Improvement and application of a non-viral episome for mammalian cells

• Antragsteller: Professor Dr. Hans Joachim Lipps
• Fachliche Zuordnung: Hämatologie, Onkologie
• Förderung: Förderung von 2006 bis 2014
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 22053309

This project will exploit a non-viral episomal vector system recently constructed in the Institute of Cell Biology, University Witten/Herdecke to analyze the epigenetic features required for selection of a mammalian origin of replication, to stably establish an episome and to provide mitotic stability in the absence of a centromere sequence. The vector used replicates in all mammalian cell lines tested so far, including primary cells and was used to generate genetically modified animals. Its minimal functional elements have been described and therefore it can be manipulated with ease. Selection of an origin and establishment as a stable episome require probably similar or the same mechanisms. The correct chromatin structure has to be adopted and the episome, respectively origin has to enter a specific nuclear region. In the frame of this project the relevance of chromatin structure, i.e. histone code and DNase I hypersensitive sites, and of nuclear localization will be analyzed. By the incorporation of c/.v-acting sequences targeting the vector to specific compartments of the nucleus combined with transfection of the vector as chromatin will increase establishment as an episome. Mitotic stability of the vectors in absence of a centromere sequence seems also to be closely related to the problem of stable establishment. Therefore, the behaviour of the prototype vector as well as its derivatives will be studied throughout the cell cycle. Results obtained within this project will lead to a deeper understanding of the epigenetic control of DNA replication but will also provide the basis for a rational design of vectors which may eventually be used in gene therapy.

• DFG-Verfahren: Schwerpunktprogramme
• Teilprojekt zu: SPP 1230: Mechanisms of gene vector entry and persistence