CELL PENETRATING TRANSPOSASE

Abstract

The Sleeping Beauty (SB) transposon is an efficient non-viral tool for inserting transgenes into cells. Its broad utilization in gene therapy has been hampered by uncontrolled transposase gene activity and the inability to use transposase protein directly. The present invention concerns the finding that SB transposase spontaneously penetrates mammalian cells and can be delivered with transposon DNA to gene-modify various cell lines, embryonic, hematopoietic and induced pluripotent stem cells. The invention provides methods and compounds to apply the cell penetrating function of transposase in methods of genetically engineering cells as well as using the transposase as a shuttle for delivering cargo into a target cell or even into a target cell organelle. Genomic integration frequency can be titrated using the technology of the invention, which adds an additional layer of safety, opening opportunities for advanced applications in genetic engineering and gene therapy.

Claims

1. A method for genetically engineering a target biological cell, the method comprising in any sequence the steps of: (i) introducing a transposon construct into the biological cell and/or providing a biological cell comprising a transposon construct; and (ii) contacting the target biological cell with a transposase protein in absence of, or without using, a protein transfection procedure or protein transfection reagent.

2. The method according to claim 1 or 2, wherein the transposase protein is, or is derived from, a Sleeping Beauty (SB) transposase.

3. The method according to claim 2, wherein the SB transposase is a protein comprising a sequence having at least 80% sequence identity to a sequence shown in any of SEQ ID NO: 1 to 3.

4. The method according to any one of claims 1 to 3, wherein the transposase protein consists of, or consists essentially of, and amino acid sequence shown in any one of SEQ ID NO: 1 to 3, optionally with not more than 50 amino acid substitutions, additions, insertions, deletions or inversions, compared to these sequences.

5. The method according to any one of claims 1 to 4, wherein the transposase protein is provided by adding the transposase protein to a medium in which said biological cell is contained, preferably to a cell culture medium of the target biological cell.

6. The method according to any one of claims 1 to 5, wherein the target biological cell is a mammalian cell, preferably selected from a stem cell, such as a hematopoietic stem cell, embryonic stem cell, spontaneously immortalized cell, artificial immortalized cell, primary cell (neurons, resting T cells), a cell derived from a B-cell such as plasma cells, Chinese hamster ovary (CHO) cell, induced pluripotent stem cell (iPSC), or is an immune cell, such as a T lymphocyte, preferably a CD4 or CD8 positive T cell, or is a Natural Killer (NK) cell, a macrophage, a dendritic cell or a B-cell.

7. The method according to any one of claims 1 to 6, wherein the transposon comprises a protein encoding nucleotide sequence, such as a sequence encoding for an antibody, a T cell receptor, or a chimeric antigen receptor (CAR).

8. A method for the delivery of a cargo-compound into a biological cell, the method comprising, covalently or non-covalently and directly or indirectly, attaching a cargo-compound to a shuttle protein to obtain a cargo-shuttle complex, and contacting the biological cell with the cargo-shuttle complex; characterized in that the shuttle protein comprises a transposase protein sequence, preferably a transposase protein as defined in any of claims 1 to 7.

9. The method according to any claim 8, wherein the shuttle protein is covalently or non-covalently coupled to a linker compound, preferably wherein the linker compound is suitable for covalently or non-covalently coupling the cargo-compound to the shuttle protein.

10. The method according to claim 9, wherein the linker compound is selected from a sortase donor or acceptor site, a biotin or streptavidin protein, or a functionally alternative component of a protein coupling system.

11. The method according to any one of claims 8 to 10, wherein the cargo-compound is selected from a small molecule, a macro-molecule, a peptide, a polypeptide, a protein, a nucleic acid, such as an RNA, DNA, RNA-DNA hybrid, PNA, or is a sugar compound, a fatty acid containing compound.

12. A use of a transposase protein in the delivery of a cargo-compound into a biological cell, wherein the transposase protein is used as a cellular shuttle protein and is covalently or non-covalently and directly or indirectly attached to the cargo-compound.

13. A cellular-shuttle, comprising (i) a transposase protein covalently or non-covalently coupled to a cargo compound; or (ii) a transposase protein covalently or non-covalently coupled to a linker compound, and wherein the linker compound is suitable for the covalent or non-covalent coupling of the cellular-shuttle to a cargo compound; or (iii) a transposase protein covalently or non-covalently coupled to a linker compound, and wherein the linker compound is further covalently or non-covalently coupled to a cargo-compound.

14. A method for introducing a transposase protein into a biological cell, the method comprising contacting the cell with the transposase protein in absence of a protein transfection agent or without using a protein transfection procedure, such as electroporation.

15. The method according to claim 14, wherein the transposase protein is a transposase protein as defined in any one of the preceding claims.

Description

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCES

[0062] The figures show:

[0063] FIG. 1 shows a schematic representation of genome engineering by the SB transposase. LE and RE mark the left and right transposon end sequences, respectively. Cargo gene transfer in the target genome is executed by the transposase, expressed from a plasmid vector (bent arrow) in the target cells.

[0064] FIG. 2 shows that direct hsSB delivery allows for efficient transgenesis in diverse mammalian cells and stem cells. Representative flow cytometric analysis of HeLa cells (top panel), Chinese hamster ovary (CHO) cells (middle panel) and mouse embryonic stem cells (mESCs; bottom panel) transfected with Venus-carrying transposon plasmids and electroporated with hsSB transposase. Cells stably expressing an integrated Venus gene were identified 3 weeks post-transfection. The electroporated hsSB protein amounts are indicated above. Y-axis: propidium iodide (PI) staining to exclude dead cells; x-axis: green fluorescence from Venus; NT, non-transfected.

[0065] FIG. 3 shows transgenesis efficiency of the a system containing recombinantly expressed SB protein with any transgene vector (SBprotAct) in different cell lines, quantified by flow cytometry. Errors bars indicate the standard deviation (n=2).

[0066] FIG. 4 shows a schematic representation of the cell engineering procedure of the invention, using spontaneous hsSB penetration.

[0067] FIG. 5 shows immunofluorescence imaging of hsSB-treated (top) and non-treated (bottom) HeLa cells, showing DAPI-stained nuclei (left), hsSB staining (middle) and the merge (right). Arrows mark cells with hsSB in the nucleus.

[0068] FIG. 6 shows Western blot analysis showing cellular uptake and retention of hsSB in HeLa cells upon addition to the culture media. Samples were blotted with either anti-SB antibody or anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as internal loading control.

[0069] FIG. 7 shows a representative flow cytometric analysis of HeLa cells transfected with Venus-encoding transposon MC and incubated with hsSB in the culture media. Venus positive cells were sorted after 2 days and analyzed 3 weeks post-delivery. Y-axis: 4′,6-diamidino-2-phenylindole (DAPI) staining to exclude dead cells; x-axis: green fluorescence from Venus. hsSB protein concentration in the culture media are indicated above each plot. NT, non-transfected.

[0070] FIG. 8 shows a Western blot analysis of induced pluripotent stem cells (iPSCs) with anti-SB antibody, following hsSB penetration from the culture media.

[0071] FIG. 9 shows a representative flow cytometric analysis of iPSCs 3 weeks after transfection with Venus transposon MC and incubation with hsSB.

[0072] FIG. 10 shows a schematic representation of T cell engineering procedure, using spontaneous hsSB penetration.

[0073] FIG. 11 shows immunofluorescence imaging of T cells showing DAPI-stained nuclei (left), hsSB staining (middle) and the merge (right). Cells stained in absence of primary SB antibody are shown below (IF control).

[0074] FIG. 12 shows a representative flow cytometric analysis of CD8+ T cells transfected with transposon minicircles (MC) and incubated with hsSB. CD8+ T cells from healthy donors were transfected with CD19 CAR MC and enriched for CAR-positive cells (using EGFRt as marker) by magnetic associated cell sorting (MACS). Representative FACS plots from one of 3 experiments (from 3 different T cell donors) are shown with fluorescence from CD8 and EGFRt specific antibodies (CD8-VioBlue and EGFRt-AF647, respectively) plotted. hsSB protein concentration in the culture media are indicated above each plot. NT, non-transfected.

[0075] FIG. 13 shows the cytolytic activity of CD19 CAR T cells generated by hsSB penetration or MC-MC controls. Cytolysis was calculated from the luminescence signals of ffLuc-expressing target cells in a 5 h co-culture assay in the presence of excess luciferin. NT, non-transfected. E:T ratio, effector to target ratio.

[0076] FIG. 14 shows the average number of CAR transgene insertions as measured by digital droplet PCR (ddPCR) of CAR T cell genomic DNA. Error bars show the copy number estimates of two independent ddPCR assays (performed on same genomic DNA samples) at 95% confidence intervals.

[0077] FIG. 15: shows penetration of hsSB-GFP fusion protein. (A) fluorescence imaging of HeLa cells showing hsSB-GFP (left) and DAPI-stained nuclei (right) following 1 h incubation with the protein. Scale bar 20 m. (B) shows fluorescence imaging of HeLa cells showing hsSB-GFP (left) and DAPI-stained nuclei (right) 24 h later. Scale bar 20 m.

[0078] FIG. 16 shows penetration of an hsSB catalytically inactive mutant fused to the N-terminus of GFP. (A) fluorescence imaging of HeLa cells showing hsSB-D153N-D244N-GFP (left) and DAPI-stained nuclei (right) following 1 h incubation with the protein. Scale bar 20 m. (B) fluorescence imaging of HeLa cells showing hsSB-D153N-D244N-GFP (left) and DAPI-stained nuclei (right) 24 h later. Scale bar 20 m.

[0079] FIG. 17 shows penetration of GFP-hsSB fusion protein. (A) fluorescence imaging of HeLa cells showing GFP-hsSB (left) and DAPI-stained nuclei (right) following 1 h incubation with the protein. Scale bar 20 μm. (B) fluorescence imaging of HeLa cells showing GFP-hsSB (left) and DAPI-stained nuclei (right) 24 h later. Scale bar 20 m.

[0080] FIG. 18 shows that the N-terminal DNA-binding domain (DBD) of hsSB efficiently penetrates into HeLa cells. (A) immunofluorescence imaging of HeLa cells showing SB staining (left) and DAPI-stained nuclei (right) following 3 h incubation with the protein. Scale bar 20 μm. A schematic of the construct hsSB-1-123 is shown below (B) immunofluorescence imaging of HeLa cells showing SB staining (left) and DAPI-stained nuclei (right) 24 h later. Scale bar 20 m.

[0081] The sequences show:

TABLE-US-00001 SEQ ID NO: 1 shows the hsSB MGKSKEISQDLRKRIVDLHKSGSSLGAISKRLAVPRSSVQTIVRKYKHHG TTQPSYRSGRRRVLSPRDERTLVRKVQINPRTTAKDLVKMLEETGTKVSI STVKRVLYRHNLKGHSARKKPLLQNRHKKARLRFATAHGDKDRTFWRNVL WSDETKIELFGHNDHRYVWRKKGEASKPKNTIPTVKHGGGSIMLWGCFAA GGTGALHKIDGSMDAVQYVDILKQHLKTSVRKLKLGRKWVFQHDNDPKHT SKVVAKWLKDNKVKVLEWPSQSPDLNPIENLWAELKKRVRARRPTNLTQL HQLCQEEWAKIHPNYCGKLVEGYPKRLTQVKQFKGNATKY SEQ ID NO: 2 (non-mutated SB100X) MGKSKEISQDLRKRIVDLHKSGSSLGAISKRLAVPRSSVQTIVRKYKHHG TTQPSYRSGRRRVLSPRDERTLVRKVQINPRTTAKDLVKMLEETGTKVSI STVKRVLYRHNLKGHSARKKPLLQNRHKKARLRFATAHGDKDRTFWRNVL WSDETKIELFGHNDHRYVWRKKGEACKPKNTIPTVKHGGGSIMLWGCFAA GGTGALHKIDGIMDAVQYVDILKQHLKTSVRKLKLGRKWVFQHDNDPKHT SKVVAKWLKDNKVKVLEWPSQSPDLNPIENLWAELKKRVRARRPTNLTQL HQLCQEEWAKIHPNYCGKLVEGYPKRLTQVKQFKGNATKY SEQ ID NO: 3 (hsSB for recombinant expression) MGKSKEISQDLRKRIVDLHKSGSSLGAISKRLAVPRSSVQTIVRKYK HHGTTQPSYRSGRRRVLSPRDERTLVRKVQINPRTTAKDLVKMLEETGTK VSISTVKRVLYRHNLKGHSARKKPLLQNRHKKARLRFATAHGDKDRTFWR NVLWSDETKIELFGHNDHRYVWRKKGEASKPKNTIPTVKHGGGSIMLWGC FAAGGTGALHKIDGSMDAVQYVDILKQHLKTSVRKLKLGRKWVFQHDNDP KHTSKVVAKWLKDNKVKVLEWPSQSPDLNPIENLWAELKKRVRARRPTNL TQLHQLCQEEWAKIHPNYCGKLVEGYPKRLTQVKQFKGNATKY (underlined are mutated or to-be mutated residues. Bold and italic are residues introduced for recombinant protein expression)

EXAMPLES

[0082] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the description, figures and tables set out herein. Such examples of the methods, uses and other aspects of the present invention are representative only, and should not be taken to limit the scope of the present invention to only such representative examples.

[0083] The examples show:

Example 1 (Comparative): Efficient Transgenesis in Mammalian Cells Using hsSB Transposase

[0084] A high solubility Sleeping Beauty (hsSB) transposase developed by the inventors was tested in various mammalian cells lines for its ability of genetically engineering cells. The amino acid sequence of the improved hsSB transposase is shown in SEQ ID NO: 3. To better quantify hsSB-mediated transposition, the inventors applied a fluorescent reporter system and transfected HeLa cells with a transposon plasmid containing the Venus gene, followed by hsSB protein delivery by protein electroporation. Cells that acquired the transposon plasmid were selected by fluorescence activated cell sorting 2 days post-transfection. The transposition efficiency was then quantified three weeks later by flow cytometric analysis of green fluorescent cells that stably expressed the Venus reporter gene as a consequence of genomic insertion by hsSB (FIG. 2). A clear, dose-dependent increase in the percentage of fluorescent cells, with the maximum efficiency (42%) achieved with 20 μg of hsSB protein (FIG. 2, upper panel, and FIG. 3) was detected. Also Chinese hamster ovary (CHO) cells and mouse embryonic stem cells could be efficiently transfected with the hsSB transposase of the invention (FIGS. 2 and 3).

Example 2: Transposase has Intrinsic Cell Penetrating Properties

[0085] For further developing methods for the genetic engineering of mammalian cells the inventors sought to make transposase delivery simpler and gentler. Remarkably, the inventors observed that the transposase protein autonomously penetrates HeLa cells and enters the nucleus when simply added to the culture medium (FIGS. 4 and 5). To test if hsSB can mediate transposition when delivered this way, the inventors transfected HeLa cells with a MC containing the Venus gene and then added hsSB to the culture medium without a further pulse or use of a transfection reagent (FIG. 4). Longitudinal Western blot analysis showed hsSB uptake within 4 hours, followed by clearance already 24 hours after delivery (FIG. 6). Fluorescent cell sorting 3 weeks post transfection revealed up to 12% Venus-positive cells (FIG. 7), demonstrating that hsSB mediated efficient transgene integration.

[0086] Next, a similar procedure for genetic engineering of human iPSCs was tested. iPSCs offer great potential for regenerative medicine but are among the most difficult cells to engineer due to their sensitivity to transfection procedures. The inventors first transfected the iPSCs with a Venus-carrying MC using a stem cell specific transfection reagent and then incubated them with hsSB protein-containing medium to allow protein penetration in the cells. hsSB efficiently penetrated iPSCs (FIG. 8) and flow cytometry of the treated cells after three weeks revealed remarkable transgenesis efficiencies of up to 3.31% (calculated as the percentage of stable integrants at 3 weeks over all transfected cells, FIG. 9). This shows that hsSB's non-invasive cell penetration helps to modify iPSCs.

Example 3: Novel Genetic Engineering Method can be Used to Generate CAR-T Cells

[0087] Finally, it was tested whether the intrinsic cell penetration property of hsSB can be exploited for CAR T cell manufacturing (FIG. 10). As electroporation is a stress factor for T cells, hsSB penetration could help preserve their fitness for downstream clinical use. The inventors first analyzed hsSB penetration in primary T cells by immunofluorescence imaging, which showed efficient protein uptake in both stimulated and non-stimulated cells within 3 hours (FIG. 11). hsSB efficiently entered the nucleus also in non-dividing cells, consistent with active transport using its intrinsic nuclear localization signal. To probe transposition, T cells were electroporated with CD19 CAR MC and hsSB was added to the cell culture media. This successfully generated human CD8+ CD19 CAR T cells at an overall transgenesis frequency of 5-7% (FIG. 12). CAR T cells were then enriched up to 90% purity by MACS (44) and showed potent lysis of CD19+ target cells, as well as high levels of effector cytokine secretion (FIGS. 12, and 13). Cells produced with this procedure showed an average number of four insertions, which is lower compared to the CAR MC-SB MC DNA based protocol (6-8 insertions; FIG. 14).

Example 4: Using the Self-Penetrating Transposase Protein as a Cargo Shuttle into Cells

[0088] HeLa cells were seeded onto a Nunc™ Lab-Tek™ II 8-well Chamber Slides™ (Thermo Fisher) (2×104 cells per well in 500 μL DMEM supplemented with 10% (v/v) human serum and 2 mM L-glutamine). On the next day, cells were incubated with hsSB-GFP at a concentration of 0.5 μM in a volume of 250 μL/well serum-free DMEM for 1 hour. Then, media was removed and cells were fixed with PFA 4% in PBS and incubated 30 min with DAPI to visualize the nuclei. Cells were imaged with a Zeiss LSM 780 confocal microscope (using a 63× oil submersion objective) in the ALMF core facility at EMBL Heidelberg. For imaging, the middle part of the nucleus was placed in focus to detect nuclear localization of hsSB.

[0089] FIG. 15 shows that the hsSB-GFP fusion protein (hsSB fused to the N-terminus of GFP) enters the cells' nuclei within 1 h (A) and is retained at least for the following 24 h (B) as observed by GFP fluorescence imaging. FIGS. 16 A and B show the same effect for a catalytically inactive mutant version of hsSB in HeLa cells. Further, fusing hsSB to the C-terminus of the GFP equally promotes penetration into HeLa cells (FIG. 17).

[0090] In another experiment a truncated version of the hsSB, namely a version consisting of the DNA binding domain of the protein (bottom of FIG. 18A) is probed in HeLa cells. Results show that the hsSB's DNA binding domain is sufficient for autonomous cell penetration from the culture media. hsSB DBD is detected in the cells with immunofluorescence imaging using an SB-specific antibody. The protein (peptide) enters the cells within 3 h (FIG. 18A) and is retained at least for the following 24 h (FIG. 18B).

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