Abstract
Background: We are developing a novel therapy for Duchenne muscular dystrophy (DMD), involving the transplantation of autologous, skeletal muscle-derived stem cells that have been genetically corrected to express dystrophin. Dystrophin is normally expressed in activated satellite cells and in differentiated muscle fibres. However, in past preclinical validation studies, dystrophin transgenes have generally been driven by constitutive promoters that would be active at every stage of the myogenic differentiation process, including in proliferating muscle stem cells. It is not known whether artificial dystrophin expression would affect the properties of these cells. Aims: Our aims are to determine if mini-dystrophin expression affects the proliferation or myogenic differentiation of DMD skeletal muscle-derived cells. Methods: Skeletal muscle-derived cells from a DMD patient were transduced with lentivirus coding for mini-dystrophins (R3–R13 spectrin-like repeats (ΔR3R13) or hinge2 to spectrin-like repeats R23 (ΔH2R23)) with EGFP (enhanced green fluorescence protein) fused to the C-terminus, driven by a constitutive promoter, spleen focus-forming virus (SFFV). Transduced cells were purified on the basis of GFP expression. Their proliferation and myogenic differentiation were quantified by ethynyl deoxyuridine (EdU) incorporation and fusion index. Furthermore, dystrophin small interfering ribonucleic acids (siRNAs) were transfected to the cells to reverse the effects of the mini-dystrophin. Finally, a phospho-mitogen-activated protein kinase (MAPK) array assay was performed to investigate signalling pathway changes caused by dystrophin expression. Results: Cell proliferation was not affected in cells transduced with ΔR3R13, but was significantly increased in cells transduced with ΔH2R23. The fusion index of myotubes derived from both ΔR3R13- and ΔH2R23 -expressing cells was significantly compromised in comparison to myotubes derived from non-transduced cells. Dystrophin siRNA transfection restored the differentiation of ΔH2R23-expressing cells. The Erk1/2- signalling pathway is altered in cells transduced with mini-dystrophin constructs. Conclusions: Ectopic expression of dystrophin in cultured human skeletal muscle-derived cells may affect their proliferation and differentiation capacity. Caution should be taken when considering genetic correction of autologous stem cells to express dystrophin driven by a constitutive promoter.
Highlights
Transplantation of autologous, skeletal muscle-derived stem cells that have been genetically corrected to express dystrophin is a promising strategy to treat Duchenne muscular dystrophy (DMD)
When we performed the proliferation assay with the purified GFP+ cells from cells that had been transduced with human desmin (hDes)–LVs, we found that hDes–∆H2R23–enhanced green fluorescent protein (EGFP) cells (79.47% ± 1.48%; n = 3; p < 0.001) contained significantly more ethynyl deoxyuridine (EdU)+ cells than non-transduced cells (59.47% ± 0.33%; n = 3); while there was no difference in the % of EDU+ cells transduced with hDes–∆R3R13–EGFP (62.13% ± 0.78%, n = 3) compared to non-transduced cells (p > 0.05) (Supplementary Figure S1)
We lentivirally transduced DMD skeletal muscle-derived cells with two different isoforms of mini-dystrophin, driven by constitutive promoters that are active in proliferating myoblasts [14]
Summary
Transplantation of autologous, skeletal muscle-derived stem cells that have been genetically corrected to express dystrophin is a promising strategy to treat Duchenne muscular dystrophy (DMD). Dystrophin would be expressed in cells at all stages of myogenic differentiation, including proliferating muscle cells This has raised concerns that upregulation of mini-dystrophin may change the performance of the cells, given that the isoforms of mini-dystrophin usually contain functional motifs that can elicit signalling pathways via their binding to corresponding intracellular or cell membrane components. In past preclinical validation studies, dystrophin transgenes have generally been driven by constitutive promoters that would be active at every stage of the myogenic differentiation process, including in proliferating muscle stem cells. It is not known whether artificial dystrophin expression would affect the properties of these cells. Caution should be taken when considering genetic correction of autologous stem cells to express dystrophin driven by a constitutive promoter
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