Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder affecting 1 in 7500. Symptoms typically arise in young adulthood and most patients show clinical features before age 30. FSHD is characterized by progressive wasting and weakness of facial and shoulder-girdle muscles, though all skeletal muscle can be affected. Currently there is no treatment for FSHD. The pathogenic events leading to FSHD have only recently started coming into focus. Several studies now support an FSHD pathogenesis model involving aberrant expression of the DUX4 gene, which encodes a myotoxic transcription factor. The emergence of DUX4 enabled the development of FSHD therapies. Previously our lab demonstrated proof of principle for the use of a DUX4-targeted RNA interference (RNAi)-based gene therapy. We have pursued two engineered microRNAs that showed strong therapeutic efficacy determined by their ability to suppress pathogenic levels of DUX4, and prevent myopathy in mice. To continue down a translational path this study was designed to determine the safety of microRNA therapy to muscle (which has not been previously determined) by delivering high doses for preliminary non-GLP toxicity studies. Both local intramuscular injections and systemic isolated limb perfusions were performed to identify safe sequence and dosing parameters for lead DUX4-targeted microRNAs at acute (3 week) and long-term (5 month) timepoints. To further circumvent potential off-target organ toxicity we have optimized these vectors to restrict expression to skeletal muscle. The safety parameters along with the promoter optimization provide necessary data for translating an RNAi-based therapy for FSHD.