Stem cell therapy of muscular dystrophies using exon skipping approach in GRMD dogs

In DMD, skeletal and cardiac muscles are affected. A combination of different therapeutic approaches might enhance the possibility of successful therapy. We isolated CD133+ cells from muscle biopsies of GRMD dogs. Two dogs characterized by a mild clinical phenotype and three characterized by a severe clinical phenotype, were treated with their own transduced U7exon 6-8 cells. Old GRMD dogs are well characterized in term of clinical history. All dogs received 2 arterial systemic injections through a catheter that was introduced in the left femoralis artery and reached the aortic arch at the level of the left subclavia. We performed three different functional measures: claim Stairs (Time), swimming, 6 minute walking test (6MWT). 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+ cells. All transplanted animals were analysed at different times; 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. The percentage of dystrophin expressing fibres ranged from 1 up to 7%, in 2 distant sections of different biopsies each of selected muscles from the dogs. Western blot analysis 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. Two dogs received their own CD133+ cells without lentiviral transduction. In the group of untreated GRMD dogs, two dogs died of pneumonia and other complications during the follow up. This is the first demonstration of a clinical effect in old GRMD dogs that regained walking ability after autologous transplantation of engineered stem cells.