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Numerous therapeutic approaches for Duchenne and Becker Muscular Dystrophy (DMD and BMD), the most common X-linked muscle degenerative disease, have been proposed. So far, the only one showing a clear beneficial effect is the use of corticosteroids. Recent evidence indicates an improvement of dystrophic cardiac and skeletal muscles in the presence of sustained cGMP levels secondary to a blocking of their degradation by phosphodiesterase five (PDE5). Due to these data, we performed a study to investigate the effect of the specific PDE5 inhibitor, tadalafil, on dystrophic skeletal muscle function. Chronic pharmacological treatment with tadalafil has been carried out in mdx mice. Behavioral and physiological tests, as well as histological and biochemical analyses, confirmed the efficacy of the therapy. We then performed a microarray-based genomic analysis to assess the pattern of gene expression in muscle samples obtained from the different cohorts of animals treated with tadalafil. This scrutiny allowed us to identify several classes of modulated genes. Our results show that PDE5 inhibition can ameliorate dystrophy by acting at different levels. Tadalafil can lead to (1) increased lipid metabolism; (2) a switch towards slow oxidative fibers driven by the up-regulation of PGC-1; (3) an increased protein synthesis efficiency; (4) a better actin network organization at Z-disk. J. Cell. Physiol. 230: 224-232, 2016. (c) 2015 Wiley Periodicals, Inc.

Different mRNAs associated to eEF1BÎł. (DOC 42 kb)

A. Chromatin immunoprecipitation was performed in hSH-SY5Y cells using anti-eEF1Bγ rabbit polyclonal antibodies or no-Ab as a control. Immunoprecipitates from each sample were analyzed by PCR performed with primers specific for the human Che-1 promoter and for the human TP53 promoter. The thymidine kinase human promoter was amplified as a negative control. A sample representing linear amplification of the total input chromatin (input) was included in the PCR as a control. B. Co-localization of endogenous Che-1, performed with the anti-Che-1 rat polyclonal antibody (green), and the mitochondrial marker Tom20 (red), in hSH-SY5Y cells. Extensive co-localization (yellow) between Che-1 and Tom20 is visualized by the merged-color image. The boxed area represents a high magnification image of co-localization. Nuclei were labeled with DAPI (blue). Scale bars: 10 μm. C. Western blot analysis of hSH-SY5Y whole-cell lysate and mitochondrial enriched fraction. The quality of mitochondrial-enriched fractions was monitored using anti-HSP60 monoclonal antibodies and anti-Tom20 rabbit polyclonal antibodies. D. Quantitative real time PCR (qPCR) analysis of the eEF1Bγ and Che-1 mRNAs in hSH-SY5Y cells (siRNA-Control and siRNA-eEF1Bγ). The gene expression ratio between eEF1Bγ and GAPDH and between Che-1 and GAPDH are shown as the mean ± SD from three independent experiments performed in triplicate. E. Representative Western blot of hSH-SY5Y whole-cell lysates treated or un-treated with siRNA as shown. The antibodies that were used are indicated. (PDF 199 kb)

Oligos used in the present study. (DOC 56 kb)

A. RIP assay output was analyzed by semi-quantitative RT-PCR with specific primers to validate some of mRNAs co-immunoprecipitated with eEF1Bγ and they are listed in Table S2. B. The myc-eEF1Bγ and MS2-GFP fusion proteins were expressed in HeLa cells with the report mRNA carrying both the MS2 binding site and the indicated 3′ UTR. C. RIP assay-eEF1Bγ mRNAs (Additional file 2: Table S2) analyzed by quantitative real time PCR (qPCR) in HeLa whole-cell lysates treated with siRNA as shown. The gene expression ratio between mRNAs and GAPDH are shown as the mean ± SD from three independent experiments performed in triplicate. D. Representative western blot of HeLa whole-cell lysates treated or un-treated with siRNA as shown. The antibodies that were used are indicated. Densitometric analysis represents the mean ± S.D. of 3 independent experiments (right panel). (PDF 208 kb)

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, also considering the risk of immunogenic reaction against dystrophin raised in human patients following the introduction of dystrophin genes.Many approaches have been proposed to increase utrophin levels in muscle. In particular 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 (1,2). Then we realized a transgenic mice expressing Jazz under the control of a muscle specific promoter showing that Jazz is able to up-regulate endogenous utrophin gene expression in skeletal muscle (3,4). The transgenic mouse expressing Jazz was then crossbred to the mdx (murine dystrophy X-linked) model of dystrophy and several histological and physiological evidences demonstrated a reduction of muscle waste and an impressive recovery of muscle strength and resistance in mdx-Jazz mice as compared with dystrophic littermates (5). 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 advancement towards the ZFP TFs-based approach to the cure of DMD and other genetic diseases. The project now focuses on the delivery of ZFP TFs-Jazz-based artificial transcription factors in mdx mice by recombinant Adeno-associated-virus vectors (rAAV) with high affinity for skeletal muscle and engineered to express the therapeutic genes under the control of a muscle specific promoter. We developed an efficient method to produce and purify rAA viruses able to infect muscle cells both in vitro and in vivo. Preliminary experiments of systemic injection in neonatal mice with AAV6-EGFP reporter gene, driven by a muscle specific promoter, demonstrated good levels of infection and expression of EGFP restricted to skeletal muscle as well as following experiments of injection with AAV6-Jazz which, as expected, is expressed in muscle fibres and colocalizes with nuclei. REFERENCES 1. Onori A, Desantis A, Buontempo S, Di Certo MG, Fanciulli, Salvatori ML, Passananti C and Corbi N. The artificial four zinc finger protein Bagly binds human utrophin promoter A at the endogenous chromosomal site and activates transcription. Biochem Cell Biol. Jun;85(3), 2007 2. Agata Desantis, Annalisa Onori, Maria Grazia Di Certo, Elisabetta Mattei, Maurizio Fanciulli, Claudio Passananti and Nicoletta Corbi. Novel activation domain derived from Che-1 cofactor coupled with the artificial protein Jazz drives utrophin upregulation. Neuromuscular Desorders, 19(2):158-62, 2009. 3. Passananti C, Corbi N, Onori A, Di Certo MG, Mattei E. Transgenic mice expressing an artificial zinc finger regulator targeting an endogenous gene. Methods Mol Biol.;649:183-206. 2010 4. Mattei E, Corbi N, Di Certo MG, Strimpakos G, Severini C, Onori A, Desantis A, Libri V, Buontempo S, Floridi A, Fanciulli M, Baban D, Davies KE, Passananti C. Utrophin up-regulation by an artificial transcription factor in transgenic mice. PLoS ONE. Aug 22;2:e774, 2007 5. Maria Grazia Di Certo, Nicoletta Corbi, Georgios Strimpakos, Annalisa Onori, Siro Luvisetto, Cinzia Severini, Angelo Guglielmotti, Enrico Maria Matassa, Cinzia Pisani, Aristide Floridi, Barbara Benassi, Maurizio Fanciulli, Armando Magrelli, Elisabetta Mattei, Claudio PassanantiThe artificial gene Jazz, a transcriptional regulator of Utrophin, corrects the dystrophic pathology in mdx mice. Human Molecular Genetics; 1;19(5):752-60, 2010.

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.

Up-regulation of the dystrophin-related gene utrophin represents a promising therapeutic strategy for the treatment of Duchenne Muscular Dystrophy (DMD). In order to re-program the utrophin expression level in muscle, we engineered artificial zinc finger transcription factors (ZF-ATFs) that target the utrophin ʻAʼ promoter. We have previously shown that the ZF-ATF “Jazz”, either by transgenic manipulation or by systemic adeno-associated viral delivery, induces significant rescue of muscle function in dystrophic “mdx” mice. We present the full characterization of an upgraded version of Jazz gene named “JZif1” designed to minimize any possible host immune response. JZif1 was engineered on the Zif268 gene-backbone using selective amino acid substitutions to address JZif1 to the utrophin ‘A’ promoter. Here, we show that JZif1 induces remarkable amelioration of the pathological phenotype in mdx mice. To investigate the molecular mechanisms underlying Jazz and JZif1 induced muscle functional rescue, we focused on utrophin related pathways. Coherently with utrophin subcellular localization and role in neuromuscular junction (NMJ) plasticity, we found that our ZF-ATFs positively impact the NMJ. We report on ZF-ATF effects on post-synaptic membranes in myogenic cell line, as well as in wild type and mdx mice. These results candidate our ZF-ATFs as novel therapeutic molecules for DMD treatment., Highlights • Up-regulation of Dystrophin-related gene Utrophin is a promising therapeutic strategy for Duchenne Muscular Dystrophy (DMD). • Zinc Finger Artificial Transcription Factors (ZF-ATFs) have been designed to target and to activate Utrophin ‘A’ promoter. • ZF-ATFs ‘Jazz’ and ‘JZif1’ were delivered by AAV to muscle tissue in dystrophic mdx mouse. • Jazz and JZif1 improve neuromuscular junction (NMJ) morphology and correct dystrophic pathology in mdx mice.