Autotransplantation in mdx mice of mdx myoblasts genetically corrected by an HSV-1 amplicon vector

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder, characterized by a lack of dystrophin. To eliminate the need for immunosuppressive drugs, transplantation of genetically modified autologous myoblasts has been proposed as a possible therapy for this myopathy. An HSV-1 amplicon vector (HSVDGN), containing a 17.3-kb full-length MCK-driven mouse dystrophin cDNA, an eGFP gene, and a neomycin resistance gene driven by CMV or SV40 promoters, respectively, was constructed and used to transduce mdx primary myoblasts. The presence of the eGFP and neomycin resistance genes facilitated the evaluation of the initial transduction efficiency and the permanent transduction frequency. At low multiplicities of infection (MOI 1-5), the majority of myoblasts (60-90%) expressed GFP. The GFP-positive mdx myoblasts were sorted by FACS and selected with neomycin (300 microg/ml) for 2 weeks. Up to 2% of initially infected mdx myoblasts stably expressed the three transgenes without further selection at that time. These altered cells were grafted into the tibialis anterior muscles of 18 mdx mice. Some of the mice were immunosuppressed with FK506 due to the anticipation that eGFP and the product of neomycin resistance gene might be immunogenic. One month after transplantation, numerous muscle fibers expressing mouse dystrophin were detected by immunohistochemistry, in both immunosuppressed (10-50%) and nonimmunosuppressed (5-25%) mdx mice. Our results demonstrated the capability of permanently expressing a full-length dystrophin in dystrophic myoblasts with HSV-1 amplicon vector and raised the possibility of an eventual treatment of DMD based on the transplantation of genetically modified autologous myoblasts.     

Novel Duchenne Muscular Dystrophy Treatment Through Myoblast Transplantation Tolerance with Anti-CD45RB, Anti-CD154 and Mixed Chimerism

Duchenne muscular dystrophy (DMD) is a fatal disease caused by a defect in the skeletal muscle protein, dystrophin. One potential therapy for DMD involves transplantation of myoblasts from normal individuals. Unfortunately, myoblast allografts are particularly immunogenic and transplant tolerance in dystrophic (mdx/mdx) mice has not yet been achieved despite using strategies successful in other allograft models. Here, we attempted to induce 'central tolerance' using either haplo- or fully allogeneic bone marrow after conditioning with low-dose (3 Gy) whole body irradiation and anti-CD154 or anti-CD45RB mAbs. With one exception, these mice lacked persistent chimerism, long-term survival of myoblast allografts, or tolerance. In contrast, the addition of anti-CD45RB to anti-CD154 uniformly resulted in long-lived high-level mixed chimerism, long-term (>100 days) engraftment of allogeneic myoblasts and deletion of donor-reactive cells. Moreover, all recipients exhibited tolerance to second myoblast allografts or donor-specific tolerance to skin transplants performed >80 days after the initial graft. Thus, we now report that anti-CD45RB synergizes with anti-CD40L to promote stable mixed chimerism and robust tolerance to myoblast allografts for the first time. This novel protocol may be applicable to future clinical trials in myoblast transplantation for treatment of DMD and for transplantation of other immunogenic allografts.     

Improved success of myoblast transplantation in mdx mice by blocking the myostatin signal

BACKGROUND.: Duchenne muscular dystrophy (DMD) is caused by a dystrophin gene mutation. Transplantation of normal myoblasts results in long-term restoration of dystrophin. However, the success of this approach is compromised by the limited time of regeneration following muscle damage. Myostatin is known to be responsible for limiting skeletal muscle regeneration. Our purpose is to verify whether blocking the myostatin signal in mdx host mice or in normal myoblasts transplanted in mdx host mice would increase the extent of muscle repair and thus allow the formation of more dystrophin-positive fibers. METHODS.: Transgenic mdx mice carrying a dominant negative form of myostatin receptor (dnActRIIB) were used to test the fiber resistance to damage and to act as a host for normal myoblast transplantation. Myoblasts obtained from nondystrophic transgenic mice carrying the dominant negative myostatin receptor were also transplanted in nontransgenic mdx mice. RESULTS.: Transgenic mdx mice carrying the dnActRIIB gene have bigger muscles than mdx mice with the normal gene of ActRIIB. Their fiber resistance to exercise-induced damage was also greatly improved. Moreover, the success of normal myoblast transplantation was significantly enhanced in mdx/dnActRIIB mice. Finally, nondystrophic dnActRIIB myoblasts formed more abundant and bigger dystrophin positive fibers when transplanted in mdx mice. CONCLUSIONS.: Blocking the myostatin signal in mdx mice allowed the size of muscle fibers to increase, the fiber resistance to damage induced by exercise to increase, and the success of normal myoblast transplantation to improve. The transplantation in mdx mice of dnActRIIB myoblasts formed more abundant and larger dystrophin positive fibers.     

Autologous transplantation of muscle-derived CD133+ stem cells in Duchenne muscle patients

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive muscle disease due to defect on the gene encoding dystrophin. The lack of a functional dystrophin in muscles results in the fragility of the muscle fiber membrane with progressive muscle weakness and premature death. There is no cure for DMD and current treatment options focus primarily on respiratory assistance, comfort care, and delaying the loss of ambulation. Recent works support the idea that stem cells can contribute to muscle repair as well as to replenishment of the satellite cell pool. Here we tested the safety of autologous transplantation of muscle-derived CD133+ cells in eight boys with Duchenne muscular dystrophy in a 7-month, double-blind phase I clinical trial. Stem cell safety was tested by measuring muscle strength and evaluating muscle structures with MRI and histological analysis. Timed cardiac and pulmonary function tests were secondary outcome measures. No local or systemic side effects were observed in all treated DMD patients. Treated patients had an increased ratio of capillary per muscle fibers with a switch from slow to fast myosin-positive myofibers.     

Myoblast transplantation: A possible surgical treatment for a severe pediatric disease

Duchenne muscular dystrophy (DMD) is a genetic X-linked recessive orphan disease that affects approximately 1 in 3 500 male births. Boys with DMD have progressive and predictable muscle destruction due to the absence of dystrophin, a protein present under the muscle fiber membrane. This absence induces contraction-related membrane damage and activation of inflammatory necrosis and fibrosis, leading to cardiac/diaphragmatic failure and death. The authors support the therapeutic role of myoblast transplantation in DMD, and describe the history and rationale for such an approach.     

Treatment with anti-CD154 antibody and donor-specific transfusion prevents acute rejection of myoblast transplantation

BACKGROUND: Achieving immunological tolerance to transplanted myoblasts would reduce the adverse effects associated with the sustained immunosuppression required for this experimental therapeutic approach in Duchenne muscular dystrophic patients. METHODS: Mdx mice were transplanted with fully allogeneic BALB/c myoblasts in the tibialis anterior muscles. Seven days before transplantation (-7), host mice received 107 total donor spleen cells i.v. (donor-specific transfusion, DST) with 500 microg of anti-CD154 mAb i.p. on days -7, -4, 0, +4. RESULTS: Results showed a high level of dystrophin expression in 83, 60, and 20% of the mice 1, 3, and 6 months, respectively, after transplantation of myoblasts. No antibodies against the donor cells were produced up to 3 months after transplantation. However, abundant activated cytotoxic cells were present in muscles still expressing high percentage of dystrophin positive fibers. CONCLUSIONS: In conclusion, the DST + anti-CD154 mAb treatments effectively prolonged myoblast survival, but this treatment could not develop tolerance to complete allogeneic myoblast transplantation.     

Superior survival and proliferation after transplantation of myoblasts obtained from adult mice compared with neonatal mice

BACKGROUND: Myoblast transfer therapy (MTT) is a strategy designed to compensate for the defective gene in myopathies such as Duchenne muscular dystrophy (DMD). Experimental MTT in the mdx mouse (an animal model of DMD) has used donor myoblasts derived from mice of various ages; however, to date, there has been no direct quantitative comparison between the efficacy of MTT using myoblasts isolated from adult and neonate donor muscle. METHODS: Donor normal male myoblasts were injected into Tibialis Anterior muscles of dystrophic female host mice and the survival and proliferation of male myoblasts quantitated using Y-chromosome specific real-time quantitative polymerase chain reaction. The survival of late preplate (PP6) myoblasts derived from neonatal (3-5 days old) or adult (6-8 weeks old) donor mice after MTT were compared. The influence of the number of tissue culture passages, on survival post-MTT, was also evaluated for both types of myoblasts. RESULTS: Surprisingly, superior transplantation efficiency was observed for adult-derived compared with neonatal myoblasts (both early and late passage). Extended expansion (>17 passages) in tissue culture resulted in inferior survival and proliferation of both adult and neonatal myoblasts; however, proliferation of early passage myoblasts (both adult and neonate) was evident between 3 weeks and 3 months. CONCLUSIONS: Myoblasts derived from neonatal mice were inferior for transplantation, and early passage donor myoblasts from adult mice are recommended for MTT in this model.     

Inhibiting myostatin with follistatin improves the success of myoblast transplantation in dystrophic mice

Duchenne muscular dystrophy is a recessive disease due to a mutation in the dystrophin gene. Myoblast transplantation permits to introduce the dystrophin gene in dystrophic muscle fibers. However, the success of this approach is reduced by the short duration of the regeneration following the transplantation, which reduces the number of hybrid fibers. Our aim was to verify whether the success of the myoblast transplantation is enhanced by blocking the myostatin signal with an antagonist, follistatin. Three different approaches were studied to overexpress follistatin in the muscles of mdx mice transplanted with myoblasts. First, transgenic follistatin/mdx mice were generated; second, a follistatin plasmid was electroporated in mdx muscles, and finally, follistatin was induced in mdx mice muscles by a treatment with a histone deacetylase inhibitor. The three approaches improved the success of the myoblast transplantation. Moreover, fiber hypertrophy was also observed in all muscles, demonstrating that myostatin inhibition by follistatin is a good method to improve myoblast transplantation and muscle function. Myostatin inhibition by follistatin in combination with myoblast transplantation is thus a promising novel therapeutic approach for the treatment of muscle wasting in diseases such as Duchenne muscular dystrophy.     

Thymic myoid cells as a source of cells for myoblast transfer

Transplantation of disaggregated myoblasts from normal donor to the muscles of a diseased host, or reimplantation of genetically modified host myoblasts, has been suggested as a possible route to therapy for inherited myopathies such as Duchenne muscular dystrophy, or to supply missing proteins that are required systemically in diseases such as hemophilia. With two exceptions, studies of myoblast transfer in the mouse have involved transplantation of donor myoblasts isolated from adult or neonatal skeletal muscle satellite cells. In this study we present evidence that thymic myoid cells are capable of participating in the regeneration of postnatal skeletal muscle, resulting in the expression of donor-derived proteins such as dystrophin and retrovirally encoded proteins such as beta-galactosidase within host muscles. This leads us to conclude that thymic myoid cells may provide an alternative to myoblasts derived from skeletal muscle as a source of myogenic cells for myoblast transfer.     

A Synthetic Mechano Growth Factor E Peptide Enhances Myogenic Precursor Cell Transplantation Success

Myogenic precursor cell (MPC) transplantation is a good strategy to introduce dystrophin expression in muscles of Duchenne muscular dystrophy (DMD) patients. Insulin-like growth factor (IGF-1) promotes MPC activities, such as survival, proliferation, migration and differentiation, which could enhance the success of their transplantation. Alternative splicing of the IGF-1 mRNA produces different muscle isoforms. The mechano growth factor (MGF) is an isoform, especially expressed after a mechanical stress. A 24 amino acids peptide corresponding to the C-terminal part of the MGF E domain (MGF-Ct24E peptide) was synthesized. This peptide had been shown to enhance the proliferation and delay the terminal differentiation of C(2)C(12) myoblasts. The present study showed that the MGF-Ct24E peptide improved human MPC transplantation by modulating their proliferation and differentiation. Indeed, intramuscular or systemic delivery of this synthetic peptide significantly promoted engraftment of human MPCs in mice. In vitro experiments demonstrated that the MGF-Ct24E peptide enhanced MPC proliferation by a different mechanism than the binding to the IGF-1 receptor. Moreover, MGF-Ct24E peptide delayed human MPC differentiation while having no outcome on survival. Those combined effects are probably responsible for the enhanced transplantation success. Thus, the MGF-Ct24E peptide is an interesting agent to increase MPC transplantation success in DMD patients.     

Losartan enhances the success of myoblast transplantation

Duchenne muscular dystrophy is a recessive X-linked genetic disease caused by dystrophin gene mutations. Cell therapy can be a potential approach aiming to introduce a functional dystrophin in the dystrophic patient myofibers. However, this strategy produced so far limited results. Transforming growth factor-β (TGF-β) is a negative regulator of skeletal muscle development and is responsible for limiting myogenic regeneration. The combination of TGF-β signaling inhibition with myoblast transplantation can be an effective therapeutic approach in dystrophin-deficient patients. Our aim was to verify whether the success of human myoblast transplantation in immunodeficient dystrophic mice is enhanced with losartan, a molecule that downregulates TGF-β expression. In vitro, blocking TGF-β activity with losartan increased proliferation and fusion and decreased apoptosis in human myoblasts. In vivo, human myoblasts were transplanted in mice treated with oral losartan. Immunodetection of human dystrophin in tibialis anterior cross sections 1 month posttransplantation revealed more human dystrophin-positive myofibers in these mice than in nontreated dystrophic mice. Thus, blocking the TGF-β signal with losartan treatment improved the success of myoblast transplantation probably by increasing myoblast proliferation and fusion, decreasing macrophage activation, and changing the expression of myogenic regulator factors.     

Blood loss in Duchenne muscular dystrophy: vascular smooth muscle dysfunction?

Patients with Duchenne muscular dystrophy (DMD) tend to bleed more during surgery than do patients with other conditions. A retrospective analysis of blood loss after spinal surgery for scoliosis was therefore undertaken in 102 patients undergoing surgery in the senior author's unit. These included 48 patients with DMD, 26 patients with spinal muscular atrophy, and a miscellaneous group of 28 other patients most of whom had idiopathic scoliosis. For each patient the age at surgery, estimated blood volume, duration of operation, Cobb angle, and number of vertebrae fused were recorded and compared. Expression of dystrophin in skeletal muscle and the underlying gene mutation were also determined. The estimated blood loss in patients with DMD was significantly higher than that in patients with spinal muscular atrophy undergoing the same or similar procedure (P < 0.005) and was also significantly greater than that of the third group, which consisted mostly of patients with idiopathic scoliosis (P < 0.0005). Blood loss in the patient group with DMD showed a significant relationship with duration of surgery (P < 0.05). As most patients expressed no dystrophin, this did not correlate with the estimated blood loss. There was also no correlation between the estimated blood loss and the type of gene mutation found causing DMD. The authors' previous observations confirm the increased blood loss in patients with DMD who undergo scoliosis surgery. Because children with DMD lack dystrophin in all muscle types, including smooth muscle, the excessive blood loss may be because of a poor vascular smooth muscle vaso-constrictive response due to a lack of dystrophin.     

Myoblast survival enhancement and transplantation success improvement by heat-shock treatment in mdx mice

BACKGROUND: Duchenne muscular dystrophy is a disease caused by the incapacity to synthesize dystrophin, which is implicated in the maintenance of the sarcolemma integrity. Myoblast transplantation is a potential treatment of this disease. However, most of the transplanted cells die very rapidly after their injection. Heat-shock proteins (HSPs) are over-expressed when cells undergo various types of stresses. Our goal was thus to investigate whether the expression of HSPs (HSP70 in particular) could protect myoblasts from death after intramuscular injection. METHODS: HSP70 expression was induced by warming the cells at 42 degrees C for 60 minutes. HSP70 over-expression was quantified by Western blot analysis. The in vitro effect of HSPs on cell survival was evaluated by fluorescence-activated cell sorter analysis using the Hoescht/propidium iodide-labeling technique, and their in vivo effects were investigated by transplanting TnI-LacZ myoblasts labeled with [methyl-14C] thymidine. RESULTS: Western blots indicated a sevenfold over-expression of the HSP70 after the heat-shock treatment. In vitro, the heat-shock treatment protected 18% of the cells from staurosporine- (1 microM) induced apoptosis. HSPs also protected 10% of the cells from death induced by either tumor necrosis factor-alpha (30 ng/mL) or glucose oxydase (0.1 U/mL). In vivo, the treatment improved the cell survival by twofold 5 days after the graft and increased by fourfold the long-term graft success. CONCLUSIONS: The heat-shock treatment is a practical approach for improving the success of myoblast transplantation; in fact, using this kind of treatment, there is no need to genetically modify the cells before their transplantation.     

杜兴肌营养不良症治疗进展及前景

目的综述杜兴肌营养不良症（Duchenne muscular dystrophy，DMD）治疗的最新研究进展。方法广泛查阅近年来相关文献，对DMD治疗的研究进展进行综述。结果目前对DMD的治疗主要有基因治疗、细胞治疗和药理学治疗，基因治疗和细胞治疗通过传递正常基因或修正突变基因以及移植正常细胞，如干细胞来进行治疗；而药理学治疗主要针对dystrophin基因缺陷引起的下游事件，运用各种药物来减缓DMD病理进程，从而改善患者的生活质量，延长寿命。结论针对DMD的各种治疗手段均存在一定困难，目前尚不能有效治疗此病，还需要研究探索更有效的治疗浍格     

Central tolerance to myogenic cell transplants does not include muscle neoantigens

BACKGROUND: Duchenne muscular dystrophy is a fatal genetic disease caused by lack of dystrophin. Myogenic cell transplantation (MT), a potential therapy for Duchenne muscular dystrophy, can restore dystrophin expression in muscles. Because allogeneic MT is highly resistant to peripheral tolerance, we proposed to induce central tolerance. However, given its immunogenicity, we asked whether central tolerance to donor major histocompatibility complex would allow long-term expression of dystrophin, a tissue-specific neoantigen in dystrophic recipients. METHODS: Central tolerance was induced in C57BL/10J mdx (dystrophic) mice by allogeneic bone marrow transplantation (BMT) after conditioning with either lethal total body irradiation (TBI) or an established nonmyeloablative protocol (anti-CD154, anti-CD8 mAbs, and low-dose TBI). Recipients subsequently received donor-strain MT or skin grafts. RESULTS: Long-term hematopoietic chimeras generated using either lethal TBI or the nonmyeloablative regimen were tolerant to donor skin grafts and both primary and secondary donor MT (>90 days). Myogenic cell transplantation survival was decreased when chimerism was transient, which was most common with nonmyeloablative conditioning and fully rather than haplo-mismatched donors. Interestingly, regardless of conditioning, MT was associated with localized muscle infiltration with Foxp3CD4, CD25CD4, and PerforinCD8 cells, whereas skin grafts lacked infiltration. CONCLUSIONS: Central tolerance achieved using regimens that eliminate nearly all endogenous peripheral lymphocytes (i.e., lethal irradiation) or a nonmyeloablative protocol that depleted peripheral CD8 cells, results in lymphocytic infiltration in muscles that received MT but not in skin allografts. This suggests that muscle-specific infiltration may result from lack of negative selection for peripheral neoantigens in the thymus after BMT and that tolerance after MT may rely on peripheral regulatory mechanisms.     

Induction of Anoikis Following Myoblast Transplantation into SCID Mouse Muscles Requires the Bit1 and FADD Pathways

Seventy-five percent of the myoblasts transplanted in the mouse muscle die during the first 4 days following transplantation. The purpose of this study was to determine if anoikis plays a role in this phenomenon. Survival and proliferation of myoblasts in vitro were determined by Hoescht-PI labeling and cell counts respectively. In vivo cell survival and proliferation were quantified by injecting human male myoblasts labeled with (14)C-thymidine in SCID mouse muscles. Survival and proliferation of the transplanted myoblasts were evaluated by scintigraphy and quantitative PCR of human Y chromosomal DNA. Inclusion of the extracellular matrix protein fibronectin enhanced transplanted myoblast survival by 1.7-fold while vitronectin improved their proliferation by 1.8-fold. Reductions in FADD and Bit1 expression reduced anoikis in vitro and improved the injected myoblast survival in vivo. Ectopic expression of the anti-apoptotic protein Bcl-2 completely abolished myoblast anoikis in vitro and enhanced cell survival by 3.1-fold in vivo. Cell death following transplantation appears to me mediated in part by anoikis. Inclusion of extracellular matrix proteins enhanced both survival and proliferation. Reduced expression of the proapoptotic proteins Bit1 and FADD or overexpression of Bcl-2 improved myoblast survival.     

Systemic human minidystrophin gene transfer improves functions and life span of dystrophin and dystrophin/utrophin‐deficient mice

Duchenne muscular dystrophy (DMD) is the most common and lethal genetic muscle disease, caused by mutations in the dystrophin gene. No efficacious treatment is currently available. Here we report AAV vector systemic delivery and therapeutic benefits of the functional human minidystrophin gene in a severe and more reliable DMD mouse model, the dystrophin/utrophin double deficiency mouse (dys?/?:utrn?/?, dKO). These mice show many pathologic and phenotypic signs typical of DMD in humans including kyphosis and shorter life span, all of which are not seen in the mdx mice due to their utrophin upregulation that partially compensates the loss of dystrophin functions and leads to mild phenotypes. The therapeutic value of this new approach was demonstrated in both mdx and dKO murine models, in which we observed highly efficient minidystrophin gene expression, ameliorated muscle pathologies, improvement in growth and motility, inhibition of spine and limb deformation, and prolongation of life span. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 421–426, 2009     

Growth Hormone Increases Bone Toughness and Decreases Muscle Inflammation in Glucocorticoid-Treated Mdx Mice,Model of Duchenne Muscular Dystrophy

The absence of functional dystrophin with mutations of the dystrophin-encoding gene in Duchenne muscular dystrophy (DMD) results in muscle inflammation and degeneration, as well as bone fragility. Long-term glucocorticoid therapy delays the muscular disease progression but suppresses growth hormone secretion, resulting in short stature and further deleterious effects on bone strength. This study evaluated the therapeutic potential of daily growth hormone therapy in growing mdx mice as a model of DMD. Growth hormone treatment on its own or in combination with glucocorticoids significantly improved muscle histology and function and decreased markers of inflammation in mdx mice. Glucocorticoid treatment thinned cortical bone and decreased bone strength and toughness. Despite the minimal effects of growth hormone on bone microarchitecture, it significantly improved biomechanical properties of femurs and vertebrae, even in the presence of glucocorticoid treatment. Together these studies suggest that the use of growth hormone in DMD should be considered for improvements to muscle and bone health. © 2019 American Society for Bone and Mineral Research.