Autologous Transplantation of Muscle Precursor Cells Modified with a Lentivirus for Muscular Dystrophy: Human Cells and Primate Models

Abstract

Duchenne muscular dystrophy (DMD) is characterized by the absence of dystrophin. We tested the ability of lentiviral vectors to deliver a transgene into myogenic cells before their transplantation. Enhanced green fluorescent protein (eGFP) transgene was efficiently transferred into cells and eGFP-positive fibers were generated following transplantation. An eGFP-micro-dystrophin transgene under the control of a cytomegalovirus promoter was then transferred with the same viral vector but caused some toxicity to the mono-nucleated cells. We then used instead a muscle creatine kinase promoter. Dystrophin expression was observed in the muscle fibers after the transplantation of such genetically modified cells into mdx and severe combined immunodeficient mice. Micro-dystrophin expression was also observed in monkey muscles a month after allogenic or autologous transplantation of genetically modified myoblasts. Therapeutic exon skipping was induced by infecting myoblasts of a DMD patient, deleted for dystrophin exons 49 and 50, with a lentivirus expressing a U7 small nuclear RNA containing antisense sequences against exon 51. The modification led to correct exon skipping and to the expression of a quasi-dystrophin in vitro and in vivo. These results demonstrate the feasibility of lentiviral-based ex vivo gene therapy for DMD.

Full‐length dysferlin expression driven by engineered human dystrophic blood derived CD133+ stem cells

“…Our lentivirus construction recapitulates the transduction efficiency of previously tested and published lentivirus vectors [38,39]. We performed FACS analysis to determine the expression of GFP in infected CD133+ normal blood derived cells and we showed a percentage of 60.9% of GFP positive cells demonstrating a good efficiency of transduction as previously described [40,41] (Fig. 3B).…”

“…The PCR product woas digested with NheI and subcloned into pRRL-cPPT-hPGK-eGFP-WPRE vector. The transduction efficiency of lentiviral vectors containing eGFP and the dysferlin cassette was tested using the same multiplicity of infection as previously described [40,41]. Both lentiviral vectors showed the same transduction efficiency.…”

Cell Transplantation and “Stem Cell Therapy” in the Treatment of Myopathies: Many Promises in Mice, Few Realities in Humans

“…With this technique, the amount of muscle expressing a donor-derived protein depends on the number of injections per muscle volume; that is, the more injections done, the better result is achieved in terms of donor-protein expression in the muscle, such as -galactosidase or dystrophin in monkeys [122,132] or dystrophin in DMD patients [11][12][13]. Looking to make a difference with the earlier clinical trials in which myoblast transplantation was performed by few injections too far apart from each other, the author has called this strategy a high-density injections protocol [124].…”

“…specially useful, which becomes his standard instrument for intramuscular cell transplantations in nonhuman primates [123,132] (Figure 6). The same device was used by the author for myoblast transplantation in several muscles of an adult DMD patient [12], and another research group had tested it for myoblast transplantation in rabbits [68].…”

Stem Cell Based Therapy for Muscular Dystrophies: Cell Types and Environmental Factors Influencing Their Efficacy

“…Considering the cloning capacity of lentiviral vectors (up to 10kb (M. Kumar et al, 2001)), it should be possible to further optimise a vector so that it accommodates most of the functionally relevant coding region of dystrophin. An optimal dystrophin construct in a lentiviral vector could be used to treat patients with different mutations, in contrast to the U7 constructs, w h i c h , a l t h o u g h t h e y c an b e p l a c e d i n a lentiviral vector and induce dystrophin expression in stem cells in vitro and following their transplantation in vivo (Quenneville et al, 2007), are mutation-specific.…”