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

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.