title: Avoiding cytotoxicity of transposases by dose-controlled mRNA delivery.
abstract: The Sleeping Beauty (SB) transposase and its newly developed hyperactive variant, SB100X, are of increasing interest for genome modification in experimental models and gene therapy. The potential cytotoxicity of transposases requires careful assessment, considering that residual integration events of transposase expression vectors delivered by physicochemical transfection or episomal retroviral vectors may lead to permanent transposase expression and resulting uncontrollable transposition. Comparing retrovirus-based approaches for delivery of mRNA, episomal DNA or integrating DNA, we found that conventional SB transposase, SB100X and a newly developed codon-optimized SB100Xo may trigger premitotic arrest and apoptosis. Cell stress induced by continued SB overexpression was self-limiting due to the induction of cell death, which occurred even in the absence of a co-transfected transposable element. The cytotoxic effects of SB transposase were strictly dose dependent and heralded by induction of p53 and c-Jun. Inactivating mutations in SB's catalytic domain could not abrogate cytotoxicity, suggesting a mechanism independent of DNA cleavage activity. An improved approach of retrovirus particle-mediated mRNA transfer allowed transient and dose-controlled expression of SB100X, supported efficient transposition and prevented cytotoxicity. Transposase-mediated gene transfer can thus be tuned to maintain high efficiency in the absence of overt cell damage.
authors: Department of Experimental Hematology, Institute of Transplantation Immunology, Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany.
authors: Melanie
authors: M
authors: Galla
authors: Axel
authors: A
authors: Schambach
authors: Christine S
authors: CS
authors: Falk
authors: Tobias
authors: T
authors: Maetzig
authors: Johannes
authors: J
authors: Kuehle
authors: Kathrin
authors: K
authors: Lange
authors: Daniela
authors: D
authors: Zychlinski
authors: Niels
authors: N
authors: Heinz
authors: Martijn H
authors: MH
authors: Brugman
authors: Gudrun
authors: G
authors: Göhring
authors: Zsuzsanna
authors: Z
authors: Izsvák
authors: Zoltán
authors: Z
authors: Ivics
authors: Christopher
authors: C
authors: Baum
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