Cell-lineage regulated myogenesis for dystrophin replacement: a novel therapeutic approach for treatment of muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is characterized in skeletal muscle by cycles of myofiber necrosis and regeneration leading to loss of muscle fibers and replacement with fibrotic connective and adipose tissue. The ongoing activation and recruitment of muscle satellite cells for myofiber regeneration results in loss of regenerative capacity in part due to proliferative senescence. We explored a method whereby new myoblasts could be generated in dystrophic muscles by transplantation of primary fibroblasts engineered to express a micro-dystrophin/enhanced green fluorescent protein (muDys/eGFP) fusion gene together with a tamoxifen-inducible form of the myogenic regulator MyoD [MyoD-ER(T)]. Fibroblasts isolated from mdx(4cv) mice, a mouse model for DMD, were efficiently transduced with lentiviral vectors expressing muDys/eGFP and MyoD-ER(T) and underwent myogenic conversion when exposed to tamoxifen. These cells could also be induced to differentiate into muDys/eGFP-expressing myocytes and myotubes. Transplantation of transduced mdx(4cv) fibroblasts into mdx(4cv) muscles enabled tamoxifen-dependent regeneration of myofibers that express muDys. This lineage control method therefore allows replenishment of myogenic stem cells using autologous fibroblasts carrying an exogenous dystrophin gene. This strategy carries several potential advantages over conventional myoblast transplantation methods including: (i) the relative simplicity of culturing fibroblasts compared with myoblasts, (ii) a readily available cell source and ease of expansion and (iii) the ability to induce MyoD gene expression in vivo via administration of a medication. Our study provides a proof of concept for a novel gene/stem cell therapy technique and opens another potential therapeutic approach for degenerative muscle disorders.

Prosurvival Factors Improve Functional Engraftment of Myogenically Converted Dermal Cells into Dystrophic Skeletal Muscle

“…Coupled with the finding that higher engrafted sections are associated with local increases in CSA, data suggest that PSC promoted donor-engrafted de novo fiber formation. However, these small fibers are likely developmentally immature, and as in the previous work we detected developmental myosin heavy chain in the majority of small-caliber donor cell-engrafted fibers [6]. The increase in CSA with cell injections may also reflect tissue remodeling that is not due to needle track injury alone, since all muscles received injections.…”

“…Previous work shows that lentiviral-modified dermal fibroblasts (dFbs) are viable candidates for autologous cell therapy; they are accessible and readily expand in culture, can be converted into the myogenic lineage in vivo, and engraft after syngeneic transplantation in dystrophic mouse muscle [5][6][7]. Delivery of cells into muscle remains an issue for most cell therapies, and with many cell types, engraftment has been insufficient to see improvements in whole muscle function.…”

Preclinical study of an ex vivo gene therapy protocol for hepatocarcinoma

“…Since then, the transplantation of 'healthy cells' has become standard therapy not only for hematopoietic Elaboration of an ex vivo gene therapy protocol B Lortal et al disorders, but also for acquired skin, heart or bone damage, 25,36 and more recently, for use as potent drug delivery vehicles. 27 Several cell types can be used as drug vehicles (keratinocytes, bone marrow-derived mesemchymal stem cells, hepatocytes, dendritic cell and fibroblasts) 21,37-39 but more and more frequently, however, primary cells must be extensively processed ex vivo before they can be used as pharmaceutical tools.…”

“…23,24 Numerous clinical trials have involved the use of human fibroblasts as drug delivery systems. They can be used, for example, to cure diseases such as junctional epidermolysis bulbosa, 25 treat venous, diabetic and pressure ulcers by the release of growth factors or cytokines (vascular endothelial growth factor (VEGF), hepatocyte growth factor, basic fibroblast growth factor (FGF), transforming growth factor (TGF) b1 and IL-8), 26 increase tissue repair in acute or chronic wounds and in aesthetic and reconstructive surgery, 27 and treat Duchenne muscular dystrophy, 25 Alzheimer's disease 28 or cancer by the induction of an immune response. 21,29 Fibroblasts are mostly obtained from skin biopsies varying in size from one clinical trial to another.…”

Muscle Satellite Cells and Duchenne Muscular Dystrophy

“…This was the first example of transcription factor-based reprogramming of the cell, but this technology has not been successfully applied to regenerative medicine. Recently, Kimura et al reported that MyoD mediated conversion of fibroblasts in situ (Kimura et al 2008). The authors first introduced a tamoxifen-inducible MyoD expression cassette together with a muscle promoter-derived dystrophin expression cassette into mdx-derived fibroblasts, and then transplanted them into mdx muscle.…”