Cell-permeable transposase

Abstract

The Sleeping Beauty (SB) transposon is an efficient, non-viral tool for inserting transgenes into cells. Its widespread use in gene therapy has been hindered by the lack of control over transposase gene activity and the inability to directly utilize the transposase protein. The present invention relates to the discovery that SB transposase spontaneously penetrates mammalian cells and can be delivered along with transposon DNA to genetically modify various cell lineages, embryonic stem cells, hematopoietic stem cells, and induced pluripotent stem cells. The present invention provides methods and compounds that apply the cell-penetrating function of transposase in methods for genetically manipulating cells, as well as methods and compounds that use transposase as a shuttle to deliver cargo into target cells or even target organelles. Using the technology of the present invention, genomic integration frequency can be tuned, adding an additional layer of safety and opening up opportunities for advanced applications in genetic engineering and gene therapy.

Description

[Technical field]

[0001] The Sleeping Beauty (SB) transposon is an efficient non-viral tool for inserting transgenes into cells. Its widespread use in gene therapy has been hindered by the lack of control of transposase gene activity and the inability to use transposase protein directly. The present invention relates to the discovery that SB transposase spontaneously penetrates mammalian cells and can be delivered with transposon DNA to genetically modify various cell lineages, embryonic stem cells, hematopoietic stem cells, and induced pluripotent stem cells. The present invention provides methods and compounds that apply the cell penetration function of transposase in methods of genetically engineering cells, and methods and compounds that use transposase as a shuttle to deliver cargo into target cells or even target organelles. Using the technology of the present invention, genome integration frequency can be adjusted, which adds an additional layer of safety, opening up the opportunity for advanced use in gene engineering and gene therapy. [Background technology]

[0002] Genetic engineering has become a crucial technique in research, biotechnology, and therapy. Viral vectors are widely used for efficient insertion of gene cargo. However, viral gene delivery is cumbersome, expensive, and carries the risk of inflammatory responses to vector-encoded epitopes (Non-Patent Document 1), and adverse genomic changes due to preferential integration into transcribed regions (Non-Patent Document 2). Non-viral genome editing nucleases (e.g., zinc finger nucleases, TALEN, or CRISPR / Cas9) allow programmed knockouts and small edits by initiating DNA repair-mediated changes in the target cell genome. However, their dependence on host repair compromises their use for the insertion of large transgenes, especially in medically relevant primary cells. The mutagenic potential of inherent DNA breaks has also recently been found to create risks of genome rearrangements (Non-Patent Document 3) and malignant transformation (Non-Patent Documents 4, 5).

[0003] Transposons offer a non-viral alternative for efficient gene delivery, and their use in research and clinical trials is rapidly increasing. Transposons elicit gene transfer rates comparable to retroviral and lentiviral vectors, but with reduced immunogenicity, unlimited cargo size, and unbiased genome distribution (Non-Patent Documents 6-8), and have advantageous attributes in terms of complexity and cost for clinical implementation.

[0004] The application of transposons to genetic engineering in vertebrates was first achieved by reconstructing an active transposon from an inactive copy in the genome of a fish called Sleeping Beauty (SB) (Non-Patent Document 9). Traditionally, the SB system includes two components provided as a plasmid DNA vector: one encoding the transposase and the other containing the gene cargo flanked by transposon end DNA sequences. To achieve gene transfer, both vectors must be transfected and the transposase gene must be expressed in the target cell. After expression, the SB transposase protein specifically binds to the transposon end of the cargo vector, excises the transgene, and integrates it at any TA dinucleotide site in the genome of the target cell (transposition) (Figure 1A). In contrast to genome editing nucleases, SB inserts its gene cargo via a direct transesterification reaction without relying on double-stranded DNA breaks and host cell DNA repair mechanisms. Due to its high insertion efficiency in vertebrates (Non-Patent Document 10), SB is a valuable tool for applications in cancer gene discovery, gene transfer, and gene therapy (recently reviewed elsewhere (Non-Patent Documents 7, 11-13)). In fact, SB is the most advanced virus-free gene delivery tool already used in clinical phase I / II trials for ex vivo manipulation of therapeutic cells (Non-Patent Documents 6, 7, 11, 13).

[0005] The majority of these trials aim to reprogram T cells by introducing genetic information for chimeric antigen receptors (CARs). CARs are artificial receptors that confer new specificity to malignant tumor-associated antigens to T cells, and CAR T cells have shown unprecedented response rates in the treatment of leukemia and lymphoma (Non-Patent Documents 14, 15). The first two completed clinical trials using SB for CAR gene insertion have already provided clinical proof of concept (Non-Patent Documents 16, 17). Compared to approved CAR T cell products that rely on virus-based gene transfer, the use of SB provides comparable efficacy with the added advantage of reduced manufacturing complexity and costs, which is crucial to increase the availability of the technology.

[0006] However, the current SB system has a significant defect in that the use of DNA encoding the transposase can lead to increased protein expression (Non-Patent Document 17) and even to the acquisition of the transposase gene in the target cell. This lack of control over the timing and kinetics of SB transposase exposure risks ongoing uncontrolled metastasis (Non-Patent Documents 18-20), raising safety concerns regarding adverse transformation of therapeutic cell products. To ensure clearance of the transposase and avoid infusion of abnormal or unstable cell products, engineered T cells in ongoing trials are cultured for 2-4 weeks after CAR gene delivery, thereby reducing cell fitness and therapeutic efficacy (Non-Patent Documents 16, 17). Thus, there is a pressing need to improve the control and safety of SB, which is also a key requirement for cell and gene therapy in general.

[0007] Previous attempts to control transposase exposure have focused on mRNA-based approaches, which shorten the time of protein expression (Non-Patent Documents 18, 21, 22) and reduce cytotoxicity in hematopoietic stem and progenitor cells (HSPCs) (Non-Patent Document 23). However, to achieve maximum control of activity, it would be desirable to use the protein directly, which is prohibited by problems in recombinant protein production (Non-Patent Document 24). In fact, direct delivery of genome editing nucleases has been demonstrated to improve their precision and control (Non-Patent Documents 25, 26). On the other hand, transposases are generally difficult to produce recombinantly and are characterized by low solubility in physiological conditions, which prevents efficient protein delivery. Recent reports have described the transfection of Mos1 transposase, Mboumar-9 transposase, and Mu transposase-DNA complexes (Non-Patent Documents 27-29), but the low efficiency of these enzymes in mammalian cells limits their therapeutic use. In medically relevant situations, delivery of piggyBac transposase fused to the viral capsid has been achieved with moderate efficiency, but with the disadvantage of retaining the viral delivery component (Non-Patent Documents 30, 31). For SB, protein aggregation, low stability, and solubility remain major bottlenecks for protein production and delivery to date (Non-Patent Document 24).

[0008] US Patent No. 5,999,943 relates to the development of an improved SB transposase (hsSB) with enhanced solubility, which discloses the improved properties of hsSB compared to other SB transposases and its use as a tool for gene delivery, for example in the context of therapeutic approaches.

[0009] It was therefore an object of the present invention to improve genetic engineering approaches based on transposable elements, in particular SB constructs. [Prior art documents] [Patent documents]

[0010]

Patent Document 1

Non-licensed literature

[0011]

Non-licensed literature 1

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

[0012] In general, by way of a brief description, the main aspects of the present invention can be described as follows.

[0013] In a first aspect, the present invention relates to a method for genetically engineering a target biological cell comprising, in any order, 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 the absence or without the use of a protein transfection procedure or protein transfection reagent.

[0014] In a second aspect, the present invention relates to a method for delivering a cargo compound into a biological cell, 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.

[0015] In a third aspect, the present invention relates to the 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.

[0016] In a fourth aspect, the present invention relates to a cell shuttle comprising a transposase protein covalently or non-covalently coupled to a cargo compound, or a transposase protein covalently or non-covalently coupled to a linker compound, which is suitable for covalently or non-covalently coupling the cell shuttle to a cargo compound, or a transposase protein covalently or non-covalently coupled to a linker compound, which is further covalently or non-covalently coupled to a cargo compound.

[0017] In a fifth aspect, the present invention relates to a kit for use in the delivery of a cargo compound into a cell, comprising a shuttle protein as defined in connection with the method of the second aspect of the invention or in connection with a shuttle according to the fourth aspect.

[0018] In a sixth aspect, the present invention relates to a method for introducing a transposase protein into an organism cell, comprising contacting the cell with the transposase protein in the absence of a protein transfection agent or without the use of a protein transfection procedure such as electroporation. [Brief description of the drawings]

[0019] [Figure 1] Schematic diagram of genome engineering by SB transposase. LE and RE represent the left and right transposon end sequences, respectively. Cargo gene transfer in the target genome is carried out by the transposase expressed from a plasmid vector (curved arrow) in the target cell. [Diagram 2]FIG. 1 shows that direct hsSB delivery enables efficient gene transfer in a variety of mammalian and stem cells. Representative flow cytometry 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 plasmid and electroporated with hsSB transposase. Three weeks after transfection, cells stably expressing the integrated Venus gene were identified. The amount of electroporated hsSB protein is indicated above. y-axis: propidium iodide (PI) staining to exclude dead cells; x-axis: green fluorescence from Venus; NT, not transfected. [Diagram 3] Figure 1 shows the transduction efficiency of a system containing SB protein recombinantly expressed with any transgene vector (SBprotAct) in various cell lines, as quantified by flow cytometry. Error bars indicate standard deviation (n=2). [Figure 4] FIG. 1 shows a schematic diagram of the cell engineering procedure of the present invention using spontaneous hsSB permeabilization. [Diagram 5] Immunofluorescence imaging of hsSB-treated (top) and untreated (bottom) HeLa cells showing DAPI stained nuclei (left), hsSB staining (middle), and merge (right). Arrows indicate cells with hsSB in the nucleus. [Figure 6] Western blot analysis showing cellular uptake and retention of hsSB in HeLa cells upon addition to culture medium. Samples were blotted with either anti-SB antibody or anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as an internal loading control. [Figure 7]Representative flow cytometry analysis of HeLa cells transfected with transposon MC encoding Venus and incubated with hsSB in culture medium. Venus positive cells were sorted after 2 days and analyzed 3 weeks after delivery. y-axis: 4',6-diamidino-2-phenylindole (DAPI) staining to exclude dead cells; x-axis: green fluorescence from Venus. hsSB protein concentration in culture medium is indicated above each plot. NT, not transfected. [Figure 8] FIG. 1 shows Western blot analysis of induced pluripotent stem cells (iPSCs) using anti-SB antibody after permeabilization with hsSB from the culture medium. [Figure 9] FIG. 13 shows a representative flow cytometry analysis of iPSCs after 3 weeks of transfection with Venus transposon MC and incubation with hsSB. [Figure 10] FIG. 1 shows a schematic of the T cell engineering procedure using spontaneous hsSB penetration. [Figure 11] Immunofluorescence imaging of T cells showing DAPI stained nuclei (left), hsSB staining (middle) and merge (right). Cells stained in the absence of primary SB antibody are shown below (IF control). [Figure 12] Representative flow cytometry analysis of CD8+ T cells transfected with transposon minicircles (MC) and incubated with hsSB. CD8+ T cells from a healthy donor were transfected with CD19 CAR MC and enriched for CAR-positive cells (using EGFRt as a marker) by magnetic associated cell sorting (MACS). Shown is a representative FACS plot from one of three experiments (from three different T cell donors) with the fluorescence from plotted CD8- and EGFRt-specific antibodies (CD8-VioBlue and EGFRt-AF647, respectively). The hsSB protein concentration in the culture medium is indicated above each plot. NT, not transfected. [Figure 13] Figure 1 shows the cytolytic activity of CD19 CAR T cells generated by permeabilization with hsSB or MC-MC control. Cytolysis was calculated from the luminescence signal of target cells expressing ffLuc in a 5-hour co-culture assay in the presence of excess luciferin. NT, not transfected. E:T ratio, effector to target ratio. [Figure 14] Average number of CAR transgene insertions measured by droplet digital PCR (ddPCR) of genomic DNA of CAR T cells. Error bars indicate copy number estimates of two independent ddPCR assays (performed on the same genomic DNA sample) with 95% confidence intervals. [Figure 15] Figure 1. Transmembrane transfer of hsSB-GFP fusion protein. (A) Fluorescence imaging of HeLa cells showing hsSB-GFP (left) and DAPI stained nuclei (right) after 1 hour of incubation with protein. Scale bar 20 μm. (B) Fluorescence imaging of HeLa cells showing hsSB-GFP (left) and DAPI stained nuclei (right) after 24 hours. Scale bar 20 μm. [Figure 16] Figure 1. Transfection of a catalytically inactive mutant of hsSB fused to the N-terminus of GFP. (A) Fluorescence imaging of HeLa cells showing hsSB-D153N-D244N-GFP (left) and DAPI stained nuclei (right) after 1 hour of incubation with the protein. Scale bar 20 μm. (B) Fluorescence imaging of HeLa cells showing hsSB-D153N-D244N-GFP (left) and DAPI stained nuclei (right) after 24 hours. Scale bar 20 μm. [Figure 17] Figure 1. Permeabilization of GFP-hsSB fusion protein. (A) Fluorescence imaging of HeLa cells showing GFP-hsSB (left) and DAPI stained nuclei (right) after 1 hour of incubation with the protein. Scale bar 20 μm. (B) Fluorescence imaging of HeLa cells showing GFP-hsSB (left) and DAPI stained nuclei (right) after 24 hours. Scale bar 20 μm. [Figure 18] Figure 1: The N-terminal DNA-binding domain (DBD) of hsSB is efficiently penetrated into HeLa cells. (A) Immunofluorescence imaging of HeLa cells showing SB staining (left) and DAPI stained nuclei (right) after 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) after 24 h. Scale bar 20 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The elements of the present invention are described below. Although these elements are listed with specific embodiments, it is understood that these elements can be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments are not to be construed as limiting the invention to only the exemplary described embodiments. It is to be understood that the specification supports and encompasses embodiments combining two or more exemplary described embodiments or one or more exemplary described embodiments with any number of the disclosed and / or preferred elements. Furthermore, unless the context indicates otherwise, all permutations and combinations of all elements described in this application are considered to be disclosed by the specification of this application.

[0021] In a first aspect, the present invention relates to a method for genetically engineering a target biological cell comprising, in any order, 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 the absence or without the use of a protein transfection procedure or protein transfection reagent.

[0022] According to a first aspect, a target cell is genetically engineered by performing a transposition reaction with a target genome, which occurs automatically in the presence of a transposable element (transposon construct or transposon unit) and a transposase protein that catalyzes the transposition reaction.

[0023] The present invention is primarily concerned with the finding that transposases, preferably hsSB, can automatically cross the cell membrane and enter the cell nucleus, thereby mediating genome modification by transposition. Such activity is unusual for macromolecules such as transposase proteins, since in prior art methods transposases required active transfection into the cell, for example using protein transfection reagents or procedures such as electroporation. In the context of the present invention, a method for genetic manipulation of cells is now provided that does not include a step of protein transfection, and in particular, it is preferred that the method does not include the use of protein transfection reagents or procedures for introducing transposase proteins into the cell. In other words, the method of the present invention comprises a step of introducing a transposase protein without the use of any carrier, reagent or method that alters the permeation of the protein through the cell membrane. However, if the method includes, for unrelated reasons, a step of introducing into the cell another protein that is not a transposase required for genetic manipulation, and such introduction of such another protein is performed by using protein transfection, such a step does not differ from the present invention, which relates to the transfection (delivery) of the transposase protein. Therefore, if the purpose is to introduce a protein other than the transposase protein required for genetic manipulation, such an additional step can be included. Also preferred is the method of the present invention in which the transposase protein is not indirectly introduced into the cell by introducing a gene expression construct encoding the transposase protein and expressing said construct in the target cell.

[0024] The term "protein transfection" in the context of the present invention shall be understood to broadly relate to any method or reagent sufficient to introduce into a target cell a protein that would not otherwise efficiently enter said target cell. Common protein transfection systems and protein transfection reagents include PULSin™, ProteoJuice™, Xfect™, and commercially available protein transfection reagents such as BioPorter™, Pierce™ Protein Transfection Reagent (ThermoFisher), TransPass™, and methods such as protein electroporation.

[0025] Therefore, in the context of the present invention, for the genetic engineering method, it is preferred to provide (introduce into) the transposase protein by adding it directly to the medium in which said biological cells are contained, preferably to the cell culture medium of the target biological cells. Thus, the transposase protein according to the invention is directly contacted with the target cells, without the use of any carrier or method that alters the permeation of the protein through the cell membrane.

[0026] The term "transposase" as used herein refers to an enzyme that is capable of transposition and is a component of a functional nucleic acid-protein complex that mediates transposition. The term "transposase" also refers to an integrase derived from a retrotransposon or of retroviral origin. A "transposition reaction" as used herein refers to a reaction in which a transposon is inserted into a target nucleic acid. The main components of the transposition reaction are a transposon and a transposase or integrase enzyme. For example, the transposase system according to the present invention is preferably the so-called "Sleeping Beauty (SB)" transposase. In certain embodiments, the transposase is an engineered enzyme with improved attributes, such as increased enzyme function. Some specific examples of engineered SB transposases include, but are not limited to, SB10, SB11, or SB100X SB transposases (see, e.g., Mates et al., Nat. Gen. 2009, which is incorporated herein by reference). Other transposition systems can be used, such as Ty1 (Devine and Boeke, 1994, and WO 95 / 23875), Tn7 (Craig, 1996), Tn10, and IS10 (Kleckner et al. 1996), Himar1 mariner transposase (Lampe et al., 1996), Mos1 (Tosi and Beverley, 2000), Tc1 (Vos et al., 1996), Tn5 (Park et al., 1992), P elements (Kaufman and Rio, 1992), and Tn3 (Ichikawa and Ohtsubo, 1990), bacterial insertion sequences (Ohtsubo and Sekine, 1996), retroviruses (Varmus and Brown 1989), and yeast retrotransposons (Boeke, 1989).

[0027] In a preferred embodiment of the invention, the transposase is a Sleeping Beauty (SB) transposase, preferably SB100X (SEQ ID NO: 2) or an enzyme derived from SB100X.

[0028] Thus, a transposase polypeptide according to the present invention is a polypeptide having transposase activity, wherein at least one mutated amino acid residue is a residue located between amino acid 150 and amino acid 250 of an SB transposase, preferably an SB100X transposase.

[0029] In some embodiments, the at least one mutated amino acid residue is preferably at least two mutated amino acid residues, or at least three, four, five or more amino acids. The transposase polypeptide of the present invention is preferably mutated at any one of amino acids 170 to 180 and / or amino acids 207 to 217 when its sequence is aligned with the sequence of an SB transposase, preferably SB100X. More preferably, the at least one mutated amino acid residue is selected from amino acid 176 and / or amino acid 212 of an SB transposase, preferably SB100X. Most preferably, the at least one mutated amino acid residue is mutated to a serine residue, preferably C176S or C176S and I212S.

[0030] In other embodiments, the transposase polypeptide of the invention further comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, and most preferably 100% sequence identity to the amino acid sequence between residues 150 and 250 shown in SEQ ID NO:1 (hsSB). Preferably, the transposase polypeptide comprises at least a mutation at C176, preferably C176S, compared to the sequence of SEQ ID NO:2. Even more preferably, the transposase polypeptide further comprises a mutation at position I212, preferably I212S.

[0031] In some embodiments, a transposase polypeptide of the invention comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, and most preferably 100% sequence identity to the full length amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 (hsSB). Preferably, the degree of sequence identity is less than 100% in some embodiments, provided that at least one of the mutations described above is present in a transposase polypeptide of the invention.

[0032] In another embodiment, the self-permeable transposase protein of the invention is a fragment of a transposase. Preferably, the fragment comprises the DNA binding domain of hsSB (FIG. 18). Preferably, the DNA binding domain of the transposase comprises 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more additional amino acids at the N-terminus and / or C-terminus.

[0033] As used herein, the term "identical" or percent "identity," when used elsewhere herein in the context of two or more nucleic acid sequences or protein / polypeptide sequences, refers to two or more sequences or subsequences that are the same, or that have (or have at least) a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity or at least that, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% identity or at least that, more preferably about 95%, 96%, 97%, 98%, 99% or more identity or at least that, over a specified region, preferably over their full length sequences, when compared and aligned for maximum correspondence over a comparison window or designated region), as measured using a sequence comparison algorithm or by manual alignment and visual inspection (see, e.g., the NCBI website). In certain embodiments, for example, when comparing the protein sequence or nucleic acid sequence of a transposase of the present invention to, for example, a reference (non-mutated transposase), the percentage identity can be determined by a Blast search provided at NCBI, and in particular for amino acid identity, a Blast search using BLASTP 2.2.28+ with the following parameters: matrix: BLOSUM62; gap penalty: presence: 11, extension: 1; neighboring word threshold: 11; window for multiple hits: 40.

[0034] Further, in some embodiments, the transposase polypeptides of the present invention have enhanced solubility compared to a reference non-mutated transposase polypeptide, preferably the reference non-mutated transposase polypeptide is the SB100X transposase, preferably as set forth in SEQ ID NO:2 (non-mutated SB100X).

[0035] In some aspects and embodiments, the transposon protein comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to the amino acid sequence of any of the indicated transposase proteins. Such transposase proteins consist of, or consist essentially of, the amino acid sequence as set forth in any one of SEQ ID NO:1-3, optionally with no more than 50, no more than 20, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, no more than 1 amino acid substitution, addition, insertion, deletion, or inversion compared to these sequences. However, such mutant transposase proteins still retain their transposase activity and / or their cell penetrating activity according to the invention.

[0036] In a particular embodiment of the present invention, the transposon construct comprises a genetic sequence to be genetically introduced into the target genome. A transposon construct or a transposon unit, in the context of the present invention disclosed herein, refers to a nucleic acid (i.e. gene) construct that comprises a target sequence intended to be the subject of transposition operably linked to a transposon genetic element required for successful transposition of said unit mediated by a transposase protein. In a particularly preferred embodiment, the transposon construct does not comprise a nucleotide sequence encoding a transposase protein. Thus, the transposon construct or transposon unit of the present invention preferably comprises inverted terminal repeats (ITRs) or direct terminal repeats (DTRs) flanking the sequence of interest to be inserted into the genome of the target cell (the target sequence to be transposed). Typically, the transposon unit is a nucleic acid and can be any form of vector suitable for transposition.

[0037] In certain embodiments of the invention, the transposable element or transposon construct or transposon unit is introduced into the target cell, for example by using known nucleic acid transfection systems. However, the method of the invention can also be carried out in a target cell that already contains a transposon construct or transposon unit, and thus the transposition reaction is initiated by introducing the transposase protein according to the invention into the target cell.

[0038] As used herein, the term "inverted terminal repeat" refers to a sequence located at one end of a transposon unit that can be cleaved by a transposase polypeptide when used in combination with a complementary sequence located at the opposite end of a vector or transposon unit. A pair of inverted terminal repeats is involved in the transposon transposition activity of the transposon unit of the present disclosure, in particular in the addition or removal and excision of DNA and the integration of DNA of interest. In one example, at least one pair of inverted terminal repeats is believed to be the minimum sequence required for transposition activity. In another example, the transposon unit of the present disclosure may contain at least two, three, or four pairs of inverted terminal repeats. As will be appreciated by those skilled in the art, to facilitate cloning, the necessary terminal sequence may be as short as possible and therefore may contain as few inverted repeats as possible. Thus, in one example, the transposon unit of the present disclosure may contain no more than one, no more than two, no more than three, or no more than four pairs of inverted terminal repeats. In one example, the transposon unit of the present disclosure may contain only one pair of inverted terminal repeats. Without wishing to be bound by theory, it is believed that having more than one pair of inverted terminal repeats may be disadvantageous, since it may result in non-specific transposase binding to multiple inverted terminal repeats, leading to removal of desired sequences or insertion of undesired sequences. The inverted terminal repeats of the present disclosure may form either perfect inverted terminal repeats (or referred to interchangeably as "perfect inverted repeats") or imperfect inverted terminal repeats (or referred to interchangeably as "imperfect inverted repeats"). As used herein, the term "perfect inverted repeats" refers to two identical DNA sequences arranged in opposite orientations. The above description of transposon units with ITRs also applies to transposon units containing DTRs.

[0039] Transposon systems (or units) that can be used with the systems and components of the invention are disclosed, for example, in WO 2017 / 050448, which is hereby incorporated by reference in its entirety.

[0040] The transposon construct according to the present invention is preferably provided with said transposon unit in the form of minicircle.However, said transposon unit may also be other nucleic acid system.However, in the context of T cell engineering, for example, to introduce CAR into T cell, minicircle is preferred.

[0041] In preferred aspects and embodiments of the invention, the target sequence introduced into the genome of the target cell by transfer is a sequence encoding a CAR, an antibody, or a T cell receptor, or any variant of such molecules. Thus, in some embodiments, the methods and compounds of the invention are preferably used to genetically engineer T cells to generate CAR T cells. As used herein, the term "chimeric antigen receptor T cells", also referred to as CAR T cells, refers to lymphocytes expressing a chimeric antigen receptor (CAR). Thus, the methods of the invention include introducing all the genetic elements necessary for the expression of a CAR in a target cell. The term "chimeric antigen receptor" or "CAR" has its general meaning in the art and refers to an artificially constructed hybrid protein or hybrid polypeptide (e.g., scFv) that comprises the antigen-binding domain of an antibody linked to a T cell signaling domain. The hallmarks of CARs include their ability to exploit the antigen-binding properties of monoclonal antibodies to redirect T cell specificity and T cell reactivity to a selected target in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition confers CAR-expressing T cells the ability to circumvent a major mechanism of tumor escape, since they recognize antigens independent of antigen processing. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains. Strategies for designing and producing such CARs are known in the art and references can be found, for example, in Bonini and Mondino, Eur. J. Immunol. 2015(19); Srivastava and Riddell, Trends Immunol. 2015(20); Jensen and Riddell, Curr. Opin. Immunol. 2015(21); Gill and June, Immunol. Rev. 2015(22).

[0042] In a preferred embodiment, the transposon system of the present invention is the SB transposon system.

[0043] The target cells according to the present invention are preferably selected from mammalian cells, preferably stem cells such as hematopoietic stem cells, embryonic stem cells, spontaneously immortalized cells, artificially immortalized cells, primary cells (neurons, resting T cells), B cells such as plasma cells, Chinese Hamster Ovary (CHO) cells, cells derived from induced pluripotent stem cells (iPSCs), or are immune cells such as T lymphocytes, preferably CD4+ or CD8+ T cells, or are natural killer (NK) cells, macrophages, dendritic cells or B cells.

[0044] In a second aspect, the present invention relates to a method for delivering a cargo compound into a biological cell, 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.

[0045] In the second aspect of the present invention, the use of the cell-penetrating activity of transposase protein is used as a cell shuttle to transport any kind of cargo into target cells. By simply attaching such cargo to the transposase protein of the present invention, any compound can be efficiently transported into cells. Therefore, the transposase protein according to the present invention is used as a cell transfection carrier.

[0046] In a preferred embodiment of the present invention, the cargo compound is delivered into the organism cell and into the cell nucleus of the organism cell. Alternatively, however, it is possible to target the cargo-shuttle complex to different organelles such as mitochondria, endoplasmic reticulum, Golgi, etc., by altering the organelle targeting sequence in the transposase, for example by exchanging the nuclear localization signal with a signal peptide of a different organelle. Thus, in certain embodiments, the shuttle protein comprises a deletion or mutation of the nuclear localization signal, or does not comprise a nuclear localization signal, and optionally comprises a signal sequence for intracellular delivery to organelles other than the cell nucleus.

[0047] The transposase used in this aspect is preferably a transposase described herein for other aspects and embodiments.

[0048] The cell shuttle of the present invention in certain embodiments comprises a transposase protein covalently or non-covalently coupled to a linker compound, preferably the linker compound is suitable for covalently or non-covalently coupling the cargo compound to the shuttle protein. The linker can be a simple peptide linker or can contain any functionality that facilitates conjugating the cargo to the shuttle protein. For example, the linker compound can be selected from the donor or acceptor sites of sortase, biotin or streptavidin proteins, or components that functionally replace protein coupling systems. Many such systems are known to those skilled in the art and include the introduction of specific functionality for chemical cross-linking, such as cysteine ​​residues, inteins, or unnatural amino acids. Alternatively, it can also be a specific peptide (i.e., binding domain specific for DNA / RNA / chemicals / lipids, etc.) suitable for non-covalently attaching the cargo compound.

[0049] In principle, the cell-penetrating activity of the transposase of the present invention can be used to transport any protein across the cell membrane, such cargo compounds being selected from small molecules, macromolecules, peptides, polypeptides, proteins, nucleic acids such as RNA, DNA, RNA-DNA hybrids, PNA, or sugar compounds, fatty acid-containing compounds.

[0050] As with the above embodiments of the first aspect of the invention, the method of the second aspect is also preferably a method that does not require the addition of a protein transfection agent or procedure, and preferably the method does not involve the use of a protein transfection reagent or a protein transfection procedure such as electroporation.

[0051] In a third aspect, the present invention relates to the 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.

[0052] Preferably, for delivery no protein transfection reagents or protein transfection procedures such as electroporation are required or included. In this context, reference is made herein to the above explanations regarding the first and second aspects of the invention, as well as to the embodiments in which no protein transfection is required to introduce the transposase protein into a cell or organelle.

[0053] In a fourth aspect, the present invention relates to a cell shuttle comprising a transposase protein covalently or non-covalently coupled to a cargo compound, or a transposase protein covalently or non-covalently coupled to a linker compound, which is suitable for covalently or non-covalently coupling the cell shuttle to a cargo compound, or a transposase protein covalently or non-covalently coupled to a linker compound, which is further covalently or non-covalently coupled to a cargo compound.

[0054] In a fifth aspect, the present invention relates to a kit for use in the delivery of a cargo compound into a cell, comprising a shuttle protein as defined in connection with the method of the second aspect of the invention or in connection with a shuttle according to the fourth aspect.

[0055] In a sixth aspect, the present invention relates to a method for introducing a transposase protein into an organism cell, comprising contacting the cell with the transposase protein in the absence of a protein transfection agent or without the use of a protein transfection procedure such as electroporation.

[0056] In addition to the above aspects and embodiments, the present invention further relates to the following series of items:

[0057] Item 1: A method for genetically engineering a target biological cell, comprising, in any order, (i) introducing a transposon construct into the biological cell and / or providing a biological cell containing a transposon construct, and (ii) contacting the target biological cell with a transposase protein in the absence or without the use of a protein transfection procedure or protein transfection reagent.

[0058] Item 2: The method according to item 1, wherein the transposon construct comprises a gene sequence to be genetically introduced into the target genome.

[0059] Item 3: The method according to item 1 or 2, wherein the transposase protein is or is derived from Sleeping Beauty (SB) transposase.

[0060] Item 4: The method of item 3, wherein the SB transposase is SB100X, preferably SB100X with the amino acid sequence set forth in SEQ ID NO:2.

[0061] Item 5: The method of item 3, wherein the SB transposase is a highly soluble SB100X (hsSB) that contains at least one mutated amino acid residue compared to the amino acid sequence between amino acid 150 and amino acid 250 of a reference non-mutated SB transposase, for example, the reference non-mutated SB transposase comprises the sequence set forth in SEQ ID NO:2.

[0062] Item 6: The method of item 5, wherein the at least one mutated amino acid residue is at least two mutated amino acid residues.

[0063] Item 7: The method according to item 5 or 6, wherein the at least one mutated amino acid residue is a mutation in any one of amino acids 170 to 180 and / or amino acids 207 to 217 of the SB transposase, preferably SB100X (sequence number 2).

[0064] Item 8: The method according to any one of items 5 to 7, wherein at least one mutated amino acid residue is selected from amino acid 176 and / or amino acid 212 of the SB transposase, preferably SB100X (SEQ ID NO: 2).

[0065] Item 9: The method according to any one of items 5 to 8, wherein at least one mutated amino acid residue is mutated to a serine residue, preferably C176S and I212S.

[0066] Item 10: The method according to any one of Items 5 to 9, wherein the transposase protein further comprises an amino acid sequence between residue 150 and residue 250 shown in SEQ ID NO: 1 or SEQ ID NO: 3, preferably an amino acid sequence having at least 60% sequence identity to the full-length sequence.

[0067] Item 11: The method according to any one of items 1 to 10, wherein the shuttle protein comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the transposase protein.

[0068] Item 12: The method according to any one of Items 1 to 11, wherein the shuttle protein consists of or substantially consists of the amino acid sequence as set forth in any one of SEQ ID NOs: 1 to 3, optionally with not more than 20, not more than 10, not more than 9, not more than 8, not more than 7, not more than 6, not more than 5, not more than 4, not more than 3, not more than 2, not more than 1 amino acid substitution, addition, insertion, deletion, or inversion compared to these sequences.

[0069] Item 13: The method according to any one of Items 1 to 12, wherein the transposase protein is provided by adding the transposase protein to a medium in which the biological cells are contained, preferably a cell culture medium of the target biological cells.

[0070] Item 14: The method according to any one of Items 1 to 13, wherein the target biological cell is a mammalian cell selected from stem cells such as hematopoietic stem cells and embryonic stem cells, spontaneously immortalized cells, artificially immortalized cells, primary cells (neurons, resting T cells), B cells such as plasma cells, Chinese hamster ovary (CHO) cells, cells derived from induced pluripotent stem cells (iPSCs), or is an immune cell such as a T lymphocyte, preferably a CD4+ T cell or a CD8+ T cell, or is a natural killer (NK) cell, a macrophage, a dendritic cell, or a B cell.

[0071] Item 15: The method according to any one of items 1 to 14, wherein the transposon comprises a nucleotide sequence encoding a protein, such as a sequence encoding an antibody, a T cell receptor, or a chimeric antigen receptor (CAR).

[0072] Item 16: A method for delivering a cargo compound into a biological cell, comprising covalently or non-covalently, and directly or indirectly, attaching the cargo compound to a shuttle protein to obtain a cargo-shuttle complex, and contacting the biological cell with the cargo-shuttle complex, wherein the shuttle protein comprises a transposase protein sequence.

[0073] Item 17: The method of item 16, wherein the cargo compound is delivered into the biological cell and into the cell nucleus of the biological cell.

[0074] Item 18: The method of item 16 or 17, wherein the transposase protein sequence is derived from Sleeping Beauty (SB) transposase.

[0075] Item 19: The method of item 18, wherein the SB transposase is SB100X, preferably SB100X with the amino acid sequence set forth in SEQ ID NO:2.

[0076] Item 20: The method of item 18, wherein the SB transposase is a highly soluble SB100X (hsSB) that contains at least one mutated amino acid compared to the amino acid sequence between amino acid 150 and amino acid 250 of a reference non-mutated SB transposase, such as the sequence set forth in SEQ ID NO:2.

[0077] Item 21: The method of Item 20, wherein the at least one mutated amino acid residue is at least two mutated amino acid residues.

[0078] Item 22: The method according to item 20 or 21, wherein the at least one mutated amino acid residue is a mutation in any one of amino acids 170 to 180 and / or amino acids 207 to 217 of the SB transposase, preferably SB100X (sequence number 2).

[0079] Item 23: The method according to any one of items 20 to 22, wherein the at least one mutated amino acid residue is selected from amino acid 176 and / or amino acid 212 of the SB transposase, preferably SB100X (SEQ ID NO: 2).

[0080] Item 24: The method according to any one of Items 20 to 23, wherein at least one mutated amino acid residue is mutated to a serine residue, preferably C176S and I212S.

[0081] Item 25: The method according to any one of Items 20 to 24, wherein the shuttle protein further comprises an amino acid sequence between residue 150 and residue 250 as shown in SEQ ID NO: 1 or SEQ ID NO: 3, preferably an amino acid sequence having at least 60% sequence identity to the full-length sequence.

[0082] Item 26: The method according to any one of Items 16 to 25, wherein the shuttle protein comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the transposase protein.

[0083] Item 27: The method according to any one of Items 16 to 20, wherein the shuttle protein comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to a sequence of at least 50, preferably 100, 150, 200, preferably at least 300 contiguous amino acids of the transposase protein.

[0084] Item 28: The method according to any one of Items 16 to 27, wherein the shuttle protein consists of or substantially consists of the amino acid sequence as set forth in any one of SEQ ID NO:1 to SEQ ID NO:3, optionally with not more than 20, not more than 10, not more than 9, not more than 8, not more than 7, not more than 6, not more than 5, not more than 4, not more than 3, not more than 2, not more than 1 amino acid substitution, addition, insertion, deletion, or inversion compared to these sequences.

[0085] Item 29: The method according to any one of items 16 to 28, wherein the shuttle protein is covalently or non-covalently coupled to a linker compound, preferably the linker compound is suitable for covalently or non-covalently coupling a cargo compound to the shuttle protein.

[0086] Item 30: The method according to item 29, wherein the linker compound is selected from a donor or acceptor site of a sortase, a biotin protein or a streptavidin protein, or a component that functionally replaces a protein coupling system.

[0087] Item 31: The method according to any one of Items 16 to 30, wherein the cargo compound is selected from small molecules, macromolecules, peptides, polypeptides, proteins, nucleic acids such as RNA, DNA, RNA-DNA hybrids, and PNA, or is a sugar compound or a fatty acid-containing compound.

[0088] Item 32: The method of any one of the preceding items, wherein the shuttle protein comprises a deletion or mutation of a nuclear localization signal, or does not comprise a nuclear localization signal, and optionally comprises a signal sequence for intracellular delivery into an organelle other than the cell nucleus.

[0089] Item 33: The method according to any one of the preceding items, wherein the method does not require the addition of a protein transfection agent or a protein transfection procedure, preferably, the method does not include the use of a protein transfection reagent or a protein transfection procedure such as electroporation.

[0090] Item 34: The method according to any one of the preceding items, wherein the biological cell is a mammalian cell.

[0091] Item 35: Use of a transposase protein in the delivery of a cargo compound into a biological cell, wherein the transposase protein is used as a cell shuttle protein and is covalently or non-covalently and directly or indirectly attached to the cargo compound.

[0092] Item 36: The use according to Item 35, wherein for delivery, no protein transfection reagent or protein transfection procedure such as electroporation is required or included.

[0093] Item 37: The use according to item 35 or 36, wherein the transposase protein is a shuttle protein as defined in any one of items 16 to 34 of the method.

[0094] Item 38: A cell 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, the linker compound being suitable for covalently or non-covalently coupling the cell shuttle to a cargo compound; or (iii) a transposase protein covalently or non-covalently coupled to a linker compound, which in turn is covalently or non-covalently coupled to a cargo compound; , cell shuttle.

[0095] Item 39: The cell shuttle according to Item 16, wherein the transposase protein is a shuttle protein defined in any one of Items 16 to 34.

[0096] Item 40: The cell shuttle according to item 38 or 39, wherein the cargo compound is selected from small molecules, macromolecules, peptides, polypeptides, proteins, nucleic acids such as RNA, DNA, RNA-DNA hybrids, PNA, or is a sugar compound, a fatty acid-containing compound.

[0097] Item 41: A kit for use in delivery of a cargo compound into a cell, comprising a shuttle protein defined in any one of items 16 to 34 or a cell shuttle defined in any one of items 38 to 40.

[0098] Item 42: A method for introducing a transposase protein into a biological cell, comprising contacting the cell with the transposase protein in the absence of a protein transfection agent or without the use of a protein transfection procedure such as electroporation.

[0099] Item 43: The method according to Item 42, wherein the transposase protein is a transposase protein defined in any one of Items 16 to 34.

[0100] Item 44: The method according to Items 42 or 43, wherein the transposase protein is a recombinantly expressed protein and is added to a cell culture medium of the biological cell.

[0101] As used herein, the terms "of the invention," "in accordance with the invention," "according to the invention," and the like are intended to refer to all aspects and embodiments of the invention described and / or claimed herein.

[0102] As used herein, the term "comprising" should be interpreted as embracing both "including" and "consisting of," both of which are specifically contemplated and thus mean embodiments according to the present invention that are separately disclosed. As used herein, "and / or" should be considered as a specific disclosure of each of the two specified features or components, with or without the other. For example, "A and / or B" should be considered as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were separately presented herein. In the context of the present invention, the terms "about" and "approximately" represent an interval of precision that is understood by the skilled artisan to still ensure the technical effect of the feature in question. This term typically indicates a deviation of ±20%, ±15%, ±10%, e.g. ±5% from the specified numerical value. As will be understood by the skilled artisan, the deviation of such a specific numerical value for a given technical effect depends on the nature of the technical effect. For example, natural or biological technical effects may generally have a greater variation than man-made or engineered technical effects. As will be understood by one of ordinary skill in the art, such specific numerical variation for a given technical effect will depend on the nature of the technical effect. For example, natural or biological technical effects may generally have a greater variation than man-made or engineered technical effects. When an indefinite or definite article is used in reference to a singular noun, such as "a," "an," or "the," the plural of that noun is also included unless something specifically specified otherwise.

[0103] It should be understood that application of the teachings of the present invention to a particular problem or environment, and the inclusion of variations of the present invention or additional features thereof (such as further aspects and embodiments), are within the capabilities of one of ordinary skill in the art in light of the teachings contained herein.

[0104] Unless the context dictates otherwise, the feature descriptions and definitions presented above are not limited to any particular aspect or embodiment of the invention, but apply equally to all aspects and embodiments described.

[0105] All references, patents and publications cited herein are hereby incorporated by reference in their entirety.

[0106] The sequence indicates:

[0107] SEQ ID NO:1 shows the hsSB. MGKSKEISQDLRKRIVDLHKSGSSLGAISKRLAVPRSSVQTIVRKYKHHGTTQPSYRSGRRRVLSPRDERTLVRKVQINPRTTAKDLVKMLEETGTKVSISTVKRVLYRHNLKGHSARKKPLLQNRHKKARLRFATAHGDKDRTFWRNVLWSDETKIELFGHNDHRYVWRKKGEA S KPKNTIPTVKHGGGSIMLWGCFAAGGTGALKIDG S MDAVQYVDILKQHLKTSVRKLKLGRKWVFQHDNDPKHTSKVVAKWLKDNKVKVLEWPSQSPDLNPIENLWAELKKRVRARRPTNLTQLHQLCQEEWAKIHPNYCGKLVEGYPKRLTQVKQFKGNATKY

[0108] SEQ ID NO:2 (non-mutated SB100X) MGKSKEISQDLRKRIVDLHKSGSSLGAISKRLAVPRSSVQTIVRKYKHHGTTQPSYRSGRRRVLSPRDERTLVRKVQINPRTTAKDLVKMLEETGTKVSISTVKRVLYRHNLKGHSARKKPLLQNRHKKARLRFATAHGDKDRTFWRNVLWSDETKIELFGHNDHRYVWRKKGEA C KPKNTIPTVKHGGGSIMLWGCFAAGGTGALKIDG I MDAVQYVDILKQHLKTSVRKLKLGRKWVFQHDNDPKHTSKVVAKWLKDNKVKVLEWPSQSPDLNPIENLWAELKKRVRARRPTNLTQLHQLCQEEWAKIHPNYCGKLVEGYPKRLTQVKQFKGNATKY

[0109] SEQ ID NO:3 (hsSB for recombinant expression) GPMMGKSKEISQDLRKRIVDLHKSGSSLGAISKRLAVPRSSVQTIVRKYKHHGTTQPSYRSGRRRVLSPRDERTLVRKVQINPRTTAKDLVKMLEETGTKVSISTVKRVLYRHNLKGHSARKKPLLQNRHKKARLRFATAHGDKDRTFWRNVLWSDETKIELFGHNDHRYVWRKKGEA S KPKNTIPTVKHGGGSIMLWGCFAAGGTGALKIDG S MDAVQYVDILKQHLKTSVRKLKLGRKWVFQHDNDPKHTSKVVAKWLKDNKVKVLEWPSQSPDLNPIENLWAELKKRVRARRPTNLTQLHQLCQEEWAKIHPNYCGKLVEGYPKRLTQVKQFKGNATKY (Underlined are residues that have been mutated or will be mutated. In bold and italics (first GPM of SEQ ID NO:3) are residues that have been introduced for recombinant protein expression).

[0110] Certain aspects and embodiments of the invention will now be described, by way of example, with reference to the description, figures and tables provided herein. Such examples of methods, uses and other aspects of the invention are merely representative and should not be considered as limiting the scope of the invention solely to such representative examples. EXAMPLES

[0111] The examples show the following:

[0112] Example 1 (comparison): Efficient gene transfer in mammalian cells using hsSB transposase The highly soluble Sleeping Beauty (hsSB) transposase developed by the present inventors was tested for its ability to genetically engineer cells in various mammalian cell lines. The amino acid sequence of the improved hsSB transposase is shown in SEQ ID NO: 3. To better quantify hsSB-mediated transposition, the present inventors applied a fluorescent reporter system and transfected HeLa cells with a transposon plasmid carrying the Venus gene, followed by hsSB protein delivery by protein electroporation. Cells that acquired the transposon plasmid were selected by fluorescence-activated cell sorting two days after transfection. Transposition efficiency was then quantified after 3 weeks by flow cytometry analysis of green fluorescent cells stably expressing the Venus reporter gene as a result of genome insertion by hsSB (Figure 2). A clear dose-dependent increase in the percentage of fluorescent cells was detected, with maximum efficiency (42%) achieved at 20 μg of hsSB protein (Figure 2, upper panel, and Figure 3). Chinese hamster ovary (CHO) cells and mouse embryonic stem cells could also be efficiently transfected with the hsSB transposase of the present invention (FIGS. 2 and 3).

[0113] Example 2: Transposases have inherent cell-penetrating properties To further develop methods for genetically engineering mammalian cells, we sought to make transposase delivery simpler and gentler. Remarkably, we observed that the transposase protein autonomously penetrated HeLa cells and entered the nucleus when simply added to the culture medium (Figures 4 and 5). To test whether hsSB could mediate transposition when delivered in this way, we transfected HeLa cells with MC containing the Venus gene and then added hsSB to the culture medium without further pulses or transfection reagents (Figure 4). Longitudinal Western blot analysis showed uptake of hsSB within 4 hours, followed by clearance already 24 hours after delivery (Figure 6). Fluorescent cell sorting 3 weeks after transfection revealed up to 12% Venus-positive cells (Figure 7), confirming that hsSB mediates efficient transgene integration.

[0114] We next tested a similar procedure to genetically engineer human iPSCs. Although iPSCs offer great potential for regenerative medicine, they are among the most difficult cells to manipulate because they are sensitive to transfection procedures. We first transfected Venus-bearing MCs into iPSCs using a stem cell-specific transfection reagent, and then incubated them with hsSB protein-containing medium to allow protein penetration into the cells. hsSB efficiently penetrated iPSCs (Figure 8), and flow cytometry of treated cells after 3 weeks revealed a remarkable gene transfer efficiency of up to 3.31% (calculated as the percentage of stable integrants at 3 weeks relative to all transfected cells, Figure 9). This indicates that the non-invasive cell penetration of hsSB is useful for modifying iPSCs.

[0115] Example 3: Novel genetic engineering methods can be used to generate CAR-T cells Finally, we tested whether the inherent cell penetration properties of hsSB could be exploited for the production of CAR T cells (Figure 10). As electroporation is a stressor for T cells, penetration with hsSB may help maintain their suitability for downstream clinical use. We first analyzed hsSB penetration in primary T cells by immunofluorescence imaging, which showed efficient protein uptake in both stimulated and unstimulated cells within 3 h (Figure 11). hsSB also efficiently enters the nucleus in non-dividing cells, consistent with active transport using its inherent nuclear localization signal. To explore transfer, we electroporated T cells with CD19 CAR MC and added hsSB to the cell culture medium. This resulted in the successful generation of human CD8+CD19 CAR T cells with an overall gene transfer frequency of 5%-7% (Figure 12). CAR T cells were then enriched to 90% purity by MACS (44) and demonstrated potent lysis of CD19+ target cells as well as high levels of effector cytokine secretion (Figures 12 and 13). Cells generated by this procedure showed fewer insertions, an average of 4, compared to the CAR MC-SB MC DNA-based protocol (6-8 insertions; Figure 14).

[0116] Example 4: Using a self-permeable transposase protein as a cargo shuttle into cells HeLa cells were seeded onto Nunc™ Lab-Tek™ II 8-well Chamber Slides™ (Thermo Fisher) (2 × 10 cells per well in 500 μL of DMEM supplemented with 10% (vol / vol) human serum and 2 mM L-glutamine). 4cells). The next day, cells were incubated with hsSB-GFP at a concentration of 0.5 μM in serum-free DMEM in a volume of 250 μL per well for 1 h. The medium was then removed and cells were fixed with PFA (4%) in PBS and incubated with DAPI for 30 min to visualize nuclei. Cells were imaged with a Zeiss LSM 780 confocal microscope (using a 63x oil immersion objective) at the ALMF Core Facility at EMBL Heidelberg. For imaging, the focus was on the central part of the nucleus to detect nuclear localization of hsSB.

[0117] Figure 15 shows that hsSB-GFP fusion protein (hsSB fused to the N-terminus of GFP) enters the cell nucleus within 1 hour (A) and is retained for at least the next 24 hours (B) as observed by GFP fluorescence imaging. Figures 16A and 16B show the same effect for a catalytically inactive mutant of hsSB in HeLa cells. Furthermore, fusing hsSB to the C-terminus of GFP similarly enhances penetration into HeLa cells (Figure 17).

[0118] In another experiment, we test a truncated version of hsSB in HeLa cells, consisting of the DNA-binding domain of the protein (Figure 18A, bottom). The results show that the DNA-binding domain of hsSB is sufficient for autonomous cell penetration from the culture medium. Using immunofluorescence imaging with an SB-specific antibody, the hsSB DBD is detected intracellularly. The protein (peptide) enters the cells within 3 hours (Figure 18A) and is retained for at least the next 24 hours (Figure 18B).

Claims

1. A method for genetically engineering a target cell, the method comprising: (a) a transposon unit comprising inverted terminal repeats (ITRs) or direct terminal repeats (DTRs) flanking a sequence to be inserted into the genome of the target cell; and (b) a protein form of a "mutant Sleeping Beauty (SB) transposase (hereinafter also referred to as mutant transposase). 2) wherein the mutant transposase has a plurality of mutations that increase the solubility of the mutant transposase compared to SEQ ID NO:2, but retains transposase activity, comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:2, and comprises the mutations C176S and I212S, thereby adapting the mutant transposase to promote recombinant integration of the sequence of interest in a target cell in protein form when used as the sole transfection agent, and wherein the genetic engineering is performed without the use of a protein transfection procedure or reagent.

2. 1. A method for delivering a cargo compound into a target cell, the method comprising: attaching a cargo compound to a shuttle protein to obtain a cargo-shuttle complex; and contacting a target cell with the cargo-shuttle complex, the shuttle protein comprising a mutant transposase, the mutant transposase having at least two mutant amino acids between amino acids 150-250 of SEQ ID NO:2, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:2, and comprising C176S and I212S mutations, wherein the mutations increase the solubility of the mutant transposase compared to SEQ ID NO:2, thereby making the mutant transposase suitable for shuttle of cargo compounds into a target cell.

3. The method of claim 2 , wherein the shuttle protein is attached to the cargo compound via a linker moiety.

4. The method of claim 3, wherein the linker moiety is a donor site-acceptor site combination of a sortase, or a biotin protein-streptavidin protein combination.

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