PABPN1 gene therapy for oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant, late-onset muscle disorder characterized by ptosis, swallowing difficulties, proximal limb weakness and nuclear aggregates in skeletal muscles. OPMD is caused by a trinucleotide repeat expansion in the PABPN1 gene that results in an N-terminal expanded polyalanine tract in polyA-binding protein nuclear 1 (PABPN1). Here we show that the treatment of a mouse model of OPMD with an adeno-associated virus-based gene therapy combining complete knockdown of endogenous PABPN1 and its replacement by a wild-type PABPN1 substantially reduces the amount of insoluble aggregates, decreases muscle fibrosis, reverts muscle strength to the level of healthy muscles and normalizes the muscle transcriptome. The efficacy of the combined treatment is further confirmed in cells derived from OPMD patients. These results pave the way towards a gene replacement approach for OPMD treatment.

Satellite cell dysfunction contributes to the progressive muscle atrophy in myotonic dystrophy type 1.

AIMS: Myotonic dystrophy type 1 (DM1), one of the most common forms of inherited neuromuscular disorders in the adult, is characterized by progressive muscle weakness and wasting leading to distal muscle atrophy whereas proximal muscles of the same patients are spared during the early phase of the disease. In this report, the role of satellite cell dysfunction in the progressive muscular atrophy has been investigated. METHODS: Biopsies were obtained from distal and proximal muscles of the same DM1 patients. Histological and immunohistological analyses were carried out and the past regenerative history of the muscle was evaluated. Satellite cell number was quantified in vivo and proliferative capacity was determined in vitro. RESULTS: The size of the CTG expansion was positively correlated with the severity of the symptoms and the degree of muscle histopathology. Marked atrophy associated with typical DM1 features was observed in distal muscles of severely affected patients whereas proximal muscles were relatively spared. The number of satellite cells was significantly increased (twofold) in the distal muscles whereas very little regeneration was observed as confirmed by telomere analyses and developmental MyHC staining (0.3-3%). The satellite cells isolated from the DM1 distal muscles had a reduced proliferative capacity (36%) and stopped growing prematurely with telomeres longer than control cells (8.4 vs. 7.1 kb), indicating that the behaviour of these precursor cells was modified. CONCLUSIONS: Our results indicate that alterations in the basic functions of the satellite cells progressively impair the muscle mass maintenance and/or regeneration resulting in gradual muscular atrophy.

Heat shock treatment increases engraftment of transplanted human myoblasts into immunodeficient mice.

Myoblast transfer therapy (MTT) is a strategy that has been proposed to treat some striated muscle pathologies. However, the first therapeutic trials using this technique were unsuccessful due to the limited migration and early cell death of the injected myoblasts. Various strategies have been considered to increase myoblast survival in the host muscle after MTT. Overexpression of heat shock proteins (HSPs) in mouse myoblasts has been shown to improve cell resistance against apoptosis in vitro and in vivo. Our objective was to determine whether heat shock (HS) treatment increased the survival of human myoblasts leading to better participation of the injected cells in muscle regeneration. For this study, HS-treated human myoblasts were injected into the tibialis anterior (TA) muscles of immunodeficient RAG-/- gammaC-/- mice. TA muscles were excised at 24 hour and at 1 month after injection. Our results showed that HS treatment increased the expression of the hsp70 protein and protected the cells from apoptosis in vitro. HS treatment dramatically increased the number of human fibers present at 1 month after injection when compared with nontreated cells. Interestingly, HS treatment decreased apoptosis at 24 hour after human myoblast injection, but no differences were observed concerning proliferation, suggesting that the increased fiber formation among the HS-treated group was probably due to decreased cell death. These data suggested that HS treatment might be used in the clinical context to improve the success of MTT.

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.

Impact on oxidative phosphorylation of immortalization with the telomerase gene.

Skin fibroblasts are essential tools for biochemical, genetic and physiopathological investigations of mitochondrial diseases. Their immortalization has been previously performed to overcome the limited number of divisions of these primary cells but it has never been systematically evaluated with respect to efficacy and impact on the oxidative phosphorylation (OXPHOS) characteristics of the cells. We successfully immortalized with the human telomerase gene 15 human fibroblasts populations, 4 derived from controls and 11 from patients with diverse respiratory chain defects. Immortalization induced significant but mild modification of the OXPHOS characteristics of the cells with lower rates of oxygen consumption and ATP synthesis associated with their loose coupling. However, it never significantly altered the type and severity of any genetic OXPHOS defect present prior to immortalization. Furthermore, it did not significantly modify the cells' dependence on glucose and sensitivity to galactose thus showing that immortalized cells could be screened by their nutritional requirement. Immortalized skin fibroblasts with significant OXPHOS defect provide reliable tools for the diagnosis and research of the genetic cause of mitochondrial defects. They also represent precious material to investigate the cellular responses to these defects, even though these should afterwards be verified in unmodified primary cells.

Myogenic stem cells: regeneration and cell therapy in human skeletal muscle.

Human skeletal muscle has been considered as an ideal target for cell-mediated therapy. However, the positive results obtained in dystrophic animal models using the resident precursor satellite cell population have been followed by discouraging evidences obtained in the clinical trials involving Duchenne muscular dystrophy patients. This text reviews the recent advances that many groups have achieved to identify from the stem cell compartment putative candidates for cell therapy. We focused our attention on stem cells with myogenic potential which might be able to improve transplantation efficiency and therefore could be used as a therapeutic tool for neuromuscular diseases.

The mitotic clock in skeletal muscle regeneration, disease and cell mediated gene therapy.

The regenerative capacity of skeletal muscle will depend on the number of available satellite cells and their proliferative capacity. We have measured both parameters in ageing, and have shown that although the proliferative capacity of satellite cells is decreasing during muscle growth, it then stabilizes in the adult, whereas the number of satellite cells decreases during ageing. We have also developed a model to evaluate the regenerative capacity of human satellite cells by implantation into regenerating muscles of immunodeficient mice. Using telomere measurements, we have shown that the proliferative capacity of satellite cells is dramatically decreased in muscle dystrophies, thus hampering the possibilities of autologous cell therapy. Immortalization by telomerase was unsuccessful, and we currently investigate the factors involved in cell cycle exits in human myoblasts. We have also observed that insulin-like growth factor-1 (IGF-1), a factor known to provoke hypertrophy, does not increase the proliferative potential of satellite cells, which suggests that hypertrophy is provoked by increasing the number of satellite cells engaged in differentiation, thus possibly decreasing the compartment of reserve cells. We conclude that autologous cell therapy can be applied to specific targets when there is a source of satellite cells which is not yet exhausted. This is the case of Oculo-Pharyngeal Muscular Dystrophy (OPMD), a late onset muscular dystrophy, and we participate to a clinical trial using autologous satellite cells isolated from muscles spared by the disease.

Myoblast transfer therapy: is there any light at the end of the tunnel?

Myoblast transfer therapy (MTT) was proposed in the 70's as a potential treatment for muscular dystrophies, based upon the early results obtained in mdx mice: dystrophin expression was restored in this model by intramuscular injections of normal myoblasts. These results were quickly followed by clinical trials for patients suffering from Duchenne Muscular Dystrophy (DMD) in the early 90's, based mainly upon intramuscular injections of allogenic myoblasts. The clinical benefits obtained from these trials were minimal, if any, and research programs concentrated then on the various pitfalls that hampered these clinical trials, leading to numerous failures. Several causes for these failures were identified in mouse models, including a massive cell death of myoblasts following their injection, adverse events involving the immune system and requiring immunosuppression and the adverse events linked to it, as well as a poor dispersion of the injected cells following their injection. It should be noted that these studies were conducted in mouse models, not taking into account the fundamental differences between mice and men. One of these differences concerns the regulation of proliferation, which is strictly limited by proliferative senescence in humans. Although this list is certainly not exhaustive, new therapeutic venues were then explored, such as the use of stem cells with myogenic potential, which have been described in various populations, including bone marrow, circulating blood or muscle itself. These stem cells presented the main advantage to be available and not exhausted by the numerous cycles of degeneration/regeneration which characterize muscle dystrophies. However, the different stem candidates have shown their limits in terms of efficiency to participate to the regeneration of the host. Another issue was raised by clinical trials involving the injection of autologous myoblasts in infacted hearts, which showed that limited targets could be aimed with autologous myoblasts, as long as enough spared muscle was available. This resulted in a clinical trial for the pharyngeal muscles of patients suffering from Oculo-Pharyngeal Muscular Dystrophy (OPMD). The results of this trial will not be available before 2 years, and a similar procedure is being studied for Fascio-Scapulo-Humeral muscular Dystrophy (FSHD). Concerning muscular dystrophies which leave very few muscles spared, such as DMD, other solutions must be found, which could include exon-skipping for the eligible patients, or even cell therapy using stem cells if some cell candidates with enough efficiency can be found. Recent results concerning mesoangioblasts or circulating AC133+ cells raise some reasonable hope, but still need further confirmations, since we have learned from the past to be cautious concerning a transfer of results from mice to humans.

Human muscle precursor cell regeneration in the mouse host is enhanced by growth factors.

The aim of this study was to optimize human muscle formation in vivo from implanted human muscle precursor cells. We transplanted donor muscle precursor cells (MPCs) prepared from postnatal or fetal human muscle into immunodeficient host mice and showed that irradiation of host muscle significantly enhanced muscle formation by donor cells. The amount of donor muscle formed in cryodamaged host muscle was increased by exposure of donor cells to growth factors before their implantation into injured host muscle. Insulin-like growth factor type I (IGF-I) significantly increased the amount of muscle formed by postnatal human muscle cells, but not by fetal human MPCs. However, treatment of fetal muscle cells with IGF-I, in combination with basic fibroblast growth factor and plasmin, significantly increased the amount of donor muscle formed. In vivo, human MPCs formed mosaic human-mouse muscle fibers, in which each human myonucleus was associated with a zone of human sarcolemmal protein spectrin.

IGF-1 induces human myotube hypertrophy by increasing cell recruitment.

Insulin-like growth factor-1 (IGF-1) has been shown in rodents (i) in vivo to induce muscle fiber hypertrophy and to prevent muscle mass decline with age and (ii) in vitro to enhance the proliferative life span of myoblasts and to induce myotube hypertrophy. In this study, performed on human primary cultures, we have shown that IGF-1 has very little effect on the proliferative life span of human myoblasts but does delay replicative senescence. IGF-1 also induces hypertrophy of human myotubes in vitro, as characterized by an increase in the mean number of nuclei per myotube, an increase in the fusion index, and an increase in myosin heavy chain (MyHC) content. In addition, muscle hypertrophy can be triggered in the absence of proliferation by recruiting more mononucleated cells. We propose that IGF-1-induced hypertrophy can involve the recruitment of reserve cells in human skeletal muscle.

Extended amplification in vitro and replicative senescence: key factors implicated in the success of human myoblast transplantation.

The limited success of human myoblast transplantation has been related to immune rejection, poor survival, and limited spread of injected myoblasts after transplantation. An important issue that has received little attention, but is nevertheless of fundamental importance in myoblast transplantation protocols, is the proliferative capacity of human satellite cells. Previous studies from our laboratory have demonstrated that the maximum number of divisions that a population of satellite cells can make decreases with age during the first two decades of life then stabilizes in adulthood. These observations indicate that when satellite cells are used as vectors in myoblast transplantation protocols it is important to consider donor age and the number of divisions that the cells have made prior to transplantation as limiting factors in obtaining an optimal number of donor derived muscle fibers. In this study, myoblasts derived from donors of different ages (newborn, 17 years old, and 71 years old) were isolated and amplified in culture. Their potential to participate in in vivo muscle regeneration in RAG2(-/-)/gamma(c)/C5 triple immunodeficient hosts after implantation was evaluated at 4 and 8 weeks postimplantation. Our results demonstrate that prolonged amplification in culture and the approach to replicative senescence are both important factors that may condition the success of myoblast transplantation protocols.

Satellite cells and training in the elderly.

In the present review, we describe the effects of ageing on human muscle fibres, underlining that each human muscle is unique, meaning that the phenotype becomes specifically changed upon ageing in different muscles, and that the satellite cells are key cells in the regeneration and growth of muscle fibres. Satellite cells are closely associated with muscle fibres, located outside the muscle fibre sarcolemma but beneath the basement lamina. They are quiescent cells, which become activated by stimulation, like muscle fibre injury or increased muscle tension, start replicating and are responsible for the repair of injured muscle fibres and the growth of muscle fibres. The degree of replication is governed by the telomeric clock, which is affected upon excessive bouts of degeneration and regeneration as in muscular dystrophies. The telomeric clock, as in dystrophies, does not seem to be a limiting factor in ageing of human muscle. The number of satellite cells, although reduced in number in aged human muscles, has enough number of cell divisions left to ensure repair throughout the human life span. We propose that an active life, with sufficient general muscular activity, should be recommended to reduce the impairment of skeletal muscle function upon ageing.

A discrepancy resolved: human satellite cells are not preprogrammed to fast and slow lineages.

Satellite cells from chicken and mouse muscle when differentiated in vitro have been shown to display a myosin heavy chain phenotype that corresponds to the fibre from which they originated. Indirect evidence has suggested that this might not be the case for human satellite cells. In the present study we have compared the myosin heavy chain (MHC) profile expressed by differentiated cultures of satellite cells isolated from single fast or slow muscle fibres. The MHC composition of the isolated fibres was determined by sodium dodecyl sulfate glycerol gel electrophoresis and Western blotting. The MHC profile expressed by the differentiated myotubes was identified by immunostaining using specific antibodies. Our results show that all human satellite cells isolated from either fast or slow fibres form myotubes in vitro which co-express both fast and slow MHCs independently of the fibre type from which they originated. These results confirm that human satellite cells, in contrast to those of birds and rodents, are not confined to distinct fast and slow lineages.

Defective satellite cells in congenital myotonic dystrophy.

In this study we have developed an in vitro cell culture system which displays the majority of the defects previously described for congenital myotonic dystrophy (CDM) muscle in vivo. Human satellite cells were isolated from the quadriceps muscles of three CDM fetuses with different clinical severity. By Southern blot analysis all three cultures were found to have approximately 2300 CTG repeats. This CTG expansion was found to progressively increase in size during the proliferative life span, confirming an instability of this triplet in skeletal muscle cells. The CDM myoblasts and myotubes also showed abnormal retention of mutant RNA in nuclear foci, as well as modifications in their myogenic program. The proliferative capacity of the CDM myoblasts was reduced and a delay in fusion, differentiation and maturation was observed in the CDM cultures compared with unaffected myoblast cultures. The clinical severity and delayed maturation observed in the CDM fetuses were closely reflected by the phenotypic modifications observed in vitro. Since the culture conditions were the same, this suggests that the defects we have described are intrinsic to the program expressed by the myoblasts in the absence of any trophic factors. Altogether, our results demonstrate that satellite cells are defective in CDM and are probably implicated in the delay in maturation and muscle atrophy that has been described previously in CDM fetuses.

A new immunodeficient mouse model for human myoblast transplantation.

Design of efficient transplantation strategies for myoblast-based gene therapies in humans requires animal models in which xenografts are tolerated for long periods of time. In addition, such recipients should be able to withstand pretransplantation manipulations for enhancement of graft growth. Here we report that a newly developed immunodeficient mouse carrying two known mutations (the recombinase activating gene 2, RAG2, and the common cytokine receptor gamma, gammac) is a candidate fulfilling these requirements. Skeletal muscles from RAG2(-/-)/gammac(-/-) double mutant mice recover normally after myotoxin application or cryolesion, procedures commonly used to induce regeneration and improve transplantation efficiency. Well-differentiated donor-derived muscle tissue could be detected up to 9 weeks after transplantation of human myoblasts into RAG2(-/-)/gammac(-/-) muscles. These results suggest that the RAG2(-/-)/gammac(-/-) mouse model will provide new opportunities for human muscle research.

Modulation of acetylcholine receptor expression in seronegative myasthenia gravis.

In a previous study, we demonstrated a compensatory mechanism for regulating acetylcholine receptor (AChR) gene expression in muscle biopsies from seropositive and seronegative (SN) myasthenia gravis (MG) patients. To further characterize the AChR regulation mechanisms involved in SNMG disease, we investigated the effects of MG sera on nicotinic AChR expression (at the protein and messenger RNA [mRNA] levels) in cultured human muscle cells. Sera from SNMG patients induced an in vitro increase in the level of nicotinic AChR beta-subunit mRNA but did not cause a decrease in AChR protein level. This apparent discrepancy was not due to a higher level of AChR synthesis as demonstrated by analysis of AChR turnover. In SN patients, the increase in beta-subunit mRNA level was followed after 48 hours by a slight increase in the amount of AChR surface protein. This regulation of nicotinic receptor expression was due to the purified IgG-containing fraction. Thus, sera from SNMG patients contain an immunoglobulin that induces an increase in AChR mRNA without causing a decrease in AChR protein level, suggesting an indirect regulatory mechanism involving another surface molecule. This model is therefore useful for defining the targets involved in the pathogenesis of SNMG disease.

Skeletal muscle regeneration and the mitotic clock.

Regeneration of muscle fibers following damage requires activation of quiescent satellite cells, their proliferation and finally their differentiation and fusion into multinucleated myotubes, which after maturation will replace the damaged fiber. The regenerative potential of human skeletal muscle will be determined, at least partly, by the proliferative capacity of the satellite cells. In this study, we have measured the proliferative life span of human satellite cells until they reach senescence. These analyses were performed on cell populations isolated from old and young donors as well as from one child suffering from Duchenne muscular dystrophy, where extensive regeneration had occurred. In order to see if there are any age-related changes in the myogenic program we have also compared the program of myogenic differentiation expressed by satellite cells from these subjects at different stages of their proliferative lifespan.

Transplantation of normal and DMD myoblasts expressing the telomerase gene in SCID mice.

The limited proliferative capacity of dystrophic human myoblasts severely limits their ability to be genetically modified and used for myoblast transplantation. The forced expression of the catalytic subunit of telomerase can prevent telomere erosion and can immortalize different cell types. We thus tested the ability of telomerase to immortalize myoblasts and analyzed the effect of telomerase expression on the success of myoblast transplantation. Telomerase expression did not significantly extend the human myoblast life span. The telomerase expressing myoblasts were nonetheless competent to participate in myofiber formation after infection with the retroviral vector. Although the new fibers obtained are less numerous than after the transplantation of normal myoblasts, these results demonstrate that the forced expression of telomerase does not block the ability of normal or dystrophic myoblasts to differentiate in vivo. It will be now necessary to determine the factors that prevent telomerase from extending the life span of human myoblasts before the potential of this intervention can be fully examined.

Shorter telomeres in dystrophic muscle consistent with extensive regeneration in young children.

Muscular dystrophies are characterised by continuous cycles of degeneration and regeneration resulting in an eventual diminution of the muscle mass and extensive fibrosis. In somatic cells chromosomal telomeres shorten with each round of cell division and telomere length is considered to be a biomarker of the replicative history of the cell. We have previously shown that human myoblasts have a limited proliferative capacity, and that normal skeletal muscle has a very low level of nuclear turnover. However, in patients suffering from muscular dystrophy the satellite cells will be forced to make repeated rounds of cell division, driving the cells towards senescence. In this study we have used the telomere length to quantify the intensity of the muscle cell turnover in biopsies from dystrophic patients of different ages. Our results show that as soon as the first clinical symptoms become apparent the muscle has already undergone extensive regeneration and the rate of telomere loss is 14 times greater than that observed in controls. This confirms that the decline in regenerative capacity is due to the premature senescence of the satellite cells induced by their excessive proliferation during muscle repair.