Simon P. Quenneville

Expression of Dog Microdystrophin in Mouse and Dog Muscles by Gene Therapy

Duchenne muscular dystrophy (DMD) is characterized by the absence of dystrophin. Several previous studies demonstrated the feasibility of delivering microdystrophin complementary DNA (cDNA) into mouse and normal nonhuman primate muscles by ex vivo gene therapy. However, these animal models do not reproduce completely the human DMD phenotype, while the dystrophic dog model does. To progress toward the use of the best animal model of DMD, a dog microdystrophin was transduced into human and dystrophic dog muscle precursor cells (MPCs) with a lentivirus before their transplantation into mouse muscles. One month following MPC transplantation, myofibers expressing the dog microdystrophin were observed. We also used another approach to introduce this transgene into myofibers, i.e., the electrotransfer of a plasmid coding for the dog microdystrophin. The plasmid was injected into mouse and dog muscles, and brief electric pulses were applied in the region of injection. Two weeks later, the transgene was detected in both animals. Therefore, ex vivo gene therapy and electrotransfer are two possible methods to introduce a truncated version of dystrophin into myofibers of animal models and eventually into myofibers of DMD patients.

Dystrophin expression following the transplantation of normal muscle precursor cells protects mdx muscle from contraction-induced damage.

Duchenne muscular dystrophy (DMD) is the most frequent muscular dystrophy. Currently, there is no cure for the disease. The transplantation of muscle precursor cells (MPCs) is one of the possible treatments, because it can restore the expression of dystrophin in DMD muscles. In this study, we investigated the effects of myoblasts injected with cardiotoxin on the contractile properties and resistance to eccentric contractions of transplanted and nontransplanted muscles. We used the extensor digitorum longus (EDL) as a model for our study. We conclude that the sole presence of dystrophin in a high percentage of muscle fibers is not sufficient by itself to increase the absolute or the specific force in the EDL of transplanted mdx muscle. This lack of strength increase may be due to the extensive damage that was produced by the cardiotoxin, which was coinjected with the myoblasts. However, the dystrophin presence is sufficient to protect muscle from eccentric damage as indicated by the force drop results.

Ex vivo modification of cells to induce a muscle-based expression.

Ex vivo gene therapy is a possible treatment for several muscular dystrophies. The best transgene to be expressed and the appropriate cell type to be used currently remain the subject of many investigations. The most adequate gene modification technique also remains to be established. Different transgenes have already been tested in animal models and transgenic mice. Several cell types were evaluated during the last decades and several vectors or transfection methods were analysed. From these essays, over time, several proofs of principles were made to demonstrate the feasibility of this type of therapy. For DMD, it is possible to express several truncated versions of dystrophin or exon skipping molecules. It is also possible to express other molecules that would mitigate the phenotype. Different cell types are also available. From the well documented myoblasts to the AC133 positive cells, the choice of cell types is exploding. Gene modification also evolved during the last decade. Efficient transfection technique and viral vectors are currently available. Given all these possibilities, the researcher has to make several choices. This review is trying to give clues of how to make those choices.

257. Ex Vivo Gene Therapy for Duchenne Muscular Dystrophy: Lentiviral and PhiC31 Integrase Approaches

Duchenne muscular dystrophy is the most severe muscular dystrophy. It is caused by the absence of dystrophin in muscle fibers. This absence lead to increased muscle damage, loss of muscle mass, loss of strength, respiratory and heart failure. This disease as currently no treatment. Myogenic cells transplantation is a possible cure for Duchenne muscular dystrophy. However, allogeneic graft success relies on the use of efficient but toxic immunosuppressive drugs. The use of these drugs is a major problem that could be solved by the use of ex vivo gene therapy. This method consists in genetically modifying patient myoblasts before their auto-transplantation. In this study, a viral and a non viral method were tested to perform the genetic modification. The co-transfection (nucleofection) of a PhiC31 integrase and a dystrophin expressing plasmid as already led to a stable expression of the full length dystrophin1. This was the biggest expression cassette ever stabilized in primary cultured human myoblasts. Using this method, we are now capable of transferring transgene expressing cells into a muscle, leading to expression into the muscle fibers. However, this method is not very efficient. This problem could be solved using viral vectors. We have generated eGFP and eGFP-micro-dystrophin expressing lentiviral vectors under the control of the CMV and MCK promoters. Following in vitro infection of human myoblasts, the cells were engrafted into SCID mice muscles. We were able to detect the expression of both transgenes into these muscle fibers one month after the engraftment. Evans bleu will be injected in the blood of the mice before an eccentric exercise. We will verify whether the expression of dystrophin in the muscle fibers reduces their damage during the exercise and thus the incorporation of Evans blue. This work indicates that ex vivo gene therapy is a possible approach to treat Duchenne muscular dystrophy.

537. Further characterisation of myo-neurospheres

Adult stem cells have been isolated from several tissues like bone marrow, brain and skin. We report the isolation and characterisation of neuronal precursor cells, potentially stem cells, from primate and mouse adult muscle and their differentiation in neuronal cells. These cells could be an excellent alternative source of neuronal cells for a therapeutic approach for neurodegenerative diseases. Following an enzymatic dissociation of the tissue, cells are growing in suspension in a proliferation medium. In these conditions, neurosphere-like cells (stated myo-neurospheres) appear after 7 to 10 days. Mouse spheres were further characterised for different surface markers, like CD34, CD90, CD45 and Sca-1. Moreover, expression of different neural crest markers (p75, Snail, Slug and Twist) were detected in the proliferated cells. To induce neuronal differentiation, cells were plated on poly-D-lysine/gelatine coated petri dishes in a differentiation medium for 12 days. The differentiated cells were characterized by immunostaining for two neuronal markers (beta-III- tubulin and neurofilament M), for glial cells marker (glial cells fibrillary acidic protein) and for a myoblast marker (desmin). 15–20% of differentiated cells are expressing neuronal markers. Moreover, all differentiated neuron-like cells were negative for desmin. Interestingly, in spheres derived from mouse tissue, the expression of the limiting enzyme for the synthesis of GABA was detected in the differentiated cells only. In conclusion, we were able to isolate neural precursors from adult muscle tissue and differentiate them into neuron-like cells. These cells could be used as an autologous source of cell for neurodegenerative disease.