610. Translating DUX4-Targeted RNAi Therapy for FSHD (2015)

OBJECTIVE: Develop a DUX4-targeted RNAi-based gene therapy for Facioscapulohumeral muscular dystrophy (FSHD) BACKGROUND: FSHD, the third most prevalent muscular dystrophy, is an autosomal dominant disorder that most commonly causes progressive weakness in muscles of the face, shoulder girdle, and limbs. FSHD was formally classified as a major form of muscular dystrophy in 1954, but the pathogenic events leading to the disease have only recently started coming into focus. Several studies now support an FSHD model involving aberrant expression of the DUX4 gene, which encodes a myotoxic transcription factor. The emergence of DUX4 represented a momentum shift in the FSHD field as it provided an important target for therapy design. As FSHD is currently untreatable, developing effective therapies is a critical need in the field. We hypothesized that an FSHD treatment should center on inhibiting DUX4 expression and/or activity, and have implemented several strategies to accomplish this goal. In the first published study on DUX4 inhibition, we demonstrated that AAV-mediated RNAi gene therapy could effectively suppress DUX4 expression and protect mouse muscles from its toxic effects. Specifically, our therapeutic DUX4-targeted artificial microRNAs improved histological, molecular, and functional outcomes associated with DUX4 expression in muscle. We are now working to translate this therapy through optimization of vector and delivery route. In addition, as over-expression of inhibitory RNAs have been associated with off-target effects, we are assessing the safety of this approach using dose-escalation and toxicology studies. The latter experiment is particularly important since the potential toxicity of RNAi therapy has not been previously assessed in muscles. DESIGN/METHODS: We addressed 4 issues in this study, as we proceed down a path toward translation: (1) removed the GFP reporter from our 1st-generation AAV vectors; (2) tested additional DUX4-targeted miRNA sequences; (3) developed muscle-restrictive promoters for miRNA expression; and (4) determined the safety of muscle-targeted miRNA therapy through dose-escalation toxicology studies, using direct intramuscular injection and vascular delivery. RESULTS: Removal of the GFP reporter gene had no deleterious effect on therapeutic efficacy and positioned our new vectors for potential clinical usage. Our alternative DUX4 miRNA proved to be equally effective at preventing DUX4-induced toxicity as the original sequence, and we have established safety thresholds by determining the maximum tolerable AAV. miRNA dose in mice for IM and IV delivery. At very high levels, all AAV-delivered miRNAs showed some toxicity, but importantly we found no deleterious overt pathology related to miRNA treatment at vector doses proven effective for DUX4 suppression in mice, over several timepoints. This toxicology data is promising for translating this strategy, especially considering DUX4 is markedly less abundant in human FSHD muscle than in our mouse model (which expresses the gene at very high levels). CONCLUSIONS: This study provides important data necessary for translating an RNAi-based therapy for FSHD.