New in vitro models of DMD by induced pluripotency in patient biopsies and gene knockdown in hESCs

Duchenne muscular dystrophy (DMD) is a genetic disorder that ultimately leads to the death of 1 in every 3,000 newborn males. Boys become wheelchair bound in their early teens and succumb to the condition in their twenties to thirties. Heart failure is a major cause of fatality and there is no cure. Years of research have identified the gene that causes DMD and the effects on patients? health have been widely documented. However, the basic understanding of how the condition affects the cells as humans grow and develop is not well understood, particularly in organs such as the heart. In part, our lack of understanding is because we cannot examine the detailed processes that go on either in the cells of young DMD patients or in the cells of their hearts.
This grant application is aimed at combining the knowledge of researchers at the University of Nottingham, who have expertise in human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC) biology, and those at the University of Newcastle, who have clinical and research experience in studying DMD and how it affects function of muscle cells. Combining these sources of expertise will provide new ways to study DMD and in the future it is hoped that this will lead to new treatments. How will this be achieved?
Whereas hESCs are derived from embryos given by consenting couples undergoing fertility treatment, iPSCs are produced by treating skin cells special proteins. This means that hESCs grown in the laboratory can be made to carry the mutations found in patients with DMD. Alternatively, skin cells from DMD patients can be converted into iPSCs that carry the patient-specific mutation. Remarkably, both hESCs and iPSCs can be grown in a plastic dish in the lab and made to mimic a very early stage of human development, including formation of beating heart cells (cardiomyocytes). This provides a way for the effect of the mutations that cause DMD on early development and cardiomyocyte function to be studied in a carefully controlled laboratory setting. The benefit of this is two-fold. First, it will provide a clearer understanding of the disease process. Second, it will provide a lab-based system that can be used in the future to test new drugs for alleviation and treatment of the symptoms of DMD that affect the heart.

The prevalence of Duchenne muscular dystrophy (DMD) is ~1 in 3,000 in newborn males and all die as young adults, usually in their twenties to thirties. Although heart failure accounts for at least 25% of fatalities, little is known about how the disorder affects development or function of human cardiomyocytes. Limited knowledge arises from lack of DMD cardiac biopsies, mouse models that show only mild heart anomalies and a complex causative DMD gene, which encodes many dystrophin isoforms. In this proposal we will exploit our wealth of knowledge of culture, genetic modification and cardiomyocyte differentiation of hESCs to produce transgenic lines in which variable numbers of the dystrophin isoforms are knocked down by RNAi. In parallel, we will extend our initial work in which pluripotency has been induced in fibroblasts by lentiviral transduction of transcription factors to now reprogramme fibroblasts from DMD patients. These cellular resources will be used to evaluate the effect of perturbed dystrophin expression on structure and location of the dystrophin-glycoprotein complex (DGC) during differentiation, as well as the impact on cardiomyocyte development, function, survival and electrophysiology. Development of this platform will have far-reaching applications for in vitro modelling of numerous genetic disorders of the heart, providing new routes to innovate and test intervention strategies.