Volumetric muscle loss (VML) is characterized by the loss of a significant portion of skeletal muscle, leading to permanent damage to muscle structure and function. VML results from major traumatic injury, and it is becoming increasingly more frequent in military Veterans as a result of roadside explosions, gunshot wounds, and motor vehicle crashes. VML contributes to long-term disability and $400 billion in economic burden in the US annually. Traumatic injuries leading to VML are associated with impaired endogenous muscle regeneration and revascularization capacity. Current surgical interventions such as muscle flap grafting or scar tissue debridement are associated with significant donor site morbidity and functional deficiency. Experimental approaches using decellularized extracellular matrix scaffolds show limited benefit in muscle recovery. Accordingly, a tissue engineering system that can restore normal skeletal muscle structure and function remains lacking for treatment of VML. Since skeletal muscle is composed generally of a bundle of parallel-aligned myofibers interspersed with blood vessels that provide blood and oxygen to the myofibers, the long-term goal of this proposal is to engineer vascularized skeletal muscle tissue constructs that mimic the native muscle and vessel structure, in order to restore muscle function after VML. The purpose of this study is to bioengineer skeletal muscle tissue composed of skeletal muscle precursor cells and vascular endothelial cells in a parallel-aligned nanofibrillar scaffold that augments cell survival, myofiber formation, and vascular perfusion recovery in a murine model of VML. Owing to the importance of vascular perfusion recovery, the scaffolds will also be engineered to release angiogenic growth factors in the form of modified mRNA (mmRNA), which obviates genomic alterations. The proposed objectives are designed to advance the understanding of how intercellular interactions with parallel-aligned nanofibrillar scaffolds, along with transient delivery of therapeutic mmRNA, can promote muscle and vascular regeneration. Accordingly, the Specific Aims are: (1) To engineer endothelialized aligned skeletal muscle composed of muscle precursor cells and endothelial cells in an aligned nanofibrillar scaffold that augments cell survival, myotube formation, and contractile function in vitro; (2) To enhance the angiogenic capacity of endothelialized and parallel- aligned engineered skeletal muscle using scaffold-mediated mmRNA delivery; and (3) To quantify the therapeutic efficacy of endothelialized and aligned engineered skeletal muscle with transient therapeutic mmRNA delivery in a murine model of VML. The proposed studies are highly significant because they seek to improve the therapeutic benefit of cell transplantation for treatment of VML, shifting away from the transplantation of acellular scaffolds to pre-formed endothelialized muscle tissue constructs with transient gene delivery for improved cli...