Successful Myoblast Transplantation in Primates Depends on Appropriate Cell Delivery and Induction of Regeneration in the Host Muscle 1 Daniel Skuk, Brigitte Roy, Marlyne Goulet, and Jacques P. Tremblay Unite ´ de Recherche en Ge ´ne ´tique Humaine, Centre Hospitalier de l’Universite ´ Laval, Que ´bec, Canada Received April 8, 1998; accepted October 8, 1998 Myoblast transplantation (MT) may be a potential treatment for severe recessive hereditary myopathies. The limited results of MT in clinical trials led us to improve this technique in monkeys, an animal model phylogenetically similar to humans. Three Macaca mulata monkeys were used as donors and six as receiv- ers for MT. Myoblasts were grown in culture from muscle biopsies of adult monkeys and infected with a retroviral vector encoding the LacZ gene. Different numbers of cells (i.e., 4  10 6 ,8  10 6 , and 24  10 6 cells) were transplanted into different muscles and 8  10 6 cells (resuspended in a notexin solution) were injected in one muscle of four monkeys. For these transplanta- tions, the cell suspension (in a volume of about 100 l) was injected at 35 sites less than 1 mm apart. Two other monkeys received 100  10 6 myoblasts resuspended in 1 ml of HBSS or 1 ml of notexin. For these two monkeys, the myoblasts were injected at 200–250 sites within a small portion of the muscle. All monkeys were immuno- suppressed with daily injections of FK506. Four weeks after MT, the transplanted muscle portions were biop- sied and the presence of -galactosidase-positive (- Gal) muscle fibers was investigated. The number of -Gal fibers was 822  150 (site grafted with 4  10 6 cells), 1253  515 (8  10 6 cells), 1084  278 (24  10 6 ), and 2852  1211 (notexin). In the monkeys grafted with 100  10 6 myoblasts, the number of -Gal fibers was 4850 (site without notexin) and 9600 (site with notexin). We demonstrated that a precise mechanical distribution of myoblasts into the tissue improves substantially MT in primates. The presence of notexin with the transplanted cells further increased the success of their transplanta- tion. These are the best results obtained either with MT or gene therapy in primates and they encourage the possibil- ity to human MT trials.  1999 Academic Press Key Words: myoblast; monkey; notexin; FK506; trans- plantation. INTRODUCTION Myoblast transplantation (MT) is an experimental approach originally proposed to treat severe and deadly recessive myopathies like Duchenne muscular dystro- phy (34, 44). Other applications also suggested for MT are the replacement of infarcted myocardium (41, 50), the improvement of the levator palpebrae superioris in congenital ptosis (3), and the systemic delivery of recombinant proteins to treat a variety of acquired and inherited diseases (4, 10). After the first promising results of MT in mice (34, 40, 44), several clinical trials were conducted with dystrophic patients (20, 27, 35, 38, 39, 49). All these MT clinical trials were unsuccessful in terms of significant muscle function improvement. Only the histological demonstration of a significant but small increase in dystrophin-positive fibers was observed by some re- searchers (20, 35, 38, 49). Because of the limited results, the clinical trials were discontinued and experi- mentation in animals was pursued only by some groups to explain the reasons of the failure. Animal research demonstrated that immune-specific reactions against the donor cells and hybrid muscle fibers take place some days after MT (15, 16, 24). Different immunosuppressive treatments such as cyclos- porine A (21), cyclophosphamide (53), rapamycin (52), and FK506 (29) were tested in mice by our team. The degree of success of MT varied depending on the immunosuppressive treatment (51), the best results being obtained with the FK506 (29). It is noteworthy that cyclosporine A was not sufficient to suppress the cellular immune reaction even at high doses (21), and cyclophosphamide was toxic for the transplanted myo- blasts (53). This observation was important because cyclosporine A and cyclophosphamide were the only immunosuppressive treatments used in the clinical trials. FK506 was also observed to be effective for MT in monkeys (30, 32). These results confirmed the role of the immune-specific reaction in the failure of MT clinical trials and the importance of the choice of the 1 This work was supported by grants from the Association Fran- c ¸aise contre les Myopathies, the Muscular Dystrophy Association, and the Muscular Dystrophy Association of Canada. Experimental Neurology 155, 22–30 (1999) Article ID exnr.1998.6973, available online at http://www.idealibrary.com on 22 0014-4886/99 $30.00 Copyright  1999 by Academic Press All rights of reproduction in any form reserved.