562. Ex Vivo Gene Therapy for Duchenne Muscular Dystrophy: Lentiviral Vectors, Exon Skipping and PhiC31 Integrase Approaches
Authors: Simon P. Quenneville, Pierre Chapdelaine, Joel Rousseau, Jacques P. Tremblay
Journal: Molecular Therapy (2006)

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 (lentiviral) and a non viral (integrase PhiC31) method were tested to perform the genetic modification. Therapeutic sequences included microdystrophin, dystrophin and exon skipping cassettes.Co-transfection (nucleofection) of a PhiC31 integrase and a dystrophin expressing plasmid was used to perform the genetic modification. We have also generated eGFP, eGFP-microdystrophin and exon skipping lentiviral vectors with CMV and MCK promoters. Following in vitro modification of dystrophic cells (mdx mice derived) and normal human myoblasts, the cells were engrafted into mdx and SCID mice muscles.The nucleofection led to a stable expression of the full length dystrophin. This was the biggest expression cassette ever stabilized in primary cultured human myoblasts. Following transplantation, the dystrophin expression was observed into a muscle, leading to the apparition of the dystrophin associated proteins a-sarcoglycan and b-dystroglycan. However, this method is not very efficient. We have also 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. Both transgenes were expressed into the muscles one month after the engraftment. The expression of microdystrophin also led to the apparition of a-sarcoglycans in the dystrophic model mice. We have also used a lentiviral vectors coding for an exon skipping cassette allowing us to skip the exon 51. In vivo experiments in SCID mice as shown that it is possible to express a quasi dystrophin protein with cells originating from a DMD patient. This work indicates that ex vivo gene therapy is a possible approach to treat Duchenne muscular dystrophy. 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 (lentiviral) and a non viral (integrase PhiC31) method were tested to perform the genetic modification. Therapeutic sequences included microdystrophin, dystrophin and exon skipping cassettes. Co-transfection (nucleofection) of a PhiC31 integrase and a dystrophin expressing plasmid was used to perform the genetic modification. We have also generated eGFP, eGFP-microdystrophin and exon skipping lentiviral vectors with CMV and MCK promoters. Following in vitro modification of dystrophic cells (mdx mice derived) and normal human myoblasts, the cells were engrafted into mdx and SCID mice muscles. The nucleofection led to a stable expression of the full length dystrophin. This was the biggest expression cassette ever stabilized in primary cultured human myoblasts. Following transplantation, the dystrophin expression was observed into a muscle, leading to the apparition of the dystrophin associated proteins a-sarcoglycan and b-dystroglycan. However, this method is not very efficient. We have also 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. Both transgenes were expressed into the muscles one month after the engraftment. The expression of microdystrophin also led to the apparition of a-sarcoglycans in the dystrophic model mice. We have also used a lentiviral vectors coding for an exon skipping cassette allowing us to skip the exon 51. In vivo experiments in SCID mice as shown that it is possible to express a quasi dystrophin protein with cells originating from a DMD patient. This work indicates that ex vivo gene therapy is a possible approach to treat Duchenne muscular dystrophy.