Transposase and use thereof

Abstract

Disclosed is a transposase polypeptide. The transposase polypeptide is derived from a Sleeping Beauty (SB) transposase, and compared to the amino acid sequence at positions 100-180 of the unmutated SB transposase shown in SEQ ID NO: 2, the amino acid sequence of the transposase polypeptide comprises at least one mutated amino acid residue.

Description

Transposase and its use Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a transposase polypeptide and its use. Background Art

[0002] Genetic engineering and gene therapy in most cases involve transferring nucleic acids into target cells or modifying the genes of target cells. Currently, this process can be performed using viral vectors, non-viral vectors (such as naked DNA plasmids, RNA, or RNPs), and transposon systems.

[0003] Compared with viral vector-mediated gene delivery, the transposon system has some advantages, such as low cost and safety. The transposon system consists of a transposon element and a transposase, which can recognize special sequences in the transposon and mediate the insertion of DNA into the cell genome. The Sleeping Beauty transposon system has been widely used in various applications of in vitro, in vitro and in vivo genetic engineering. Several polypeptides derived from the original SB transposase have been identified and determined to have different transposition activities. These transposase variants were obtained by random mutagenesis and then screened, so the mechanism of the source of different transposition activities is not well understood. Generally speaking, the protein stability of the transposase, the efficiency of transport from the cytoplasm to the nucleus, the efficiency of steps such as recognition of target transposons, cutting of target sequences, and integration of transposons may all affect the final transposition efficiency (Z Izsvak and Zoltan Ivics. Sleeping Beauty Transposition: Biology and Applications for Molecular Therapy. MOLECULAR THERAPY Vol. 9, No. 2, February 2004). There have been reports of obtaining highly efficient transposases by optimizing a specific step in the transposition process, such as improving their binding to the target DNA (Voigt, F. et al. Sleeping beauty transposase structure allows rational design of hyperactive variants for genetic engineering. Nat. Commun. 7:11126. doi:10.1038 / ncomms11126(2016)). While randomly selected transposases have high transposition efficiencies, their unclear mechanism may affect integration location and the permissible transgene size.

[0004] Therefore, there is still a need in the art to develop methods with clear mechanisms and high certainty for obtaining new transposases, as well as new transposases with different transposition activities.

[0005] Summary of the Invention

[0006] This application screens for transposases with varying transposition activities based on their activity in the key step of transposon cleavage. The mechanism is clear, and the resulting enzymes can also be used to screen for corresponding transposon sequences, thereby establishing an efficient and safe transposition system. Improving a specific step to obtain a target transposase polypeptide provides greater certainty and facilitates safety assessment of the resulting product.

[0007] This application uses an alanine scanning method to generate mutants of SB transposase, specifically in a region that has not been actively studied before. These mutants were evaluated for different excision activities, and the screening method was quick and simple. In addition, these mutants with single or combined mutations (Tri-FITase, a refined transposase for in vitro, in vivo, and in vivo) were determined to have different transposition activities and can therefore be used in different applications, such as T cell genetic engineering, which may require transposases with higher or lower activity, carry genetic material of different sizes, and / or have different insertion characteristics.

[0008] In one aspect, the present application provides a transposase polypeptide, the amino acid sequence of which comprises at least one mutated amino acid residue compared to the amino acid sequence of positions 100 to 180 of the unmutated SB transposase as shown in SEQ ID NO: 2.

[0009] In certain embodiments, the amino acid sequence of the transposase polypeptide comprises 1 to 20 mutated amino acid residues.

[0010] In certain embodiments, the amino acid sequence of the transposase polypeptide comprises at least two mutated amino acid residues.

[0011] In certain embodiments, the mutation site of the at least one mutated amino acid residue is located at positions 110 to 120 and / or positions 170 to 180 of the amino acid sequence shown in SEQ ID NO: 2.

[0012] In certain embodiments, the mutated amino acid residue is located at position 114, 116, 177 and / or 178 of the amino acid sequence shown in SEQ ID NO: 2.

[0013] In certain embodiments, the amino acid sequence of the transposase polypeptide comprises a mutation at amino acid position G114 compared to the unmutated SB transposase amino acid sequence shown in SEQ ID NO:2.

[0014] In certain embodiments, the amino acid sequence of the transposase polypeptide comprises a mutation at amino acid position S116 compared to the unmutated SB transposase amino acid sequence shown in SEQ ID NO:2.

[0015] In certain embodiments, the amino acid sequence of the transposase polypeptide comprises a mutation at amino acid position K177 compared to the unmutated SB transposase amino acid sequence shown in SEQ ID NO:2.

[0016] In certain embodiments, the amino acid sequence of the transposase polypeptide comprises a mutation at amino acid position P178 compared to the unmutated SB transposase amino acid sequence shown in SEQ ID NO:2.

[0017] In certain embodiments, the at least one mutated amino acid residue is mutated to an alanine residue.

[0018] In certain embodiments, the amino acid residues that are mutated are all alanine residues.

[0019] In certain embodiments, the amino acid sequence of the transposase polypeptide comprises an amino acid mutation selected from the group consisting of G114A, S116A, K177A, and P178A, compared to the unmutated SB transposase as shown in SEQ ID NO: 2.

[0020] In certain embodiments, the transposase polypeptide amino acid sequence comprises a mutation at amino acid position G114 to alanine (G114A) compared to the unmutated SB transposase amino acid sequence shown in SEQ ID NO: 2.

[0021] In certain embodiments, the transposase polypeptide amino acid sequence comprises a mutation at amino acid position S116 to alanine (S116A) compared to the unmutated SB transposase amino acid sequence shown in SEQ ID NO: 2.

[0022] In certain embodiments, the transposase polypeptide amino acid sequence comprises a mutation at amino acid position K177 to alanine (K177A) compared to the unmutated SB transposase amino acid sequence shown in SEQ ID NO: 2.

[0023] In certain embodiments, the transposase polypeptide amino acid sequence comprises a mutation at amino acid position P178 to alanine (P178A) compared to the unmutated SB transposase amino acid sequence shown in SEQ ID NO: 2.

[0024] In certain embodiments, compared to the unmutated SB transposase as shown in SEQ ID NO: 2, the transposase polypeptide amino acid sequence comprises a combination of amino acid mutations selected from the group consisting of:

[0025] i) G114A, S116A;

[0026] ii) G114A, K177A;

[0027] iii) G114A, P178A;

[0028] iv) S116A, K177A;

[0029] v) S116A, P178A; and

[0030] vi)K177A, P178A.

[0031] In another aspect, the present application provides a transposase polypeptide comprising a sequence having at least 90% identity to SEQ ID NO: 2, wherein the transposase polypeptide comprises an alanine at a position corresponding to position 114 and / or an alanine at a position corresponding to position 116.

[0032] In certain embodiments, the transposase polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO: 1 and SEQ ID NO: 3-11.

[0033] In another aspect, the present application provides a polynucleotide molecule comprising a nucleic acid sequence encoding the transposase polypeptide described herein.

[0034] In certain embodiments, it comprises RNA or DNA.

[0035] In certain embodiments, the DNA comprises a promoter for expressing the transposase polypeptide.

[0036] In certain embodiments, the DNA comprises a promoter for expressing the transposase polypeptide in mammalian cells.

[0037] In certain embodiments, the molecule is mRNA.

[0038] In certain embodiments, the polynucleotide molecule comprises a 5' cap, an IRES motif and / or a poly (A) sequence.

[0039] In certain embodiments, the polynucleotide molecule comprises a poly(A) sequence of 20 to 300 nucleotides.

[0040] In another aspect, the present application provides a construct comprising the polynucleotide molecule described herein and a regulatory sequence, wherein the regulatory sequence is operably linked to the expressible polynucleotide to allow expression of the polynucleotide.

[0041] In another aspect, the present application provides a cell comprising the transposase polypeptide described herein, the polynucleotide molecule described herein, and / or the construct described herein.

[0042] In certain embodiments, the cells comprise mammalian cells.

[0043] In certain embodiments, the cells comprise human cells.

[0044] In certain embodiments, the cells are autologous.

[0045] In certain embodiments, the cells are allogeneic.

[0046] In certain embodiments, the cells comprise stem cells or induced pluripotent stem (iPS) cells.

[0047] In certain embodiments, the cells comprise immune effector cells.

[0048] In certain embodiments, the cells comprise natural killer (NK) cells, T cells, or precursors of NK cells or T cells.

[0049] In certain embodiments, the cell comprises a transposase polypeptide as described herein.

[0050] In certain embodiments, the cells contain mRNA or DNA encoding the transposase polypeptide described herein.

[0051] In another aspect, the present application provides a transposon system comprising:

[0052] (a) a transposon unit containing inverted terminal repeats (ITRs) or direct terminal repeats (DTRs) flanking a target sequence to be inserted into the genome of a target cell; and

[0053] (b) the transposase polypeptide described herein, the polynucleotide molecule described herein, and / or the construct described herein.

[0054] In another aspect, the present application provides an in vitro use of the transposon system described herein for delivering genes to target cells.

[0055] In another aspect, the present application provides a method for genetically engineering cells, comprising the following steps:

[0056] (a) transfecting target cells with the transposon system described in the present application;

[0057] (b) culturing the target cell under conditions that allow the target cell to be cultured, thereby producing an engineered cell.

[0058] In certain embodiments, the method further comprises: (c) isolating the engineered cell.

[0059] In certain embodiments, the target sequence encodes a screenable or selectable marker.

[0060] In certain embodiments, the target sequence encodes a therapeutic polypeptide or an inhibitory nucleic acid.

[0061] In certain embodiments, the target sequence encodes an antibody, a T cell receptor (TCR), or a chimeric antigen receptor (CAR).

[0062] In certain embodiments, the target sequence encodes a CAR.

[0063] In certain embodiments, the CAR targets a tumor antigen.

[0064] In certain embodiments, the tumor antigen is CD19, CD20, ROR1, CD22 carcinoembryonic antigen, alpha-fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate-specific antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2 / Neu, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Rα, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, combined HER2-HER3 or combined HER1-HER2.

[0065] In certain embodiments, the target cell comprises a mammalian cell.

[0066] In certain embodiments, the target cell comprises a human cell.

[0067] In certain embodiments, the target cells are autologous.

[0068] In certain embodiments, the target cells are allogeneic.

[0069] In certain embodiments, the target cells comprise stem cells or induced pluripotent stem (iPS) cells.

[0070] In certain embodiments, the target cells comprise immune effector cells or precursors thereof.

[0071] In certain embodiments, the target cells include natural killer (NK) cells, T cells, or precursors of NK cells or T cells.

[0072] In certain embodiments, transfecting the target cell comprises using a polymer-, polypeptide-, or lipid-based transfection reagent.

[0073] In certain embodiments, transfecting the target cell comprises electroporating the cell.

[0074] In certain embodiments, the method further comprises:

[0075] (a) transfecting a cell population with the transposon system described herein;

[0076] (b) incubating the population under conditions suitable for transposase activity, thereby integrating the target sequence in the genome of the cells and generating a population of engineered cells.

[0077] In certain embodiments, the method further comprises:

[0078] (a) transfecting a population of T cells or T cell precursors with the transposon system described herein, wherein the target sequence encodes a CAR or TCR;

[0079] (b) incubating the population under conditions suitable for transposase activity, thereby integrating the CAR in the genome of the cells and generating a population of engineered T cells or T cell precursors.

[0080] In certain embodiments, the method further comprises: (c) culturing the engineered cells in a medium that selectively enhances the proliferation of CAR or TCR-expressing T cells.

[0081] On the other hand, the present application provides a pharmaceutical composition comprising the transposase polypeptide described herein, the polynucleotide molecule described herein, and / or the construct described herein, and a pharmaceutically acceptable carrier and / or excipient.

[0082] On the other hand, the present application provides a kit comprising

[0083] (a) a transposon unit containing inverted terminal repeats (ITRs) or DTRs flanking a target sequence to be inserted into the genome of a target cell; and

[0084] (b) the transposase polypeptide described herein, the polynucleotide molecule described herein, and / or the construct described herein.

[0085] On the other hand, the present application provides a use of the transposase polypeptide described herein, the polynucleotide molecule described herein, the construct described herein, the cell described herein, the transposase system described herein and / or the pharmaceutical composition described herein in the preparation of a medicament for treating a disease or condition.

[0086] In certain embodiments, the disease or condition is cancer.

[0087] In certain embodiments, the disease or condition is an infectious disease.

[0088] On the other hand, the present application provides a compound for treating a disease or condition, wherein the compound comprises the transposase polypeptide described herein, the polynucleotide molecule described herein, the construct described herein, the cell described herein, the transposase system described herein, the pharmaceutical composition described herein and / or the kit described herein.

[0089] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0091] FIG1A shows a schematic diagram of the excision assay used in the present application to distinguish SB transposases with known transposition activities.

[0092] Figure 1B shows the results of excision experiments using three commonly used SB transposases.

[0093] FIG2 shows that the transposase mutants prepared by the alanine scanning method have different transposase cleavage activities.

[0094] FIG3 shows the results of a transposase transposition assay using the Tri-FITase of the present application in Hela cells.

[0095] FIG. 4A-FIG 4B show that the Tri-FITase of the present application achieves chimeric antigen receptor expression in T cells. DETAILED DESCRIPTION

[0096] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0097] Definition of terms

[0098] In the present application, the term "transposase" generally refers to an enzyme that is a component of a functional nucleic acid-protein complex capable of transposition and mediates transposition. The term "transposase" may also include integrases derived from retrotransposons or retroviral sources. "Transposition reaction" as used herein generally refers to the reaction in which a transposon is inserted into a target nucleic acid. The main components in the transposition reaction are transposons and transposases or integrases. For example, the transposase system according to the present invention is preferably derived from the so-called "Sleeping Beauty (SB)" transposase. In some aspects, transposases are engineered enzymes with improved properties such as enhanced enzymatic function. Some specific examples of engineered SB transposases include, but are not limited to, SB10, SB11 or SB100×SB transposases (see, for example, Mates et al., Nat. Gen. 2009, 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), Mariner transposase (Lampe et al., 1996), 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).

[0099] In the present application, the term "transposon unit" refers to a nucleic acid construct comprising a transposon gene sequence and a target sequence to be introduced into the genome of a target cell. Typically, the transposon unit is a nucleic acid and can be any form of vector suitable for transposition.

[0100] In this application, the term "inverted terminal repeat" refers to a sequence located at one end of a transposon unit that, when used in combination with a complementary sequence located at the opposite end of a vector or transposon unit, can be cleaved by a transposase polypeptide. The pair of inverted terminal repeats is involved in the transposition activity of the transposon of the transposon unit of the present disclosure, particularly with respect to DNA addition or removal, as well as excision and integration of target DNA. In one example, at least one pair of inverted terminal repeats appears to be the minimum sequence required for transposition activity in a plasmid. In another example, the transposon unit of the present disclosure may include at least two, three, or four pairs of inverted terminal repeats. As will be appreciated by those skilled in the art, in order to facilitate easy cloning, the necessary terminal sequences may be as short as possible, thereby containing 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 include only one inverted terminal repeat. Although not wishing to be bound by theory, it is envisioned that having more than one inverted terminal repeat may be disadvantageous because it may result in non-specific transposases binding to multiple inverted terminal repeats and resulting in removal of the desired sequence or insertion of an undesired sequence. The inverted terminal repeats of the present disclosure can form perfect inverted terminal repeats (or interchangeably referred to as "perfect inverted repeats") or imperfect inverted terminal repeats (or interchangeably referred to as "imperfect inverted repeats"). As used herein, the term "perfect inverted repeat" refers to two identical DNA sequences placed in opposite orientations. The above description of transposon units having ITRs also applies to transposon units having DTRs.

[0101] Transposon systems that can be used with the transposon polypeptides / nucleic acids of the present invention are disclosed, for example, in WO 2017 / 050448 A1, which is included in the present disclosure by reference.

[0102] In the present application, the term "chimeric antigen receptor" or "CAR" generally refers to a group of polypeptides, which are generally two in the simplest embodiment, and when in immune effector cells, provide cell specificity to target cells (usually cancer cells) and generate intracellular signals. In some embodiments, CAR includes at least one extracellular antigen binding domain (such as VHH, scFv or part thereof), a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as "intracellular signaling domain"), which includes a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule as defined below. In some embodiments, the group of polypeptides is in the same polypeptide chain (for example, comprising a chimeric fusion protein). In some embodiments, the group of polypeptides is discontinuous with each other, for example, in different polypeptide chains. In some aspects, the group of polypeptides includes a dimerization switch, which can couple polypeptides to each other in the presence of a dimerization molecule, for example, the antigen binding domain can be coupled to the intracellular signaling domain. On the one hand, the stimulatory molecule of CAR is a ζ chain associated with a T cell receptor complex. In one aspect, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., a primary signaling domain of CD3-ζ). In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule defined below. On the one hand, the costimulatory molecule can be selected from 4-1BB (i.e., CD137), CD27, ICOS and / or CD28. On the one hand, CAR comprises a chimeric fusion protein that may include an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. On the one hand, CAR comprises a chimeric fusion protein that may include an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, and the intracellular signaling domain comprises a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. On the one hand, CAR includes a chimeric fusion protein, which may include an extracellular antigen recognition domain, a transmembrane domain and an intracellular signal transduction domain, and the intracellular signal transduction domain includes a functional signal transduction domain derived from one or more costimulatory molecules and a functional signal transduction domain derived from a stimulatory molecule. On the one hand, CAR includes a chimeric fusion protein, which may include an extracellular antigen recognition domain, a transmembrane domain and an intracellular signal transduction domain, and the intracellular signal transduction domain includes at least two functional signal transduction domains derived from one or more costimulatory molecules and a functional signal transduction domain derived from a stimulatory molecule. In one aspect, CAR includes an optional leader sequence on the amino terminus (N-ter) of the CAR fusion protein.In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., VHH) during cellular processing and localizes the CAR to the cell membrane.

[0103] In this application, the term "antibody" is generally used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170: 4854-4861). Antibodies can be mouse, human, humanized, chimeric, or derived from other species.

[0104] In this application, the term "homology" can generally be equated with sequence "identity". Homologous sequences can include amino acid sequences that are at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the subject sequence. Typically, a homologue will comprise an active site, etc., identical to the subject amino acid sequence. Homology can be considered in terms of similarity (i.e., amino acid residues having similar chemical properties / functions), or it can be expressed in terms of sequence identity. In this application, a sequence with a percentage identity to any one of the SEQ ID NOs of a mentioned amino acid sequence or nucleotide sequence refers to a sequence with the percentage identity over the entire length of the mentioned SEQ ID NO.

[0105] To determine sequence identity, a sequence alignment can be performed, which can be performed in various ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software, etc. Those skilled in the art can determine appropriate parameters for the alignment, including any algorithms needed to achieve optimal alignment over the full-length sequences being compared.

[0106] In this application, the terms "upstream" and "downstream" are functional definitions and generally refer to the direction or polarity of a chain of coding nucleotide sequences. The "upstream" direction refers to nucleotides located in the 5' direction of a given polynucleotide sequence, i.e., toward the starting nucleotide. With respect to amino acid sequences, the term "upstream" is interpreted as / refers to amino acids located in the N-terminal direction, i.e., toward the start of the polypeptide chain.

[0107] In this application, the term "isolated nucleic acid molecule" generally refers to isolated forms of nucleotides, deoxyribonucleotides or ribonucleotides of any length or their analogs, separated from their natural environment or artificially synthesized.

[0108] In this application, the term "construct" generally refers to a nucleic acid molecule that can self-replicate in a suitable host, which transfers the inserted nucleic acid molecule into and / or between host cells. The construct may include a vector that is primarily used for inserting DNA or RNA into a cell, a vector that is primarily used for replicating DNA or RNA, and a vector that is primarily used for expression of the transcription and / or translation of DNA or RNA. The vector also includes a vector with multiple of the above functions. The construct may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce the desired expression product by cultivating a suitable host cell that contains the vector.

[0109] In this application, the term "host cell" or "cell" generally refers to an individual cell, cell line or cell culture that can or already contains a vector comprising the isolated nucleic acid molecule described herein, or that is capable of expressing the isolated antigen-binding fragment described herein. The host cell may include the progeny of a single host cell. Due to natural, accidental or intentional mutations, the progeny cells may not necessarily be completely identical in morphology or genome to the original parent cell, but they may be capable of expressing the isolated antigen-binding fragment described herein. The host cell can be obtained by in vitro transfection of cells using the vectors described herein. The host cell can be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., a yeast cell, e.g., a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NSO cell or a myeloma cell). For example, the host cell can be an Escherichia coli cell. For example, the host cell can be a yeast cell. For example, the host cell can be a mammalian cell. For example, the mammalian cell can be a CHO-K1 cell.

[0110] In this application, the term "T cell" or "T lymphocyte" can be any T cell, such as a cultured T cell, for example a primary T cell, or a T cell from a cultured T cell line, such as Jurkat, SupTI, etc., or a T cell obtained from a mammal (preferably a primate, species including monkeys, dogs, or humans). If obtained from a mammal, the T cell can be obtained from many sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cell can also be enriched or transfected. The T cell can be obtained by maturing hematopoietic stem cells into T cells in vitro or in vivo. In exemplary aspects, the T cell is a human T cell. In exemplary aspects, the T cell is a T cell isolated from humans. T cells can be any type of T cell, including NKT cells, and can be of any developmental stage, including but not limited to CD4+ / CD8+ double positive T cells; CDA+ helper T cells; such as Th1 and Th2 cells, CD8+ T cells (such as cytotoxic T cells); peripheral blood mononuclear cells (PBMC); peripheral blood leukocytes (PBL); tumor infiltrating cells (TIL); memory T cells; untreated T cells and the like. Preferably, T cells are CD8+ T cells or CD4+ T cells. In some alternatives, the T cells are allogeneic (different donors from the same species) to the recipient of the cells or the cells to be received (such as the cells are in the form of a therapeutic composition); in some alternatives, the T cells are autologous (the donor and the recipient are the same); in some alternatives, the T cells are syngeneic (the donor and the recipient are different, but are identical twins).

[0111] In this application, the term "immune effector cell" generally refers to an immune cell that participates in an immune response and performs an effector function. For example, the effector function may include clearing foreign antigens or promoting immune effector responses. Immune effector cells may include plasma cells, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.

[0112] The immune effector cells of the present application can be autologous / self (autologous / autogeneic) ("one's own") or non-autologous ("non-one's own", such as allogeneic, isogenic or allogeneic). In the present application, the term "autologous" generally refers to cells from the same subject. "Allogenic" generally refers to cells that are of the same species but genetically different from the compared cells. "Isogenic" generally refers to cells of a different subject that are genetically identical to the compared cells. "Allogenic" generally refers to cells of a species different from the compared cells. In some embodiments, the cells of the present application are autologous or allogenic.

[0113] In this application, the term "modification" generally refers to changing the state or structure of a cell and / or a change in the state or structure of a cell. The change is usually compared to the state or structure of a corresponding cell without the modification, and the change may include a change in the expression level or function of an endogenous gene, such as downregulating, upregulating or not expressing the expression level of an endogenous gene in the cell by genetic engineering means, and the genetic engineering means may include homologous recombination, CRISPR / Cas9 system gene editing, etc.; the change may also include a change in the expression, structure or function of a cell protein, such as a change in the expression level or function of a corresponding protein achieved by a change in the expression level or function of the endogenous gene, such as a change in protein expression, structure or function achieved by regulating protein translation or post-translational modification; the change may also include the introduction of exogenous genes, the expression of exogenous proteins, etc.

[0114] In this application, the term "nucleic acid" or "polynucleotide" or "nucleic acid molecule" generally refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term may include nucleic acids containing analogs of natural nucleotides, which have similar binding properties to a reference nucleic acid (e.g., sequence information is shown) and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, the sequence of a nucleic acid may include variants thereof modified in a conservative manner, such as degenerate codon substitutions, alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated.

[0115] In this application, the term "operably linked" generally refers to placing regulatory sequences necessary for expression of a coding sequence at an appropriate position relative to the coding sequence so as to achieve expression of the coding sequence. The term "operably linked" may also refer to the arrangement of coding sequences and regulatory sequences (e.g., promoters, enhancers, and termination elements) in an expression vector. This definition sometimes also applies to the arrangement of nucleic acid sequences of a first and a second nucleic acid molecule in which a hybrid nucleic acid molecule is generated.

[0116] In this application, the term "regulatory sequence" generally includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that regulate the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Those skilled in the art will appreciate that the design of expression vectors, including the selection of regulatory sequences, depends on factors such as the choice of host cells to be transformed and the desired level of protein expression. Regulatory sequences for mammalian host cell expression can include viral elements that direct high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., adenovirus major late promoter (AdMLP)), and polyoma virus. Alternatively, non-viral regulatory sequences such as the ubiquitin promoter or the β-globin promoter can be used. Still further, regulatory elements may contain sequences from different sources, such as the SRα promoter system, which includes sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472).

[0117] In this application, the term "expression" generally refers to the transcription and / or translation of a specific nucleotide sequence.

[0118] In this application, the terms "tumor" and "cancer" are used interchangeably and generally refer to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers are described herein and include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. The terms "cancer" or "tumor" include pre-malignant and malignant cancers and tumors, and also encompass solid tumors and non-solid tumors.

[0119] "Tumor antigen" as used herein includes meanings known in the art, including any molecule expressed on tumor cells (or associated with tumor cell development), known or believed to have an effect on the tumorigenic properties of tumor cells. Many tumor antigens are known in the art. Whether a molecule is a tumor antigen can also be determined according to techniques and assays well known to those skilled in the art, such as clonogenic assays, transformation assays, in vitro or in vivo tumor formation assays, gel migration assays, gene knockout analysis, and the like. Preferably, the term "tumor antigen" when used herein refers to a human transmembrane protein, i.e., a cell membrane protein anchored in the lipid bilayer of the cell. The human transmembrane protein used herein will typically include an "extracellular domain" that can bind a ligand, a lipophilic transmembrane domain, a conserved intracellular domain, such as a tyrosine kinase domain, and a carboxyl-terminal signaling domain having several tyrosine residues that can be phosphorylated. Tumor antigens include molecules such as EGFR, HER2 / neu, HER3, HER4, EpCAM, CEA, TRAIL, TRAIL receptor 1, TRAIL receptor 2, lymphotoxin beta receptor, CCR4, CD19, CD20, CD22, CD28, CD33, CD40, CD80, CSF-1R, CTLA-4, fibroblast activation protein (FAP), hepsin, melanoma-associated chondroitin sulfate proteoglycan (MCSP), prostate-specific membrane antigen (PSMA), VEGF receptor 1, VEGF receptor 2, IGF1-R, TSLP-R, TIE-1, TIE-2, TNF-α, TNF-like weak inducer of apoptosis (TWEAK), or IL-1R.

[0120] As used herein, the term "pharmaceutically acceptable" generally refers to those compounds, materials, compositions and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0121] In this application, the term "pharmaceutically acceptable carrier" generally refers to any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH regulators, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc. that are compatible with drug administration. The use of such media and agents for pharmaceutical active substances is well known in the art. The composition may contain other active compounds that provide supplementary, additional or enhanced therapeutic functions.

[0122] As used herein, the term "effective amount" or "effective dose" generally refers to an amount sufficient to achieve, or at least partially achieve, a desired effect. A "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent is generally any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes regression of a disease as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or the prevention of impairment or disability resulting from the disease.

[0123] In this application, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "to be", "to be composed of..."

[0124] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0125] In this application, the term "subject" generally refers to humans or non-human animals, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats or monkeys.

[0126] In the present application, the term "marker gene" refers to a nucleic acid sequence that is capable of expressing an activity that causes a positive or negative selection to occur in a host cell, such as to the presence or absence of a sequence. The marker sequence or gene may optionally display positive and negative selectivity. Important positive selection markers are, for example, antibiotic resistance genes. Selective markers may be positive, negative, or bifunctional. Positive selection markers allow the selection of cells that carry the marker, while negative selection markers allow selective elimination of cells that carry the marker. Typically, selective markers will confer resistance to drugs or confer metabolic or catabolism defects in the compensatory cell. Selectable markers that can be used in eukaryotic cells include, for example, genes for aminoglycoside phosphotransferases (APHs), such as hygromycin phosphotransferase (hyg), neomycin and G418 APHs, dihydrofolate reductase (DHFR), thymidine kinase (tk), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (selective agent indole), histidinol dehydrogenase (selective agent histidinol D), and genes that provide resistance to puromycin, bleomycin, phleomycin, chloramphenicol, Zeocin, and mycophenolic acid. Other selectable markers are described, for example, in WO 92 / 08796 and WO 94 / 28143.

[0127] Detailed Description of the Invention

[0128] On the one hand, the present application provides a transposase polypeptide, wherein the transposase polypeptide is derived from Sleeping Beauty (SB) transposase, and the amino acid sequence of the transposase polypeptide comprises at least one mutated amino acid residue compared to the amino acid sequence of positions 100 to 180 of the unmutated SB transposase as shown in SEQ ID NO:2.

[0129] In certain embodiments, the number of amino acid residues mutated is 1 to 20. For example, the number of amino acid residues mutated is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0130] In certain embodiments, the at least one mutated amino acid residue is at least two mutated amino acid residues. For example, the mutated amino acid residues are 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4 or 2 to 3. For example, the mutated amino acid residues are 2.

[0131] In certain embodiments, the mutation site of the at least one mutated amino acid residue is located at positions 110 to 120 and 170 to 180 of the amino acid sequence as shown in SEQ ID NO: 2. For example, the mutation site is located at position 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179 and / or 180 of the amino acid sequence as shown in SEQ ID NO: 2.

[0132] In certain embodiments, the mutated amino acid residue is located at position 114, 116, 177 and / or 178 of the amino acid sequence shown in SEQ ID NO: 2.

[0133] For example, the amino acid sequence of the transposase polypeptide comprises a mutation at the G114 amino acid position compared to the unmutated SB transposase amino acid sequence as set forth in SEQ ID NO: 2. For example, the amino acid sequence of the transposase polypeptide comprises a mutation at the S116 amino acid position compared to the unmutated SB transposase amino acid sequence as set forth in SEQ ID NO: 2. For example, the amino acid sequence of the transposase polypeptide comprises a mutation at the K177 amino acid position compared to the unmutated SB transposase amino acid sequence as set forth in SEQ ID NO: 2. For example, the amino acid sequence of the transposase polypeptide comprises a mutation at the P178 amino acid position compared to the unmutated SB transposase amino acid sequence as set forth in SEQ ID NO: 2.

[0134] For example, the amino acid sequence of the transposase polypeptide comprises mutations at amino acid positions G114 and S116, compared to the unmutated SB transposase amino acid sequence as set forth in SEQ ID NO: 2. For example, the amino acid sequence of the transposase polypeptide comprises mutations at amino acid positions G114 and K177, compared to the unmutated SB transposase amino acid sequence as set forth in SEQ ID NO: 2. For example, the amino acid sequence of the transposase polypeptide comprises mutations at amino acid positions G114 and P178, compared to the unmutated SB transposase amino acid sequence as set forth in SEQ ID NO: 2. For example, the amino acid sequence of the transposase polypeptide comprises mutations at amino acid positions S116 and K177, compared to the unmutated SB transposase amino acid sequence as set forth in SEQ ID NO: 2. For example, the amino acid sequence of the transposase polypeptide comprises mutations at amino acid positions S116 and P178, compared to the unmutated SB transposase amino acid sequence as set forth in SEQ ID NO: 2. For example, compared to the unmutated SB transposase amino acid sequence shown in SEQ ID NO: 2, the amino acid sequence of the transposase polypeptide comprises mutations at amino acid positions K177 and P178.

[0135] In certain embodiments, the at least one mutated amino acid residue is mutated to an alanine residue.

[0136] In certain embodiments, the amino acid residues that are mutated are all mutated to alanine residues.

[0137] In certain embodiments, the amino acid sequence of the transposase polypeptide comprises an amino acid mutation selected from the group consisting of G114A, S116A, K177A, and P178A, compared to the unmutated SB transposase as shown in SEQ ID NO: 2.

[0138] For example, compared to the unmutated SB transposase amino acid sequence as shown in SEQ ID NO: 2, the transposase polypeptide amino acid sequence comprises a mutation at amino acid position G114 to alanine (G114A). For example, compared to the unmutated SB transposase amino acid sequence as shown in SEQ ID NO: 2, the transposase polypeptide amino acid sequence comprises a mutation at amino acid position S116 to alanine (S116A). For example, compared to the unmutated SB transposase amino acid sequence as shown in SEQ ID NO: 2, the transposase polypeptide amino acid sequence comprises a mutation at amino acid position K177 to alanine (K177A). For example, compared to the unmutated SB transposase amino acid sequence as shown in SEQ ID NO: 2, the transposase polypeptide amino acid sequence comprises a mutation at amino acid position P178 to alanine (P178A).

[0139] In certain embodiments, compared to the unmutated SB transposase as shown in SEQ ID NO: 2, the transposase polypeptide amino acid sequence comprises a combination of amino acid mutations selected from the group consisting of:

[0140] i) G114A, S116A;

[0141] ii) G114A, K177A;

[0142] iii) G114A, P178A;

[0143] iv) S116A, K177A;

[0144] v) S116A, P178A; and

[0145] vi)K177A, P178A.

[0146] In another aspect, the present application provides a transposase polypeptide comprising an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 2, wherein the transposase polypeptide comprises an alanine at a position corresponding to position 114 and / or an alanine at a position corresponding to position 116.

[0147] In certain embodiments, the transposase polypeptide comprises the amino acid sequence of SEQ ID NO: 1, any one of SEQ ID NOs: 3-11, or a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1, any one of SEQ ID NOs: 3-11.

[0148] On the other hand, the present application provides a polynucleotide molecule comprising a nucleic acid sequence encoding the aforementioned transposase polypeptide.

[0149] In some embodiments, it includes RNA or DNA. In some aspects, the molecule can be a DNA expression vector. For example, the DNA expression vector can include a transposase encoding sequence operably connected to a promoter (such as T7 or SP6 promoter) for expressing polypeptides in vitro or a promoter for expressing polypeptides in mammalian cells. In some aspects, the polynucleotide molecule can be RNA or mRNA. In other aspects, RNA can include a 5'-cap, an IRES motif, (heterologous) 5'UTR, (heterologous) 3'UTR and / or a poly (A) sequence. In some aspects, RNA can additionally include a poly (A) sequence of 20 to 300 nucleotides.

[0150] On the other hand, the application provides a method for preparing a transposase polypeptide as described above, comprising transfecting cells with a polynucleotide encoding a transposase polypeptide and expressing the polypeptide from the polynucleotide. In another embodiment, the invention provides a host cell comprising the polypeptide or polynucleotide molecule of the embodiment. In some cases, the cell is a mammalian cell, such as a human cell. In some aspects, the cell is a stem cell or an induced pluripotent stem (iPS) cell. In other aspects, the cell is a natural killer (NK) cell, a precursor of a NK cell, a T cell, a precursor of a T cell, or an immune cell. In some cases, the cell may comprise an RNA encoding the transposase polypeptide of the embodiment. In another embodiment, a cell colony is provided, the cell comprising the polypeptide or polynucleotide molecule of the embodiment.

[0151] On the other hand, the application provides a method for genetically engineered cells, comprising: transfecting cells with a DNA vector comprising a sequence of a transposase polypeptide as described above or a nucleic acid encoding a transposase polypeptide and comprising coding flanking transposon repeats, then incubating the cells under conditions suitable for (transient or stable) transposase activity, thereby integrating the genetic elements of selection in the genome of the cell and producing engineered cells. In some aspects, the DNA vector encoding the selected genetic elements of transposon repeats also comprises the sequence of the transposase polypeptide encoding the embodiment. Therefore, in some aspects, the method of the embodiment comprises transfecting cells with a DNA vector comprising a sequence of a transposase polypeptide encoding flanking transposon repeats and the sequence of the transposase encoding the embodiment (which is under the control of a promoter sequence), then incubating the cells under conditions suitable for transposase expression and activity, thereby integrating the genetic elements of selection in the genome of the cell and producing engineered cells. In some aspects, the method comprises a third step of separating or cultivating engineered cells. In some aspects, the genetic elements selected are screenable or selectable markers. In other aspects, the genetic elements selected can encode antibodies, inhibitory nucleic acids (for example, small interfering RNA (siRNA)), therapeutic polypeptides, T cell receptors (TCR), chimeric antigen receptors (CAR) or enhancers of immune cell function. In specific aspects, the genetic elements encoding CAR or TCR of selection. In other aspects, the genetic elements selected can be genes or parts thereof for replacing or modifying the corresponding genes from cells (for example, changing the sequence or expression of genes or "knockout" gene expression in cells). In some aspects, the cells of transfection are mammalian cells, such as human cells. In some cases, cells can be stem cells or iPS cells. In some aspects, cells can be immune system cells or their precursors, such as NK cells, T cells, precursors of NK cells or precursors of T cells.

[0152] In some aspects, transfection cells can include the use of chemical-based transfection reagents, electroporation of cells or other technologies for providing nucleic acid and / or protein to the cytoplasm and nucleus of cells. For example, salt precipitates (such as CaPO4 precipitates), lipids (such as charged or non-polar lipids), cationic polymers, PEG-complexes and / or protein complexes (such as cationic polypeptides) can be used to transfect cells. In some aspects, transfection can involve the use of liposomes, such as phospholipid liposomes (for example, liposomes incorporating glycerophospholipids or sphingolipids). In other aspects, cells can be transduced with viral vectors (for example, adenovirus, adeno-associated virus, retrovirus (such as slow virus) or vaccinia virus vectors). In some aspects, the viral vector used according to the embodiment is a non-integrating viral vector. Those skilled in the art will recognize that in some aspects, the transposase of the embodiment can be delivered to cells together with or separately from the nucleic acid encoding the transposon repetition. For example, in some aspects, protein transfection reagents can be used to deliver transposase to cells as recombinant polypeptides, and nucleic acid transfection systems or viral vectors can be used to deliver the nucleic acid molecules comprising the transposon repetitions. In additional aspects, RNA encoding the transposase is co-transfected with DNA comprising the transposon repeats and the selected genetic element.

[0153] In other aspects, the method further includes a DNA vector transfection cell population with a transposase polypeptide or a nucleic acid encoding a transposase polypeptide and a sequence of a selected genetic element comprising a transposon repeat sequence on the coding flank, and the colony is hatched under conditions suitable for transposase activity, so as to integrate the genetic element selected in the genome of the cell and produce an engineered cell colony. In a specific aspect, the method includes a colony of a DNA vector transfecting T cell or a T cell precursor with a transposase polypeptide or a nucleic acid encoding a transposase polypeptide and a sequence of a CAR comprising a transposon repeat on the coding flank, and hatching colony under conditions suitable for transposase activity, so as to integrate CAR in the genome of the cell and produce a colony of engineered T cell or a T cell precursor. In other aspects, the method includes a colony of a DNA vector transfecting T cell or a T cell precursor with a sequence (operably connected to a promoter) of a CAR sequence and a coding embodiment transposase comprising a transposon repeat on the coding flank, and hatching colony under conditions suitable for transposase activity, so as to integrate CAR in the genome of the cell and produce a colony of engineered T cell or a T cell precursor. In some aspects, the engineered cells can be additionally cultured in a medium that selectively enhances the proliferation of CAR-expressing T cells.

[0154] In another embodiment, a method of providing a T cell response in a human subject having a disease is provided, comprising first obtaining a population of engineered T cells or T cell precursors according to the embodiments, optionally culturing the cells in a medium that selectively enhances the proliferation of CAR-expressing T cells, and then administering an effective amount of CAR-expressing T cells to the subject to provide a T cell response.

[0155] In some aspects, the method of the embodiment includes:(a) obtaining a cell sample from a subject, the sample comprising T cells or T cell progenitors;(b) DNA transfection cells are transfected with a chimeric antigen receptor (CAR) having a transposon flanking and a transposase encoding the embodiment of the invention integrated into the cell genome to provide a transgenic CAR expressing cell colony;(c) optionally, ex vivo culture transgenic CAR cell colonies in a culture medium that selectively enhances CAR expression T cell proliferation;And (d) applying an effective amount of transgenic CAR cells to the subject to provide T cell responses. Therefore, in some aspects, transgenic CAR cells are cultured in vitro for less than 21 days, for example, less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days or less. In some aspects, CAR cells are cultured in vitro for no more than 3 to 5 days. In other aspects, the steps (a)-(d) of the present method (i.e., obtaining a cell sample to administering CAR T cells) are completed in no more than 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 days. In other aspects, the cell sample from the subject can be a sample of peripheral blood or umbilical cord blood less than about 200ml. In some aspects, samples can be collected by plasmapheresis. In some aspects, samples are collected by methods not involving plasmapheresis (e.g., by venipuncture). In further aspects, the initial volume of the cell sample is less than 175 ml, less than about 175 ml, less than 150 ml, less than about 150 ml, less than 125 ml, less than about 125 ml, less than 100 ml, less than about 100 ml, less than 75 ml, less than about 75 ml, less than 50 ml, less than about 50 ml, less than 25 ml, or less than about 25 ml (e.g., when obtained from a subject, the cell sample has an initial volume of between about 50 to about 200 ml, between about 50 to about 100 ml, or between about 100 to about 200 ml).

[0156] In some aspects, the method of the embodiment relates to transfecting cells with DNA encoding a chimeric antigen receptor (CAR) and a transposase. Cell transfection methods are well known in the art, but in some aspects, efficient transfection methods such as electroporation are used. For example, a nuclear transfection device can be used to introduce nucleic acid into cells. In certain embodiments, the transfection step does not involve infecting or transducing cells with viruses, which can cause genotoxicity and / or lead to an immune response for cells containing viral sequences in treated subjects.

[0157] Other aspects of the embodiment relate to transfecting cells with expression vectors encoding CAR. A large number of CAR constructs and their expression vectors are known in the art. For example, in some aspects, the CAR expression vector is a DNA expression vector, such as a plasmid, a linear expression vector or an episome. In some aspects, the vector comprises additional sequences, such as sequences that promote CAR expression, such as promoters, enhancers, poly A signals and / or one or more introns. In some aspects, the CAR coding sequence is flanked by transposon sequences so that the presence of transposase allows the coding sequence to be integrated into the genome of the transfected cell.

[0158] As described in detail above, in some aspects, the transposase of the embodiment of the transfected cell genome is further transfected with promoting CAR encoding sequence to be integrated.In some aspects, transposase is provided as DNA expression vector.In some aspects, transposase is provided as expressible RNA or protein so that the long-term expression of transposase does not occur in transgenic cells.For example, in some aspects, there is provided a transposase encoded by mRNA (for example, mRNA comprising cap and poly-A tail).

[0159] The method of the embodiment can be used to prepare (for example, for clinical trials) CAR+T cells with binding specificity to various tumor antigens (such as CD19, ROR1, CD56, EGFR, CD33, CD123, c-met, GD2). The CAR+T cells produced using this technology can be used to treat patients with leukemia (such as AML, ALL, CML), infection and / or solid tumors. For example, the method of the embodiment can be used to treat cell proliferative diseases, fungi, viruses, bacteria or parasitic infections. Pathogens that can be targeted include but are not limited to Plasmodium, Trypanosoma, Aspergillus (Aspergillus), Candida (Candida), HSV, RSV, EBV, CMV, JC virus, BK virus or Ebola pathogens. Other examples of antigens that can be targeted by the CAR cells of the embodiments include, but are not limited to, CD19, CD20, carcinoembryonic antigen, alpha-fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, melanoma-associated antigen, mutant p53, mutant ras, HER2 / Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Rα, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, or VEGFR2.

[0160] In certain embodiments of the present invention, CAR cells are delivered to individuals in need thereof, such as individuals with cancer or infection. The cells then enhance the individual's immune system to attack the corresponding cancer or pathogenic cells. In some cases, one or more doses of antigen-specific CAR T cells are provided to the individual. In the case of providing two or more doses of antigen-specific CAR T cells to the individual, the duration between administrations should be sufficient to allow for the time of proliferation in the individual, and in a specific embodiment, the duration between doses is 1, 2, 3, 4, 5, 6, 7 days or more.

[0161] The source of allogeneic or autologous T cells modified to contain a chimeric antigen receptor (and in some cases, lack a functional TCR) can be of any kind, but in specific embodiments, for example, the cells are obtained from a reservoir of umbilical cord blood, peripheral blood, human embryonic stem cells, or induced pluripotent stem cells. A suitable dose for therapeutic effect will be at least 10 cells per dose. 5 or between about 10 5 to about 10 10 An exemplary dosing regimen consists of four weekly dosing cycles of increasing doses, starting on day 0 with at least about 10 5Starting with 10 cells, for example, gradually increasing to approximately 10 within a few weeks of starting an intrapatient dose escalation regimen. 10 Suitable modes of administration include intravenous, subcutaneous, intracavitary (e.g., via a reservoir access device), intraperitoneal, and direct injection into the tumor mass.

[0162] The pharmaceutical composition of the present invention can be used alone or in combination with other confirmed agents that can be used to treat cancer. Regardless of whether it is delivered alone or in combination with other agents, the pharmaceutical composition of the present invention can be delivered to various parts of mammals, particularly the human body, to achieve a specific effect by various routes. Those skilled in the art will recognize that although more than one route can be used for administration, a specific route can provide a more direct and more effective reaction than another route. For example, intradermal delivery can be more advantageously used to treat melanoma relative to inhalation. It can be delivered by administration, including applying or dripping the preparation into the body cavity, inhalation or insufflation of an aerosol, or by parenteral introduction, including intramuscular, intravenous, intraportal, intrahepatic, peritoneal, subcutaneous or intradermal administration to achieve local or systemic delivery.

[0163] The composition of the embodiment can be provided in unit dosage form, wherein each dosage unit (e.g., injection) contains a predetermined amount of the composition alone or in appropriate combination with other active agents. As used herein, the term unit dosage form refers to a physically discrete unit suitable as a unit dose for human and animal subjects, each unit containing a predetermined amount of the composition of the present invention alone or in combination with other active agents, which is calculated in an amount sufficient to produce a desired effect in combination with a pharmaceutically acceptable diluent, carrier, or vehicle where appropriate. The specification of the unit dosage form of the present invention depends on the specific pharmacodynamics associated with the pharmaceutical composition in a particular subject.

[0164] An effective amount or sufficient number of isolated transduced T cells are present in a composition and introduced into a subject such that a long-term, specific anti-tumor response is established to reduce the size of the tumor or eliminate tumor growth or regrowth than would otherwise result in the absence of such treatment. Ideally, the amount of transduced T cells reintroduced into the subject results in a reduction in tumor size of about or at least about 10%, about or at least about 20%, about or at least about 30%, about or at least about 40%, about or at least about 50%, about or at least about 60%, about or at least about 70%, about or at least about 80%, about or at least about 90%, about or at least about 95%, about or at least about 98%, or about 100% or 100% when compared to the original or initial (e.g., "treatment day 0") size of the tumor.

[0165] Any composition described herein may be included in a kit. In some aspects, the transposase polypeptide of the embodiment or the nucleic acid encoding the transposase polypeptide is provided in the kit. Such kits may include a variety of additional elements, such as DNA vectors encoding transposon repeats, transfection reagents, cells, CAR expression constructs, culture media, aAPCs, growth factors, antibodies (e.g., for sorting or characterizing CAR T cells) and / or plasmids encoding CAR or transposase.

[0166] In a non-limiting example, the kit comprises a transposase polypeptide of the embodiment or a nucleic acid encoding a transposase polypeptide, one or more reagents for producing a CAR-expressing construct (with flanking transposon repeats), cells for transfecting the expression construct, and / or one or more instruments for obtaining cells for transfection expressing the construct (such instruments may be syringes, pipettes, tweezers, and / or any such medically approved device). In another aspect, a transfection device such as an electroporation device is included.

[0167] The test kit may comprise one or more suitable aliquots of the composition of the present invention or reagents for producing the composition of the present invention. The components of the test kit may be packaged in aqueous media or lyophilized form. The container means of the test kit may comprise at least one vial, test tube, flask, bottle, syringe or other container means that can be placed and preferably suitable aliquots of the composition. In the case of a plurality of components in the test kit, the test kit will generally also comprise a second, third or other additional container that can be separately placed in other components. However, various combinations of components may be included in the vial. The test kit of the present invention will generally also comprise a device for comprising the chimeric receptor construct and any other reagent container in a sealed constraint for commercial sale. For example, such container may comprise an injection or blow-molded plastic container that retains the desired vial.

[0168] Without intending to be bound by any theory, the following examples are merely intended to illustrate the transposase polypeptide, preparation method, and use of the present invention, and are not intended to limit the scope of the present invention.

[0169] Example

[0170] Example 1 Transposase enzyme cleavage activity test

[0171] Figure 1A shows a schematic diagram of a transposase enzyme activity assay. The streptomycin resistance gene in the plasmid is split by a transposon encoding a zeocin resistance gene. This plasmid and the transposase plasmid are co-transfected into HEK293T cells. Plasmid DNA is extracted from the HEK293T cells and used to transform E. coli. Plasmids cleaved by the transposase lose zeocin resistance but gain streptomycin resistance.

[0172] Figure 1B shows the results of testing the enzymatic activity of different transposases. HEK293T cells were seeded on a 6-well plate and cultured overnight. The cells were then transfected with equal amounts of transposon plasmids, plasmids containing no transposase, an inactivated transposase mutant (E279D), SB11 transposase, and SB100X transposase. After 72 hours, the cells were harvested, plasmid DNA was extracted using a Plasmid Mini Kit, and Escherichia coli was transformed. The transformed bacteria were plated on streptomycin-containing plates, cultured overnight, and single colonies were counted. The number of colonies reflects the activity of the transposase. The enzymatic activity of SB100X transposase was higher than that of SB11, while the transposon alone or the inactivated transposase mutant had no enzymatic activity. Plasmid DNA extracted from the colonies and sequenced further demonstrated that the streptomycin-separated reading frame on the transposon was restored under the action of the transposase.

[0173] Example 2 Transposase mutants prepared by alanine scanning method have different enzymatic activities

[0174] Transposase mutants were generated by site-directed mutagenesis and chain amplification. Following sequencing and identification, plasmids encoding the various transposase mutants were co-transfected with the transposon plasmid into HEK293T cells, and transposase cleavage activity was assayed using the method described in Example 1. The E279D transposase mutant served as a negative control. Results were normalized to those of the SB32X transposase (SEQ ID NO: 2). All error bars represent the mean average (SEM) of three independent replicates. The results demonstrated that the multiple transposase mutants provided herein exhibited higher cleavage activity than SB32X.

[0175] Example 3 Transposition activity test of transposase mutants

[0176] HeLa cells were cultured in DMEM (Gibco, NY, USA) containing fetal bovine serum. The cells were co-transfected with 250 ng of plasmid DNA encoding each transposase mutant and 750 ng of transposon plasmid DNA encoding the Zeocin resistance gene. After 24 hours, the cells were transferred to a 10-cm culture dish. Zeocin-resistant cell clones were selected using a culture medium containing Zeocin. Two weeks later, the cells were fixed with 4% paraformaldehyde, stained, and the number of cell colonies was counted. The results were normalized to SB32X transposase (SEQ ID NO: 2). All error bars represent the SEM of three independent replicates. The results show that the multiple transposase mutants provided in this application have higher enzymatic activity than SB32X.

[0177] Example 4 Preparation of CAR-T cells using transposase mutants

[0178] T cells were sorted from PBMC using magnetic beads, and minicircle DNA expressing CD19-CAR was electroporated into T cells simultaneously with three transposase plasmids, SB11, SB100X, or Tri-FITase, using an electroporator (Lonza). After culturing overnight in RPMI-1640 medium containing 10% FBS and 0.1% DMSO, live cells were enriched by OptiPrep (Axis-Shield) gradient density centrifugation. CAR-T cells were then expanded and cultured in X-VIVO15 medium containing 10% FBS and cytokines (50 IU / mL IL-2, 50 IU / mL IL-7, and 100 IU / mL IL-15), and the culture medium was refreshed every 2 days. The number of live cells was counted at different times after electroporation, and the CAR-T positive rate and MFI were detected by flow cytometry. Dead cells were excluded with 7-AAD, T cells were labeled with PE-Cy7-Anti-CD3, and CAR-T cells were labeled with FITC-CD19-Fc. T cells transfected with different transposases maintained high viability. The CAR-positive rate in T cells transfected with Tri-FITase transposase was higher than that in T cells transfected with SB11 transposase, and similar to that in T cells transfected with SB100X.

Claims

1. A transposase polypeptide comprising at least one mutated amino acid residue compared to the amino acid sequence of positions 100 to 180 of the unmutated SB transposase as shown in SEQ ID NO:

2. 2 . The transposase polypeptide according to claim 1 , wherein the amino acid sequence of the transposase polypeptide comprises 1 to 20 mutated amino acid residues.

3. The transposase polypeptide of claim 1, wherein the amino acid sequence of the transposase polypeptide comprises at least two mutated amino acid residues.

4. The transposase polypeptide according to any one of claims 1 to 3, wherein the mutation site of the at least one mutated amino acid residue is located at positions 110 to 120 and / or positions 170 to 180 of the amino acid sequence as shown in SEQ ID NO:

2.

5. The transposase polypeptide according to any one of claims 1 to 4, wherein the mutated amino acid residue is located at position 114, 116, 177 and / or 178 of the amino acid sequence shown in SEQ ID NO:

2.

6. The transposase polypeptide according to any one of claims 1 to 5, wherein the amino acid sequence of the transposase polypeptide comprises a mutation at amino acid position G114 compared to the unmutated SB transposase amino acid sequence shown in SEQ ID NO:

2.

7. The transposase polypeptide according to any one of claims 1 to 6, wherein the amino acid sequence of the transposase polypeptide comprises a mutation at amino acid position S116 compared to the unmutated SB transposase amino acid sequence shown in SEQ ID NO:

2.

8. The transposase polypeptide according to any one of claims 1 to 7, wherein the amino acid sequence of the transposase polypeptide comprises a mutation at amino acid position K177 compared to the unmutated SB transposase amino acid sequence shown in SEQ ID NO:

2.

9. The transposase polypeptide according to any one of claims 1 to 8, wherein the amino acid sequence of the transposase polypeptide comprises a mutation at amino acid position P178 compared to the unmutated SB transposase amino acid sequence shown in SEQ ID NO:

2.

10. The transposase polypeptide of any one of claims 1-9, wherein the at least one mutated amino acid residue is mutated to an alanine residue.

11. The transposase polypeptide of any one of claims 1-10, wherein the amino acid residue mutations are all mutated to alanine residues.

12. The transposase polypeptide according to any one of claims 1 to 11, wherein the amino acid sequence of the transposase polypeptide comprises an amino acid mutation selected from the group consisting of G114A, S116A, K177A, and P178A compared to the unmutated SB transposase as shown in SEQ ID NO:

2.

13. The transposase polypeptide according to any one of claims 1 to 12, wherein the amino acid sequence of the transposase polypeptide comprises a mutation at amino acid position G114 to alanine (G114A) compared to the unmutated SB transposase amino acid sequence shown in SEQ ID NO:

2.

14. The transposase polypeptide according to any one of claims 1 to 13, wherein the amino acid sequence of the transposase polypeptide comprises a mutation at amino acid position S116 to alanine (S116A) compared to the unmutated SB transposase amino acid sequence shown in SEQ ID NO:

2.

15. The transposase polypeptide according to any one of claims 1 to 14, wherein the amino acid sequence of the transposase polypeptide comprises a mutation at amino acid position K177 to alanine (K177A) compared to the unmutated SB transposase amino acid sequence shown in SEQ ID NO:

2.

16. The transposase polypeptide according to any one of claims 1 to 15, wherein the amino acid sequence of the transposase polypeptide comprises a mutation at amino acid position P178 to alanine (P178A) compared to the unmutated SB transposase amino acid sequence shown in SEQ ID NO:

2.

17. The transposase polypeptide according to any one of claims 1 to 16, wherein the amino acid sequence of the transposase polypeptide comprises a combination of amino acid mutations selected from the group consisting of: i) G114A, S116A; ii) G114A, K177A; iii) G114A, P178A; iv) S116A, K177A; v) S116A, P178A; and vi)K177A, P178A.

18. A transposase polypeptide comprising a sequence having at least 90% identity to SEQ ID NO: 2, wherein the transposase polypeptide comprises an alanine at a position corresponding to position 114 and / or an alanine at a position corresponding to position 116.

19. The transposase polypeptide according to any one of claims 1-18, comprising the amino acid sequence shown in any one of SEQ ID NO: 1 and SEQ ID NO: 3-11.

20. A polynucleotide molecule comprising a nucleic acid sequence encoding the transposase polypeptide of any one of claims 1-19.

21. The polynucleotide molecule of claim 20, comprising RNA or DNA.

22. The polynucleotide molecule of any one of claims 20-21, wherein the DNA comprises a promoter for expressing the transposase polypeptide.

23. The polynucleotide molecule of any one of claims 20-22, wherein the DNA comprises a promoter for expressing the transposase polypeptide in mammalian cells.

24. The polynucleotide molecule of any one of claims 20-21, wherein the molecule is mRNA.

25. The polynucleotide molecule of claim 24, comprising a 5' cap, an IRES motif and / or a poly (A) sequence.

26. The polynucleotide molecule according to any one of claims 20 to 25, comprising a poly(A) sequence of 20 to 300 nucleotides.

27. A construct comprising the polynucleotide molecule of any one of claims 20-26 and a regulatory sequence, wherein the regulatory sequence is operably linked to the expressible polynucleotide to allow expression of the polynucleotide.

28. A cell comprising the transposase polypeptide of any one of claims 1-19, the polynucleotide molecule of any one of claims 20-26, and / or the construct of claim 27.

29. The cell of claim 28, wherein the cell comprises a mammalian cell.

30. The cell of any one of claims 28-29, wherein the cell comprises a human cell.

31. The cell of any one of claims 28-30, wherein the cell is autologous.

32. The cell of any one of claims 28-31, wherein the cell is allogeneic.

33. The cell of any one of claims 28-32, wherein the cell comprises a stem cell or an induced pluripotent stem (iPS) cell.

34. The cell of any one of claims 28-33, wherein the cell comprises an immune effector cell.

35. The cell of any one of claims 28-34, wherein the cell comprises a natural killer (NK) cell, a T cell, or a precursor of a NK cell or a T cell.

36. The cell of any one of claims 28-35, wherein the cell comprises the transposase polypeptide of any one of claims 1-19.

37. The cell of any one of claims 28-36, wherein the cell comprises mRNA or DNA encoding the transposase polypeptide of any one of claims 1-19.

38. A transposon system comprising: (a) a transposon unit containing inverted terminal repeats (ITRs) or direct terminal repeats (DTRs) flanking a target sequence to be inserted into the genome of a target cell; and (b) The transposase polypeptide of any one of claims 1-19, the polynucleotide molecule of any one of claims 20-26, and / or the construct of claim 27.

39. Use of the transposon system of claim 38 for delivering genes to target cells in vitro.

40. A method for genetically engineering cells, comprising the steps of: (a) transfecting a target cell with the transposon system according to claim 38; (b) culturing the target cell under conditions that allow the target cell to be cultured, thereby producing an engineered cell.

41. The method of claim 40, further comprising: (c) isolating the engineered cells.

42. The method of any one of claims 40-41, wherein the target sequence encodes a screenable or selectable marker.

43. The method of any one of claims 40-42, wherein the target sequence encodes a therapeutic polypeptide or an inhibitory nucleic acid.

44. The method of any one of claims 40-43, wherein the target sequence encodes an antibody, a T cell receptor (TCR), or a chimeric antigen receptor (CAR).

45. The method of any one of claims 40-44, wherein the target sequence encodes a CAR.

46. The method of claim 45, wherein the CAR targets a tumor antigen.

47. The method of claim 46, wherein the tumor antigen is CD19, CD20, ROR1, CD22 carcinoembryonic antigen, alpha-fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate specific antigen, melanoma-associated antigen, mutated p53, mutated ras, HER2 / Neu, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11Rα, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, combined HER2-HER3, or combined HER1-HER2.

48. The method of any one of claims 40-47, wherein the target cell comprises a mammalian cell.

49. The method of any one of claims 40-48, wherein the target cell comprises a human cell.

50. The method of any one of claims 40-49, wherein the target cell is autologous.

51. The method of any one of claims 40-50, wherein the target cells are allogeneic.

52. The method of any one of claims 40-51, wherein the target cells comprise stem cells or induced pluripotent stem (iPS) cells.

53. The method of any one of claims 40-52, wherein the target cell comprises an immune effector cell or a precursor thereof.

54. The method of any one of claims 40-53, wherein the target cell comprises a natural killer (NK) cell, a T cell, or a precursor of a NK cell or a T cell.

55. The method of any one of claims 40-54, wherein transfecting the target cell comprises using a polymer-, polypeptide-, or lipid-based transfection reagent.

56. The method of any one of claims 40-55, wherein transfecting the target cell comprises electroporating the cell.

57. The method according to any one of claims 40 to 56, further comprising: (a) transfecting a cell population with the transposon system of claim 38; (b) incubating the population under conditions suitable for transposase activity, thereby integrating the target sequence in the genome of the cells and generating a population of engineered cells.

58. The method of any one of claims 40-57, further comprising: (a) allowing the transposon system of claim 38 to transfect a population of T cells or T cell precursors, wherein the target sequence Column encoding CAR or TCR; (b) incubating the population under conditions suitable for transposase activity, thereby integrating the CAR in the genome of the cells and generating a population of engineered T cells or T cell precursors.

59. The method of claim 58, further comprising: (c) culturing the engineered cells in a medium that selectively enhances the proliferation of CAR- or TCR-expressing T cells.

60. A pharmaceutical composition comprising the transposase polypeptide of any one of claims 1-19, the polynucleotide molecule of any one of claims 20-26, and / or the construct of claim 27, and a pharmaceutically acceptable carrier and / or excipient.

61. A kit comprising (a) a transposon unit containing inverted terminal repeats (ITRs) or DTRs flanking a target sequence to be inserted into the genome of a target cell; and (b) The transposase polypeptide of any one of claims 1-19, the polynucleotide molecule of any one of claims 20-26, and / or the construct of claim 27.

62. Use of the transposase polypeptide of any one of claims 1-19, the polynucleotide molecule of any one of claims 20-26, the construct of claim 27, the cell of any one of claims 28-37, the transposase system of claim 38 and / or the pharmaceutical composition of claim 60 in the preparation of a medicament for treating a disease or condition.

63. The use according to claim 62, wherein the disease or disorder is cancer.

64. The use according to claim 62, wherein the disease or disorder is an infectious disease.

65. A compound for treating a disease or condition, wherein the compound comprises the transposase polypeptide of any one of claims 1-19, the polynucleotide molecule of any one of claims 20-26, the construct of claim 27, the cell of any one of claims 28-37, the transposase system of claim 38, the pharmaceutical composition of claim 60, and / or the kit of claim 61.

Images