there is an urgent need to develop new approaches and safer therapies for DMD patients. We have previously reported functional improvements which correlated with increased dystrophin expression following transplantation of dystrophin expressing chimeric (DEC) cells of myoblast origin to the mdx mouse models of DMD. In this study, we demonstrated that systemic-intraosseous transplantation of DEC human cells derived from myoblasts of normal and DMD-affected donors, increased dystrophin expression in cardiac, respiratory, and skeletal muscles of the mdx/scid mouse model of DMD. DEC transplant correlated with preservation of ejection fraction and fractional shortening on echocardiography, improved respiratory function on plethysmography, and improved strength and function of the limb skeletal muscles. Enhanced function was associated with improved muscle histopathology, revealing reduced mdx pathology, fibrosis, decreased inflammation, and preserved muscle morphology and architecture. Our findings confirm that DECs generate a systemic protective effect in DMD-affected target organs. Therefore, DECs represents a novel therapeutic approach with the potential to preserve or enhance multiorgan function of the skeletal, cardiac, and respiratory muscles critical for the well-being of DMD patients.

Cardiac Protection after Systemic Transplant of Dystrophin Expressing Chimeric (DEC) Cells to the mdx Mouse Model of Duchenne Muscular Dystrophy.

Duchenne Muscular Dystrophy (DMD) is a progressive lethal disease caused by X-linked mutations of the dystrophin gene. Dystrophin deficiency clinically manifests as skeletal and cardiac muscle weakness, leading to muscle wasting and premature death due to cardiac and respiratory failure. Currently, no cure exists. Since heart disease is becoming a leading cause of death in DMD patients, there is an urgent need to develop new more effective therapeutic strategies for protection and improvement of cardiac function. We previously reported functional improvements correlating with dystrophin restoration following transplantation of Dystrophin Expressing Chimeric Cells (DEC) of myoblast origin in the mdx and mdx/scid mouse models. Here, we confirm positive effect of DEC of myoblast (MBwt/MBmdx) and mesenchymal stem cells (MBwt/MSCmdx) origin on protection of cardiac function after systemic DEC transplant. Therapeutic effect of DEC transplant (0.5 × 106) was assessed by echocardiography at 30 and 90 days after systemic-intraosseous injection to the mdx mice. At 90 days post-transplant, dystrophin expression in cardiac muscles of DEC injected mice significantly increased (15.73% ± 5.70 -MBwt/MBmdx and 5.22% ± 1.10 - MBwt/MSCmdx DEC) when compared to vehicle injected controls (2.01% ± 1.36) and, correlated with improved ejection fraction and fractional shortening on echocardiography. DEC lines of MB and MSC origin introduce a new promising approach based on the combined effects of normal myoblasts with dystrophin delivery capacities and MSC with immunomodulatory properties. Our study confirms feasibility and efficacy of DEC therapy on cardiac function and represents a novel therapeutic strategy for cardiac protection and muscle regeneration in DMD.

Dystrophin Expressing Chimeric (DEC) Human Cells Provide a Potential Therapy for Duchenne Muscular Dystrophy.

Duchenne Muscular Dystrophy (DMD) is a progressive and lethal disease caused by mutations of the dystrophin gene. Currently no cure exists. Stem cell therapies targeting DMD are challenged by limited engraftment and rejection despite the use of immunosuppression. There is an urgent need to introduce new stem cell-based therapies that exhibit low allogenic profiles and improved cell engraftment. In this proof-of-concept study, we develop and test a new human stem cell-based approach to increase engraftment, limit rejection, and restore dystrophin expression in the mdx/scid mouse model of DMD. We introduce two Dystrophin Expressing Chimeric (DEC) cell lines created by ex vivo fusion of human myoblasts (MB) derived from two normal donors (MBN1/MBN2), and normal and DMD donors (MBN/MBDMD). The efficacy of fusion was confirmed by flow cytometry and confocal microscopy based on donor cell fluorescent labeling (PKH26/PKH67). In vitro, DEC displayed phenotype and genotype of donor parent cells, expressed dystrophin, and maintained proliferation and myogenic differentiation. In vivo, local delivery of both DEC lines (0.5 × 106) restored dystrophin expression (17.27%±8.05-MBN1/MBN2 and 23.79%±3.82-MBN/MBDMD) which correlated with significant improvement of muscle force, contraction and tolerance to fatigue at 90 days after DEC transplant to the gastrocnemius muscles (GM) of dystrophin-deficient mdx/scid mice. This study establishes DEC as a potential therapy for DMD and other types of muscular dystrophies.

Filaria associated clinical manifestations in children in an endemic area and morbidity control by immunomonitoring and optimal DEC therapy: Sevagram experience.

Lymphatic filariasis is a major public health problem in India with 412 million people living in bancroftian endemic areas and is a major cause of clinical morbidity. Twenty million people are reported to suffer from chronic disease manifestations such as lymphoedema, hydrocele or elephantiasis. At least twice the number have been shown to suffer from acute and occult filarial infections in an endemic area without diagnosis. Due to non-availability of suitable diagnostic test for confirming filaria aetiology other than parasitological examination, no significant study on filariasis in children has been reported earlier. Studies in our laboratory for more than a decade showed usefulness of microfilarial excretory-secretory antigen in confirming filarial aetiology in acute and occult infections in adults as well as in children. This study reports acute and atypical manifestations such as lymphadenopathy, asthmatic bronchitis, pulmonary eosinophilia, mono-arthritis, recurrent URI, pneumonia, nutritional anemia, pain in abdomen etc. in children living in filaria endemic area having no microfilaraemia but showing filaria aetiology by immunomonitoring for the presence of antibody or antigen and responding to optimal DEC therapy.

Immunomonitoring followed by optimal dec therapy for successful management of clinical filariasis in an endemic area.

Lymphatic filariasis continues to be the major cause of clinical morbidity in India and other developing tropical countries. One of the major lacunae in the effective management of clinical filarial cases is the non-availability of a suitable diagnostic test for confirming filaria aetiology in acute, chronic and occult clinical cases where microfilariae (mf) are not usually seen in peripheral circulation. Studies in our laboratory have shown the usefulness of filarial antibody and antigen assays using microfilarial excretory-secretory (mf ES) antigen in detecting microfilaraemic, acute and chronic filarial cases and in confirming filarial aetiology in occult infections. Diethylcarbamazine citrate (DEC) is the drug of choice for lymphatic filariasis. Different regimens of DEC have been explored in the treatment of microfilaraemic cases. Immunomonitoring has shown that the seroconversion of antigen and antibody positivity was found to be very helpful in determining appropriate period of DEC treatment for clinical relief and cure in clinical filarial patients and further they did not have recurrence in most of the cases. Optimal DEC (6mg/kg body wt/day for 21 days each month for 3-12 months) therapy was found to be very effective in acute and atypical clinical manifestations such as asthmatic bronchitis, pulmonary eosinophilia, monoarthritis, recurrent upper respiratory tract infections (URI), pneumonia (super imposed infections) in children and minimal hydrocele, epididymoorchitis, lymphangitis, lymphadenitis, acute abdomen, central serous retinopathy, tenosynovitis, pain and swelling in limbs and joints in adults living in filaria endemic areas.