Experimental gene therapy of Duchenne Muscular Dystrophy by artificial transcription factors upregulating the dystrophin-related gene Utrophin

The cure of Duchenne muscular dystrophy (DMD) depends on the development of innovative therapies able to extend the survival and improve the quality of life of dystrophic patients. Over-expression of the dystrophin-related gene utrophin represents one of this therapeutic strategies, due to its ability to replace defective dystrophin without triggering immunological reactions. One of the major problems related to the delivery of utrophin gene to muscular tissues is its high molecular weight. Thus it is crucial to develop small molecules, easy to be inserted in the vectors most widely used for gene therapy. For this purpose we developed small synthetic zinc finger transcription factors (ZFP TFs) that specifically bind the utrophin promoter. We already demonstrated that the expression of an artificial three-zinc finger transcription factor (Jazz) up-regulates utrophin and induces a significant recovery of muscle strength in dystrophic mdx mice. Here we demonstrate the feasibility and efficacy of an experimental gene therapy based on the systemic delivery of the ZFP TF-Jazz in mdx mice by a recombinant adeno-associated-virus vector (rAAV) selected for its known high affinity for skeletal muscle (AAV8) and engineered to express the therapeutic genes under the control of the muscle specific alpha-Actin promoter. Guideline experiments with AAV 8-alpha-Actin-EGFP vectors revealed a widespread and durable expression restricted to skeletal and cardiac muscles of mdx mice. Systemic intraperitoneal injection of a myc tagged AAV 8-alpha-Actin-Jazz expression vector in mdx mice resulted in the expression of myc-Jazz and utrophin upregulation in several muscular districts accompanied by a significant recovery from dystrophic phenotype. This was as assessed by histological analysis revealing reduction of fiber necrosis and inflammatory cell infiltration in Jazz-treated mice. Physiological data also provided evidence for functional recovery of muscle contractile force in the mdx mice expressing the artificial gene Jazz, as shown both by electrophysiological tests on isolated muscles and by improved performance in treadmill exercise. These results are, to our knowledge, the first to establish the feasibility and efficacy of a body-wide viral-mediated gene therapy of DMD based on ZF ATFs able to upregulate utrophin in a very specific way in skeletal and cardiac muscle of dystrophic mice, counteracting the symptoms of the disease.