Genetically modified systems to study muscular dystrophies

Abstract

We developed different approaches to utilize genetically modified cells and animal models to understand molecular mechanism of Facioscapulohumeral muscular dystrophy (FSHD).Main obstacle with primary cell lines is to obtain sufficient number of tissue specific progenitors that will be able extensively to proliferate and maintain the phenotype to be used for relevant in vitro studies. As an alternative solution for obtaining appropriate cells are induced pluripotent stem (IPS) cells. IPS cells can be made from different somatic cells by cell reprogramming. Patient specific IPS cells retain the genotype of the patient, they are good models to study molecular mechanism of diseases and they are suitable for autologous cell transplantations. We generated IPS cells from myoblasts from FSHD patients using common reprogramming factors (Oct4, Sox2, KLF4 and c-Myc), incorporated in viral vectors. Mesenchymal stem cells were obtained by differentiation of IPS cells in 3D culture conditions in presence of cocktail of growth factors. Myogenic progenitors were obtained by overexpressing Myf5 in mesenchymal cell fraction. Generated cells revealed typical characteristics of myoblast and were able functionally to recover damaged mouse muscle. Furthermore, we were able to genetically correct affected locus in FSHD IPS cells by removing 4qA161 allele using zinc finger nucleases. Gene corrected cells did not express DUX4 and displayed normal myogenic properties compared to the affected ones.To be able to study FSHD in vivo and test various therapy approaches we developed transgenic mouse model that has incorporated DUX4 gene on X chromosome. IDUX4 mouse expressed DUX4 in different tissue. Male pups have runt phenotype, with prominent skin, testis and retina pathological changes.In this talk, advantages and methods for cell manipulations for generating in vitro and in vivo diseases models will be discussed.