Re-programming utrophin gene expression in DMD using artificial transcription factors

Up-regulation of the dystrophin-related gene utrophin represents a promising therapeutic strategy for the treatment of Duchenne muscular dystrophy (DMD). The strategy is based on the capacity of utrophin to functionally replace defective dystrophin. In order to re-program the utrophin expression level in muscle, we engineered artificial zinc finger transcription factors (ZFATFs) on Zif268/Egr1 backbone. Using selective amino acid substitutions, the natural Zif268 transcription factor was specifically re-directed to the human and mouse conserved regions of the utrophin promoter ''A''. Furthermore, these minimally modified ZF-ATFs were designed to minimize any host immune-response against these proteins. Our present focus is the delivery of ZFATFs using recombinant Adeno-associated viral vectors engineered to express the therapeutic genes under the control of the human alpha-actin muscle specific promoter (mAAV-vector), combined with the use of the muscle-targeting AAV-serotype 8. Upon intraperitoneal injection of mAAV8- ZF-ATFs into dystrophin-deficient (mdx) mice, a significant amelioration of the pathological phenotype was observed. Histological and physiological analysis revealed a reduction of fiber necrosis and cell infiltration, as well as the functional recovery in muscle contractile force. Since utrophin is also a key determinant of the neuromuscular junction (NMJ), ongoing studies will characterize the NMJ in mAAV8-ZF-ATF treated mice. In summary, the development of ZF-ATF-re-directed technology, coupled with the mAAV delivery, highlights the potential of this novel therapeutic strategy for treatment of DMD.