Utrophin up-regulation mediated by an artificial transcription factor ameliorates the dystrophic symptoms in mdx mice.

Duchenne Muscular Dystrophy (DMD) is a severe muscle degenerative disease, due to the absence of functional dystrophin, for which there is not currently an effective treatment. A promising cure for DMD is based on increasing levels of utrophin, a cytoskeletal protein similar to dystrophin and able to compensate for its absence. Many approaches have been proposed to increase utrophin levels in muscle but the size of the gene (one of the largest known) restrict its use in pre-clinical studies based on gene therapy. The development of small molecules able to up-regulate utrophin in vivo is then crucial. To this end we designed a synthetic three zinc finger based transcription factors (TF), named "Jazz", which has been demonstrated to drive in vitro the transcription of a test gene from the utrophin promoter (1) and then we realized a transgenic mice expressing "Jazz" under the control of a muscle specific promoter. We demonstrated by Chromatin Immunoprecipitation assay (ChIP) that Jazz selectively binds its DNA target sequence on the utrophin promoter and is able to up-regulate endogenous utrophin gene expression in the skeletal muscle as demonstrated by real-time PCR. Immunohistochemistry and western blot analyses (2). The transgenic mouse expressing Jazz was then crossbred to the mdx (murine dystrophy X-linked) model of dystrophy and several experiments were performed to check the effects of Jazz-induced utrophin up-regulation on the progression of the disease. Histological and immunochemical analysis of muscles from mdx-Jazz mice demonstrated a reduced degree of necrosis and inflammatory infiltration, together with lower serum levels of creatine kinases (a specific marker of muscle destruction), as compared with the dystrophic mdx mice. Moreover, we observed an enhancement of muscular strength in Jazz-mdx mice, as demonstrated both in vitro by electrophysiological analysis and in vivo by exercises performance tests (tread mill). To our knowledge, this is the first example of a transgenic mouse expressing an artificial gene coding for a zinc finger based transcription factor and the results obtained expressing the Jazz-TF in a dystrophic background, indicate that the strategy of transcriptional targeting of endogenous genes could represents an useful tool for the treatment of genetic disease, which still lack a cure.