The main limitation of non-viral episomal vectors, like pEPI, for their use in gene therapy is their low establishment efficiency in transfected cells. Within this project we focus on increasing establishment efficiency by inserting cis acting elements. For that purpose UCOE and 5'HS4 insulator sequence have been inserted so far with promising results. Furthermore we will determine the relevance of an ongoing transcription for the replication and long term maintenance of pEPI using two approaches: (1) We constructed an inducible plasmid (pEPI-TetON) in which expression of the transgene can be repressed and thus the consequences of repressed transcription on vector maintenance can be investigated. (2) We attempt to construct a self destructing vector basing on transgene silencing by an inserted, inducible shRNA cassette according to the "nascent transcript model". To prove the practical use of pEPI we will insert the transcription factors (under the control of a single promoter) needed for reprogramming adult mouse fibroblasts and analyze the efficiency of non viral reprogramming. Moreover, we try to repopulate the haematopoietic system by transfecting CD34+ cells with pEPI derivates. If successful, therapeutic sequences may be inserted and tested in an animal model system.

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