ANIMAL MODELS TO VALIDATE THE USE OF NEW SMALL SYNTHETIC TRANSCRIPTION FACTORS IN THE GENE THERAPY OF MUSCLE DYSTROPHIES

Duchenne Muscular Dystrophy (DMD) affects 1 out of 3,000 boys and is a severe X-linked genetic disease for which there is not currently an effective treatment. DMD causes progressive muscle degeneration due to the absence of functional dystrophin. The over-expression of dystrophin-related genes such as utrophin, a cytoskeletal protein similar to dystrophin and able to compensate for its absence, represents a promising therapeutic strategy for the cure of DMD. 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 (ZFP-TF), named "Jazz", which has been demonstrated to drive in vitro the transcription of a test gene from the utrophin promoter (1, 4). Then we realized a transgenic mice expressing Jazz under the control of a muscle specific promoter. We demonstrated by Chromatin Immunoprecipitation assay that Jazz selectively binds its DNA target sequence on the utrophin promoter in vivo 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 the Jazz- induced utrophin up-regulation on the progression of the disease. Histological and immunochemical analysis demonstrated a reduced degree of inflammatory infiltration, necrosis and regeneration in muscles of mdx-Jazz mice, as compared with the non-transgenic dystrophic littermates. Lower serum levels of creatine kinases (a specific marker of muscle destruction) were also observed in mdx-Jazz mice and an increased contractility was revealed by electrophysiological analysis on isolated muscles of dystrophic mice expressing Jazz. Even more significant results were obtained in vivo by exercise performance tests (tread mill), demonstrating an impressive recovery of muscle strength and resistance in mdx-Jazz mice, which perform two fold better than mdx non-transgenic littermates, approaching the results attained by wild type mice. 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. Starting from these positive results, we are developing even more specific and efficient zinc finger transcription factors (3) that will be used to test the feasibility and efficacy of the gene therapy of DMD with ZFP-TFs.