Reversible Immortalization and Transfer of a Dystrophin Human Artificial Chromosome into Human Mesoangioblasts: Towards Autologous Cell Therapy of Duchenne Muscular Dystrophy

Human mesoangioblasts (MABs) are vessel–associated progenitors able to differentiate into skeletal muscle and to reconstitute the damaged muscle fibers when injected into a Duchenne Muscular Dystrophy (DMD) mouse model (mdx), an incurable myopathy for which no therapy currently exists. Because of these features, human MABs are now entered clinical experimentation based upon allogeneic transplantation. However autologous cell therapy for DMD still faces significant limitations, first of all the large size of the dystrophin gene that hampers its allocation into conventional gene-delivery tools such as viral vectors. In order to overcome these limitations, a novel approach of autologous cell therapy to treat DMD was developed by transplanting dystrophic MABs genetically-corrected with a Human Artificial Chromosome (HAC) containing the entire dystrophin locus (DYS-HAC). The feasibility of this strategy was already proved: MABs derived from mdx mouse and genetically corrected with the DYSHAC, were able to engraft skeletal muscle and restore dystrophin positive fibers in dystrophic mice, resulting into a significant morphological and functional amelioration of the phenotype. In the case of DMD MABs, an additional step of immortalization is fundamental before DYS-HAC transferring, which requires clonal expansion and analysis, since they undergo replicative senescence. To reversibly immortalize normal and dystrophic human MABs, lentiviral vectors encoding floxed hTERT IRES-HSV1-TK and Bmi-1 have been used. Normal clones have been characterized for proliferation and proper expression of hTERT and Bmi-1; they remained growth factor-dependent, contact-inhibited, 10 non tumorigenic and myogenic in vitro. Notably, upon transplantation into dystrophic mice, they engrafted skeletal muscle and restored dystrophin expression. I then moved to the immortalization of DMD MABs in order to transfer the DYS-HAC, obtaining immortalized DMD DYS-HAC MABs. These data set the conditions for future clinical translation of this experimental strategy for the treatment of the DMD patients.