Medicina Molecular e Genética

Test of Critical Steps towards a Combined Cell and Gene Therapy Approach for the Treatment of Duchenne Muscular Dystrophy

Abstract

Tine Q Kajhoj, Mogens Duch, Finn Skou Pedersen, Henrik Løvschall and Ernst-Martin Füchtbauer

Background: Therapies for muscular dystrophies remain a major challenge in spite of advanced strategies using either cell or gene therapy. We here propose a combined approach of cell and gene therapy. As gene delivery vehicles with specific homing potential we have chosen mesoangioblasts which are stem cells with mesodermal potential. This study specifically tests the maintenance of myogenic properties by EGFP-transduced mesoangioblasts and their ability to function as retroviral packaging cells in transient culture.

Methods: Mesoangioblasts transduced to express EGFP (EGFP-MA) were tested for myogenic properties in coculture. Survival and in situ myogenic differentiation were studied upon injection into degenerating M. gastrocnemius of athymic mice. In situ participation in muscle regeneration was confirmed on cryo-sections using EGFP fluorescence as marker. The ability of mesoangioblasts to serve as retroviral packaging cells was tested using the murine cell line NIH 3T3 fibroblasts as recipients in vitro and evaluation of transduction by fluorescence microscopy.

Results: EGFP-MA retained the ability to differentiate into skeletal muscle myotubes upon co-culture with C2C12 cells. In vivo, mouse M. gastrocnemius exhibited EGFP-signals within and at the basal lamina of skeletal muscle fibers 3, 10 and 24 days after injection of EGFP-MA. In culture, target cells could be transduced with EGFPencoding virions produced by mesoangioblasts.

Conclusions: Introduction of a retroviral vector into mesoangioblasts did not interfere with the myogenic properties of mesoangioblasts in culture or in vivo. Mesoangioblasts are able to function as retroviral packaging cells in vitro. While a possible therapeutic application of this new gene delivery system will require further detailed analysis of the long-term efficiency and the quantitative aspects of the method, our proof of principle study shows that the approach is feasible.