PROJECT ABSTRACT Duchenne muscular dystrophy is a genetic disease caused by defects in a protein known as dystrophin. Dystrophin functions to provide structural and mechanical stability to the muscle cells; and the absence of functional dystrophin in Duchenne muscular dystrophy leads to progressive muscle degeneration, weakness, and a significantly reduced life expectancy due to cardiorespiratory failure. There is no cure. As a potential curative therapy, restoration of dystrophin through muscle stem cell transplantation is actively being explored, but the clinical translation of these efforts is hampered by the inability to generate large quantities of muscle stem cells in dishes without compromising their therapeutic potency. In addition, it is also very challenging to expand muscle stem cells derived from dystrophic muscles in dishes due to their inherent functional deficits. This further limits strategies to gene correct patient-derived muscle stem cells outside the body, grow them in a dish, and transplant the functional muscle stem cells in an autologous manner. In this Diversity Supplement application, we will develop a Notch-modulatory muscle stem cell culture platform that enables the generation of large quantities of therapeutically potent muscle stem cells. This research strengthens and expands the scope of the parent R01 project by engineering a hydrogel platform to support dystrophic muscle cell expansion for cell therapy applications. Towards this goal, Aim 1 will engineer a Notch-modulatory hydrogel for rejuvenating dystrophic muscle stem cells ex vivo. Aim 2 will determine the effects of suppressing p38α/β mitogen-activated protein kinase (MAPK) activity of dystrophic muscle stem cells maintained within the Notch-modulatory hydrogel long-term. The outcomes of this research will trailblaze a pathway towards personalized (autologous) muscle stem cell therapy for safely treating Duchenne muscular dystrophy, where such technologies will also enable ex vivo gene correction on the muscle stem cells outside the patient's body before being re-introduced to restore dystrophin.