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A highly soluble Sleeping Beauty transposase improves control of gene insertion

Nature Biotechnology volume 37, pages 1502–1512 (2019)Cite this article

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Abstract

The Sleeping Beauty (SB) transposon system is an efficient non-viral gene transfer tool in mammalian cells, but its broad use has been hampered by uncontrolled transposase gene activity from DNA vectors, posing a risk of genome instability, and by the inability to use the transposase protein directly. In this study, we used rational protein design based on the crystal structure of the hyperactive SB100X variant to create an SB transposase (high-solubility SB, hsSB) with enhanced solubility and stability. We demonstrate that hsSB can be delivered with transposon DNA to genetically modify cell lines and embryonic, hematopoietic and induced pluripotent stem cells (iPSCs), overcoming uncontrolled transposase activity. We used hsSB to generate chimeric antigen receptor (CAR) T cells, which exhibit potent antitumor activity in vitro and in xenograft mice. We found that hsSB spontaneously penetrates cells, enabling modification of iPSCs and generation of CAR T cells without the use of transfection reagents. Titration of hsSB to modulate genomic integration frequency achieved as few as two integrations per genome.

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Fig. 1: Design and characterization of the high-solubility SB protein variant.
Fig. 2: Efficient cell engineering by direct hsSB delivery.
Fig. 3: Direct hsSB delivery allows for efficient transgenesis in diverse mammalian cells and stem cells.
Fig. 4: CD19 CAR T cell generation by SBprotAct and functional analysis of the cell product.
Fig. 5: hsSB transposase has intrinsic cell-penetrating properties, allowing engineering of HeLa cells and induced pluripotent stem cells.
Fig. 6: Generation of CAR T cells by spontaneous hsSB penetration.

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Data generated or analyzed during this study and all unique materials can be made available by the corresponding authors upon reasonable request.

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Acknowledgements

The authors thank F. Dyda, K.R. Patil, M. Beck and members of the Barabas lab for helpful discussions and the Nature Editing Service for editing assistance. We also thank the Flow Cytometry Core Facility, the Genomics Core Facility, the Advanced Light Microscopy Facility and the Protein Expression and Purification Core Facility at EMBL Heidelberg for materials and support. We thank Z. Izsvak (Max Delbrück Center) for providing the CMV(CAT)T7-SB100X plasmid and F. Spitz (EMBL Heidelberg) for the pT2/PGK-neo construct; A. Aulehla for providing access to the Neon transfection system and reagents; S. Henkel for providing reagents and assistance for mESC culture and immunostaining; H. Bönig (Blutspendedienst des Deutschen Roten Kreuz) for providing human HSPCs; K.-M. Noh and N. Diaz for providing materials and advice with iPSC culturing; V. Rybin for assistance and advice with the CD spectroscopy experiments; and V. Benes for assistance and support with sequence analysis. This work was supported by the EMBL, the Paul Ehrlich Institute, the EMBL International PhD Programme (fellowship to I.Q.), the Deutsche Forschungsgemeinschaft (project number 324392634, TRR 221 to M.H. and H.E.) and German Cancer Aid (Deutsche Krebshilfe, Max Eder Program Award 70110313 to M.H.). M.H. was supported by the Young Scholar Program of the Bavarian Academy of Sciences (Junges Kolleg, Bayerische Akademie der Wissenschaften) and the m4 Award in Personalized Medicine (Free State of Bavaria, BIO-1601-0002). Z.I. was supported by the Center for Cell and Gene Therapy of the LOEWE (Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz) program in Hessen, Germany, and by a grant from the Deutsche Forschungsgemeinschaft (IV 21/11-1).

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Author notes

  1. These authors contributed equally: Irma Querques, Andreas Mades, Cecilia Zuliani.

  2. These authors jointly supervised this work: Michael Hudecek, Orsolya Barabas.

Authors and Affiliations

  1. Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany

    Irma Querques, Cecilia Zuliani & Orsolya Barabas

  2. Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany

    Andreas Mades, Miriam Alb, Markus Machwirth, Hermann Einsele & Michael Hudecek

  3. Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany

    Csaba Miskey, Esther Grueso & Zoltán Ivics

  4. Genomics Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany

    Tobias Rausch

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  1. Irma Querques
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Contributions

I.Q., A.M., C.Z., M.H. and O.B. designed the research. All authors contributed to analysis and discussion of the results and read and approved the manuscript. I.Q., A.M., C.Z., M.H. and O.B. wrote the manuscript with input from all authors. I.Q. designed, screened and characterized transposase variants. A.M., M.A. and M.M. performed T cell engineering and characterization. C.Z. conducted transposition assays and engineering of HeLa, CHO, mESC and iPSC lines. C.M. performed integration site profiling in T cells and copy number analyses. E.G. performed HSPC engineering. T.R. analyzed integration sites in HeLa cells. H.E. provided expert advice and support during the project. Z.I. supervised integration profiling, copy number analyses and HSPC engineering and participated in coordinating the research. M.H. supervised T cell engineering and analyses and oversaw the project. O.B. supervised protein work and cell line engineering and oversaw the project.

Corresponding authors

Correspondence to Michael Hudecek or Orsolya Barabas.

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Competing interests

Z.I. is an inventor on patents concerning the development and use of the Sleeping Beauty technology (proprietor Max-Delbrück-Centrum für Molekulare Medizin; patents US9228180B2 and EP2160461B1), and two patent applications have been filed with the European Patent Office concerning the hsSB transposase (EP17187128 and EP19158066.1).

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Integrated supplementary information

Supplementary Figure 1 Biochemical characterization of the hsSB transposase.

a, SDS-PAGE analysis of samples collected during recombinant production and purification of the hsSB transposase variant. hsSB was produced in E. coli (fused to N-terminal purification and solubility tags) at high yields and was highly pure (>95%) after tag removal and size exclusion chromatography. b, SDS-PAGE analysis of purified SB proteins after concentration (conc.). hsSB can be concentrated up to 50 fold (corresponding to 20 mg per ml), whereas SB100X undergoes precipitation at concentrations higher than 7 mg per ml. The vast majority of hsSB remains in the soluble fraction in the low-salt R buffer used for electroporation, even at high protein concentration. c, CD spectra of the SB100X (blue curve) and hsSB (red curve) proteins. a–c, Experiments were repeated independently two b and three a,c times with similar results.

Supplementary Figure 2 Transposition assays in HeLa cells.

a, Representative transposition assay. Transgene (neor) insertions generated G418-resistant colonies after hsSB transposase transfection. Experiments were repeated independently three times with similar results. b, Number of resistant colonies with various amounts of hsSB provided on a plasmid vector. Mean values (n = 2 independent experiments).

Supplementary Figure 3

Retention time of hsSB delivered into HeLa cells as a protein or expressed from plasmid DNA. Western blot analysis was performed on lysates from HeLa cells transfected with 0.5 µg of transposon DNA (pT2/PGK-neo) and electroporated with 10 μg of the hsSB protein or transfected with 500 ng of the hsSB expression plasmid. Samples were collected at the indicated time points, and 20 µg of the total cell lysate was separated by electrophoresis and transferred to a nitrocellulose membrane. The SB transposase was detected with an anti-SB antibody. The internal loading control was GAPDH, detected with an anti-GAPDH antibody. a, Full scan of the blots shown in Fig. 2d. b, Measurement of the intensities of the bands allowed for the quantification of hsSB persistence in HeLa cells over time. Experiments were repeated independently two times with similar results.

Supplementary Figure 4 Transposition assays in mESCs.

a, Representative transposition assay. Transgene (neor) insertions generated G418-resistant colonies after transfection of increasing amounts of hsSB transposase. Transposition assay cultures were diluted 1:10 for plating. Colony counts were corrected for the dilution and plotted. b, Flow cytometry plots showing the percentage of cells expressing the Oct4 pluripotency marker versus the Venus-positive cells. a,b, Experiments were performed once.

Supplementary Figure 5 Characterization of CAR T cell products.

a, Cytokine secretion of CAR T cells produced by transfection with CD19 CAR MC and hsSB protein (MC-hsSB) or CD19 CAR MC and SB100X MC (MC-MC). The concentrations of IL-2 and IFN-γ were measured by ELISA in cell culture media after a 24-h co-culture with target cells (E:T = 4:1). b, CAR T cells were labelled with carboxyfluorescein succinimidyl ester and co-cultured with irradiated target cells (E:T = 4:1). Proliferation was measured by the CFSE dilution of CAR T cells after 72 h (NT = non-transfected). a,b, Data were obtained from n = 3 independent experiments with n = 3 different T cell donors. Mean values; error bars represent s.d. c, Consensus sequence of SB insertion sites in T cells taken from the batch used for injection into the Raji xenograft mouse model. d, Heatmap demonstrating the insertion frequencies into genes of various expression levels (expr.lev).

Supplementary Figure 6 Supplementary data from the Raji xenograft mouse model.

a, Ventral and dorsal bioluminescence images of mice treated with CD19 CAR T cells or non-transfected (NT) control T cells (as in Fig. 4) taken at the indicated time points. b, Group analysis of tumor growth. Mean values; error bars represent s.d. Group sizes are identical to the images displayed in a: n = 2, 3, 5 and 5 animals for untreated, NT, MC-MC and MC-hsSB samples, respectively. c, Kaplan–Meier survival curves.

Supplementary Figure 7 Full scans of the Western blots analyzing hsSB uptake.

a, Blots showing cellular uptake and retention of hsSB in HeLa cells upon addition to the culture medium shown in Fig. 5c. The SB transposase was detected with anti-SB antibody. The internal loading control was GAPDH, blotted with an anti-GAPDH antibody. b, Blots for hsSB penetration from the culture media in iPSCs, blotted with anti-SB or anti-lamin B1/B2 (loading control) antibody as shown in Fig. 5e.

Supplementary Figure 8 Characterization of T cells after spontaneous hsSB penetration.

a, hsSB penetration in stimulated and non-stimulated CD4+ T cells. Immunofluorescence imaging of T cells showing DAPI-stained nuclei (blue), hsSB staining (green) and the merge. Cells stained in the absence of primary SB antibody are shown below (IF control). Experiments were repeated two times using cells from different donors with similar results. b, Functional analysis of CD19 CAR T cells generated with spontaneous hsSB penetration (MC-hsSB) or by transfection of SB100X MC (MC-MC). Cytokine secretion of CAR T cells. CD8+ T cells from healthy donors were transfected with CD19 CAR MC, incubated with hsSB, enriched for CAR-modified cells (EGFRt positive) and expanded with irradiated CD19+ B-LCL cells before functional assays. Results from one experiment with one T cell donor are shown. The concentrations of IL-2 and IFN-γ were measured by ELISA in cell culture media after a 24-h co-culture with target cells (E:T = 4:1). Mean values; error bars represent s.d. (n = 3 independent measurements with 1 T cell donor). NT = non-transfected.

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Querques, I., Mades, A., Zuliani, C. et al. A highly soluble Sleeping Beauty transposase improves control of gene insertion. Nat Biotechnol 37, 1502–1512 (2019). https://doi.org/10.1038/s41587-019-0291-z

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