PROJECT SUMMARY Congenital Myasthenic Syndromes (CMS) are a group of genetically and phenotypically heterogeneous, neuromuscular transmission disorders characterized by muscle weakness (myasthenia) that worsens with physical exertion. DoK-7 (Downstream of tyrosine kinase 7) is a key regulator of neuromuscular junction (NMJ) formation. Homozygous loss-of or reduction of-function mutations in the human DOK7 gene underlie a limb-girdle type of CMS characterized by NMJs that are about half the normal size. DoK-7 CMS is an orphan disease estimated to affect 3,600 people worldwide. Patients with DoK-7 CMS have a decreased Quality of Life (QoL) due to exercise intolerance, dependency on intermittent respiratory support, and/or tube feeding by adulthood (2/3 of the patients). Moreover, about half of the patients will need a wheelchair for ambulation, and the other half will require walking aids. No cure nor standardized treatment has been yet developed for DoK-7 CMS. While forms of CMS are managed through the administration of acetylcholine (AChE) inhibitors, DoK-7 CMS is refractory and can deteriorate if treated with AChE inhibitors. Symptoms ameliorations for DoK-7 CMS is achieved by recurrent administration of Ephedrine and Albuterol, 2-adrenergic receptor agonists, which provide suboptimal symptom management for some patients. Moreover, prolonged exposure to 2-adrenergic receptor can cause tachycardia cardiac ischemia, heart failure, cardiomyopathy, and increased inflammatory response. Amplo Biotechnology is developing AMP-101, the first gene therapy product (GTP) for DoK-7 CMS. The treatment is based on a recombinant adeno-associated virus vector carrying the human DOK7 gene. AMP-101 development has so far progressed exceptionally well. Extensive testing in a DoK-7 CMS mouse model (DoK-7 KI/KI) during Phase I activities (SBIR project 1R44NS124351- 01) has shown that the GTP is able to rescue the extreme DoK-7 KI/KI phenotype, extending survival and restoring normal motor activity. GLP Toxicology studies in C57BL/6 mice have shown that AMP-101 is safe and well tolerated, with no macroscopic observations, and no liver toxicity observed in the histopathology analysis conducted on D29 and D183 samples. Moreover, activities conducted during Phase II project (4R44NS124351-02) have demonstrated that AMP-101 can be manufactured at 50 L scale with high purity and high yield. The new product will enable a shift in the current clinical practice from chronic administration of drugs to alleviate symptoms to a one-off treatment, administered through a single intravenous injection. The solution will allow physicians to treat the entire affected population, curing adult disease, and stopping disease progression in children. The goal of this SBIR Phase II B project is to ensure smooth transition to the clinic supporting the development of assays to detect cellular immune responses against viral antigens and viral shedding and the manufacturing of a 500L GMP run.