WO2024061314A1 - Système de transposon et son application - Google Patents

Système de transposon et son application Download PDF

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WO2024061314A1
WO2024061314A1 PCT/CN2023/120383 CN2023120383W WO2024061314A1 WO 2024061314 A1 WO2024061314 A1 WO 2024061314A1 CN 2023120383 W CN2023120383 W CN 2023120383W WO 2024061314 A1 WO2024061314 A1 WO 2024061314A1
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transposase
transposon
cells
cell
dna
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PCT/CN2023/120383
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English (en)
Chinese (zh)
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张平静
刘韬
宋成义
高波
孙艳
钱其军
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上海吉量医药工程有限公司
浙江吉量科技有限公司
上海细胞治疗集团有限公司
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Publication of WO2024061314A1 publication Critical patent/WO2024061314A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the invention relates to the field of genetic engineering, and specifically to a transposon system and its application.
  • Transposable genetic elements also known as transposons, are segments of DNA that can move from one genomic location to another within a single cell. Transposons can be divided into two major categories based on their transposition mechanisms: (1) for elements called retrotransposons, transposition can occur through reverse transcription of RNA intermediates, and (2) For DNA transposons, transposition can occur through direct transposition of DNA flanked by inverted terminal repeats (ITRs). Active transposons encode one or more proteins required for transposition, and natural active DNA transposons carry transposase genes. As gene transfer vectors, transposons have been widely used in research in the fields of transgenesis, gene capture, and gene therapy, and have achieved good results.
  • Tc1/Mariner transposons are the most widely distributed DNA transposon superfamily in nature, including bacteria, invertebrates and vertebrates. Among vertebrates, the Tc1/Mariner transposon is most widely distributed in teleost fishes. Through bioinformatics and experimental verification, 15 Tc1/Mariner superfamily transposon members with natural transposition activity have been reported, namely Tc1, Tc3, Minos, Mos1, Bari3, Fot1, impala, Famar1, Osmar5, ISY100, Mboumar-9, Passport, Tana1, Thm3, SB, ZB transposons.
  • Naturally active transposons may cause genome instability. Therefore, during evolution, transposons will accumulate mutations and gradually reduce or even lose their activity. For example, most of the Tc1/Mariner superfamily transposons found in higher animals (such as mammals) The transposition activity is lost due to defects in the open reading frame of the transposase (such as mutations, frameshifts, insertions, deletions, or the presence of stop codons).
  • the Sleeping Beauty (SB) transposon and PiggyBac transposon which are currently the most widely used in clinical gene therapy, are often highly efficient mutations that have undergone bioinformatics molecular reconstruction and/or engineering transformation. type transposase/transposon system.
  • the mammalian innate immune system has the ability to recognize and direct responses against foreign DNA.
  • the main signal that triggers this reaction is unmethylated CpG motifs in DNA sequences of microbial origin. These motifs are rare in vertebrate DNA but abundant in bacterial and some viral DNA. Since gene therapy often involves the delivery of DNA from bacterially derived plasmids or recombinant viruses, it inevitably results in varying degrees of severity acute inflammatory response. For example, non-viral gene delivery vectors composed of cationic lipid-DNA complexes have the most severe reactions, producing side effects at lower doses and lethality at higher doses.
  • plasmid DNA in gene therapy contains unmethylated CG dinucleotides (CpG), it will cause an inflammatory response in targeted tissues or cells and reduce the duration of transgene expression. Retention of even a single CpG in pDNA is sufficient to trigger an inflammatory response, whereas pDNA vectors without CpGs are not.
  • the expression efficiency of transposon-integrated genes can be improved by using a CpG-free pDNA expression vector as a transposon system.
  • the present invention attempts to eliminate or reduce the CpG motif of TIR in the transposon plasmid DNA vector, reduce CpG and improve the safety and effectiveness of the non-viral vector.
  • the present invention relates to a transposon system, including a transposase or a polynucleotide containing its coding sequence, and/or a transposon DNA containing a terminal inverted repeat sequence; the transposase is a mutant transposase or Fusion transposase, the fusion transposase is a wild-type ZB transposase or its mutant transposase fused with a functional polypeptide.
  • the invention also relates to the use of transposon systems in genetic modification.
  • the terminal inverted repeat sequence may or may not contain mutations compared to the wild-type terminal inverted repeat sequence.
  • the mutant transposase in the present invention is an engineered ZB transposase, which is called BZ transposase.
  • the BZ transposase or transposon system of the present invention has significantly improved integration efficiency and transgene expression level compared with the wild-type ZB transposase or ZB transposition system.
  • the BZ transposase of the present invention can be used in stably transfected cells, cell line development, genome modification, gene therapy, cell therapy, transgenic animals, etc. in the form of virus, plasmid DNA, mRNA or protein.
  • the BZ transposase of the invention comprises at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% compared to full-length SEQ ID NO: 1 An amino acid sequence with sequence identity and one or more amino acid substitution mutations compared to SEQ ID NO: 1.
  • the BZ transposase has increased transposition efficiency compared to a wild-type ZB transposase with an amino acid sequence as set forth in SEQ ID NO: 1.
  • the BZ transposase has one or more amino acid substitutions in its DNA binding and oligomerization domains, DDE catalytic domain, or combinations thereof.
  • the BZ transposase of the invention comprises an amino acid sequence having mutations at one or more of the following positions compared to full-length SEQ ID NO: 1: 5, 21, 35, 38, 56, 61, 71, 73, 110, 125, 134, 137, 138, 144, 186, 188, 189, 204, 215, 216, 217, 218, 215-218, 235, 251 and 289, among which, The position number is the position number of SEQ ID NO:1.
  • the BZ transposase also has mutations at one or more of the following positions: 79, 120, and 208, wherein the position number is that of SEQ ID NO: 1.
  • the amino acid sequence of the BZ transposase provided by the invention has at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least Amino acid sequences that are 99% sequence identical and have mutations at one or more of the following positions: 5, 21, 35, 38, 56, 61, 71, 73, 110, 125, 134, 137, 138 , 144, 186, 188, 189, 204, 215, 216, 217, 218, 215-218, 235, 251 and 289, wherein the position number is the position number of SEQ ID NO: 1.
  • the BZ transposase also has mutations at one or more of the following positions: 79, 120, and 208, wherein the position number is that of SEQ ID NO: 1.
  • the mutations at each position are independently selected from the mutations shown in Table A below:
  • the mutations at positions 79, 120 and 208 are independently selected from the substitution mutations shown in Table B below:
  • the one or more amino acid mutations include mutations of aspartic acid or glutamic acid to neutral or basic amino acids. In some embodiments, the one or more amino acid substitutions are based on conservative principles of the Tcl/Mariner transposon family.
  • the BZ transposase of the invention is a BZ transposase having the mutations shown at the following positions of SEQ ID NO: 1: 5S, 21C, 21K, 35I, 38R, 56L, 61R, 71H , 71R, 73L, 110K, 110R, 125L, 134A, 137T, 138G, 138K, 144A, 144E, 186N, 188Y, 189A, 204V, 215-218DAVQ, 216A, 217V, 218K, 235R, 251T or K289A, where, the value Indicates the position of mutation, and the letter after the numerical value indicates the amino acid residue after mutation.
  • the BZ transposase of the invention has substitution mutations at at least positions 71 and 110 compared to SEQ ID NO: 1.
  • the BZ transposase of the invention in addition to the substitution mutations at positions 71 and 110, is selected from the group consisting of 5, 21, 22, 35, 38, 56, 61, 73, 79, 125 , 134, 137, 138, 144, 186, 188, 189, 204, 215-218, 216, 217, 218, 235, 251, 289, 120 and 208 have substitution mutations at one or more positions, the positions The position number is SEQ ID NO:1.
  • the BZ transposase of the invention in addition to the substitution mutations at positions 71 and 110, is selected from the group consisting of 5, 21, 22, 79, 120, 125, 134, 137, 138, 144 , 189, 208, 216 and 251 have substitution mutations at one or more positions, and the position number is the position number of SEQ ID NO: 1. In some embodiments, the BZ transposase of the invention has substitution mutations at at least positions 71, 79 and 110 compared to SEQ ID NO: 1.
  • the BZ transposase of the invention has substitution mutations at positions 71 and 110, as well as substitution mutations at one or more of the following positions: 5, 21, 22, 134, 137, 138, 144, 189, 208, 216 and 251; preferably, at the same time, there is a substitution mutation at one or more of the following positions: 134, 137, 138, 144, 189 , 208, 216 and 251.
  • the BZ transposase of the invention has substitution mutations at position 71, position 110, position 208, and at one or more of the following positions Have substitution mutations: 5, 21, 22, 134, 137, 138, 144, 189, 216 and 251; preferably, have substitution mutations at one or more of the following positions: 134, 137, 138, 144 ,189, 216 and 251.
  • the BZ transposase of the invention has substitution mutations at positions 71, 110, 208 and 216 compared to SEQ ID NO: 1, while at one of the following positions or There are substitution mutations at multiple positions: 5, 21, 22, 134, 137, 138, 144, 189 and 251; preferably, there are substitution mutations at one or more of the following positions: 134, 137, 138 , 144, 189 and 251.
  • the BZ transposase of the invention has substitution mutations at positions 71, 110, 208, 216 and 251, while at the following positions There are substitution mutations at one or more positions: 5, 21, 22, 134, 137, 138, 144 and 189; preferably, there are substitution mutations at one or more of the following positions: 134, 137 , 138, 144 and 189.
  • the BZ transposase of the invention has substitution mutations at positions 71, 110, 189, 208, 216 and 251 compared to SEQ ID NO: 1, At the same time, there is a substitution mutation at one or more of the following positions: 5, 21, 22, 134, 137, 138, and 144; preferably, at the same time, there is a substitution mutation at one or more of the following positions: 134 , 137, 138 and 144.
  • the BZ transposase of the invention is at position 71, 110, 138, 189, 208, 216 and 251 compared to SEQ ID NO: 1
  • substitution mutations and at the same time have substitution mutations at one or more of the following positions: 5, 21, 22, 134, 137 and 144; preferably, at the same time have substitution mutations at one or more of the following positions :134, 137 and 144.
  • the BZ transposase of the invention has substitution mutations at positions 71, 79 and 110 as compared to SEQ ID NO: 1, while at one or more of the following positions Having substitution mutations: 5, 21, 22, 120, 125, 134, 137, 138, 144, 189, 208, 216 and 251; preferably having substitution mutations at one or more of the following positions: 134, 137 , 138, 144, 189, 208, 216 and 251.
  • the BZ transposase of the invention has substitution mutations at positions 71, 79, 110 and 208 compared to SEQ ID NO: 1, while at one of the following positions or There are substitution mutations at multiple positions: 5, 21, 22, 120, 125, 134, 137, 138, 144, 189, 216 and 251; preferably at the same time, there are substitution mutations at one or more of the following positions: 134 , 137, 138, 144, 189, 216 and 251.
  • the BZ transposase of the invention has substitution mutations at positions 71, 79, 110, 208 and 216, while at the following positions There are substitution mutations at one or more positions: 5, 21, 22, 120, 125, 134, 137, 138, 144, 189 and 251; preferably, there are substitution mutations at one or more of the following positions at the same time :134, 137, 138, 144, 189 and 251.
  • the BZ transposase of the invention has substitution mutations at positions 71, 79, 110, 208, 216 and 251 compared to SEQ ID NO: 1, Also have substitution mutations at one or more of the following positions: 5, 21, 22, 120, 125, 134, 137, 138, 144 and 189; It is preferred to have substitution mutations at one or more of the following positions: 134, 137, 138, 144 and 189.
  • the BZ transposase of the invention is at position 71, 79, 110, 189, 208, 216 and 251 compared to SEQ ID NO: 1
  • substitution mutations and have substitution mutations at one or more of the following positions: 5, 21, 22, 120, 125, 134, 137, 138 and 144; preferably at one or more of the following positions at the same time
  • substitution mutations on: 134, 137, 138 and 144 There are substitution mutations on: 134, 137, 138 and 144.
  • the BZ transposase of the invention is at positions 71, 79, 110, 138, 189, 208, 216 and There is a substitution mutation at position 251, and at the same time, there is a substitution mutation at one or more of the following positions: 5, 21, 22, 120, 125, 134, 137 and 144; preferably at one or more of the following positions at the same time There are substitution mutations at positions: 134, 137 and 144.
  • the substitution mutation at position 71 is Q mutation into R, K or H
  • the substitution mutation at position 79 is Q mutation into R, K or H
  • the substitution mutation at position 110 is H mutation. is R or K.
  • the substitution mutations at each position are selected from the substitution mutations shown in Table A and Table B respectively.
  • the mutation at position 22 is D to A, L, I, G or V.
  • the substitution mutations at each of the above positions are independently selected from: 5S, 21K, 22A, 120S, 125L, 35I, 56L, 61R, 71H, 71R, 73L, 79R, 94E, 110K, 110R, 120G, 120S , 125L, 125M, 134A, 137R, 137T, 138G, 138K, 138R, 144A, 144E, 186N, 188H, 188Y, 189A, 204V, 208V, 216A, 217A, 217V, 218K, 235R and 251T, where the numerical values refer to The amino acid position number of SEQ ID NO: 1, the letter after the numerical value indicates the amino acid residue after substitution mutation.
  • the BZ transposase of the invention is a BZ transposase having any one or more of the following sets of mutations compared to SEQ ID NO: 1:
  • Q71R ⁇ H110R refers to having multiple mutations Q71R and H110R at the same time, and others are similar.
  • the BZ transposase of the invention has substitution mutations at at least positions 208 and 216 compared to SEQ ID NO: 1.
  • the BZ transposase of the invention in addition to the substitution mutations at positions 208 and 216, is selected from the group consisting of 5, 21, 22, 35, 38, 56, 61, 71, 73, 110 ,125, There are substitution mutations at one or more positions of 134, 137, 138, 144, 186, 188, 189, 204, 215-218, 217, 218, 235, 251, 79 and 120, and the position number is SEQ ID NO : Position number of 1.
  • the BZ transposase of the invention is selected from the group consisting of 5, 21, 22, 79, 120, 125, 134, 137, 138, 144 There are substitution mutations at one or more positions of , 189 and 251, and the position number is the position number of SEQ ID NO: 1.
  • the BZ transposase of the present invention has substitution mutations at positions 208, 216 and 251, while at the same time, it is selected from the group consisting of 5, 21, 22, One or more of 35, 38, 56, 61, 71, 73, 110, 125, 134, 137, 138, 144, 186, 188, 189, 204, 215-218, 217, 218, 235, 79 and 120
  • substitution mutations at the position and the position number is the position number of SEQ ID NO: 1; preferably, there is a substitution mutation at one or more of the following positions: 134, 137, 138, 144 and 189.
  • the BZ transposase of the present invention has substitution mutations at positions 189, 208 and 216, while at the same time, it is selected from the group consisting of 5, 21, 22, 35, 38, 56, 61, 71, 73, 110, 125, 134, 137, 138, 144, 186, 188, 204, 215-218, 217, 218, 235, 79 and 120
  • the position number is the position number of SEQ ID NO: 1; preferably, there are substitution mutations at one or more of the following positions: 134, 137, 138, 144 and 251.
  • the BZ transposase of the present invention has substitution mutations at positions 189, 216, 208 and 251, while at the same time, at positions 5, 21 , 22, 35, 38, 56, 61, 71, 73, 110, 125, 134, 137, 138, 144, 186, 188, 204, 215-218, 217, 218, 235, 79 and one or more of 120
  • substitution mutations at three positions and the position number is the position number of SEQ ID NO: 1; preferably, there are substitution mutations at one or more of the following positions: 134, 137, 138 and 144.
  • the BZ transposase of the invention has substitution mutations at positions 138, 189, 216, 208 and 251, while being selected from One of 5, 21, 22, 35, 38, 56, 61, 71, 73, 110, 125, 134, 137, 144, 186, 188, 204, 215-218, 217, 218, 235, 79 and 120
  • the position number is the position number of SEQ ID NO: 1; preferably, there are substitution mutations at one or more of the following positions: 134, 137 and 144.
  • the substitution mutations at each position are selected from the substitution mutations shown in Table A and Table B respectively.
  • the mutation at position 22 is D to A, L, I, G or V.
  • the BZ transposase is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, Positions 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 have mutations.
  • the BZ transposase is as described in any of the above At most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, Positions 24, 25, 26, and 27 have mutations.
  • the substitution mutations at each of the above positions are independently selected from: 5S, 21K, 22A, 120S, 125L, 35I, 56L, 61R, 71H, 71R, 73L, 79R, 94E, 110K, 110R, 120G, 120S , 125L, 125M, 134A, 137R, 137T, 138G, 138K, 138R, 144A, 144E, 186N, 188H, 188Y, 189A, 204V, 208V, 216A, 217A, 217V, 218K, 235R and 251T, where the numerical values refer to The amino acid position number of SEQ ID NO: 1, the letter after the value indicates the amino acid residue after substitution mutation.
  • the BZ transposase can have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27. In various embodiments, a BZ transposase can have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27.
  • the BZ transposase of the invention is a BZ transposase having any one or more of the following sets of mutations compared to SEQ ID NO: 1:
  • the transposase of the present invention is a fusion transposase, which includes the BZ transposase described in any embodiment herein and a functional polypeptide, which is preferably a DNA sequence-specific binding domain. and/or nuclear localization signal domain (NLS).
  • a functional polypeptide which is preferably a DNA sequence-specific binding domain. and/or nuclear localization signal domain (NLS).
  • the DNA sequence-specific binding domain comprises a leucine zipper domain, a CRISPR/Cas domain, a TALE domain, a zinc finger domain, an AAV Rep DNA binding domain, or any combination thereof.
  • the amino acid sequence of the leucine zipper domain is as shown in any one of SEQ ID NOs: 13-15.
  • the nuclear localization signal domain includes SV40 NLS, C-myc NLS, TAF1 NLS, TP53 NLS, STAT3 NLS, or any combination thereof.
  • the functional polypeptide is connected to the wild-type ZB transposase or a mutant thereof with or without a connector.
  • the BZ transposase and the DNA sequence-specific binding domain are separated by a connector.
  • the connector comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or at least 50 amino acids.
  • the sequence of the connector is (GGGS) n or A(EAAAK) n A, where n is a positive integer greater than 0, such as 1, 2, 3, 4 or 5; preferably, the sequence of the connector is GGGS.
  • the transposon can be recognized by the transposase described in any embodiment of the present invention.
  • the DNA sequence of the transposon includes a gene expression element containing terminal inverted repeat sequences at both ends.
  • the terminal inverted repeat sequence is a wild-type terminal inverted repeat sequence or a mutant thereof.
  • the terminal inverted repeat sequence does not contain a CpG motif compared with the wild-type terminal inverted repeat sequence SEQ ID NO: 2, 11 or 12; more preferably, the CpG motif in the terminal inverted repeat sequence is deleted or mutated; further preferably, the mutation is to mutate the CpG motif to ApG, GpG, TpG, CpA or CpT, preferably to TpG or CpA.
  • the transposon includes a gene expression cassette located between terminal inverted repeats.
  • the gene of interest encodes a protein selected from the group consisting of cell receptors, immune checkpoint proteins, cytokines, T cell receptors, B cell receptors, chimeric antigen receptors, and any combination thereof.
  • the invention provides a polynucleotide molecule encoding a transposon system as described in any embodiment herein, including a polynucleotide encoding the transposase, and/or an ITR sequence containing a mutation of transposon DNA.
  • the polynucleotide comprises DNA encoding the BZ transposase or the fusion transposase.
  • the polynucleotide comprises messenger RNA (mRNA) encoding the BZ transposase or the fusion transposase.
  • mRNA messenger RNA
  • the mRNA is chemically modified, including but not limited to pseudouridine ( ⁇ ), N1-methylpseudouridine (m1 ⁇ ), 5-methylcytosine nucleoside (m5C), 5 -Methoxyuridine (5moU).
  • the terminal inverted repeat sequence of the transposon is set forth in SEQ ID NO: 2.
  • the polynucleotide molecule is present in a nucleic acid construct, such as a DNA vector.
  • the DNA vector includes minicircle plasmids, nanoplasmids, doggybones, and other DNA forms that do not contain antibiotics or/and replicon DNA sequences.
  • the transposase polypeptide The nucleotides and/or the transposon are present in a viral vector, for example, the viral vector is selected from an adenovirus vector, an adeno-associated virus vector, a retroviral vector, a herpes simplex virus vector or a vaccinia virus vector.
  • the polynucleotide encoding the transposase and the transposon are present in the same vector or in different vectors.
  • Yet another aspect of the present disclosure provides cells producing transposon systems as described herein. Yet another aspect of the present disclosure provides cells containing polynucleotide molecules of a transposon system as described herein.
  • Yet another aspect of the present disclosure provides a method comprising the step of introducing a transposon system as described herein into a cell.
  • said introducing comprises contacting said cell with a polynucleotide encoding said BZ transposase or said fusion transposase.
  • the polynucleotide comprises DNA encoding the BZ transposase or the fusion transposase.
  • the polynucleotide comprises messenger RNA (mRNA) encoding the BZ transposase or the fusion transposase.
  • mRNA messenger RNA
  • the mRNA is chemically modified.
  • said introducing includes contacting said cell with a nucleic acid construct, such as a DNA vector, containing said transposon.
  • a nucleic acid construct such as a DNA vector
  • the DNA vector includes minicircle plasmids, nanoplasmids, doggybones, and other DNA forms that do not contain antibiotics or/and replicon DNA sequences.
  • the introduction includes contacting the cell with a nucleic acid construct (such as a plasmid vector) containing the transposon and a polynucleotide encoding the BZ transposase or the fusion transposase .
  • said introducing comprises contacting said cell with said BZ transposase or said fusion transposase, preferably by adding said BZ transposase or said fusion transposase directly to a solution containing The transposase protein is provided in the culture medium of the cells (preferably added to the cell culture medium of the cells of the target organism).
  • a solution containing The transposase protein is provided in the culture medium of the cells (preferably added to the cell culture medium of the cells of the target organism).
  • no reagents, vectors or methods that alter the penetration of the protein across the cell membrane may be used.
  • the present invention provides a method for genetically engineering cells, wherein the method does not include a protein transfection step.
  • the method does not include a transposase protein transfection step.
  • the method of the present invention includes the step of introducing the transposase protein without using any vector, reagent or method that alters the penetration of the protein across the cell membrane.
  • the transposon comprises a combination of DNA elements located between two terminal inverted repeats, including but not limited to promoters, enhancers, expressed genes, 5-UTRs, 3-UTR and other sequence elements well known to those skilled in the art.
  • the two termini are reversed Either of the repeating sequences (both reverse complements) comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99 of SEQ ID NO:2 % identity of the sequence.
  • the introduction includes via electroporation, microinjection, calcium phosphate precipitation, cationic polymers, dendrimers, liposomes, microparticle bombardment, fugene, direct sonic loading, cell extrusion, optical
  • the cells are transfected by transfection, protoplast fusion, impalefection, magnetofection, nucleofection or any combination thereof.
  • said introducing includes electroporating said cells.
  • the cells are primary cells isolated from the subject.
  • the subject is human.
  • the subject is a patient suffering from a disease.
  • the subject has been diagnosed with cancer or tumor.
  • the cells are isolated from the blood of the subject.
  • the cells comprise primary immune cells.
  • the cells comprise primary leukocytes.
  • the cells comprise primary T cells.
  • the primary T cells comprise ⁇ T cells, helper T cells, memory T cells, natural killer T cells, effector T cells, or any combination thereof.
  • the primary immune cells comprise CD3+ cells.
  • the cells comprise stem cells.
  • the stem cells are selected from: embryonic stem cells, hematopoietic stem cells, epidermal stem cells, epithelial stem cells, bronchoalveolar stem cells, mammary stem cells, mesenchymal stem cells, intestinal stem cells, endothelial stem cells, neural stem cells, olfactory adult stem cells, neural Crest stem cells, testicular cells, and any combination thereof.
  • the stem cells comprise induced pluripotent stem cells.
  • the present disclosure provides a pharmaceutical composition comprising pharmaceutically acceptable excipients and cells according to any aspect of the present invention.
  • Another aspect of the present disclosure provides a method of treatment, comprising: (a) introducing a transposon and a BZ transposase or a fusion transposase as described herein that recognizes the transposon into a cell, thereby generating a genetically modified cell; (b) administering the genetically modified cell to a patient in need of the treatment.
  • the genetically modified cell comprises a transgene introduced by the transposon.
  • the patient has been diagnosed with cancer or a tumor.
  • the administration comprises infusing the genetically modified cell into a blood vessel of the patient.
  • transposase polynucleotide, transposon, transposon system, cell or pharmaceutical composition according to any aspect of the present invention, which is selected from the following (1 )-(6) any one:
  • Engineered BZ transposase in the form of mRNA is used in cell therapy and transgenesis, further improving safety.
  • the engineered BZ transposase protein in a non-transfectable form is used in cell therapy and transgenics, further improving safety.
  • Figure 1 shows the ZB transposase expression plasmid used for screening BZ transposase and the transposon plasmid containing the EGFP gene expression cassette.
  • Figure 2 shows the results of transposition efficiency screening of the BZ transposase single mutant library.
  • Figure 3 shows that BZ transposase combinatorial mutants improve transposition and expression efficiency in CHO.
  • Figure 4 shows that BZ transposase combinatorial mutants improve transposition and expression efficiency in PBMC.
  • FIG. 5 shows that some embodiments of the BZ transposase combinatorial mutants have increased transposition efficiency (EGFP%).
  • FIG 6 shows that some embodiments of BZ transposase combinatorial mutants improve transposon gene expression efficiency (gMFI).
  • FIG. 7 shows that additional embodiments of BZ transposase combinatorial mutants have increased transposition efficiency (EGFP%).
  • FIG 8 shows that BZ transposase combinatorial mutants of other embodiments improve transposon gene expression efficiency (gMFI).
  • Figure 9 shows that the transposition efficiency of ZB-based fusion transposase is not affected by the type of fusion protein.
  • FIG. 10 shows the transposition efficiency of transposase added in the form of protein.
  • Figure 11 shows that the level of cell proliferation is not affected when the engineered BZ transposase is used for CAR-T cell therapy. The effect can reach the level of high-efficiency transposase of PiggyBac.
  • Figure 12 shows that when the engineered BZ transposase is used for CAR-T cell therapy, the transposition efficiency can reach the level of PiggyBac high-efficiency transposase.
  • Figure 13 shows that when the engineered BZ transposase is used for CAR-T cell therapy, there is no significant difference in the killing effect of the prepared Car-T cells on tumor cell L363.
  • Figure 14 shows the effect of CpG motif mutations in ITR on gene integration of the BZ transposon system.
  • Figure 15 shows the effect of fusing different ZIP structures on transposition efficiency.
  • the present invention optimizes the transposase through engineering methods. amino acid sequence and/or transposon DNA sequence to improve the overall activity of the ZB transposition system.
  • the engineered ZB transposase or ZB transposon system is called BZ transposase or BZ transposon system.
  • transgenes via DNA transposons Compared with viral transduction of immune cells (such as T lymphocytes), delivery of transgenes via DNA transposons has several advantages: ease of use, potential for delivery of large gene fragments, rapid clinical application and low production costs, long-term and high-level stable expression of transgenes, significantly less mutagenicity, non-carcinogenicity and reversibility compared to retroviruses.
  • Ex vivo genetic modification of non-transformed primary human T lymphocytes using gene transfer delivery systems based on non-viral vectors is extremely difficult.
  • the mature cases reported are limited to Sleeping beauty transposon, PiggyBac transposon, and TcBuster transposon.
  • the present invention improves the efficiency of BZ transposase-mediated transposition to make it suitable for clinical application in cellular immunotherapy.
  • CN105018523A proves that ZB transposon is an effective non-viral tool for inserting transgenes into cells, but it still has safety risks for cell or gene therapy.
  • the use of transposase-encoding DNA results in prolonged expression of the transposase protein in target cells.
  • the lack of controlled timing and kinetics of transposase exposure poses the risk of sustained and uncontrolled transposition, which raises safety concerns regarding the conversion of adverse therapeutic cell products.
  • engineered T cells in ongoing trials are cultured for 2-4 weeks after CAR gene delivery, which can reduce cell fitness and therapeutic efficacy.
  • some embodiments of the present invention use a method of engineering highly active BZ transposase based on mRNA expression. This method shortens the time of protein expression and reduces the risk of immune cells, hematopoietic stem cells and Cytotoxicity of progenitor cells (HSPC) and other cells.
  • HSPC progenitor cells
  • Transposases are generally difficult to produce recombinantly and exhibit low solubility under physiological conditions, hampering efficient protein delivery.
  • PiggyBac and TcBuster transposases which are known to be widely used, have not reported methods using proteins. Only the patent CN113661247A reported that the solubility of the sleeping beauty transposase was improved through mutation of the enzyme, overcoming the shortcomings of SB protein aggregation, low stability and solubility, thereby achieving higher application value.
  • the present invention unexpectedly discovered that the purified protein of engineered highly active BZ transposase is naturally highly stable, soluble, and can autonomously cross the cell membrane and enter the nucleus to mediate genome modification through transposition. This activity is not applicable to macromolecular proteins. Common.
  • the transposase needs to be efficiently transfected into cells using, for example, protein transfection reagents or procedures (eg, electroporation).
  • a purified engineered highly active BZ transposase protein is used to realize transposition integration of foreign genes in cells.
  • the present invention relates to the discovery that BZ transposase spontaneously penetrates mammalian cells and can be delivered with transposon DNA to genetically modify various cell lines, embryonic, hematopoietic and induced pluripotent stem cells.
  • the present invention provides methods and compounds for utilizing the cell-penetrating functions of transposases in methods of genetically engineering cells and using transposases as shuttles to deliver combinations of DNA elements into target cells and even target organelles.
  • the above methods and discoveries improve the safety of genetic engineering and gene therapy.
  • DNA transposons can transpose via a non-replicative "cut and paste" mechanism. This requires recognition of two terminal inverted repeats by a catalytic enzyme, a transposase, which cleaves its target, thereby releasing the DNA transposon from its donor template. After excision, the DNA transposon can then integrate into the recipient DNA cleaved by the same transposase.
  • DNA transposons are flanked by two terminal inverted repeats and may contain genes encoding transposase enzymes that catalyze transposition.
  • Genome editing applications based on DNA transposons include binary systems containing transposase components and transposon components.
  • the transposase components are transposase or its encoding nucleic acid (mRNA or DNA), and the transposon components are containing Transposon DNA of the gene of interest flanked by terminal inverted repeats.
  • Co-delivery of transposon components and transposase components into target cells achieves transposition integration effects that rely on the cutting and pasting mechanism.
  • This article discusses various devices, systems, and methods related to the use of engineered highly active BZ transposases for synergistic approaches to cellular gene integration, particularly to enhance gene transfer into human hematopoietic and/or immune system cells.
  • the present disclosure relates to improved engineered highly active BZ transposases, transposon systems, transposon vector sequences, transposase delivery methods, and transposon delivery methods.
  • this study identifies specific universal sites for the production of highly active BZ transposases.
  • improved methods for delivering highly active BZ transposase as chemically modified in vitro transcribed mRNA are described.
  • a method is described to deliver BZ transposon vectors as "mini" loops of DNA in which nearly all prokaryotic sequences have been removed by recombinant methods.
  • improvements in direct delivery of purified highly active BZ transposase protein are described method.
  • a method of fusing a nuclear localization signal domain using BZ transposase is described. The above embodiments can improve the efficiency of gene transfer by transposons to various target cells individually or in combination.
  • BZ transposase Compared to wild-type ZB transposase (SEQ ID NO: 1), BZ transposase may contain one or more amino acid substitutions.
  • the BZ transposase may comprise an amino acid sequence having at least 70% sequence identity with the full-length sequence of wild-type ZB transposase (SEQ ID NO: 1).
  • the BZ transposase can comprise at least 40%, at least 50%, at least 60%, at least 70%, at least 80% identical to the full-length sequence of wild-type ZB transposase (SEQ ID NO: 1) , an amino acid sequence with at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.
  • one or more amino acid substitutions of the BZ transposase are mutated to a charged acidic or basic amino acid (e.g., K, R, and H) compared to SEQ ID NO: 1.
  • the BZ transposase may comprise an amino acid sequence having at least one amino acid that is different from the full-length sequence of the wild-type ZB transposase (SEQ ID NO: 1).
  • the BZ transposase can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more
  • the amino acid sequence of multiple amino acids differs from the full-length sequence of wild-type ZB transposase (SEQ ID NO: 1).
  • BZ transposases can contain one or more in any of the three domains of the ZB transposase (duplex DNA binding and oligomerization domain, DDE catalytic domain) as well as in the interdomain region between the domains.
  • a BZ transposase may contain one or more amino acid substitutions in the duplex DNA binding and oligomerization domain, the DDE catalytic domain, the interdomain region between domains, or a combination thereof.
  • the invention provides a BZ transposase comprising an amino acid sequence having mutations at one or more of the following positions compared to full-length SEQ ID NO: 1: 5 ,21,35,38,56,61,71,73,110,125,134,137,138,144,186,188,189,204,215,216,217,218,215-218,235,251 and 289.
  • the BZ transposase may also have mutations at one or more of the following positions: 79, 120 and 208.
  • the mutations at each position are independently selected from the mutations shown in Table A and Table B.
  • the one or more amino acid mutations include mutations of aspartic acid or glutamic acid to neutral or basic amino acids. In some embodiments, the one or more amino acid substitutions are based on conservative principles of the Tcl/Mariner transposon family.
  • the BZ transposase of the invention has substitution mutations at at least positions 71 and 110 compared to SEQ ID NO: 1.
  • the BZ transposase of the present invention can also be selected from the group consisting of 5, 21, 22, 35, 38, 56, 61, 73, 79, 125, 134, 137, 138, 144, 186, 188, 189, 204, 215, 216, 217, 218, 215-218, 235, 251, 120 and 208 have substitution mutations at one or more positions.
  • the BZ transposase of the invention in addition to the substitution mutations at positions 71 and 110, is selected from the group consisting of 5, 21, 22, 120, 125, 134, 137, 138, 144, 189 , 208, 216 and 251 have substitution mutations at one or more positions.
  • the BZ transposase of the invention has substitution mutations at at least positions 71, 79 and 110 compared to SEQ ID NO: 1.
  • the substitution mutation at position 71 is Q mutation into R, K or H
  • the substitution mutation at position 79 is Q mutation into R, K or H
  • the substitution mutation at position 110 is H mutation. is R or K.
  • the BZ transposase of the invention has substitution mutations at positions 71 and 110, as well as substitution mutations at one or more of the following positions: 5, 21, 22, 134, 137, 138, 144, 189, 208, 216 and 251; preferably, at the same time, there is a substitution mutation at one or more of the following positions: 134, 137, 138, 144, 189 , 208, 216 and 251.
  • the BZ transposase of the invention has substitution mutations at position 71, position 110, position 208, and at one or more of the following positions Have substitution mutations: 5, 21, 22, 134, 137, 138, 144, 189, 216 and 251; preferably, have substitution mutations at one or more of the following positions: 134, 137, 138, 144 , 189, 216 and 251.
  • the BZ transposase of the invention has substitution mutations at positions 71, 110, 208 and 216 compared to SEQ ID NO: 1, while at one of the following positions or There are substitution mutations at multiple positions: 5, 21, 22, 134, 137, 138, 144, 189 and 251; preferably, there are substitution mutations at one or more of the following positions: 134, 137, 138 , 144, 189 and 251.
  • the BZ transposase of the invention has substitution mutations at positions 71, 110, 208, 216 and 251, while at the following positions There are substitution mutations at one or more positions: 5, 21, 22, 134, 137, 138, 144 and 189; preferably, there are substitution mutations at one or more of the following positions: 134, 137 , 138, 144 and 189.
  • the BZ transposase of the invention has substitution mutations at positions 71, 110, 189, 208, 216 and 251 compared to SEQ ID NO: 1, At the same time, there is a substitution mutation at one or more of the following positions: 5, 21, 22, 134, 137, 138, and 144; preferably, at the same time, there is a substitution mutation at one or more of the following positions: 134 , 137, 138 and 144.
  • the BZ transposase of the invention is at position 71, 110, 138, 189, 208, 216 and 251 compared to SEQ ID NO: 1 Having substitution mutations, simultaneously having substitution mutations at one or more of the following positions: 5, 21, 22, 134, 137 and 144; preferably, simultaneously at one or more of the following positions There are substitution mutations on the positions: 134, 137 and 144.
  • the BZ transposase of the invention has substitution mutations at positions 71, 79 and 110 as compared to SEQ ID NO: 1, while at one or more of the following positions Having substitution mutations: 5, 21, 22, 120, 125, 134, 137, 138, 144, 189, 208, 216 and 251; preferably having substitution mutations at one or more of the following positions: 134, 137 , 138, 144, 189, 208, 216 and 251.
  • the BZ transposase of the invention has substitution mutations at positions 71, 79, 110 and 208 compared to SEQ ID NO: 1, while at one of the following positions or There are substitution mutations at multiple positions: 5, 21, 22, 120, 125, 134, 137, 138, 144, 189, 216 and 251; preferably at the same time, there are substitution mutations at one or more of the following positions: 134 , 137, 138, 144, 189, 216 and 251.
  • the BZ transposase of the invention has substitution mutations at positions 71, 79, 110, 208 and 216, while at the following positions There are substitution mutations at one or more positions: 5, 21, 22, 120, 125, 134, 137, 138, 144, 189 and 251; preferably, there are substitution mutations at one or more of the following positions at the same time :134, 137, 138, 144, 189 and 251.
  • the BZ transposase of the invention has substitution mutations at positions 71, 79, 110, 208, 216 and 251 compared to SEQ ID NO: 1, At the same time, there are substitution mutations at one or more of the following positions: 5, 21, 22, 120, 125, 134, 137, 138, 144, and 189; preferably at one or more of the following positions. Substitution mutations: 134, 137, 138, 144 and 189.
  • the BZ transposase of the invention is at position 71, 79, 110, 189, 208, 216 and 251 compared to SEQ ID NO: 1
  • substitution mutations and have substitution mutations at one or more of the following positions: 5, 21, 22, 120, 125, 134, 137, 138 and 144; preferably at one or more of the following positions at the same time
  • substitution mutations on: 134, 137, 138 and 144 There are substitution mutations on: 134, 137, 138 and 144.
  • the BZ transposase of the invention is at positions 71, 79, 110, 138, 189, 208, 216 and There is a substitution mutation at position 251, and at the same time, there is a substitution mutation at one or more of the following positions: 5, 21, 22, 120, 125, 134, 137 and 144; preferably at one or more of the following positions at the same time There are substitution mutations at positions: 134, 137 and 144.
  • the BZ transposase of the invention has substitution mutations at at least positions 208 and 216 compared to SEQ ID NO: 1.
  • the BZ transposase of the invention in addition to the substitution mutations at positions 208 and 216, is selected from the group consisting of 5, 21, 22, 35, 38, 56, 61, 71, 73, 110 , 125, 134, 137, 138, 144, 186, 188, 189, 204, 215-218, 217, 218, 235, 251, 79 and 120 have substitution mutations at one or more positions.
  • the BZ transposase of the present invention also has one or more positions selected from the group consisting of 5, 21, 22, 79, 120, 125, 134, 137, 138, 144, 189 and 251 There is a substitution mutation on, and the position number is the position number of SEQ ID NO: 1.
  • the BZ transposase of the present invention has substitution mutations at positions 208, 216 and 251, while at the same time, it is selected from the group consisting of 5, 21, 22, One or more of 35, 38, 56, 61, 71, 73, 110, 125, 134, 137, 138, 144, 186, 188, 189, 204, 215-218, 217, 218, 235, 79 and 120
  • substitution mutations at the position and the position number is the position number of SEQ ID NO: 1; preferably, there is a substitution mutation at one or more of the following positions: 134, 137, 138, 144 and 189.
  • the BZ transposase of the present invention has substitution mutations at positions 189, 208 and 216, while at the same time, it is selected from the group consisting of 5, 21, 22, 35, 38, 56, 61, 71, 73, 110, 125, 134, 137, 138, 144, 186, 188, 204, 215-218, 217, 218, 235, 79 and 120
  • the position number is the position number of SEQ ID NO: 1; preferably, there are substitution mutations at one or more of the following positions: 134, 137, 138, 144 and 251.
  • the BZ transposase of the present invention has substitution mutations at positions 189, 216, 208 and 251, while at the same time, it has substitution mutations at positions 5, 21 , 22, 35, 38, 56, 61, 71, 73, 110, 125, 134, 137, 138, 144, 186, 188, 204, 215-218, 217, 218, 235, 79 and one or more of 120
  • substitution mutation at a position and the position number is the position number of SEQ ID NO: 1; preferably, there is a substitution mutation at one or more of the following positions: 134, 137, 138 and 144.
  • the BZ transposase of the invention has substitution mutations at positions 138, 189, 216, 208 and 251, while being selected from One of 5, 21, 22, 35, 38, 56, 61, 71, 73, 110, 125, 134, 137, 144, 186, 188, 204, 215-218, 217, 218, 235, 79 and 120 or have substitution mutations at multiple positions; preferably at the same time, there are substitution mutations at one or more of the following positions: 134, 137 and 144.
  • the mutation at each position is to a positively charged amino acid.
  • the amino acids at positions S38, Q71 and H110 are mutated to positively charged amino acids, such as H, K or R.
  • the substitution mutations at each position are selected from the substitution mutations shown in Table A and Table B respectively.
  • the mutation at position 22 is D to A, L, I, G or V.
  • the substitution mutations at each of the above positions are independently selected from: 5S, 21K, 22A, 120S, 125L, 35I, 56L, 61R, 71H, 71R, 73L, 79R, 94E, 110K, 110R, 120G, 120S, 125L, 125M, 134A , 137R, 137T, 138G, 138K, 138R, 144A, 144E, 186N, 188H, 188Y, 189A, 204V, 208V, 216A, 217A, 217V, 218K, 235R, 251T and 289A, where the numerical value refers to SEQ ID NO: The amino acid position number of 1, the letter after the value indicates the amino acid residue after substitution mutation.
  • Exemplary BZ transposases may contain one or more amino acid substitutions from Table C.
  • a BZ transposase may contain at least one amino acid substitution from Table C.
  • the BZ transposase may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30 or More amino acid substitutions from Table C.
  • the BZ transposase contains one or more amino acid substitutions selected from N5S, F21K, S38R, Q71H, Q71R, Q79R, H110R, H110K, K134A, K137T, Q138K, V144E, G189A, G216A and K251T. More preferably, the BZ transposase contains one or more mutations selected from: S38R, Q71H, Q71R, H110K and H110R.
  • Exemplary BZ transposases comprise one or more amino acid substitutions or combinations of substitutions from Table D and Table E.
  • a BZ transposase may contain at least one amino acid substitution or combination of substitutions from Table D and Table E.
  • the BZ transposase may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30 or More amino acid substitutions or combinations of substitutions from Table D and Table E.
  • the BZ transposase comprises one or more amino acid substitutions or substitution combinations selected from the following: Q71R ⁇ H110R, Q71R ⁇ Q79R ⁇ H110R, G216A ⁇ Q71R ⁇ Q79R ⁇ H110R, H208V ⁇ Q71R ⁇ Q79R ⁇ H110R, H208V ⁇ G216A ⁇ Q71R ⁇ Q79R ⁇ H110R.
  • a "highly active" BZ transposase may refer to any BZ transposase that has increased transposition efficiency compared to a wild-type ZB transposase having the amino acid sequence of SEQ ID NO: 1.
  • Transposition efficiency can be measured as the percentage of successful transposition events that occur in a host cell population, normalized by the amounts of transposons and transposases introduced into that host cell population. In many cases, when comparing the transposition efficiency of two or more transposases, the same transposon construct is compared with two or more transposases for transfection of host cells under the same or similar transfection conditions. Each of the multiple transposases performs pairing. The amount of transposition events in a host cell can be examined by various methods.
  • a transposon construct can be designed to contain a reporter gene located between the terminal inverted repeats, and transfected cells positive for the reporter gene can be counted as cells in which a successful transposition event occurred, which can be concluded An estimate of the amount of transposition events.
  • the same or similar transfection conditions can be performed on the host cells. For transfection, the same transposase was paired with each different transposon. Similar methods can be used to measure transposition efficiency. Other methods known to those skilled in the art can also be performed to compare transposition efficiency.
  • An exemplary method may include systematically targeting the amino acids of a BZ transposase to increase the net charge of the amino acid sequence.
  • Methods may include systematically mutating the DNA binding and oligomerization domains to positively charged histidine (H), lysine (K) or arginine (R) residues.
  • H histidine
  • K lysine
  • R arginine
  • an increase in the net charge of the DNA binding and oligomerization domains at neutral pH may increase the stability of the transposase/transposon and transposase/transposase interaction complexes. , enhance the binding ability between target DNA and transposase protein and transposase, thereby improving the integration efficiency of transposase.
  • Highly active BZ transposases may contain one or more amino acid substitutions.
  • the one or more amino acid substitutions may be substitutions of non-conservative amino acids in the wild-type ZB sequence (SEQ ID NO: 1) with conservative amino acids.
  • BZ transposases include BZ transposases comprising at least one amino acid substitution from Table E.
  • the BZ transposase may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30 or More amino acid substitutions from Table E.
  • the BZ transposase may comprise amino acid substitutions S38R, Q71R, Q79R, H110R, G216A.
  • the BZ transposase may also comprise one or more of the amino acid substitutions H208V, K251T, G189A, Q138K, Q138R, K134A, K137T, V144E, N005S, F21K, K120S, N125L.
  • the transposase of the present invention may also be a fusion transposase comprising the ZB or BZ transposase, and the fusion transposase further comprises a functional polypeptide.
  • the "functional polypeptide” described herein refers to a polypeptide having its own function, such as a DNA sequence-specific binding domain and/or a nuclear localization signal domain (NLS).
  • the DNA sequence-specific binding domain refers to a domain that functions based on DNA sequence specificity, such as a leucine zipper domain, a CRISPR/Cas domain, a TALE domain, a zinc finger domain, an AAV Rep DNA binding domain, or any combination thereof.
  • the nuclear localization signal domain includes SV40 NLS, C-myc NLS, TAF1 NLS, TP53 NLS, STAT3 NLS, or any combination thereof.
  • the leucine zipper domain is derived from the Basic_leucine-zipper_C protein family of IPR020983 in the InterPro protein database (https://www.ebi.ac.uk/interpro/), the bZIP protein family of IPR004827, and the bZIP_sf protein family of IPR046347. The domain portion that mediates homodimerization.
  • the leucine zipper domain is selected from C/EBP ⁇ (also referred to herein as C-EBPO or C/EBPO, whose amino acid sequence is shown in SEQ ID NO: 13), CREPT (whose amino acid sequence is shown in SEQ ID NO: 14), The amino acid sequence is shown in SEQ ID NO: 14) and LZIP (the amino acid sequence is shown in SEQ ID NO: 15), both of which can improve the transposition efficiency.
  • C/EBP ⁇ also referred to herein as C-EBPO or C/EBPO, whose amino acid sequence is shown in SEQ ID NO: 13
  • CREPT whose amino acid sequence is shown in SEQ ID NO: 14
  • LZIP the amino acid sequence is shown in SEQ ID NO: 15
  • the DNA sequence-specific or non-specific binding domain can be located at the C-terminus or N-terminus of the transposase.
  • the nuclear localization signal domain can be located at the C-terminus or N-terminus of the transposase, preferably the N-terminus.
  • the leucine zipper domain is located in the N segment of the transposase.
  • the DNA sequence-specific or non-specific binding domain and the nuclear localization signal domain can be located at the same end or different ends of the transposase.
  • the DNA sequence-specific or non-specific binding domain and the nuclear localization signal domain can be located at the N-terminal and C-terminal, or the C-terminal and N-terminal of the transposase, respectively.
  • the BZ transposase and the DNA sequence-specific binding domain may be directly connected or separated by a linker.
  • the linker contains at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or at least 50 amino acids .
  • the sequence of the linker is (GGGS) n or A(EAAAK) n A, n is a positive integer greater than 0, such as 1, 2, 3, 4 or 5; preferably, the linker's The sequence is GGGS.
  • mutants of the BZ transposase or fusion transposase of any embodiment also includes mutants of the BZ transposase or fusion transposase of any embodiment.
  • mutant or variant includes a mutant of a transposase, as long as the mutant retains the respective biological functions of the antibody, transmembrane region and intracellular domain.
  • suitable mutants of the antibodies of the invention include mutants that have at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the antibody being compared. Sequence identity between two aligned sequences can be calculated using, for example, NCBI's BLASTp.
  • the mutant of the invention has one or more (such as within 20, within 15, within 10, within 8, within 5 or within 3, such as 1 -20, 1-10, etc.) amino acid residue insertion, substitution or deletion.
  • conservative substitutions with amino acids with similar or similar properties generally do not change the function of the protein or polypeptide.
  • amino acids with similar or similar properties include, for example, families of amino acid residues with similar side chains.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • amino acids with acidic side chains chain amino acids (e.g., aspartic acid, glutamic acid)
  • amino acids with uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine amino acids
  • amino acids with non-polar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chain amino acids e.g., threonine, valine, isoleucine
  • amino acids with aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • the mutant retains 5, 21, 22, 35, 38, 56, 61, 71, 73, 79, 110, 125, 134, Amino acid substitutions at positions 137, 138, 144, 186, 188, 189, 204, 215-218, 215, 216, 217, 218, 235, 251, 289, 120 or 208.
  • the sequence of the transposase described herein may be a modified polypeptide sequence.
  • Modified forms (which generally do not alter the primary structure) include chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications of the polypeptide during its synthesis and processing or during further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility properties.
  • the amino terminus or carboxyl terminus of the transposase of the present invention may also contain one or more polypeptide fragments as protein tags.
  • Any suitable tag may be used for this article.
  • the tag can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6. These tags can be used to purify proteins.
  • transposases described herein can spontaneously penetrate cells through direct contact with cells and can be delivered with transposon DNA to genetically modify various cell lines, embryonic, hematopoietic and induced pluripotent stem cells.
  • the invention provides polynucleotide molecules encoding the polynucleotide of the transposase (BZ transposase or fusion transposase) of the invention, and/or transposon DNA containing mutated or unmutated ITR sequences. .
  • the present invention also provides the complement of the coding sequence for the transposase.
  • the polynucleotide can be a recombinant nucleic acid molecule or a synthetic one; it can include DNA, RNA, and PNA (peptide nucleic acid) and can be a hybrid thereof.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the present invention also encompasses degenerate variants of the polynucleotide sequence encoding the fusion protein, ie, nucleotide sequences encoding the same amino acid sequence but with different nucleotide sequences.
  • the polynucleotide molecule comprises encoding the BZ transposase or the fusion transposase Enzyme DNA.
  • the polynucleotide may also comprise a nucleic acid sequence encoding a transposon recognized by the BZ transposase or the fusion transposase.
  • the polynucleotide is present in a DNA vector.
  • the vector can be a cloning vector or an expression vector.
  • the expression vector contains a transposase expression cassette, which is a nucleic acid construct that contains a promoter, a transposase coding sequence and a PolyA tailing signal sequence.
  • the expression vector usually also contains other elements that are usually included in the vector, such as multiple cloning sites, resistance genes, replication origin sites, etc.
  • the nucleic acid construct may also contain other elements required for expression, including but not limited to enhancers, etc.
  • the DNA vector includes minicircle plasmid, nanoplasmid, doggybone and other DNA forms that do not contain antibiotics or/and replicon DNA sequences.
  • the polynucleotide comprises messenger RNA (mRNA) encoding the BZ transposase or the fusion transposase.
  • mRNA messenger RNA
  • the mRNA is chemically modified, including but not limited to pseudouridine ( ⁇ ), N1-methylpseudouridine (m1 ⁇ ), 5-methylcytosine nucleoside (m5C), 5 -Methoxyuridine (5moU).
  • the present invention also relates to polynucleotides that hybridize to the polynucleotide sequences described above and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides that hybridize under stringent conditions to the polynucleotides of the invention.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding water during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90%, more It is best when hybridization occurs only when the ratio is above 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • recombination can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, transforming it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.
  • Recombinant vectors can be constructed using methods well known to those skilled in the art, see for example the techniques described in Sambrook et al., Ausubel (1989) or other standard texts.
  • Vectors containing nucleic acid molecules of the invention can be transferred into host cells by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is usually used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cell hosts, see Sambrook et al.
  • the protein encoding the protein of the invention (or its fragment, or its derivative) can be obtained entirely by chemical synthesis.
  • DNA sequence The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the invention through chemical synthesis.
  • the present invention also relates to nucleic acid constructs containing the nucleic acid sequences of the polynucleotide molecules described herein, and one or more regulatory sequences operably linked to these sequences.
  • the nucleic acid constructs of the present invention can be manipulated in various ways to ensure the expression of the transposase. Before inserting the nucleic acid construct into the vector, the nucleic acid construct can be manipulated according to the differences or requirements of the expression vector.
  • the regulatory sequences and operating methods required to express proteins via DNA or mRNA are known in the art. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.
  • the nucleic acid construct is a vector, such as a cloning vector, an expression vector, and an integration vector.
  • Expression of the polynucleotide sequence of the present invention is usually achieved by operably connecting the polynucleotide sequence of the present invention to an expression vector.
  • Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.
  • Integration vectors contain components that integrate the target sequence into the cell genome. These vectors can be used to transform appropriate host cells to enable them to express proteins. Vectors usually contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences.
  • sequences usually include one or more of the following nucleotide sequences: promoters, one or more enhancer sequences, replication origins, transcription termination sequences, complete intron sequences containing donor and acceptor splice sites, sequences encoding leader sequences for polypeptide secretion, ribosome binding sites, polyadenylation sequences, multiple linker regions for inserting nucleic acids encoding antibodies to be expressed, and selectable marker elements.
  • the type of vector is not limited, for example, plasmids, phagemids, phage derivatives, animal viruses, and cosmids, and may vary depending on the host cell to be introduced.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.
  • the vector introduced into the cell may also contain either or both a selectable marker gene or a reporter gene to facilitate identification and Select expressing cells.
  • the transposon includes a combination of DNA elements located between two terminal inverted repeat sequences.
  • the transposon can be recognized by the transposase according to any aspect of the present invention.
  • the combination of DNA elements includes but is not limited to a promoter, Enhancers, expressed genes, 5-UTR, 3-UTR and other sequence elements well known to those skilled in the art.
  • the two terminal inverted repeats each independently comprise SEQ ID NO: 2, 11 or 12 having to Sequences that are less than 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical.
  • the combination of DNA elements comprises an expressed gene.
  • the DNA element combination may also include a promoter selected from CMV, EFS, MND, EF1 ⁇ , CAGC, PGK, UBC, U6, H1 and Cumate.
  • the expressed gene encodes a protein selected from the group consisting of cellular receptors, immune checkpoint proteins, cytokines, and any combination thereof.
  • the expressed gene encodes a cell receptor selected from T cell receptor (TCR), B cell receptor (BCR), chimeric antigen receptor (CAR), or any combination thereof.
  • the CAR can sequentially include a polypeptide (such as scFv) that binds tumor cell membrane antigens, a hinge region, a transmembrane region, and an intracellular signal region.
  • a polypeptide such as scFv
  • the hinge region, transmembrane region and intracellular signal region well known in the art for constructing CAR can be used to construct the CAR of the present invention.
  • polypeptides that bind tumor cell membrane antigens can bind to membrane antigens widely expressed by tumor cells with medium affinity.
  • the polypeptides are usually inserted with antigenic epitopes, and the inserted positions are selected from any 1, 2, or 3 of the following 3 positions. :
  • the polypeptide that binds to tumor cell membrane antigens is a natural polypeptide or a synthetic polypeptide; preferably, the synthetic polypeptide is a single-chain antibody or Fab fragment.
  • the chimeric antigen receptor of the present invention can target one or more of the following antigens: CD19, CD20, CEA, GD2, FR, PSMA, PMEL, CA9, CD171/L1-CAM, IL-13RL1, MART-1, ERBB2, NY-ESO-1, AFP, MUC1, CD22, CD23, CD30, CD33, CD44v7/8, CD70, VEGFR1, VEGFR2, IL-11R/, EGP-2, EGP-40, FBP, GD3, PSCA, FSA , PSA, HMGA2, LeY, EpCAM, MSLN, IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125, CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1
  • the expressed gene encodes an antigen recognition domain.
  • the antigen recognition domain may comprise an antibody, an antibody mimetic, a protein scaffold, or a fragment thereof.
  • the antibody is a chimeric antibody, a recombinant antibody, a humanized antibody, or a human antibody.
  • the antibody is affinity-tuned.
  • Non-limiting examples of antibodies of the invention include single chain variable fragments (scFv), VHHs, single domain antibodies (sdAB), small modular immunopharmaceutical (SMIP) molecules, or Nanobodies.
  • the VHH is Camelidae. Alternatively or additionally, in certain embodiments, the VHH is humanized.
  • Non-limiting examples of antibody fragments of the invention include complementarity determining regions, variable regions, heavy chains, light chains, or any combination thereof.
  • Non-limiting examples of antibody mimetics of the present invention include: affibody, Afflilin molecule, affimer, Affitin molecule, alphabody, anticalin, and Avimer molecule, DARPin , Fynomer, Kunitz domain peptide, or monobody.
  • Non-limiting examples of protein scaffolds of the invention include Centyrin.
  • Transposons are usually provided in the form of nucleic acid constructs, especially DNA vectors.
  • any DNA vector in the art that facilitates the introduction of transposons into cells can be used, such as minicircle plasmids, nanoplasmids, Doggybone and other DNA plasmids.
  • the DNA plasmid may or may not contain antibiotic and/or replicon sequences.
  • host cell refers to a prokaryotic or eukaryotic cell capable of replicating the vector and/or expressing the heterologous gene encoded by the vector.
  • the host cell can be used as a vector or as a recipient of the mRNA.
  • a host cell may be "transfected” or “transformed,” which refers to the process of transfection or transduction of exogenous nucleic acid into the host cell. Transformed cells include primary subject cells and their progeny.
  • engineered” and “recombinant” cells or host cells as used herein generally refer to cells into which exogenous nucleic acid sequences, such as vectors or mRNA, have been introduced. Thus, recombinant cells can be distinguished from naturally occurring cells that do not contain introduced recombinant nucleic acid.
  • host cells include cells that carry the polynucleotide molecules described herein and/or produce the transposase.
  • the invention provides cells carrying the transposase of the invention and/or its coding sequence.
  • the cell may further comprise a nucleic acid sequence encoding a transposon recognized by the transposase.
  • the polynucleotide molecule may also comprise a nucleic acid sequence encoding a transposon recognized by the BZ transposase or the fusion transposase.
  • the type of cells is not limited, as long as they can express the transposase described herein or can utilize the transposase described herein to achieve transposition of the transposon.
  • the cells are primary cells isolated from the subject.
  • the subject is a healthy subject or has been diagnosed with a disease (eg, cancer or tumor).
  • the cells are isolated from the subject's blood, such as PBMCs or cells derived therefrom.
  • the cells may comprise primary immune cells, such as primary leukocytes.
  • the cells comprise primary T cells.
  • the primary T cells include ⁇ T cells, helper T cells, memory T cells, natural killer T cells, effector T cells, or any combination thereof.
  • the primary immune cells comprise CD3+ cells.
  • the cells comprise stem cells.
  • the stem cells are selected from: embryonic stem cells, hematopoietic stem cells, epidermal stem cells, epithelial stem cells, bronchoalveolar stem cells, mammary stem cells, mesenchymal stem cells, intestinal stem cells, endothelial stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells, testicular cells and any combination thereof.
  • the stem cells include induced pluripotent stem cells.
  • the nucleic acid constructs, vectors, and mRNA of the invention can be introduced into cells of interest.
  • the methods introduced include electroporation, microinjection, calcium phosphate precipitation, cationic polymers, dendrimers, liposomes, microparticle bombardment, fugene, direct acoustic loading, cell extrusion, optical transfection,
  • the cells are transfected by protoplast fusion, impalefection, magnetofection, nucleofection or any combination thereof.
  • the Nucleic acid constructs or recombinant expression vectors are used to be introduced.
  • the obtained transformants can be cultured using conventional methods to express the BZ transposase or fusion transposase of the present invention.
  • the medium used in culture can be selected from various conventional media. Cultivate under conditions suitable for host cell growth.
  • an inducible promoter is used to express the transposase, after the host cells have grown to an appropriate cell density, the selected promoter is induced using an appropriate method (such as temperature switching or chemical induction), and the cells are cultured for an additional period of time. .
  • the polypeptide in the above method can be expressed within the cell, on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic sterilization, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the present invention also relates to a system for genome editing (transposon system), which comprises: (1) a BZ transposase or a fusion transposase as described herein, or a polynucleotide encoding the same, and/or (2) a transposon DNA containing a mutated or unmutated terminal inverted repeat sequence.
  • the transposon is as described elsewhere herein.
  • An exemplary wild-type terminal inverted repeat sequence is shown in SEQ ID NO: 2, 11 or 12.
  • the polynucleotide comprises DNA or messenger RNA (mRNA) encoding the BZ transposase or the fusion transposase.
  • mRNA messenger RNA
  • the transposon is present in a DNA vector.
  • the polynucleotide and the transposon are present on the same plasmid.
  • the terminal inverted repeats of transposons can be mutated to improve transposition efficiency.
  • the terminal inverted repeat of the transposon does not contain a CpG motif as compared to the wild-type terminal inverted repeat (e.g., SEQ ID NO: 2, 11, or 12).
  • the CpG motif can be deleted or mutated, for example to ApG, GpG, TpG, CpA or CpT.
  • the present invention also provides a terminal inverted repeat sequence of a transposon and a transposon containing the terminal inverted repeat sequence, where the CpG in SEQ ID NO: 11 or 12 is mutated into ApG or GpG , TpG, CpA or CpT sequence.
  • This article also relates to a method of preparing a cell, comprising the step of introducing into a cell a BZ transposase or fusion transposase as described herein and a transposon recognized by the BZ transposase or fusion transposase.
  • said introducing comprises contacting said cell with a polynucleotide encoding said BZ transposase or said fusion transposase.
  • the polynucleotide comprises a protein encoding the BZ transposase or the fusion transposase DNA or messenger RNA (mRNA).
  • said introducing comprises contacting said cell with said BZ transposase or said fusion transposase, preferably by adding said BZ transposase or said fusion transposase directly to a solution containing The transposase protein is provided in the culture medium of the cells (preferably added to the cell culture medium of the cells of the target organism).
  • a solution containing The transposase protein is provided in the culture medium of the cells (preferably added to the cell culture medium of the cells of the target organism).
  • no reagents, vectors or methods that alter the penetration of the protein across the cell membrane may be used.
  • the introduction includes contacting the cell with a DNA vector containing the transposon.
  • the DNA vector includes a minicircle plasmid.
  • the introduction includes contacting the cell with a plasmid vector containing the transposon and a polynucleotide encoding the BZ transposase or the fusion transposase.
  • the present invention also provides a method for genetic engineering of cells using transposase, wherein the method involves allowing the transposase to penetrate the cell membrane but does not include a protein transfection step. In particular, the method does not include a step for converting the transposase into the cell membrane. Proteins are introduced into cells using protein transfection reagents or procedures. The method includes a process of directly contacting the transposase with the cells to be received by the transposase, such as by incubating the cells with a cell culture medium containing the transposase. The methods of the present invention include the step of introducing the transposase protein without using any vector, reagent or method that alters the penetration of the protein across cell membranes.
  • protein transfection in the context of the present invention should be understood broadly to refer to any method or reagent sufficient to introduce a protein into a target cell that cannot efficiently enter said target cell.
  • Popular protein transfection systems and reagents include commercial protein transfection reagents such as PULSin TM , ProteoJuice TM , Xfect TM and , Pierce TM protein transfection reagent (ThermoFisher), TransPass TM , and protein electroporation and other methods.
  • Also provided herein is a method of treatment comprising: (a) introducing a transposon and a BZ transposase or fusion transposase as described herein that recognizes the transposon into a cell, thereby generating a genetically modified cell, It comprises a transgene introduced by the transposon, comprising a transgene introduced by the transposon; (b) administering the genetically modified cells to a patient in need of the treatment.
  • the patient has been diagnosed with cancer or tumor.
  • the administering includes infusing the genetically modified cells into a blood vessel of the patient.
  • the invention also relates to the use of a BZ transposase or fusion transposase described herein, its coding sequence (DNA or RNA) or a nucleic acid construct, a cell, or a genome editing system for the preparation of a product, such as gene editing Kits, engineered immune cells or pharmaceutical compositions.
  • the immune cells include ⁇ T cells, helper T cells, memory T cells, natural killer T cells, effector T cells, etc.
  • the scope of the invention also encompasses BZ transposases or fusion transposases described herein, their coding sequences (DNA or RNA) or nucleic acid constructs, cells, or genome editing systems.
  • the kit further includes a nucleic acid encoding a transposon recognized by the BZ transposase or the fusion transposase or a nucleic acid construct thereof (for example, a DNA vector comprising the transposon), a host cell, One or more of the cell culture medium, cytokines, and instructions for use suitable for the host cell.
  • transposases, nucleic acid molecules and cells of the invention may be administered alone or as pharmaceutical compositions in combination with diluents and/or with other components such as relevant cytokines or cell populations. Therefore, the present invention also provides pharmaceutical compositions comprising the transposase, nucleic acid construct, gene editing system or cell prepared by the method described herein and pharmaceutically acceptable excipients.
  • a "pharmaceutically acceptable excipient” is a pharmaceutically or food-acceptable carrier, solvent, suspending agent or excipient for delivering the transposase, nucleic acid construct, gene editing system or cell of the present invention to an animal or human.
  • a pharmaceutically acceptable excipient is non-toxic to the recipient of the composition at the dose and concentration used.
  • Various types of carriers or excipients commonly used for delivering proteins, nucleic acids or cells in treatments known in the art may be included.
  • Exemplary excipients may be liquid or solid, including but not limited to: pH regulators, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, glidants, sweeteners, dyes/colorants, flavor enhancers, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers.
  • a pharmaceutically acceptable excipient may include one or more inactive ingredients, including but not limited to: stabilizers, preservatives, additives, adjuvants, sprays, compressed air or other suitable gases, or other suitable inactive ingredients for use with pharmacodynamic compounds.
  • the optimal pharmaceutical composition can be determined depending on the intended route of administration, mode of delivery, and desired dosage.
  • compositions of the invention may be selected for parenteral delivery, for inhalation or for delivery through the gastrointestinal tract (such as orally), for example for intravenous infusion delivery.
  • the preparation of such compositions is within the skill in the art.
  • Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations comprising immune cells, particularly immune cells (eg, T cells), in sustained or controlled release delivery formulations.
  • the pharmaceutical compositions of the present invention may also be administered in a manner suitable for the disease to be treated (or prevented).
  • compositions for in vivo administration are generally provided in sterile preparations. Sterilization is achieved by filtration through a sterile filter membrane. Compositions for parenteral administration may be stored in lyophilized form or in solution (eg, cryopreserved preparations). Parenteral compositions are typically placed in containers with sterile access openings, such as intravenous solution strips or vials with a stopper pierceable by a hypodermic needle.
  • cryopreserved formulations can be stored in ready-to-use form or further formulated prior to administration.
  • suitable for delivery of pharmaceutical compositions described herein may be cryopreserved formulations that can withstand long-distance transport without damaging the cells.
  • cryopreservation preparations usually also include components such as cell cryopreservation solution and human serum albumin (HSA).
  • the frozen pharmaceutical composition Before administration (eg, intravenous infusion), the frozen pharmaceutical composition needs to be stored at low temperature (eg, placed in liquid nitrogen). After thawing, the cryopreserved preparation can be directly or formulated into an infusion composition for infusion to the patient.
  • the cryopreservation solution or infusion composition may also contain dimethyl sulfoxide, sodium chloride, glucose, sodium acetate, potassium chloride or magnesium chloride, etc., the concentration of which can be determined by those skilled in the art (for example, an experienced physician) according to the cell , diseases, patients and other conditions are determined.
  • the genetically modified cells of the invention or compositions thereof may be combined with other therapies known in the art.
  • Patient “Patient,” “subject,” “individual” and the like are used interchangeably herein to refer to a living organism, such as a mammal, that can elicit an immune response. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and transgenic species thereof.
  • the present invention obtains the mutated BZ enzyme nucleic acid sequence through PCR, connects it to a plasmid and then transforms it into E. coli to obtain an engineered BZ transposase.
  • CHO cells and/or PBMC cells were transfected with the constructed expression vector or mRNA of engineered BZ transposase and transposon containing eGFP or expression gene, and transposon expression was observed.
  • Replacing BZ transposase with a fusion transposase fused with other polypeptides does not affect the transposase or transposon gene integration functions.
  • transposon expression was also observed when cells were transfected with transposons and incubated with media containing wild-type ZB transposase or engineered BZ transposase.
  • This article adopts the following methods.
  • High-Fidelity DNA Polymerase (New England BioLabs) was used for all site-directed mutagenesis.
  • For single point mutations after performing rolling circle PCR mutagenesis, use DpnI restriction endonuclease digestion, and take 5 ⁇ L of the digestion reaction product for transformation in TOP10 E. coli competent cells.
  • For combinatorial mutagenesis after mutagenesis using multiple PCR reactions, the PCR products were purified using an agarose gel DNA recovery kit (Tianmo Biotechnology), and Hieff was used to purify the PCR products.
  • YEASEN performs 10 ⁇ L ligation reaction on the purified PCR product, and takes 5 ⁇ L of the ligation reaction product for transformation in TOP10 E. coli competent cells. For each mutant, 3 single clones were picked for culture, and half of each were taken for sequencing identification and bacterial preservation. For mutants with correct identified sequences, NucleoBond Xtra MiDi EF plasmid preparation kit (MACHEREY-NAGEL) was used to prepare them for transfection. of plasmids. The supercoil ratio and endotoxin content of plasmid samples were randomly inspected and identified.
  • Lonza2B electroporation reagent take 100ul of the electroporation fluid, mix 4ug of transposase plasmid and 4ug of transposon plasmid with the electroporation reagent, add it to the centrifuged cell pellet, resuspend it, add it to the electroporation cup, and place it in the Lonza In the 2B electroporation instrument, set the electroporation program to H-014. After electroporation, the cells were transferred to a six-well plate with complete medium and cultured. Fluorescence photography was taken on D5, D9, and D13 after electroporation, and the transposition efficiency of transposase in CHO-K1 cells was detected by flow cytometry.
  • the frozen PBMC were recovered and counted, and 1 ⁇ 10 7 cells were taken, centrifuged and the supernatant was removed.
  • Lonza 2B electroporation reagent take 100ul of the electroporation fluid, mix 4ug of transposase plasmid and 4ug of transposon plasmid with the electroporation reagent, add it to the centrifuged cell pellet, resuspend it, add it to the electroporation cup, and place it in In the Lonza 2B electroporation instrument, set the electroporation program to U-014.
  • the cells were transferred to a six-well plate with complete culture medium (AIM-V+2% FBS) and cultured, and 500 U/ml IL-2 was added.
  • the plate was transferred on the 5th day after electroporation, and fluorescence photography, counting, and flow cytometry were performed on D5, D9, and D13 to detect the transposition efficiency of transposase in PBMC cells.
  • GFP fluorescence was captured with an Olympus inverted fluorescence microscope on days D5, D9, and D13 with an exposure time of 500 ms and a photography magnification of 100 ⁇ .
  • the number of GFP fluorescent cells and fluorescence intensity were used to evaluate the presence of transposase. Transposition efficiency in cells.
  • Lonza2B electroporation reagent take 100ul of electroporation fluid, mix 4ug of different transposase mutant plasmids and 4ug of transposon plasmid (pZB-dCG-eGFP) with the electroporation reagent, add it to the centrifuged cell pellet, and resuspend Then add it to the electroporation cup, place it in the Lonza 2B electroporation instrument, and set the electroporation program to H-014. After electroporation, the cells were transferred to a six-well plate with complete medium and cultured. Take fluorescence photos on D5, D9, and D13 after electroporation. Harvest the transfected cells.
  • Lonza 2B electroporation reagent take 100ul of electroporation fluid, mix 4ug of transposase plasmid and 4ug of transposon plasmid (pZB-dCG-eGFP) with the electroporation reagent, add it to the centrifuged cell pellet, resuspend and add into the electroporation cup, place it in the Lonza 2B electroporation instrument, and set the electroporation program to U-014.
  • the cells were transferred to a six-well plate with complete culture medium (AIM-V+2% FBS) and cultured, and 500 U/ml IL-2 was added. Transfer the plate on the 5th day after electroporation, and take fluorescence photos and counts on D5, D9, and D13. Harvest the transfected cells. After washing once with 1 ⁇ DPBS, resuspend in 500ulDPBS and add to the flow tube or 96-well. The board is tested on the machine. Cells were analyzed using Cytex's SpectroFlo and GFP expression was assessed using the FITC channel. Different transposase mutants were screened and evaluated based on their final transposition efficiency.
  • Example 1 Screening of BZ transposase mutants with efficient gene integration in CHO and PBMC cells
  • This example tests the transposition efficiency of different transposase plasmids in CHO cells and/or PBMC cells, and identifies transposase mutants with efficient gene integration.
  • the vector sequence of the transposon constructed in this example is SEQ ID NO: 3, which contains the eGFP fluorescent protein expression cassette.
  • Engineered BZ transposases with amino acid substitutions at different positions were screened on CHO-K1 and/or PBMC cells and mutants with high enzyme activity were identified.
  • the vector sequence containing wild-type ZB transposase is shown in SEQ ID NO: 4. According to the sequence of SEQ ID NO: 4, the codons of the amino acids at the corresponding positions are replaced to obtain Vector sequences for different engineered BZ transposases.
  • the amino acid sequence of wild-type ZB transposase is as follows:
  • the constructed engineered transposase or transposon was transfected into CHO cells and/or PBMC cells at a certain mass ratio using electroporation.
  • CHO and/or PBMC cells were transfected together with WT ZB transposase and PiggyBac (PB) transposase (the sequence of which is described in CN105154473A) as a control transposase.
  • PB PiggyBac
  • the amino acid outside the second helix of the BZ transposase HTH domain is mutated to a positively charged amino acid such as histidine (H), lysine (K) or arginine (R) (such as S38R, Q71H , Q71R, H110K and H110R, etc.) the transposition efficiency increased more significantly.
  • Example 2 Screening of BZ transposase combinatorial mutants with efficient gene integration on CHO and PBMC
  • This example tests the transposition efficiency of different engineered BZ transposase mutant combinations to identify BZ transposase mutant combinations with efficient gene integration.
  • Example 1 Two batches of multiple groups of different engineered BZ transposase combination mutants were compared in different cells.
  • the cell transfection method and transposition efficiency detection method were the same as in Example 1: one batch was the medium-strength mutant selected in Example 1. Single point mutants and 2, 3 or more combined mutants of Q71R/H110R (Table D), and the other batch is 2 or 3 of the single point mutants of medium strength screened in Example 1 or more combinatorial mutants above (Table E).
  • 206 (Q71R/H110R) was selected for comparison with PiggyBac (PB). The results are shown in Figures 3 and 4. 206 (Q71R/H110R) was Both CHO-K1 and PBMC cells have equivalent or better effects than PiggyBac (PB)
  • Example 3 ZB or BZ transposase in the form of fusion protein has no effect on its gene integration ability
  • This example tests the transposition efficiency of exemplary fusion transposases containing NLS.
  • Different NLS tags are connected to the N-terminus of the transposase to generate different types of transposase.
  • Different NLS tags were added to the wild-type ZB transposase, and their amino acid sequences are shown in Table F.
  • the above-described exemplary fusion transposase containing NLS was transfected into PBMC cells by means of electroporation, the cells were grown in complete medium (without drug selection), and the fluorescent protein was detected by flow cytometry on day 13 after transfection. The expression level was used to evaluate the transposition efficiency of the exemplary fusion transposase containing NLS.
  • This example is used to prove that the protein form of BZ transposase can mediate gene integration of transposons.
  • Resuscitate and culture CHO-K1 cells When the cells are in good growth condition, digest and wash the cells. Use the Lonza 2B system (Lonza) to transfect the transposon (carrying the eGFP reporter gene) into the cells by electroporation. Electroporate for 4.5 hours and then culture. The protein form of BZ transposase is added to the base. Use empty cells as a viability control. After electroporated cells were proliferated and cultured for 14 days after transfection, the cells were harvested, washed once with DPBS, and resuspended in 200 ⁇ L of DPBS buffer. Cells were analyzed using a Cytek Northern Lights (Cytek Biosciences) flow cytometer, and EGFP expression was assessed using the FITC channel.
  • Lonza 2B system Lonza
  • Example 5 BZ transposase in the form of mRNA can mediate genomic integration of CAR plasmid or antibody plasmid DNA and be used for cell gene therapy
  • mRNA messenger RNA
  • BZ transposase ZB protein with Q71R, H110R mutations
  • PB transposase and its transposon system the mRNA is pseudourea Glycoside chemically modified mRNA to engineer CD4+/CD8+ T cells.
  • the CD4+/CD8+ T cells used in the examples were extracted from the peripheral blood of healthy people, separated using CD4+/CD8+ magnetic beads, and spread on the anti-CD3/anti-CD28 or anti-coated anti-CD3/anti-CD28 or anti- CD3/4-1BBL was cultured in a six-well plate in complete medium containing 100 U/ml IL-2 (without drug selection), and incubated for two days for activation.
  • the inventor uses a transgene carrying a CAR gene (information about CAR is disclosed in CN202111681582.0, the entire content of which is incorporated herein by reference. This embodiment uses the CAR shown in SEQ ID NO: 99 in CN202111681582.0).
  • transposon plasmid (257) and exemplary transposase BZ mRNA, and transgene expression was monitored by flow cytometry for 9 days after electroporation.
  • the transposon plasmid (257) carrying the CAR gene of different costimulatory domains 4-1BB and CD28 was co-transduced with PB mRNA transposase and exemplary transposase BZ mRNA respectively (Table G).
  • the transposition efficiency of mRNA using the exemplary BZ transposase was comparable to that of PB mRNA transposase at 9 days after transfection. There was no significant difference, and there were no limiting differences in T cell proliferation efficiency and fold. It shows that the BZ transposase in the form of mRNA can mediate the gene integration of CAR and/or antibody plasmid DNA used in gene therapy, and the effect is equivalent to the most efficient PiggyBac (PB) transposase in this technical field.
  • the anti-CD3/4-1BBL activation method was selected to prepare CAR-T for the killing experiment of tumor cell L363. The results also showed that the CAR-T cells prepared by the new BZ transposase were better than the CAR prepared by PB transposase. -There is no significant difference in T cell killing effect.
  • Example 6 CpG mutation in the BZ transposon ITR maintains the gene integration function of the transposon system
  • BZ transposon ITR mutation clears the CpG motif, reduces immunogenicity while maintaining the gene integration function of the transposon system.
  • the wild-type BZ transposon 5′ITR sequence and 3′ITR sequence are shown in SEQ ID NO: 11 and 12.
  • the transposon plasmid carrying ITR mutation and eGFP reporter gene and BZ mRNA were co-electrotransfected into CHO-K1 cells.
  • the cells were grown in complete culture medium (without drug selection) and detected by flow cytometry on the 14th day after transfection. The expression of fluorescent proteins was used to evaluate the transposition efficiency of ITR-mutated transposons.
  • Example 7 Fusion of leucine zipper structure can improve the transposition activity of ZB or BZ transposase
  • This example tests the transposition efficiency of an exemplary fusion transposase containing three ZIPs. Connect different ZIP structures at the N-terminus of the transposase to generate different types of transposase. Different ZIP structures were added to the mutant BZ transposase (ZB transposase with Q71R, H110R mutations), and its amino acid sequence is shown in Table H. With the help of HD transfection reagent (Promoga), the above-mentioned exemplary fusion transposase containing ZIP and the transposon donor carrying the selection gene NEO were co-transfected into Hela cells, and three duplicate wells were set up in each group.
  • HD transfection reagent Promoga

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Abstract

L'invention concerne un système de transposon, comprenant une transposase et un transposon. La transposase est une transposase mutante ou une transposase de fusion. Par comparaison avec une transposase ZB de type sauvage telle que représentée par SEQ ID NO : 1, la transposase mutante a une ou plusieurs mutations de substitution d'acide aminé et a une efficacité de transposition améliorée. Le transposon contient une séquence ITR mutante, qui est dépourvue d'un motif CpG par rapport à une séquence ITR de type sauvage SEQ ID NO : 11 ou 12, et peut réduire l'immunogénicité sans affecter l'efficacité de transposition.
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WO2010085699A2 (fr) * 2009-01-23 2010-07-29 The Johns Hopkins University Transposon piggybac de mammifère et procédés d'utilisation
CN105018523A (zh) * 2015-04-09 2015-11-04 扬州大学 一种zb转座子系统及其介导的基因转移方法
CN112159822A (zh) * 2020-09-30 2021-01-01 扬州大学 一种PS转座酶与CRISPR/dCpf1融合蛋白表达载体及其介导的定点整合方法
WO2022118237A1 (fr) * 2020-12-03 2022-06-09 Friedrich Miescher Institute For Biomedical Research Utilisation d'une combinaison d'un motif orphelin et d'une densité cpg pour réguler l'expression d'un transgène hétérologue

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US20020016975A1 (en) * 1997-03-11 2002-02-07 Regents Of The University Of Minnesota Dna-based transposon system for the introduction of nucleic acid into dna of a cell
WO2010085699A2 (fr) * 2009-01-23 2010-07-29 The Johns Hopkins University Transposon piggybac de mammifère et procédés d'utilisation
CN105018523A (zh) * 2015-04-09 2015-11-04 扬州大学 一种zb转座子系统及其介导的基因转移方法
CN112159822A (zh) * 2020-09-30 2021-01-01 扬州大学 一种PS转座酶与CRISPR/dCpf1融合蛋白表达载体及其介导的定点整合方法
WO2022118237A1 (fr) * 2020-12-03 2022-06-09 Friedrich Miescher Institute For Biomedical Research Utilisation d'une combinaison d'un motif orphelin et d'une densité cpg pour réguler l'expression d'un transgène hétérologue

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TOBIAS JURSCH;CSABA MISKEY;ZSUZSANNA IZSVÁK;ZOLTÁN IVICS : "Regulation of DNA transposition by CpG methylation and chromatin structure in human cells", MOBILE DNA, BIOMED CENTRAL LTD, LONDON, UK, vol. 4, no. 1, 15 May 2013 (2013-05-15), London, UK , pages 15, XP021152734, ISSN: 1759-8753, DOI: 10.1186/1759-8753-4-15 *

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