Résumé :
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In DMD, skeletal and cardiac muscles are affected, leading to wheelchair dependency, respiratory failure and premature death. A combination of different strategies might enhance the possibility of successful therapy. We isolated CD133+ cells from muscle biopsies of GRMD dogs. Five dogs one year old, 2 characterized by a mild clinical phenotype and 3 characterized by a severe clinical phenotype, were treated with their own trasnduced U7exon 6-8 CD133+ stem cells. Old GRMD dogs are well characterized in term of clinical history and no variation among different dogs in the progression of the disease are observed from this age. All dogs received 2 arterial systemic injections (100 x106 cells each) through a catheter that was introduced in the left femoralis artery and reached the aortic arch at the level of the left subclavia. From a clinical point of view, dogs with mild phenotype had bad performance and was walking with difficulties before injection and dogs with a severe phenotype were in worst condition since unable to walking and full of retraction. In order to test whether morphological and biochemical changes, we performed three different functional measures: claim Stairs (Time), swimming, 6 minute walking test (6MWT), quantitative values. After the injection, all treated dogs had a clinical performance improvement. After the first injection, all dogs had no detectable anti-dystrophin antibodies and its circulating lymphocytes did not react to transduced CD133+ stem cells. All transplanted animals were analysed at different times (6, 12 months after injection); most of the biopsies in all muscles had a morphological amelioration when compared to untreated dogs. Dystrophin expression in the biopsies was variable, ranging from 2 to _ 7% in several biopsies of the injected legs. Western blot analysis of extracts from different biopsies of the same muscles confirmed the presence of different amount of dystrophin, varying from an undetectable signal to around 6% of a wt canine muscle. Other two dogs received their own muscle-derived CD133+ stem cells without lentiviral transduction. No clusters of dystrophin positive myofibers and clinical modifications of the performance were observe, also they appear stable during the last year of observation. This is the first demonstration of a clinical effect in old GRMD dogs that regained walking ability after autologous transplantation of engineered stem cells.
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