ADSS1 myopathy is a rare, autosomal recessive disease caused by mutations in the ADSS1 gene, which encodes a striated muscle-specific isoform of adenylosuccinate synthetase (AdSS1). AdSS1 plays a critical role in the adenine nucleotide cycle, which is important for energy metabolism in muscle cells. Enzymatic defects, engendered by loss-of-function mutations in ADSS1, lead to a bottleneck in the adenine nucleotide cycle, causing metabolic dysfunction which ultimately results in progressive muscle weakness, mobility impairment, and cardiac and respiratory dysfunction, often requiring the use of a ventilator. Despite its debilitating nature, there is currently no cure or targeted treatments available. Unfortunately, due to the ultrarare nature of ADSS1 myopathy, very little research into possible therapeutic strategies has been done. To test the feasibility of gene replacement strategy in this disease, we first developed and characterized two mouse models of ADSS1 myopathy: Adss1 constitutive knockout, and Adss1 p.D261N knock-in (the most prevalent point mutation in patients). The phenotype of p.D261N knock-in mice is still being evaluated. Homozygous Adss1 knockout mice are viable without obvious signs of weakness, but preliminary data demonstrate abnormalities in total muscular adenine content and in ex vivo muscle contractility studies. AAV9-based delivery of a full-length human ADSS1 gene leads to robust protein expression in skeletal and cardiac muscle. Further studies to demonstrate phenotypic correction are underway. Currently, we are developing next-generation muscle-directed MyoAAV 4A capsid-based vectors for delivery of both linear and self-complementary ADSS1 cargos. This work provides substantial support for the development of gene therapy for ADSS1 myopathy and deepens our understanding of the role of purine homeostasis in muscle metabolism and function.