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Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates

Abstract

The Sleeping Beauty (SB) transposon is a promising technology platform for gene transfer in vertebrates; however, its efficiency of gene insertion can be a bottleneck in primary cell types. A large-scale genetic screen in mammalian cells yielded a hyperactive transposase (SB100X) with 100-fold enhancement in efficiency when compared to the first-generation transposase. SB100X supported 35–50% stable gene transfer in human CD34+ cells enriched in hematopoietic stem or progenitor cells. Transplantation of gene-marked CD34+ cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution. In addition, SB100X supported sustained (>1 year) expression of physiological levels of factor IX upon transposition in the mouse liver in vivo. Finally, SB100X reproducibly resulted in 45% stable transgenesis frequencies by pronuclear microinjection into mouse zygotes. The newly developed transposase yields unprecedented stable gene transfer efficiencies following nonviral gene delivery that compare favorably to stable transduction efficiencies with integrating viral vectors and is expected to facilitate widespread applications in functional genomics and gene therapy.

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Figure 1: The Sleeping Beauty (SB) transposon system.
Figure 2: Selecting phylogenetic variations in the SB transposase.
Figure 3: The high-throughput transposase screen.
Figure 4: Characterization of the SB100X transposase in transfected human HeLa cells.
Figure 5: Production of transgenic mice using SB100X.
Figure 6: Stable transposition into human CD34+ cells.
Figure 7: Long-term engraftment and lymphohematopoietic reconstitution with transposon-containing CD34+ cells.
Figure 8: In vivo hepatic gene delivery using hyperactive SB.

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Acknowledgements

We thank C. Judis, V. Gillijns and M. Shrahna for technical assistance. Z. Izsvák is an EURYI Awardee. We acknowledge the financial support of EU FP5 (JUMPY), EU FP6 (INTHER) and EU FP7 (PERSIST), grants from the Volkswagen Stiftung and from the Bundesministerium für Bildung und Forschung (NGFN-2), VIB GOA/2004/09 and FWO (G.0632.07). We thank E. Zeira, E. Galun (Goldyne Savad Institute of Gene Therapy) and M. Rhee (Chunguam University of Korea) for providing the pT2/CAGGS-GFP and the pcGlobin2 constructs, respectively. We further thank M.J. Fraser (University of Notre Dame) and A. Bradley (Sanger Centre) for providing the pXL-BacII and codon-optimized piggyBac transposase constructs, respectively, and A. Leutz and N. Rajewsky for critical reading of the manuscript. A. Schmitt is affiliated with Medical Faculty of Charité, Berlin.

Author information

Authors and Affiliations

Authors

Contributions

L.M. designed the hyperactive screen and characterization of the hyperactive transposases, generated some of the hyperactive mutations, generated and screened the hyperactive library, and characterized the hyperactives in human HeLa cells and in transgenic mice, analyzed data and wrote the paper. M.K.L.C. designed, managed and performed the HSC and liver gene transfer experiments, analyzed data, wrote the paper and supervised the project. E.B. performed the transfections on the HSC and liver, did the in vivo transplantations, ELISA and analyzed data. B.J. contributed with animal work (microinjections and in vitro embryo culture). N.M. generated some of the hyperactive mutations. A.A.-S. generated the constructs and conducted the qPCR, excision assays and integration site analysis and analyzed data. D.P.G. analyzed the subcelluler localization of SB and SB100X transposases in response to heat shock. A.S. compared SB and SB100X for protein stability. K.B. contributed with mouse microinjections. J.M. did confocal microscopical analysis on HSC, bioinformatics, ELISA and analyzed data. L.M. optimized HSC transfections and did FACS. E.S.-K. did in vivo transfections and stem cell culture experiments. C.G. did HSC transplantation experiments in vivo. D.P. compared SB and SB100X for DNA binding. C.M. isolated common insertion sites from hematopoietic lineages by LAM-PCR. B.F. generated some of the hyperactive mutations. T.V. designed and managed the experiments on the HSC and liver gene transfer, analyzed data, supervised the project and wrote the paper. Z. Ivics designed the hyperactive screen and characterization of the hyperactive transposases, analyzed and interpreted data, supervised the project and wrote the paper. Z. Izsvák designed the hyperactive screen and characterization of the hyperactive transposases, analyzed and interpreted data, supervised the project and wrote the paper.

Corresponding authors

Correspondence to Thierry VandenDriessche or Zsuzsanna Izsvák.

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Mátés, L., Chuah, M., Belay, E. et al. Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet 41, 753–761 (2009). https://doi.org/10.1038/ng.343

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