Deletion of the GAA repeats from the human frataxin gene using the CRISPR-Cas9 system in YG8R-derived cells and mouse models of Friedreich ataxia.

The Friedreich ataxia is a monogenic disease due to a hyperexpanded GAA triplet located within the first intron of the frataxin gene that causes transcriptional issues. The resulting frataxin protein deficiency leads to a Fe-S cluster biosynthesis dysfunction in the mitochondria and to oxidative stress and cell death. Here we use the CRISPR-Cas9 system to remove the mutated GAA expansion and restore the frataxin gene transcriptional activity and protein level. Both YG8R and YG8sR mouse models and cell lines derived from these mice were used to CRISPR-edited successfully the GAA expansion in vitro and in vivo. Nevertheless, our results suggest the YG8sR as a better and more suitable model for the study of the CRISPR-Cas9 edition of the mutated frataxin gene.

Development of an AAV9 coding for a 3XFLAG-TALEfrat#8-VP64 able to increase in vivo the human frataxin in YG8R mice.

Artificially designed transcription activator-like effector (TALE) proteins fused to a transcription activation domain (TAD), such as VP64, are able to activate specific eukaryotic promoters. They thus provide a good tool for targeted gene regulation as a therapy. However, the efficacy of such an agent in vivo remains to be demonstrated as the majority of studies have been carried out in cell culture. We produced an adeno-associated virus 9 (AAV9) coding for a TALEfrat#8 containing 13 repeat variable diresidues able to bind to the proximal promoter of human frataxin (FXN) gene. This TALEfrat#8 was fused with a 3XFLAG at its N terminal and a VP64 TAD at its C terminal, and driven by a CAG promoter. This AAV9_3XFLAG-TALEfrat#8-VP64 was injected intraperitoneally to 9-day-old and 4-month-old YG8R mice. After 1 month, the heart, muscle and liver were removed and their FXN mRNA and FXN protein were analyzed. The results show that the AAV9_3XFLAG-TALEfrat#8-VP64 increased the FXN mRNA and FXN protein in the three organs studied. These results corroborate our previous in vitro studies in the FRDA human fibroblasts. Our study indicates that an AAV coding for a TALE protein coupled with a TAD may be used to increase gene expression in vivo as a possible treatment not only for FRDA but also for other haploinsufficiency diseases.

Meganucleases can restore the reading frame of a mutated dystrophin.

Mutations in Duchenne muscular dystrophy (DMD) are either inducing a nonsense codon or a frameshift. Meganucleases (MGNs) can be engineered to induce double-strand breaks (DSBs) at specific DNA sequences. These breaks are repaired by homologous recombination or by non-homologous end joining (NHEJ), which results in insertions or deletions (indels) of a few base pairs. To verify whether MGNs could be used to restore the normal reading frame of a dystrophin gene with a frameshift mutation, we inserted in a plasmid coding for the dog micro-dystrophin sequences containing a MGN target. The number of base pairs in these inserted sequences changed the reading frame. One of these modified target micro-dystrophin plasmids and an appropriate MGN were then transfected in 293FT cells. The MGN induced micro-deletion or micro-insertion in the micro-dystrophin that restored dystrophin expression. MGNs also restored micro-dystrophin expression in myoblasts in vitro and in muscle fibers in vivo. The mutation of the targeted micro-dystrophin was confirmed by PCR amplification followed by digestion with the Surveyor enzyme and by cloning and sequencing of the amplicons. These experiments are thus a proof of principle that MGNs that are adequately engineered to target appropriate sequences in the human dystrophin gene should be able to restore the normal reading frame of that gene in DMD patients with an out-of-frame deletion. New MGNs engineered to target a sequence including or near nonsense mutation could also be used to delete it.

Overexpression of follistatin in human myoblasts increases their proliferation and differentiation, and improves the graft success in SCID mice.

Duchenne muscular dystrophy is caused by the absence of functional dystrophin, leading to the myofiber membrane instability and progressive muscle atrophy. Myoblast transplantation in dystrophic muscles is a potential therapy, as it permits the long-term restoration of dystrophin expression in transplanted muscles. However, the success of this approach is limited by the short period of muscle repair following myoblast transplantation. Myostatin, a powerful inhibitor of muscle growth, is involved in terminating the period of muscle repair following injury by reducing myoblast proliferation and differentiation. Follistatin forms a complex with myostatin, preventing its interaction with its receptor and thus blocking the myostatin signal. Here, we used a lentivirus to overexpress the follistatin protein in normal myoblasts to block the myostatin signaling. We measured the potential of transduced myoblasts to proliferate and to form multinucleated myotubes in vitro. And finally, we considered the engraftment success of those transduced myoblasts in comparison with control cells in vivo within SCID mice TA muscle. Our results first confirmed the overexpression of follistatin into lentivirus transduced myoblasts, and second showed that the overexpression of the follistatin in normal human myoblasts improved in vitro their proliferation rate by about 1.5-fold after 96 h and also their differentiation rate by about 1.6- and 1.8-fold, respectively, in the absence and in the presence of recombinant myostatin. Finally, our data demonstrated that the engraftment of human normal myoblasts overexpressing the follistatin protein into SCID mouse muscles was enhanced by twofold.

Vascular endothelial growth factor reduced hypoxia-induced death of human myoblasts and improved their engraftment in mouse muscles.

Muscle precursor cell (myoblasts) transplantation is considered as a potential approach to restore dystrophin expression in Duchenne muscular dystrophy (DMD) patients. The study purpose was to verify the implication of hypoxia in the myoblast death observed after their transplantation and also to evaluate the potential beneficial effects of vascular endothelial growth factor (VEGF) overexpression on myoblast engraftment in a murine model. Pimonidazole hydrochloride (hypoxyprobe-1) was used to mark selectively myoblasts to evaluate their hypoxia in vivo. In vitro, hypoxia was induced by culturing human myoblasts in hypoxic environment. In vitro effects of VEGF(165) on survival of human cells was assessed by Hoescht-PI labeling. Tibialis anterior (TA) female mouse muscles were electroporated with a plasmid containing the VEGF(165) or with an empty vector. Circulating VEGF concentration was assessed by ELISA. After 2 weeks of electroporation, severe combined immunodeficient (SCID) mice were transplanted with 800 000 human male myoblasts labeled with radioactive thymidine. Mouse muscles were harvested 2 and 4 days later and myoblast survival and proliferation were evaluated by scintigraphy and Y chromosome quantitative PCR. The long-term graft success was evaluated using gamma-radiograph imaging and by counting the dystrophin positive muscle fibers. Hypoxyprobe labeling has shown that most of the transplanted myoblasts were hypoxic. The transplantation of radioactive male myoblasts in female mice electroporated with the VEGF(165) plasmid demonstrated that VEGF reduced their death by 10% but did not improve their proliferation. VEGF(165) enhanced human myoblast survival in vitro under hypoxic conditions. Electroporation of TA muscles of SCID mouse with the vector coding for VEGF(165) promoted angiogenesis and improved by 1.5-fold the success of myoblast transplantation in comparison with the control mice that were electroporated with the empty vector. These results indicate that hypoxia is partially responsible for the death of the transplanted myoblasts. VEGF can be used to improve myoblast survival and the graft success.

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.

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.

Dystrophin expression in host muscle following transplantation of muscle precursor cells modified with the phiC31 integrase.

Duchenne muscular dystrophy (DMD) is the most severe muscular dystrophy. It is caused by the absence of dystrophin in muscle fibers. The autologous transplantation of genetically corrected muscle precursor cells (MPCs) is a possible cure for DMD. A non-viral method of genetic modification was tested in this study. The co-transfection (nucleofection) of a phiC31 integrase and a transgene expressing plasmid in MPCs led to an increased stable expression in vitro. The stable expression of a small transgene (eGFP) in muscle fibers was initially demonstrated following the transplantation of the genetically modified cells. The stable expression of a truncated version of dystrophin as well as the full-length dystrophin fused with eGFP was then demonstrated in MPCs obtained from an mdx mice. The transplantation of these cells led not only to the expression of these fusion proteins in muscle fibers but also to the reconstitution of the dystrophin complex. Human MPCs were also genetically modified with a plasmid coding for the full-length human dystrophin gene fused with eGFP and transplanted in severe combined immuno deficient mice leading to the expression of eGFP dystrophin in muscle fibers. This work indicates that cell transplantation after correction of MPCs with phiC31 integrase is a possible approach to treat DMD.

Interleukin-4 improves the migration of human myogenic precursor cells in vitro and in vivo.

Different molecules are available to recruit new neighboring myogenic cells to the site of regeneration. Formerly called B cell stimulatory factor-1, IL-4 can now be included in the list of motogenic factors. The present report demonstrates that human IL-4 is not required for fusion between mononucleated myoblasts but is required for myotube maturation. In identifying IL-4 as a pro-migratory agent for myogenic cells, these results provide a mechanism which partly explains IL-4 demonstrated activity during differentiation. Among the different mechanisms by which IL-4 might enhance myoblast migration processes, our results indicate that there are implications of some integrins and of three major components of the fibrinolytic system. Indeed, increases in the amount of active urokinase plasminogen activator and its receptor were observed following an IL-4 treatment, while the plasminogen activator inhibitor-1 decreased. Finally, IL-4 did not modify the amount of cell surface alpha5 integrin but increased the presence of beta3 and beta1 integrins. This integrin modulation might favor myogenic cell migration and its interaction with newly formed myotubes. Therefore, IL-4 co-injection with transplanted myoblasts might be an approach to enhance the migration of transplanted cells for the treatment of a damaged myocardium or of a Duchenne Muscular Dystrophy patient.

Tubulyzine, a novel tri-substituted triazine, prevents the early cell death of transplanted myogenic cells and improves transplantation success.

Myoblast transplantation (MT) is a potential therapeutic approach for several muscular dystrophies. A major limiting factor is that only a low percentage of the transplanted myoblasts survives the procedure. Recent advances regarding how and when the myoblasts die indicate that events preceding actual tissue implantation and during the first days after the transplantation are crucial. Myoseverin, a recently identified tri-substituted purine, was shown to induce in vitro the fission of multinucleated myotubes and affect the expression of a variety of growth factors, and immunomodulation, extracellular matrix-remodeling, and stress response genes. Since the effects of myoseverin are consistent with the activation of pathways involved in wound healing and tissue regeneration, we have investigated whether pretreatment and co-injection of myoblasts with Tubulyzine (microtubule lysing triazine), an optimized myoseverin-like molecule recently identified from a triazine library, could reduce myoblast cell death following their transplantation and consequently improves the success of myoblast transplantation. In vitro, using annexin-V labeling, we showed that Tubulyzine (5 microM) prevents normal myoblasts from apoptosis induced by staurosporine (1 microM). In vivo, the pretreatment and co-injection of immortal and normal myoblasts with Tubulyzine reduced significantly cell death (assessed by the radio-labeled thymidine of donor DNA) and increased survival of myoblasts transplanted in Tibialis anterior (TA) muscles of mdx mice, thus giving rise to more hybrid myofibers compared to transplanted untreated cells. Our results suggest that Tubulyzine can be used as an in vivo survival factor to improve the myoblast-mediated gene transfer approach.

Tumor necrosis factor-alpha (TNF-alpha) stimulates chemotactic response in mouse myogenic cells.

Migration of transplanted myogenic cells occurs during both embryogenesis and regeneration of skeletal muscles and is important for successful myoblast transplantation, but little is known about factors that promote chemotaxis of these cells. Tumor necrosis factor-alpha (TNF-alpha) is known to induce chemotactic effect on several cell types. In this study, we investigated its influence on the in vitro and in vivo motility of C2C12 and primary myoblasts. In the in vitro test performed in the blind-well Boyden chambers, we showed that TNF-alpha (50-400 U/ml) significantly enhanced the ability of myogenic cells to migrate. The dose-response curve for this factor was bell shaped, with maximum activity in the 200 U/ml range. In the in vivo test, intramuscular administration of TNF-alpha was performed by an Alzet pump connected to a perforated polyethylene microtube inserted in the tibialis anterior (TA) of CD1 mice. In these experiments, myoblasts were injected under the muscle epimysium. The recipient mice were immunosuppressed with FK506. Our results showed that, 5 days after myoblast transplantation, cells migrated further in the muscles infused with TNF-alpha than in the muscles not exposed to TNF-alpha. TNF-alpha not only has a chemotactic activity but may also modify cell migration via its action on matrix metalloproteinase (MMP) expression. The proteolytic activities of the MMPs secreted in the muscles were thus also assessed by gelatin zymography. The results showed an increased of MMP-2 and MMP-9 transcripts in the TNF-alpha-infused muscles injected with myogenic cells. Myoblast migration during transplantation may be enhanced by overlapping gradients of several effector molecules such as TNF-alpha, interferon-gamma (INF-gamma), and interleukins, released at the site of muscle injury. We propose that TNF-alpha may promote myoblast migration directly through chemotactic activity and indirectly by enhancing MMP activity at the site of muscle injury.

The urokinase plasminogen activator: an interesting way to improve myoblast migration following their transplantation.

Muscle cell migration plays an important role in the incorporation of transplanted myoblasts in muscle fibers. Understanding the mechanisms underlying the high migration capacity of the C(2)C(12) myoblast cell line may help to develop approaches to improve the migration of normal myoblasts and consequently to increase their participation to the host myofiber regeneration. We have previously shown that matrix metalloproteinases are implicated in the in vivo migration of C(2)C(12). Here, we studied the role of urokinase plasminogen activator (uPA) in this process. The expression of uPA mRNA and the enzymatic activity of uPA were studied in both normal myoblasts and the C(2)C(12) myoblast cell line. Reverse transcriptase polymerase chain reaction analysis showed that uPA mRNA was more strongly expressed in C(2)C(12) cells than in normal myoblasts. The enzymatic activity of secreted uPA analyzed by casein zymography is higher in medium conditioned by C(2)C(12) cells than in medium conditioned by normal myoblasts. Using our previously described microtube technique to assess in vivo cell migration, we showed that uPA is implicated in the in vivo migration of C(2)C(12) cells since this migration was abrogated in the presence of aprotinin (a general serine protease inhibitor) or amiloride (a uPA-specific inhibitor). We, therefore, hypothesized that increasing endogenous uPA expression by normal myoblasts may improve their migration capacity. Since an accumulating body of evidence has shown that growth factors regulate expression of uPA in a wide range of cells, we treated normal myoblasts with several growth factors alone or in combination with components of the extracellular matrix (ECM). All stimulants tested showed a minimal to strong effect on uPA enzymatic activity as assayed by zymography analysis. The positive effect of basic fibroblast growth factor (bFGF) on uPA enzymatic activity was slightly potentiated in the presence of fibronectin. Moreover, the pretreatment and coinjection of mouse myoblasts with bFGF alone or in combination with fibronectin improved significantly their in vivo migration throughout the tibialis anterior muscle of mdx mice. These results suggest that increasing uPA expression by an appropriate combination of growth factors and ECM components constitutes a possible approach to improving the migration of myogenic cells after transplantation.

Dysferlin expression after normal myoblast transplantation in SCID and in SJL mice.

Limb girdle muscular dystrophy type 2B form and Miyoshi myopathy are both caused by mutations in the recently cloned gene dysferlin. In the present study, we have investigated whether cell transplantation could permit dysferlin expression in vivo. Two transplantation models were used: SCID mice transplanted with normal human myoblasts, and SJL mice, the mouse model for limb girdle muscular dystrophy type 2B and Miyoshi myopathy, transplanted with allogeneic primary mouse muscle cell cultures expressing the beta-galactosidase gene under control of a muscle promoter of Troponin I. FK506 immunosuppression was used in the non-compatible allogeneic model. One month after transplantation, human and mouse dysferlin proteins were detected in all transplanted SCID and SJL muscles, respectively. Co-localization of dysferlin and human dystrophin or beta-galactosidase-positive fibers was observed following the transplantation of myoblasts. Dysferlin proteins were monitored by immunocytochemistry and Western blot. The number of dysferlin-positive fibers was 40-50% and 20-30% in SCID and SJL muscle sections, respectively. Detection of dysferlin in both SCID mice and dysferlin-deficient SJL mouse shows that myoblast transplantation permits the expression of the donor dysferlin protein.

Gene therapy: a strategy for the treatment of inherited muscle diseases?

The emergence of new vectors of viral origin (recombinant adeno-associated viruses, second and third generation adenoviruses) and a new potential source of cells for transplantation (muscle-derived stem cells) are broadening the panel of therapeutic options for myopathies. Although the perfect gene-transfer method(s) have not yet been found, recent findings will certainly constitute a strong knowledge base for future clinical trials.

[Transplantation of normal or genetically modified myoblasts for the treatment of hereditary or acquired diseases]. / La transplantation de myoblastes normaux ou génétiquement modifiés pour le traitement de maladies héréditaires ou acquises.

The clinical trials of myoblast transplantation in Duchenne Muscular Dystrophy (DMD) patients produced disappointing results. The main problems responsible for these poor results have since then been identified and partially resolved. One of them was related to the use of an inadequate immunosuppression and, since then, immunosuppression with FK506 has permitted successful myoblast transplantation not only in mice but also in monkeys. The requirement for a sustained immunosuppression may be eventually avoided by developing a state of tolerance to the allogeneic cells or by autologous transplantation of genetically corrected myoblasts or stem cells. The rapid death of 75-80% of the injected myoblasts during the first five days has also contributed to the limited success of the early trials. This death was due to an inflammatory reaction and has been compensated in animal experiments by the injection of a larger number of cells (30 millions per cc). Finally, the myoblasts migrated only 0.5 mm away from their site of injection. This problem is currently compensated in animal experiments by injecting the myoblasts at every mm. The number of injections required may eventually be reduced by transfecting myoblasts with one or several metalloproteinase genes. The very good results obtained during the last two years in primates permit us to undertake a new phase I clinical trial to verify that myoblast transplantation can lead to the formation of muscle fibers expressing normal dystrophin in muscles of DMD patients.

Transfection of large plasmids in primary human myoblasts.

The ex vivo gene therapy approach for Duchenne muscular dystrophy is promising since myoblast transplantation in primates is now very efficient. One obstacle to this treatment is the low transfection efficiency of large DNA constructs in human primary myoblasts. Small plasmids can be easily transfected with the new phosphonolipid described in this study. However, a dramatic drop in transfection efficiency is observed with plasmids of 12 kb or more containing EGFP minidystrophin and EGFP dystrophin fusion genes. The transfection of human primary myoblasts with such large plasmids could only be achieved when the DNA was linked to an adenovirus with the use of polyethylenimine (PEI), with efficiencies ranging between 3 and 5% of transitory transfection. Branched 2 kDa PEI was less toxic in PEI adenofection than branched 25 kDa PEI or linear 22 kDa PEI. The adenovirus was an absolute necessity for an efficient transfection. An integrin-binding peptide, a nuclear localization signal peptide, chloroquine, glycerol or cell cycle synchronization using aphidicolin did not enhance PEI adenofection. Following PEI adenofection, the adenoviral proteins were detected using a polyclonal antibody. The detected antigens fell below the detectable level after 12 days in culture. We thus provide in this study an efficient and reproducible method to permit efficient delivery of large plasmids to human primary myoblasts for the ex vivo gene therapy of Duchenne muscular dystrophy.

Electrotransfer of naked DNA in the skeletal muscles of animal models of muscular dystrophies.

The electrotransfer of naked DNA has recently been adapted to the transduction of skeletal muscle fibers. We investigated the short- and long-term efficacy of this methodology in wild-type animals and in mouse models of congenital muscular dystrophy (dy/dy, dy(2J)/dy(2J)), or Duchenne muscular dystrophy (mdx/mdx). Using a reporter construct, the short-term efficacy of fiber transduction reached 40% and was similar in wild-type, dy/dy and dy(2J)/dy(2J) animals, indicating that ongoing muscle fibrosis was not a major obstacle to the electrotransfer-mediated gene transfer. Although the complete rejection of transduced fibers was observed within 3 weeks in the absence of immunosuppression, the persistency was prolonged over 10 weeks when transient or continuous immunosuppressive regimens were used. Using therapeutic plasmids, we demonstrated that electrotransfer also allowed the transduction of large constructs encoding the laminin alpha2 chain in dy/dy mouse, or a chimeric dystrophin-EGFP protein in mdx/mdx mouse. The correct sarcolemmal localization of these structural proteins demonstrated the functional relevance of their expression in vivo, with a diffusion domain estimated to be 300 to 500 microm. However, degeneration-regeneration events hampered the long-term stability of transduced fibers. Given its efficacy for naked DNA transfer in these models of muscular dystrophies, and despite some limitations, gene electrotransfer methodology should be further explored as a potential avenue for treatment of muscular dystrophies.

Scale-up of a myoblast culture process.

The effects of different types of cell carriers, strategies for cell transfer on carriers, and of several fusion inhibitors on the growth kinetics of primary human myoblasts culture were studied in order to develop a bioprocess suitable for the treatment of Duchenne muscular dystrophy based on the transplantation of unfused cells. Our results indicate that myoblast production is larger on Cytodex 1 and 3 than on polypropylene or polyester fabrics and on a commercial porous macrocarrier. Myoblast growth conditions with Cytodex 1 were further investigated to establish the bioprocess operating conditions. It was found that microcarrier density of 3 g DW l(-1), inoculum density of 2x10(5) cells ml(-1), and continuous agitation speed of 30-rpm result in final myoblast production comparable to static cultures. However, for all the culture conditions used, myoblasts growth kinetics exhibited a lag phase that lasted a minimum of 1 week prior to growth, the end of the lag phase correlating with the appearance of microcarrier aggregates. Based on this observation, we propose that aggregation promotes cell growth by offering a network of very large inter-particular pores that protect cells from mechanical stress. We took advantage of the presence of these aggregates for the scale-up of the culture process. Indeed, using myoblast-loaded microcarrier-aggregates instead of myoblast suspension to inoculate a fresh suspension of microcarriers significantly reduced the duration of the lag phase and allowed the scale-up of the bioprocess at the 500-ml scale. In order to ensure the production of unfused myoblasts, the efficiency of five different fusion inhibitors was investigated. Only calpeptin (9.1 microg ml(-1)) significantly inhibited the fusion of the myoblasts, while TGFbeta (50 ng ml(-1)) and LPA (10 microg ml(-1)) increased myoblasts growth but did not affect fusion, sphingosine (30 microg ml(-1)) induced a 50% death and NMMA (25 microg ml(-1)) had no effect on either growth or fusion. Finally, transplantation trials on severe combined immunodeficient mice showed that microcarrier-cultured human myoblasts grown using the optimized bioprocess resulted in grafts as successful as myoblasts grown in static cultures. The bioprocess, therefore, prove to be suitable for the large-scale production of myoblasts required for muscular dystrophy treatment.

Combined immunosuppression of mycophenolate mofetil and FK506 for myoblast transplantation in mdx mice.

BACKGROUND: Overcoming adverse effects of immunosuppressors can be achieved by combining different drugs, thus allowing a dosage reduction. Myoblast transplantation is a potential therapy for Duchenne muscular dystrophy. Our research group previously established that FK506 (tacrolimus) is an effective immunosuppressive drug for myoblast transplantation in mice and monkeys. METHODS: In the present study, a reduced dose of FK506 at 1.0 mg/kg/day was used in combination with mycophenolate mofetil (MMF; 80 mg/kg/day) as an immunosuppressive protocol for myoblast transplantation. Graft success was evaluated by quantifying the number of dystrophin-positive fibers per muscle section that were injected with normal cells. RESULTS: MMF used alone could not prevent immune rejection of the transplanted myoblasts. MMF given in combination with FK506 immediately after transplantation reduced the success of myoblast transplantation by about 50%. A low dose of FK506 combined with MMF after the establishment of the graft (3 weeks) maintained graft success and controlled immune infiltration compared with a low dose of FK506 alone. However, lymphocyte infiltration was observed at longer term using a low dose of FK506 combined with MMF. CONCLUSIONS: The diminution of graft success when combining FK506 and MMF by the time of myoblast transplantation could be attributed to the inhibition of myoblast fusion by MMF. The use of MMF and FK506 after the establishment of the graft did not reduce graft success, however, this combination was not effective at controlling long-term immune rejection in comparison with the optimal dose of FK506 alone.

Intraarterial injection of muscle-derived CD34(+)Sca-1(+) stem cells restores dystrophin in mdx mice.

Duchenne muscular dystrophy is a lethal recessive disease characterized by widespread muscle damage throughout the body. This increases the difficulty of cell or gene therapy based on direct injections into muscles. One way to circumvent this obstacle would be to use circulating cells capable of homing to the sites of lesions. Here, we showed that stem cell antigen 1 (Sca-1), CD34 double-positive cells purified from the muscle tissues of newborn mice are multipotent in vitro and can undergo both myogenic and multimyeloid differentiation. These muscle-derived stem cells were isolated from newborn mice expressing the LacZ gene under the control of the muscle-specific desmin or troponin I promoter and injected into arterial circulation of the hindlimb of mdx mice. The ability of these cells to interact and firmly adhere to endothelium in mdx muscles microcirculation was demonstrated by intravital microscopy after an intraarterial injection. Donor Sca-1, CD34 muscle-derived stem cells were able to migrate from the circulation into host muscle tissues. Histochemical analysis showed colocalization of LacZ and dystrophin expression in all muscles of the injected hindlimb in all of five out of five 8-wk-old treated mdx mice. Their participation in the formation of muscle fibers was significantly increased by muscle damage done 48 h after their intraarterial injection, as indicated by the presence of 12% beta-galactosidase-positive fibers in muscle cross sections. Normal dystrophin transcripts detected enzymes in the muscles of the hind limb injected intraarterially by the mdx reverse transcription polymerase chain reaction method, which differentiates between normal and mdx message. Our results showed that the muscle-derived stem cells first attach to the capillaries of the muscles and then participate in regeneration after muscle damage.