Generation of improved viral hybrid-vectors for stable transduction of mammalian cells
Final Report Abstract
For a safe and successful gene therapy approach the therapeutic payload needs to be efficiently transduced and maintained in the target cell without side effects. Towards this end vector systems for somatic integration and non-viral genetic elements for episomal maintenance of the transgene were explored. However, a large number of vectors for somatic integration are associated with genotoxicity due to the risk of insertional mutagenesis and non-viral vector systems are often limited by their low transduction efficiencies. We aimed at developing novel viral hybrid-vectors which combine the best features of two different vector systems in one vector. These hybrid-vectors utilize the high transduction efficiencies of adenovirus and adeno-associated virus (AAV) in concert with improved genetic elements for stabilized transgene expression. In the context if the project we successfully generated three novel hybrid-vectors with improved features for stable transduction of mammalian cells. The first vector system combines the high-capacity adenovirus technology for efficient and safe delivery of the transgene with the hyperactive Sleeping Beauty (SB) transposase system for somatic integration. Efficacy of this adenovirus-SB hybrid-vector system was demonstrated in mice and a canine model for hemophilia B and the safety profile was evaluated including a detailed integration side analysis. The second vector system combines the high-capacity adenoviral vector technology with the plasmid replicon pEPito. After release of the plasmid replicon from the adenoviral vector genome in transduced cells, we could demonstrate that this system results in stabilized transgene expression derived from the episomally maintained plasmid replicon. The third hybrid-vector combines AAV vectors with the hyperactive SB transposase SB100X. We could show that this hybrid-vector results in significantly increased integration efficiencies compared to conventionally used AAV vectors. In summary, we established three novel hybrid-vectors which combine the best features of two different vector systems. Each of them displays special advantages and could be used in customized gene therapeutic approaches in the future.
Publications
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(2010). A hyperactive transposase system enables persistent phenotypic correction in large animals. Molecular Therapy, 8:1896-906
Martin Häusl, Wenli Zhang, Nadine Müther, Christina Rauschhuber, H. W. Franck, Timothy C. Nichols, Mark A. Kay and Anja Ehrhardt
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(2010). Critical amino acid residues within the PhiC31 integrase DNA binding domain affect recombination activities in mammalian cells. Human Gene Therapy, 21:1104-18
Raphael Liesner, Wenli Zhang, Nadja Noske, and Anja Ehrhardt
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(2011). A one-for-all real-time PCR for the detection and quantification of adeno-associated virus serotype 2 derived inverted terminal repeat sequences. Human Gene Therapy, 23:18-28
Christine Aurnhammer, Maren Haase, Nadine Muether, Christina Rauschhuber, Martin Häusl, I. Huber, Hans Nitschko, Ulrich Busch, A. Sing, Anja Ehrhardt and Baiker
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(2011). Development of adenovirus hybrid vectors for Sleeping Beauty transposition in large mammals. Current Gene Therapy, 11: 363-374
Martin Hausl, Wenli Zhang, Nadine Müther, Richard Voigtländer, Christina Rauschhuber, and Anja Ehrhardt
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(2012). A protocol for the determination of establishment efficiency of S/MAR containing non-viral episomal expression vectors. Cold Spring Harbor Protocols
Claudia Hagedorn, Armin Baiker, Jan Postberg, Anja Ehrhardt, Hans- Joachim Lipps
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(2012). Rescue of S/MAR containing non-viral episomal expression vectors. Cold Spring Harbor Protocols
Claudia Hagedorn, Armin Baiker, Jan Postberg, Anja Ehrhardt, Hans- Joachim Lipps
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(2012). Topic introduction: Handling S/MAR vectors. Cold Spring Harbor Protocols
Claudia Hagedorn, Armin Baiker, Jan Postberg, Anja Ehrhardt, Hans- Joachim Lipps
