Myoblast transplantation is one of the possible therapeutic strategies for different types of myopathy or for post-ischaemic cardiac insufficiency (Vilquin et al., 2005) and is the focus of multiple developments in collaboration with several teams. We have validated the use of satellite cells resulting from muscle biopsies of patients affected by facioscapulohumeral (FSH) muscular dystrophy, in cell transplantation protocols. In this pathology, certain muscular groups are selectively affected, taking advantage of molecular diagnosis, but no treatment is available yet. Satellites cells originating from muscles unaffected by the pathology show characteristics comparable to those of cells resulting from healthy donors (Vilquin et al., 2005). This pre-clinical testing has validated the hypothesis that it is possible to transplant these myoblasts from unaffected muscles into muscles affected by the disease. A Phase I clinical trial has been accepted and is in progress (six patients included in 2006).
We are continuing studies on cell survival after transplantation in skeletal muscular tissue, and are validating the use of cellular markers. We have shown that pre-treatment of the cells by thermal shock could improve their short term survival (Maurel et al., 2005).
We are meticulously characterizing the cell types present within adult skeletal muscle, in vitro and ex vivo. We have observed the presence of cell types expressing mesenchymatous markers (a review of their clinical application can be found in Vilquin et al., 2006) and are separating the different populations. Some of them show osteogenic, chondrogenic and/or adipogenic differentiation capacities (manuscript under revision). In addition, a population of muscle cells show the capacity to differentiate into smooth muscle cells (Lericousse et al., 2007).
We are continuing the characterization of progenitor cardiac cells, in particular cells expressing the differentiation factor Islet-1. We have analyzed the kinetics of persistence of these cells in normal and pathological murine models.
In the field of cardiac cellular therapy, we participated in the first Phase I clinical trial of autologous myoblast transplantation (Hagège et al., 2006) and the first Phase II clinical trial (Menasché et al., 2008), and we are evaluating alternate indications (Messas et al., 2006).

 

 

 

Considering a gene correction strategy, we plan to test several approaches. On one hand, the introduction of a functional allele is expected to prevent or stabilize the dystrophic process. We started to evaluate this strategy in the delta-sarcoglycan deficient hamster strain CHF147. On the other hand, at a symptomatic stage of the disease, gene correction might not be efficient enough to restore functional muscle mass and a more symptom directed approach might be useful, potentially in addition to gene correction. We have initially validated the hypothesis according to which it is possible to induce compensatory mechanisms in cardiac disease during muscular dystrophy, and thereby to delay the progression of a dilated cardiomyopathy towards terminal cardiac insufficiency. In CHF147 hamsters, it is possible to delay the degradation of the myocardial structure and to stabilize cardiac function by the administration of the recombinant protein IGF-1, as well as injection of a plasmid DNA coding for IGF1 (Serose et al, 2005). More recently, we have been able to show that the beneficial effects obtained by a treatment of short duration and with a relatively low dose had lasting effects on the structure as well as on cardiac function (Serose et al, 2006). From a clinical point of view, one can interpret these results as a change in functional class according to the NYHA classification. In the animal model, we have been able to show that this functional change was accompanied by a significant improvement in survival of the treated animals, with a 20% increase in the average survival.
As it appears more and more clearly that nucleic acids can be used as therapeutic principles, formulation remains a major field of research. We explore two complementary approaches. Virus-derived vectors might achieve dissemination through the entire muscular system, and we evaluate currently several AAV-based vectors. As an additional approach, we continue to develop inert synthetic vectors based on various polymers. A physicochemical characterization of the various formulations could be carried out. Toxicology and efficacy tests have made it possible to retain two vector types that showed an improvement in the effectiveness of gene transfer in striated muscles (Roques et al., 2007).
Moreover, considering more specifically the cardiac involvement in muscular dystrophies, we investigate the therapeutic potential of an endogenous peptide that demonstrated antifibrotic properties. This same peptide reveals proangiogenic capacity. In a skin flap model in rats, we were able to show the effectiveness of the tetrapeptide to induce an increase in angiogenesis and a clear improvement in the survival of skin flaps and wound healing (Fromes et al, 2006).

 

 

 

With the aim of better understanding the pathophysiology of muscular diseases, we are continuing our efforts to set up functional evaluation in small animals. At the level of cardiac muscle, monitoring the electrical activity of the heart muscle provides insight into its intrinsic excitatory properties, as well as its extracardiac regulation. We have implemented several tools to record the surface ECG in small animals in conditions of restrained or freely moving vigilance, based on telemetry (Mongue-Din et al, 2007). Time- and frequency-domain analysis have been developed to analyze and quantify ECG signals. Furthermore, we have implemented hemodynamic evaluation techniques by conductance catheterization, allowing to study the pressure-volume relationship in vivo, and to investigate more precisely the myocardial contractility. More recently, we developed high-resolution imaging in small animals (Fromes Y. et al, 2007(a) ; Fromes Y. et al, 2007 (b)).
Diseases of smooth muscle, in particular at the vascular level and perfusion anomalies, which result, could be studied in the model of complete delta-sarcoglycan deficiency in the CHF147 hamster. In vascular smooth cells from the aorta, we could thus show an increase in apoptosis associated with a loss in the differentiation of mature cells (Lipskaia L.et al., 2007). Functional evaluation of small resistance vessels is currently going on with ex vivo experiments and should be tested in vivo by measurements of limb muscle perfusion. Moreover, at the level neuromuscular, we have set up in vivo EMG monitoring techniques by long-term instrumentation of animals. In a limb girdle muscular dystrophy model, we intend to evaluate the proximal versus distal muscle disease.

 

 

 

We are participating on the one hand, with the development of cardiac and muscle imaging methods in small animals and on the other hand, with the optimization of imaging methods to follow the outcome of transplanted cells. We are comparing the importance of different classes of contrast agents for the non-invasive longitudinal follow-up of human muscle cells transplanted in the mouse.
From a point of view of magnetic resonance imaging, we have developed a new method of signal acquisition compatible with unsedated animals. This approach enabled studies of cardiac physiology without the influence of anesthetic drugs (Parzy E.et al.,2007). Furthermore, based on the NMR technique, we have been able to develop measurement techniques and to study the muscular perfusion of limbs in laboratory rodents (Bertoldi D et al., 2006). By following these approaches, it was possible to study the muscular oxygenation in vivo.