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Area 3: Molecular genetics of eukaryotes The influence of epigenetics, DNA repair and cell cycle pathways on the gene therapy approach of Small Fragment Homologous Replacement (SFHR). Marco Lucarelli Associate professor Department of Cellular Biotechnologies and Hematology Tel. / Fax 06‐4451784, e‐mail [email protected]
Abstract The aim of this proposal is to clarify the molecular mechanisms of recognition of cell invasion by exogenous DNA, possibly underlying a gene targeting approach called Small Fragment Homologous Replacement (SFHR). This approach is able to stably modify a genomic sequence by homologous replacement of a small DNA fragment, by a still poorly understood molecular mechanism. The modification is inheritable, physiologically expressed and long‐term maintained. SFHR potential is currently limited by a low and variable frequency of correction. This project will focus on the relationship between SFHR and chromatin structure (modifying during cell cycle), DNA methylation (a main effector of both chromatin remodelling and host defence), DNA repair and cell cycle pathways. To clarify the underlying mechanisms, the reciprocal influence of these 4 main biochemical pathways and the SFHR will be studied, in a reporter cellular system of mouse embryonic fibroblasts, in a differentiated human cellular model of Cystic Fibrosis and in a Spinal Muscular Atrophy mouse embryonic stem cell model. Drugs acting on specific target mechanisms and single‐gene targeting will be also used to dissect the pathways and to manipulate SFHR efficiency. The results will clarify the molecular mechanisms of cell invasion by exogenous DNA and its genomic integration, as well those of SFHR. The selection of strategies to increase SFHR efficiency will open up new perspective for SFHR therapeutic applications for in vivo and ex vivo treatments of inherited diseases, using differentiated and stem cells as targets. Group components COLLABORATORS (all of the Department of Cellular Biotechnologies and Hematology at Sapienza University of Rome) Dr. Giampiero Ferraguti, Researcher of Clinical Biochemistry and Clinical Molecular Biology Dr. Fabrizio Ceci, Researcher of Clinical Biochemistry and Clinical Molecular Biology Dr. Silvia Pierandrei, Doctorate student of “Scienze Pasteuriane” Dr. Sabina Maria Bruno, Postgraduate student of Clinical Pathology 1
Jonathan Di Fazio, Student of Molecular and Cellular Medical Biotechnologies Giulia Licciardello, Student of Biology Applied to Biomedical Research Gianluca Profeta, Student of Neurobiology Prof. Roberto Strom, Full Professor of Clinical Biochemistry and Clinical Molecular Biology In collaboration with Prof. Federica Sangiuolo, Associate Professor of Medical Genetics, Dip. Bio‐
Medicina e Prevenzione, Università di Roma “Tor Vergata”. Relevant publications 2008 – 2012 • Schippa S., Iebba V., Santangelo F., De Biase R.V., Stamato A., Bertasi S., Rolla M., Lucarelli M., Conte M.P., Quattrucci S. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene shapes fecal microbiota in Cystic Fibrosis patients. PLoS One (2012) Submitted. IF2011 = 4.092 • Pascale E., Lucarelli M., Passarelli F., Butler R.H., Tamellini A., Addessi E., Visalberghi E., Manciocco A., Vitale A., Laviola G. Monomorphic region of the serotonin transporter promoter gene in new world monkeys. American Journal of Primatology (2012) 74:1028‐1034. IF2011 = 2.221 • Luchetti A., Filareto A., Sanchez M., Ferraguti G., Lucarelli M., Novelli G., Sangiuolo F., Malgieri A. Small Fragment Homologous Replacement: evaluation of factors influencing modification efficiency in an eukaryotic assay system. PLoS One (2012) 7(2):e30851. IF2011 = 4.092 • Lucarelli M., Pierandrei S., Bruno S.M., Strom R. The genetics of CFTR: genotype – phenotype relationship, diagnostic challenge and therapeutic implications. In “Cystic Fibrosis – Renewed hopes through research”, Intech open access publisher (2012) Chap. 5, pp. 91 – 122. • Ferraguti G., Pierandrei S., Bruno S.M., Ceci F., Strom R., Lucarelli M. A template for mutational data analysis of the CFTR gene. Clinical Chemistry and Laboratory Medicine (2011) 49(9):1447‐1451. IF2011 = 2.150 • Fuso A., Ferraguti G., Grandoni F., Ruggeri R., Scarpa S., Strom R., Lucarelli M. Early demethylation of non‐CpG, CpC‐rich, elements in the myogenin 5'‐flanking region: a priming effect on the spreading of active demethylation? Cell Cycle (2010) 9(19):3965‐3976. IF2010 = 4.999 • Lucarelli M., Narzi L., Pierandrei S., Bruno S.M., Stamato A., d’Avanzo M., Strom R., Quattrucci S. A new complex allele of the CFTR gene partially explains the variable phenotype of the L997F mutation. Genetics in Medicine (2010) 12(9):548‐555. IF2010 = 5.280 • Auriche C., Di Domenico E.G., Pierandrei S., Lucarelli M., Castellani S., Conese M., Melani R., Zegarra‐Moran O., Ascenzioni F. CFTR expression and activity from the human CFTR locus in BAC vectors, with regulatory regions, isolated by a single step procedure. Gene Therapy (2010) 17(11):1341‐1354. IF2010 = 4.538 • Elia J., Delfino M., Imbrogno N., Capogreco F., Lucarelli M., Rossi T., Mazzilli F. Human semen hyperviscosity: prevalence, pathogenesis and therapeutic aspects. Asian Journal of Andrology (2009) 11(5):609‐615. IF2009 = 1.688 2
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