Novel approaches to humanized skeletal muscle in pig

PROJECT SUMMARY The overall objective of this Phase 1 research proposal is to significantly enhance the efficiency of generating human skeletal muscle in pig. The economic burden related to trauma and injury in the US is estimated at $400 billion annually. Skeletal muscle comprises ~40% of the body’s mass and contributes to locomotion, thermoregulation, and whole-body metabolism and, therefore, the loss of skeletal muscle due to surgery, deep burns, or injury is associated with immobility and an increased risk of all-cause morbidity and mortality. Effective therapies do not exist. Our goal is to develop novel regenerative therapies by generating human skeletal muscle in pig for reconstructive and transplantation based surgeries. Our early stage prototype, using somatic cell nuclear transfer (SCNT; cloning) and blastocyst complementation provides proof of concept for the feasibility of our platform and patented technology. With this patented technology, we have produced viable and normal intra-species chimeras and have further proven our ability to produce human skeletal muscle in mid-gestational embryos as well. Our surgical team has substantial expertise in muscle reconstruction following oncological procedures and military injuries. The problem: While our publications indicate the importance and feasibility of this platform approach, the process of SCNT is inherently inefficient, labor intensive, and technically demanding all of which limits the efficiency of muscle production. Furthermore, the generation of viable and normal offspring is challenged by abnormalities that result from failures in epigenetic reprogramming, cell cycle synchronies, chimerism, and low pregnancy rates. Our solution: In recent years, there have been numerous reports of direct CRISPR-based editing of zygotes produced by in vitro fertilization (IVF). This technique has been demonstrated to be highly efficient yet whether zygote gene editing can be combined with complementation strategies in porcine zygotes is unknown. Here, we will develop a platform for the production of intra- and inter-species chimeras using zygote gene editing together with blastocyst complementation. Our hypothesis: We hypothesize that zygote editing will allow for the production of significantly greater numbers of robust embryos, which will ultimately yield a significantly greater number of complemented embryos that will result in viable and healthy offspring with human muscle. We predict that zygote editing and embryo complementation will provide a transformational platform for the production of human skeletal muscle for the treatment musculoskeletal disorders and injury. Impact: The ability to generate large quantities of mature, human skeletal muscle holds unprecedented potential for transforming the care of patients with severe musculoskeletal injury and disease.