Final Report Summary - STEM CELLS FOR DMD (Novel strategies for the cell therapy of muscular dystrophies)
The progressive muscle loss that is the hallmark of Duchenne muscular dystrophy (DMD) has proved very difficult to halt or reverse. Although the causative mutations of DMD were identified in the X-linked gene encoding dystrophin (a structural muscle protein) several decades ago, translating this genetic discovery into new treatments has been challenging. Most therapeutic strategies aim to use gene therapy to deliver the normal dystrophin gene to the dystrophic muscles of DMD patients. However, the dystrophin gene is too large to be carried by the viral vectors gene usually used in gene therapy and all muscles in the body would have to be injected with the vector and replacement
Our team has combined stem cell therapy with a human artificial chromosome vector to overcome these two challenges in the mdx mouse model of DMD. We had previously identified a blood vessel stem cell called ''mesoangioblast'' that has the dual talents of being able to cross blood vessel walls and to differentiate into a variety of mesoderm cell types including muscle cells. Thanks to a collaboration with the team of Prof. Mitsuo Oshimura of Tottori University, Japan, our group has succeeded in transferring a human artificial chromosome containing the whole dystrophin locus (Dys-HAC), previously created in Oshimura lab (Hoshiya et al. 2009) into mouse dystrophic mesoangioblasts isolated from mdx mice; then we injected the corrected mesoangioblasts directly into the dystrophic skeletal muscles of recipient mdx-SCID (immune deficient) mice, to prevent reaction against the human protein. Transplanted mesoangioblasts were able to engraft in dystrophic muscles, express normal dystrophin, and produce functional muscle fibers with amelioration of dystrophic pathology. Moreover, transplanted mesoangioblasts contributed to the muscle satellite cell pool, which produces new muscle cells under normal conditions. Next, we showed that if injected into the arterial circulation, the corrected mesoangioblasts of mdx mice were able to cross blood vessel walls, home to dystrophic muscles and graft contribute to the formation of new dystrophin-expressing myofibers. We then showed that mice receiving the mesoangioblast transplants showed reduced fiber fragility, increased force, and greater motor capacity on treadmill and freewheel tests. Although there are still technical and regulatory hurdles to be overcome before this strategy can be used in DMD patients, stem cell-mediated transfer of the normal dystrophin gene using a human artificial chromosome shows promise as a treatment for this tragic and ultimately fatal disease. Notably a clinical trial based upon transplantation of HLA-matched donor mesoangioblasts (Eudract N. 2011-000176-33), was completed by the PI, demonstrating clinical feasibility of this approach.
The limited proliferation potential of primary human cells represents the major technical problem in order to transfer this strategy to human dystrophic mesoangioblasts. To overcome this hurdle we have reversibly immortalized human and canine dystrophic mesoangioblasts by transferring genes that promote proliferation (telomerase and Bm1) with lentiviral vectors that can be removed from the genome prior to transplantation, in order to prevent the risk of generating tumors. Our results, that are being prepared for publication (Benedetti et al.) show that the immortalizing sequences extend the lifespan of human and canine cells, do not prevent their ability of differentiate into muscle cells and, most importantly, do not promote the formation of tumors when transplanted into immune deficient mice and autologous dogs, respectively.
Our team has combined stem cell therapy with a human artificial chromosome vector to overcome these two challenges in the mdx mouse model of DMD. We had previously identified a blood vessel stem cell called ''mesoangioblast'' that has the dual talents of being able to cross blood vessel walls and to differentiate into a variety of mesoderm cell types including muscle cells. Thanks to a collaboration with the team of Prof. Mitsuo Oshimura of Tottori University, Japan, our group has succeeded in transferring a human artificial chromosome containing the whole dystrophin locus (Dys-HAC), previously created in Oshimura lab (Hoshiya et al. 2009) into mouse dystrophic mesoangioblasts isolated from mdx mice; then we injected the corrected mesoangioblasts directly into the dystrophic skeletal muscles of recipient mdx-SCID (immune deficient) mice, to prevent reaction against the human protein. Transplanted mesoangioblasts were able to engraft in dystrophic muscles, express normal dystrophin, and produce functional muscle fibers with amelioration of dystrophic pathology. Moreover, transplanted mesoangioblasts contributed to the muscle satellite cell pool, which produces new muscle cells under normal conditions. Next, we showed that if injected into the arterial circulation, the corrected mesoangioblasts of mdx mice were able to cross blood vessel walls, home to dystrophic muscles and graft contribute to the formation of new dystrophin-expressing myofibers. We then showed that mice receiving the mesoangioblast transplants showed reduced fiber fragility, increased force, and greater motor capacity on treadmill and freewheel tests. Although there are still technical and regulatory hurdles to be overcome before this strategy can be used in DMD patients, stem cell-mediated transfer of the normal dystrophin gene using a human artificial chromosome shows promise as a treatment for this tragic and ultimately fatal disease. Notably a clinical trial based upon transplantation of HLA-matched donor mesoangioblasts (Eudract N. 2011-000176-33), was completed by the PI, demonstrating clinical feasibility of this approach.
The limited proliferation potential of primary human cells represents the major technical problem in order to transfer this strategy to human dystrophic mesoangioblasts. To overcome this hurdle we have reversibly immortalized human and canine dystrophic mesoangioblasts by transferring genes that promote proliferation (telomerase and Bm1) with lentiviral vectors that can be removed from the genome prior to transplantation, in order to prevent the risk of generating tumors. Our results, that are being prepared for publication (Benedetti et al.) show that the immortalizing sequences extend the lifespan of human and canine cells, do not prevent their ability of differentiate into muscle cells and, most importantly, do not promote the formation of tumors when transplanted into immune deficient mice and autologous dogs, respectively.