Swivel sub-system and application thereof

Abstract

Provided is a transposon system comprising a transposase and a transposon, the transposase being a mutant transposase or a fusion transposase, the mutant transposase having one or more amino acid substitution mutations and having improved transposon efficiency compared to a wild-type ZB transposase as shown in SEQ ID NO: 1; the transposon contains a mutant ITR sequence, and compared with a wild type ITR sequence SEQ ID NO: 11 or 12, the transposon lacks a CpG motif, so that the immunogenicity can be reduced on the basis of not influencing the transposon efficiency.

Description

A transposon system and its application Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to a transposon system and applications thereof. Background Art

[0002] Transposable genetic elements, also known as transposons, are DNA segments 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 mechanism: (1) for elements known as retrotransposons, transposition occurs through reverse transcription of an RNA intermediate, and (2) for DNA transposons, transposition occurs through direct transposition of DNA flanked by inverted terminal repeats (ITRs). Active transposons encode one or more proteins required for transposition, and naturally active DNA transposons carry transposase genes. Transposons have been widely used as gene transfer vectors in research in the fields of transgenics, gene capture, and gene therapy, and have achieved excellent application results.

[0003] The Tc1 / Mariner transposon is the most widespread DNA transposon superfamily in nature, encompassing bacteria, invertebrates, and vertebrates. Among vertebrates, Tc1 / Mariner transposons are most widespread in bony fish. Through bioinformatics and experimental validation, 15 members of the Tc1 / Mariner superfamily have been reported to exhibit natural transposition activity: Tc1, Tc3, Minos, Mos1, Bari3, Fot1, impala, Famar1, Osmar5, ISY100, Mboumar-9, Passport, Tana1, Thm3, SB, and ZB transposons.

[0004] Naturally active transposons can cause genomic instability. Consequently, during evolution, transposons accumulate mutations, gradually reducing or even losing their activity. For example, the majority of Tc1 / Mariner superfamily transposons found in higher animals (such as mammals) lose their transposition activity due to defects in the transposase open reading frame (e.g., mutations, frameshifts, insertions, deletions, or the presence of stop codons). For these reasons, the Sleeping Beauty (SB) and PiggyBac transposons, currently the most widely used in clinical gene therapy, are highly efficient mutant transposase / transposon systems that have undergone bioinformatics molecular reconstruction and / or engineering.

[0005] The mammalian innate immune system recognizes and directs a response against foreign DNA. The primary signal triggering this response is unmethylated CpG motifs within DNA sequences of microbial origin. These motifs are rare in vertebrate DNA but are abundant in bacterial and some viral DNA. Because gene therapy typically involves delivering DNA from bacterially derived plasmids or recombinant viruses, it inevitably leads to acute inflammatory responses of varying severity. Non-viral gene delivery vectors, such as cationic lipid-DNA complexes, are the most severe, producing side effects at lower doses and lethality at higher doses. Plasmid DNA (pDNA) used in gene therapy containing unmethylated CpG dinucleotides (CpGs) can induce an inflammatory response in target tissues or cells, reducing the duration of transgene expression. Even a single CpG residue within the pDNA is sufficient to trigger an inflammatory response, whereas CpG-free pDNA vectors do not. Using CpG-free pDNA expression vectors as transposon systems can improve the efficiency of transposon-integrated gene expression. 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.

[0006] There remains a need in the art for more efficient transposase / transposon systems.

[0007] Summary of the Invention

[0008] The present invention relates to a transposon system comprising a transposase or a polynucleotide encoding the transposase, and / or a transposon DNA containing an inverted terminal repeat sequence; the transposase is a mutant transposase or a fusion transposase, wherein the fusion transposase is a wild-type ZB transposase or a mutant thereof fused with a functional polypeptide. The present invention also relates to the use of the transposon system in gene modification. The inverted terminal repeat sequence may or may not contain a mutation compared to the wild-type inverted terminal repeat sequence.

[0009] The mutant transposase of the present invention is an engineered ZB transposase, referred to as BZ transposase. The BZ transposase or transposon system of the present invention significantly improves integration efficiency and transgene expression compared to wild-type ZB transposase or ZB transposition systems. The BZ transposase of the present invention can be used in the form of viruses, plasmid DNA, mRNA, or proteins for applications in stable cell transfection, cell line development, genome modification, gene therapy, cell therapy, and transgenic animals.

[0010] In some embodiments, the BZ transposase of the present invention comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the full-length SEQ ID NO: 1, and has one or more amino acid substitution mutations compared to SEQ ID NO: 1. In some embodiments, the BZ transposase has improved transposition efficiency compared to the wild-type ZB transposase having the amino acid sequence set forth in SEQ ID NO: 1.

[0011] In one or more embodiments, the BZ transposase has one or more amino acid substitutions in its DNA binding and oligomerization domain, DDE catalytic domain, or a combination thereof.

[0012] In some embodiments, the BZ transposase of the present invention comprises an amino acid sequence having a mutation at one or more of the following positions compared to the 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, wherein the positions are numbered as in SEQ ID NO: 1. In some embodiments, the BZ transposase further comprises a mutation at one or more of the following positions: 79, 120, and 208, wherein the positions are numbered as in SEQ ID NO: 1.

[0013] In some embodiments, the amino acid sequence of the BZ transposase provided herein has at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1, and has a mutated amino acid sequence 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 positions are numbered as in SEQ ID NO: 1. In some embodiments, the BZ transposase further has a mutation at one or more of the following positions: 79, 120, and 208, wherein the positions are numbered as in SEQ ID NO: 1.

[0014] In some embodiments, the mutations at each position are independently selected from the mutations shown in Table A below:

[0015] Table A

[0016] In some embodiments, the mutations at positions 79, 120, and 208 are independently selected from the substitution mutations shown in Table B below:

[0017] Table B

[0018] In some embodiments, the one or more amino acid mutations include mutations of aspartic acid or glutamic acid to neutral amino acids or basic amino acids. In some embodiments, the one or more amino acid substitutions are based on the conservation principle of the Tc1 / Mariner transposon family.

[0019] In some embodiments, the BZ transposase of the present 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, wherein the numbers indicate the positions of the mutations and the letters after the numbers indicate the amino acid residues after the mutations.

[0020] In some embodiments, the BZ transposase of the present invention has substitution mutations at least at positions 71 and 110 compared to SEQ ID NO: 1. In some embodiments, in addition to the substitution mutations at positions 71 and 110, the BZ transposase of the present invention further has substitution mutations at one or more positions 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, where the positions are numbered as in SEQ ID NO: 1. In some embodiments, in addition to the substitution mutations at positions 71 and 110, the BZ transposase of the present invention further has a substitution mutation at one or more positions selected from 5, 21, 22, 79, 120, 125, 134, 137, 138, 144, 189, 208, 216, and 251, the positions being numbered as in SEQ ID NO: 1. In some embodiments, the BZ transposase of the present invention has substitution mutations at at least positions 71, 79, and 110 compared to SEQ ID NO: 1. In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has substitution mutations at positions 71 and 110, and has substitution mutations at one or more of the following positions: 5, 21, 22, 134, 137, 138, 144, 189, 208, 216 and 251; preferably, has substitution mutations at one or more of the following positions: 134, 137, 138, 144, 189, 208, 216 and 251. In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has substitution mutations at positions 71, 110, and 208, and has substitution mutations at one or more of the following positions: 5, 21, 22, 134, 137, 138, 144, 189, 216, and 251; preferably, has substitution mutations at one or more of the following positions: 134, 137, 138, 144, 189, 216, and 251. In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has substitution mutations at positions 71, 110, 208 and 216, and has substitution mutations at one or more of the following positions: 5, 21, 22, 134, 137, 138, 144, 189 and 251; preferably, has substitution mutations at one or more of the following positions: 134, 137, 138, 144, 189 and 251.In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has substitution mutations at positions 71, 110, 208, 216 and 251, and has substitution mutations at one or more of the following positions: 5, 21, 22, 134, 137, 138, 144 and 189; preferably, has substitution mutations at one or more of the following positions: 134, 137, 138, 144 and 189. In some embodiments, compared to SEQ ID NO: 1, the BZ transposase of the present invention has a substitution mutation at position 71, 110, 189, 208, 216, and 251, and a substitution mutation at one or more of the following positions: 5, 21, 22, 134, 137, 138, and 144; preferably, a substitution mutation at one or more of the following positions: 134, 137, 138, and 144. In some embodiments, compared to SEQ ID NO: 1, the BZ transposase of the present invention has a substitution mutation at position 71, 110, 138, 189, 208, 216, and 251, and a substitution mutation at one or more of the following positions: 5, 21, 22, 134, 137, and 144; preferably, a substitution mutation at one or more of the following positions: 134, 137, and 144. In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has substitution mutations at positions 71, 79, and 110, and has substitution mutations at one or more of the following positions: 5, 21, 22, 120, 125, 134, 137, 138, 144, 189, 208, 216, and 251; preferably, has substitution mutations at one or more of the following positions: 134, 137, 138, 144, 189, 208, 216, and 251. In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has substitution mutations at positions 71, 79, 110, and 208, and has substitution mutations at one or more of the following positions: 5, 21, 22, 120, 125, 134, 137, 138, 144, 189, 216, and 251; preferably, has substitution mutations at one or more of the following positions: 134, 137, 138, 144, 189, 216, and 251.In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has substitution mutations at positions 71, 79, 110, 208 and 216, and has substitution mutations at one or more of the following positions: 5, 21, 22, 120, 125, 134, 137, 138, 144, 189 and 251; preferably, has substitution mutations at one or more of the following positions: 134, 137, 138, 144, 189 and 251. In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has substitution mutations at positions 71, 79, 110, 208, 216, and 251, and simultaneously has substitution mutations at one or more of the following positions: 5, 21, 22, 120, 125, 134, 137, 138, 144, and 189; preferably, simultaneously has substitution mutations at one or more of the following positions: 134, 137, 138, 144, and 189. In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has a substitution mutation at position 71, 79, 110, 189, 208, 216 and 251, and has a substitution mutation at one or more of the following positions: 5, 21, 22, 120, 125, 134, 137, 138 and 144; preferably, has a substitution mutation at one or more of the following positions: 134, 137, 138 and 144. In some embodiments, compared to SEQ ID NO: 1, the BZ transposase of the present invention has substitution mutations at positions 71, 79, 110, 138, 189, 208, 216, and 251, and substitution mutations at one or more of the following positions: 5, 21, 22, 120, 125, 134, 137, and 144; preferably, substitution mutations at one or more of the following positions: 134, 137, and 144. Preferably, the substitution mutation at position 71 is from Q to R, K, or H; the substitution mutation at position 79 is from Q to R, K, or H; and the substitution mutation at position 110 is from H to R or K. Preferably, the substitution mutations at each position are selected from the substitution mutations shown in Tables A and B, respectively. Preferably, the mutation at position 22 is from D to A, L, I, G, or V.In some embodiments, the substitution mutations at each of the above positions are independently selected from the group consisting of: 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, wherein the numbers refer to the amino acid position numbers of SEQ ID NO: 1, and the letters after the numbers represent the amino acid residues after the substitution mutations.

[0021] In some embodiments, the BZ transposase of the present invention is a BZ transposase having any one or more of the following mutations compared to SEQ ID NO: 1:

[0022] Q71R\H110R,

[0023] Q71R\Q79R\H110R,

[0024] G216A\Q71R\Q79R\H110R,

[0025] H208V\Q71R\Q79R\H110R、

[0026] H208V\G216A\Q71R\Q79R\H110R、

[0027] F21K\D22A\Q71R\H110R,

[0028] N005S\F21K / D22A\Q71R\Q79R\H110R、

[0029] K120S\N125L\Q71R\Q79R\H110R、

[0030] G216A\H208V\G189A\Q71R\Q79R\H110R、

[0031] G216A\H208V\K251T\Q71R\Q79R\H110R、

[0032] G216A\H208V\K251T\G189A\Q71R\Q79R\H110R、

[0033] G216A\H208V\K251T\G189A\Q138K\Q71R\Q79R\H110R,

[0034] G216A\H208V\K251T\G189A\Q138R\Q71R\Q79R\H110R、

[0035] G216A\H208V\K251T\G189A\K134A\Q71R\Q79R\H110R、

[0036] G216A\H208V\K251T\G189A\Q138K\K134A\Q71R\Q79R\H110R、

[0037] G216A\H208V\K251T\G189A\Q138R\K134A\Q71R\Q79R\H110R、

[0038] G216A\H208V\K251T\G189A\Q138K\V144E\Q71R\Q79R\H110R、

[0039] G216A\H208V\K251T\G189A\Q138K\K137T\Q71R\Q79R\H110R、

[0040] G216A\Q71R\H110R、

[0041] H208V\Q71R\H110R、

[0042] H208V\G216A\Q71R\H110R、

[0043] G216A\H208V\G189A\Q71R\H110R、

[0044] G216A\H208V\K251T\Q71R\H110R、

[0045] G216A\H208V\K251T\G189A\Q71R\H110R、

[0046] G216A\H208V\K251T\G189A\Q138K\Q71R\H110R、

[0047] G216A\H208V\K251T\G189A\Q138R\Q71R\H110R、

[0048] G216A\H208V\K251T\G189A\K134A\Q71R\H110R、

[0049] G216A\H208V\K251T\G189A\Q138K\K134A\Q71R\H110R,

[0050] G216A\H208V\K251T\G189A\Q138R\K134A\Q71R\H110R,

[0051] G216A\H208V\K251T\G189A\Q138K\V144E\Q71R\H110R,

[0052] G216A\H208V\K251T\G189A\Q138K\Q71R\H110R,

[0053] G216A\H208V\K251T\G189A\Q138R\Q71R\H110R、

[0054] G216A\H208V\K251T\G189A\K134A\Q71R\H110R、

[0055] G216A\H208V\K251T\G189A\Q138K\K134A\Q71R\H110R,

[0056] G216A\H208V\K251T\G189A\Q138R\K134A\Q71R\H110R,

[0057] G216A\H208V\K251T\G189A\Q138K\V144E\Q71R\H110R,

[0058] G216A\H208V\K251T\G189A\Q138K\K137T\Q71R\H110R, or

[0059] N005S\F21K / D22A\Q71R\H110RR.

[0060] Among them, Q71R\H110R refers to the simultaneous presence of multiple mutations Q71R and H110R, and the others are similar.

[0061] In some embodiments, the BZ transposase of the present invention has substitution mutations at least at positions 208 and 216 compared to SEQ ID NO: 1. In some embodiments, in addition to the substitution mutations at positions 208 and 216, the BZ transposase of the present invention further has substitution mutations at one or more positions selected from the group consisting of positions 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, the positions being numbered as in SEQ ID NO: 1. In some embodiments, in addition to the substitution mutations at positions 208 and 216, the BZ transposase of the present invention further has substitution mutations at one or more positions selected from 5, 21, 22, 79, 120, 125, 134, 137, 138, 144, 189 and 251, where the position numbers are the position numbers of SEQ ID NO: 1. In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has a substitution mutation at position 208, 216 and 251, and a substitution mutation at one or more positions selected from 5, 21, 22, 35, 38, 56, 61, 71, 73, 110, 125, 134, 137, 138, 144, 186, 188, 189, 204, 215-218, 217, 218, 235, 79 and 120, wherein the positions are numbered as in SEQ ID NO: 1; preferably, the BZ transposase has a substitution mutation at one or more of the following positions: 134, 137, 138, 144 and 189. In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has a substitution mutation at positions 189, 208, and 216, and simultaneously has a substitution mutation at one or more positions selected from 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, wherein the positions are numbered as in SEQ ID NO: 1; preferably, it simultaneously has a substitution mutation at one or more of the following positions: 134, 137, 138, 144, and 251.In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has a substitution mutation at positions 189, 216, 208 and 251, and a substitution mutation at one or more positions selected from 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, wherein the positions are numbered as in SEQ ID NO: 1; preferably, the BZ transposase has a substitution mutation at one or more of the following positions: 134, 137, 138 and 144. In some embodiments, compared to SEQ ID NO: 1, the BZ transposase of the present invention has substitution mutations at positions 138, 189, 216, 208, and 251, and also has substitution mutations at one or more positions selected from 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, where the positions are numbered as in SEQ ID NO: 1; preferably, it also has substitution mutations at one or more of the following positions: 134, 137, and 144. Preferably, the substitution mutations at each position are selected from the substitution mutations shown in Tables A and B, respectively. Preferably, the mutation at position 22 is from D to A, L, I, G, or V. In one or more embodiments, the BZ transposase has a mutation 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 positions of the sites described in any of the above embodiments. In one or more embodiments, the BZ transposase has a mutation 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, 24, 25, 26, 27 positions of the sites described in any of the above embodiments.

[0062] In some embodiments, the substitution mutations at each of the above positions are independently selected from the group consisting of: 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, wherein the numbers refer to the amino acid position numbers of SEQ ID NO: 1, and the letters after the numbers represent the amino acid residues after the substitution mutations. In various embodiments, 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 of these mutations. In various embodiments, the BZ transposase can have 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, 24, 25, 26, 27 of these mutations.

[0063] In some embodiments, the BZ transposase of the present invention is a BZ transposase having any one or more of the following mutations compared to SEQ ID NO: 1:

[0064] G216A\H208V,

[0065] G216A\H208V\G189A、

[0066] G216A\H208V\K251T,

[0067] G216A\H208V\K251T\G189A、

[0068] G216A\H208V\K251T\G189A\Q138K、

[0069] G216A\H208V\K251T\G189A\Q138R、

[0070] G216A\H208V\K251T\G189A\K134A,

[0071] G216A\H208V\K251T\G189A\Q138K\K134A,

[0072] G216A\H208V\K251T\G189A\Q138R\K134A、

[0073] G216A\H208V\K251T\G189A\Q138K\V144E, or

[0074] G216A\H208V\K251T\G189A\Q138K\K137T.

[0075] In some embodiments, the transposase of the present invention is a fusion transposase, which comprises the BZ transposase described in any embodiment herein and a functional polypeptide, wherein the functional polypeptide is preferably a DNA sequence-specific binding domain and / or a nuclear localization signal domain (NLS).

[0076] In some embodiments, 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. In some embodiments, the amino acid sequence of the leucine zipper domain is as shown in any one of SEQ ID NOs: 13-15. In some embodiments, the nuclear localization signal domain comprises an SV40 NLS, a C-myc NLS, a TAF1 NLS, a TP53 NLS, a STAT3 NLS, or any combination thereof.

[0077] In some embodiments, the functional polypeptide is connected to the wild-type ZB transposase or a mutant thereof with or without a linker. In some embodiments of the fusion transposase, the BZ transposase and the DNA sequence-specific binding domain are separated by a linker. In some embodiments, the linker 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. In some embodiments, 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 sequence of the linker is GGGS.

[0078] In some embodiments, the transposon can be recognized by the transposase described in any embodiment of the present invention. In one or more embodiments, 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. Preferably, the terminal inverted repeat sequence does not contain a CpG motif compared to 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.

[0079] In some embodiments, the transposon comprises a target gene expression cassette located between terminal inverted repeats. In some embodiments, the target gene encodes a protein selected from a cell receptor, an immune checkpoint protein, a cytokine, a T cell receptor, a B cell receptor, a chimeric antigen receptor, and any combination thereof.

[0080] In some embodiments, the present invention provides a polynucleotide molecule encoding the transposon system as described in any embodiment herein, including a polynucleotide encoding the transposase, and / or a transposon DNA containing a mutant ITR sequence.

[0081] In some embodiments, the polynucleotide comprises DNA encoding the BZ transposase or the fusion transposase. In some embodiments, the polynucleotide comprises messenger RNA (mRNA) encoding the BZ transposase or the fusion transposase. In some embodiments, the mRNA is chemically modified, including but not limited to pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine nucleoside (m5C), 5-methoxyuridine (5moU). In some embodiments, the terminal inverted repeat sequence of the transposon is as shown in SEQ ID NO: 2. In some embodiments, the polynucleotide molecule is present in a nucleic acid construct such as a DNA vector. In some embodiments, the DNA vector includes a minicircle plasmid, a nanoplasmid, a doggybone, or other DNA form that does not contain antibiotics or / and replicon DNA sequences. In some embodiments, the transposase polynucleotide and / or the transposon are present in a viral vector, for example, the viral vector is selected from an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a herpes simplex virus vector, or a vaccinia virus vector.

[0082] In some embodiments, the polynucleotide encoding the transposase and the transposon are present in the same vector or in different vectors.

[0083] Yet another aspect of the present disclosure provides a cell that produces a transposon system as described herein. Yet another aspect of the present disclosure provides a cell that contains a polynucleotide molecule of a transposon system as described herein.

[0084] Yet another aspect of the present disclosure provides a method comprising the step of introducing the transposon system as described herein into a cell.

[0085] In some embodiments of the method, the introducing comprises contacting the cell with a polynucleotide encoding the BZ transposase or the fusion transposase. In some embodiments, the polynucleotide comprises DNA encoding the BZ transposase or the fusion transposase. In some embodiments, the polynucleotide comprises messenger RNA (mRNA) encoding the BZ transposase or the fusion transposase. In some embodiments, the mRNA is chemically modified.

[0086] In some embodiments of the method, the introducing comprises contacting the cell with a nucleic acid construct, such as a DNA vector, containing the transposon. In some embodiments, the DNA vector comprises a DNA form, such as a minicircle plasmid, a nanoplasmid, or a doggybone, that does not contain antibiotics and / or replicon DNA sequences. In some embodiments, the introducing comprises 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.

[0087] In some embodiments, the introducing comprises contacting the cell with the BZ transposase or the fusion transposase, preferably by adding the BZ transposase or the fusion transposase directly to a culture medium containing the cell (preferably a cell culture medium containing cells of the target organism). In direct contacting of the BZ transposase or the fusion transposase with the target cell according to the present invention, no reagents, carriers or methods that alter protein penetration across the cell membrane may be used.

[0088] In some embodiments, the present invention provides a method for genetically engineering a cell, wherein the method does not comprise a protein transfection step. In particular, it is preferred that the method does not comprise the use of a protein transfection reagent or procedure to introduce the transposase protein into the cell. In other words, the method of the present invention comprises 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.

[0089] In some embodiments of the method, the transposon comprises a combination of DNA elements located between two terminal inverted repeats, including but not limited to promoters, enhancers, expression genes, 5-UTRs, 3-UTRs, and other sequence elements known to those skilled in the art. In some embodiments, either of the two terminal inverted repeats (their reverse complements) comprises a sequence that is 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% identical to SEQ ID NO: 2. In some embodiments, the introducing comprises transfecting the cell by electroporation, microinjection, calcium phosphate precipitation, cationic polymers, dendrimers, liposomes, microparticle bombardment, fugene, direct sonication, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, nucleofection, or any combination thereof. In some embodiments, the introducing comprises electroporating the cell.

[0090] In some embodiments of the method, the cells are primary cells isolated from a subject. In some embodiments, the subject is a human. In some embodiments, the subject is a patient suffering from a disease. In some embodiments, the subject has been diagnosed with cancer or a tumor. In some embodiments, the cells are isolated from the subject's blood. In some embodiments, the cells comprise primary immune cells. In some embodiments, the cells comprise primary leukocytes. In some embodiments, the cells comprise primary T cells. In some embodiments, the primary T cells comprise γδ T cells, helper T cells, memory T cells, natural killer T cells, effector T cells, or any combination thereof. In some embodiments, the primary immune cells comprise CD3+ cells. In some embodiments, the cells comprise stem cells. In some embodiments, the stem cells are selected from the group consisting of: embryonic stem cells, hematopoietic stem cells, epidermal stem cells, epithelial stem cells, bronchioalveolar 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. In some embodiments, the stem cells comprise induced pluripotent stem cells.

[0091] The present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and the cells according to any one of the embodiments of the present invention.

[0092] Another aspect of the present disclosure provides 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; (b) administering the genetically modified cell to a patient in need of the treatment. In some embodiments, the genetically modified cell comprises a transgene introduced by the transposon. In some embodiments, the patient has been diagnosed with cancer or a tumor. In some embodiments, the administration comprises infusing the genetically modified cell into a blood vessel of the patient.

[0093] Another aspect of the present disclosure provides a use of the transposase, polynucleotide, transposon, transposon system, cell or pharmaceutical composition according to any embodiment of the present invention, which is selected from any one of the following (1) to (6):

[0094] (1) Application in the preparation of drugs or reagents for integrating the target gene expression cassette into the host cell genome;

[0095] (2) Application in the preparation of tools for integrating target gene expression cassettes into the host cell genome;

[0096] (3) Application in the preparation of transgenic animals and transgenic cells;

[0097] (4) Application in the preparation of drugs or preparations for genome research, gene therapy, cell therapy, or stem cell induction and post-induction differentiation;

[0098] (5) Application in the preparation of tools for genomic research, gene therapy, cell therapy, or stem cell induction and post-induction differentiation;

[0099] (6) Application in the preparation of kits, engineered immune cells or pharmaceutical compositions.

[0100] 3. Beneficial effects brought by the technical solution of the present invention

[0101] 1) The efficiency of wild-type transposase is too low. The engineering of wild-type ZB transposase significantly improves the transposition gene integration efficiency of transposase, making it have practical application value.

[0102] 2) After engineering the BZ transposase to improve its efficiency, the optimal TIR sequence mutation was screened, which reduced cytotoxicity and improved its feasibility for cell therapy and transgenic applications.

[0103] 3) Engineered BZ transposase in the form of mRNA is used in cell therapy and transgenics, further improving safety.

[0104] 4) The engineered BZ transposase protein in a non-transfectable form is used in cell therapy and transgenics, further improving safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1 shows the ZB transposase expression plasmid used in screening BZ transposase and the transposon plasmid containing the EGFP gene expression cassette

[0106] FIG2 shows the results of transposition efficiency screening of the BZ transposase single mutant library.

[0107] FIG3 shows that combined mutants of the BZ transposase improve transposition and expression efficiency in CHO cells.

[0108] FIG4 shows that combined mutants of the BZ transposase improve transposition and expression efficiency in PBMCs.

[0109] FIG. 5 shows that BZ transposase combination mutants according to some embodiments improve transposition efficiency (EGFP%).

[0110] FIG6 shows that BZ transposase combination mutants according to some embodiments increase transposon gene expression efficiency (gMFI).

[0111] FIG. 7 shows that BZ transposase combination mutants according to other embodiments improve transposition efficiency (EGFP%).

[0112] FIG8 shows that BZ transposase combination mutants according to other embodiments improve transposon gene expression efficiency (gMFI).

[0113] FIG9 shows that the transposition efficiency of the ZB-based fusion transposase is not affected by the type of fusion protein.

[0114] FIG10 shows the transposition efficiency when the transposase is added in the form of a protein.

[0115] Figure 11 shows that the engineered BZ transposase does not affect the cell proliferation level when used for CAR-T cell therapy and can reach the level of the high-efficiency PiggyBac transposase.

[0116] Figure 12 shows that when the engineered BZ transposase is used for CAR-T cell therapy, the transposition efficiency can reach the level of the high-efficiency PiggyBac transposase.

[0117] 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 the tumor cells L363.

[0118] FIG14 shows the effect of CpG motif mutations in ITRs on gene integration in the BZ transposon system.

[0119] FIG15 shows the effect of fusing different ZIP structures on transposition efficiency. DETAILED DESCRIPTION

[0120] Because the wild-type ZB transposase or ZB transposon system has the disadvantage of low transposition activity, it cannot achieve the expected optimal effect in real-world applications such as transgenic or cell therapy. Therefore, the present invention uses engineering methods to optimize the transposase amino acid sequence and / or transposon DNA sequence to improve the overall activity of the ZB transposase system. In the present invention, the engineered ZB transposase or ZB transposon system is referred to as the BZ transposase or BZ transposon system.

[0121] 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. In vitro genetic modification of non-transformed primary human T lymphocytes using gene transfer delivery systems based on non-viral vectors is extremely difficult. Currently, the only mature cases reported are limited to Sleeping Beauty transposon, PiggyBac transposon and TcBuster transposon. The present invention improves the transposition efficiency mediated by BZ transposase, making it suitable for clinical application in cellular immunotherapy.

[0122] CN105018523A proves that ZB transposon is an effective non-viral tool for inserting transgene into cells, but for cell or gene therapy, it still has safety risks, and the use of transposase encoding DNA causes the transposase protein to be continuously expressed in target cells for an extended period of time, and the time and kinetics of the exposure of the transposase that lacks control bring about a continuous and uncontrolled transposition risk, which has caused safety concerns about the conversion of unfavorable therapeutic cell products. To ensure that transposase is removed and to avoid inputting abnormal or unstable cell products, the engineered T cells of the ongoing experiment are cultured for 2-4 weeks after CAR gene delivery, which reduces cell adaptability and therapeutic effect. Therefore, there is an urgent need to improve the controllability and safety of the transposase / transposon system, which is also a key requirement for general cell and gene therapy. In order to control this transposase exposure risk, a method based on mRNA expression engineering of highly active BZ transposase is used in some embodiments of the present invention, which shortens the time of protein expression and reduces the cytotoxicity of cells such as immune cells, hematopoietic stem cells and progenitor cells (HSPC).

[0123] Under normal circumstances, transposases are usually difficult to recombinantly produce and exhibit low solubility under physiological conditions, hindering effective protein delivery. Known and widely used PiggyBac and TcBuster transposases have not reported solutions using proteins. Only patent CN113661247A reports that the solubility of the sleeping beauty transposase is improved by mutation, overcoming the shortcomings of SB protein aggregation, low stability and solubility, thereby achieving higher application value. The present invention unexpectedly discovered that the engineered highly active BZ transposase purified protein naturally has high stability, solubility, can autonomously cross the cell membrane and enter the cell nucleus to mediate genome modification through transposition, and this activity is uncommon for large molecular proteins. Because in the prior art methods, transposases need to be effectively transfected into cells using, for example, protein transfection reagents or procedures (such as electroporation). In a further optimization method of the present invention, the transposition integration of exogenous genes in cells is achieved using a purified engineered highly active BZ transposase protein. The present invention relates to the discovery that BZ transposase spontaneously penetrates mammalian cells and can be delivered along 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 function of transposases in methods for genetically engineering cells and using transposases as shuttles to deliver combinatorial DNA elements into target cells and even into target organelles. These methods and discoveries improve the safety of genetic engineering and gene therapy.

[0124] 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 cuts its target, thereby releasing the DNA transposon from its donor template. After excision, the DNA transposon can then integrate into a recipient DNA that has been cut by the same transposase. In some of their natural configurations, DNA transposons are flanked by two terminal inverted repeats and may contain a gene encoding a transposase that catalyzes transposition.

[0125] DNA transposon-based genome editing applications include a binary system consisting of a transposase component (transposase or its encoding nucleic acid (mRNA or DNA)) and a transposon component (transposon DNA containing a target gene flanked by inverted terminal repeats). The transposon and transposase components are co-delivered into target cells to achieve transposition integration via a cut-and-paste mechanism.

[0126] This article discusses various devices, systems, and methods related to collaborative approaches for using engineered hyperactive BZ transposases for cellular gene integration, particularly for enhancing gene transfer into human hematopoietic and / or immune system cells. This disclosure relates to improved engineered hyperactive BZ transposases, transposon systems, transposon vector sequences, transposase delivery methods, and transposon delivery methods. In one embodiment, this study identified specific universal sites for preparing hyperactive BZ transposases. In another embodiment, an improved method for delivering hyperactive BZ transposases as chemically modified in vitro transcribed mRNA is described. In another embodiment, a method for delivering BZ transposon vectors as "mini" circles of DNA is described, in which almost all prokaryotic sequences have been removed by recombinant methods. In another embodiment, an improved method for directly delivering purified hyperactive BZ transposase protein is described. In another embodiment, a method for using BZ transposases fused to a nuclear localization signal domain is described. The above embodiments can be used individually or in combination to improve the efficiency of transposon gene transfer to various target cells.

[0127] BZ transposase

[0128] One aspect of the present disclosure provides a BZ transposase. The BZ transposase may comprise one or more amino acid substitutions compared to wild-type ZB transposase (SEQ ID NO: 1). The BZ transposase may comprise an amino acid sequence having at least 70% sequence identity to the full-length sequence of wild-type ZB transposase (SEQ ID NO: 1). In some embodiments, the BZ transposase may comprise an amino acid sequence having at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the full-length sequence of wild-type ZB transposase (SEQ ID NO: 1). In some embodiments, one or more amino acid substitutions of the BZ transposase are mutated to charged acidic or basic amino acids (e.g., K, R, and H) compared to SEQ ID NO: 1.

[0129] The BZ transposase may comprise an amino acid sequence having at least one amino acid that differs from the full-length sequence of wild-type ZB transposase (SEQ ID NO: 1). In some embodiments, the BZ transposase may comprise an amino acid sequence having 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 amino acids that differ from the full-length sequence of wild-type ZB transposase (SEQ ID NO: 1). The BZ transposase may comprise one or more amino acid substitutions in any of the three domains of the ZB transposase (the duplex DNA binding and oligomerization domain, the DDE catalytic domain), and the interdomain regions between the domains, or any combination thereof. In some cases, the BZ transposase may comprise one or more amino acid substitutions in the duplex DNA binding and oligomerization domain, the DDE catalytic domain, the interdomain regions between the domains, or a combination thereof.

[0130] In one aspect, in some embodiments, the present invention provides a BZ transposase comprising an amino acid sequence having a mutation 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, compared to the full-length SEQ ID NO: 1. The BZ transposase may also have a mutation at one or more of the following positions: 79, 120, and 208. In some embodiments, the mutations at each position are independently selected from the mutations shown in Tables A and B. In some embodiments, the one or more amino acid mutations comprise a mutation from aspartic acid or glutamic acid to a neutral or basic amino acid. In some embodiments, the one or more amino acid substitutions are based on conservation principles within the Tc1 / Mariner transposon family.

[0131] In some embodiments, compared to SEQ ID NO: 1, the BZ transposase of the present invention has substitution mutations at least at positions 71 and 110. In addition, in addition to the substitution mutations at positions 71 and 110, the BZ transposase of the present invention may also have substitution mutations at one or more positions selected from the group consisting of positions 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.

[0132] In some embodiments, in addition to the substitution mutations at positions 71 and 110, the BZ transposase of the present invention further has a substitution mutation at one or more positions selected from the group consisting of positions 5, 21, 22, 120, 125, 134, 137, 138, 144, 189, 208, 216, and 251. In some embodiments, compared to SEQ ID NO: 1, the BZ transposase of the present invention has substitution mutations at at least positions 71, 79, and 110. Preferably, the substitution mutation at position 71 is Q to R, K, or H; the substitution mutation at position 79 is Q to R, K, or H; and the substitution mutation at position 110 is H to R or K.

[0133] In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has substitution mutations at positions 71 and 110, and has substitution mutations at one or more of the following positions: 5, 21, 22, 134, 137, 138, 144, 189, 208, 216 and 251; preferably, has substitution mutations at one or more of the following positions: 134, 137, 138, 144, 189, 208, 216 and 251. In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has substitution mutations at positions 71, 110, and 208, and has substitution mutations at one or more of the following positions: 5, 21, 22, 134, 137, 138, 144, 189, 216, and 251; preferably, has substitution mutations at one or more of the following positions: 134, 137, 138, 144, 189, 216, and 251. In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has substitution mutations at positions 71, 110, 208 and 216, and has substitution mutations at one or more of the following positions: 5, 21, 22, 134, 137, 138, 144, 189 and 251; preferably, has substitution mutations at one or more of the following positions: 134, 137, 138, 144, 189 and 251. In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has substitution mutations at positions 71, 110, 208, 216 and 251, and has substitution mutations at one or more of the following positions: 5, 21, 22, 134, 137, 138, 144 and 189; preferably, has substitution mutations at one or more of the following positions: 134, 137, 138, 144 and 189. In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has substitution mutations at positions 71, 110, 189, 208, 216 and 251, and has substitution mutations at one or more of the following positions: 5, 21, 22, 134, 137, 138 and 144; preferably, has substitution mutations at one or more of the following positions: 134, 137, 138 and 144.In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has substitution mutations at positions 71, 110, 138, 189, 208, 216 and 251, and has substitution mutations at one or more of the following positions: 5, 21, 22, 134, 137 and 144; preferably, has substitution mutations at one or more of the following positions: 134, 137 and 144. In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has substitution mutations at positions 71, 79, and 110, and has substitution mutations at one or more of the following positions: 5, 21, 22, 120, 125, 134, 137, 138, 144, 189, 208, 216, and 251; preferably, has substitution mutations at one or more of the following positions: 134, 137, 138, 144, 189, 208, 216, and 251. In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has substitution mutations at positions 71, 79, 110, and 208, and has substitution mutations at one or more of the following positions: 5, 21, 22, 120, 125, 134, 137, 138, 144, 189, 216, and 251; preferably, has substitution mutations at one or more of the following positions: 134, 137, 138, 144, 189, 216, and 251. In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has substitution mutations at positions 71, 79, 110, 208 and 216, and has substitution mutations at one or more of the following positions: 5, 21, 22, 120, 125, 134, 137, 138, 144, 189 and 251; preferably, has substitution mutations at one or more of the following positions: 134, 137, 138, 144, 189 and 251. In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has a substitution mutation at position 71, 79, 110, 208, 216 and 251, and has a substitution mutation at one or more of the following positions: 5, 21, 22, 120, 125, 134, 137, 138, 144 and 189; preferably, has a substitution mutation at one or more of the following positions: 134, 137, 138, 144 and 189.In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has a substitution mutation at position 71, 79, 110, 189, 208, 216 and 251, and has a substitution mutation at one or more of the following positions: 5, 21, 22, 120, 125, 134, 137, 138 and 144; preferably, has a substitution mutation at one or more of the following positions: 134, 137, 138 and 144. In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has substitution mutations at positions 71, 79, 110, 138, 189, 208, 216 and 251, and has substitution mutations at one or more of the following positions: 5, 21, 22, 120, 125, 134, 137 and 144; preferably has substitution mutations at one or more of the following positions: 134, 137 and 144.

[0134] On the other hand, in some embodiments, the BZ transposase of the present invention has substitution mutations at least at positions 208 and 216 compared to SEQ ID NO: 1. In some embodiments, in addition to the substitution mutations at positions 208 and 216, the BZ transposase of the present invention further has substitution mutations at one or more positions selected from the group consisting of positions 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. In some embodiments, in addition to the substitution mutations at positions 208 and 216, the BZ transposase of the present invention further has substitution mutations at one or more positions selected from 5, 21, 22, 79, 120, 125, 134, 137, 138, 144, 189 and 251, where the position numbers are the position numbers of SEQ ID NO: 1.

[0135] In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has a substitution mutation at position 208, 216 and 251, and a substitution mutation at one or more positions selected from 5, 21, 22, 35, 38, 56, 61, 71, 73, 110, 125, 134, 137, 138, 144, 186, 188, 189, 204, 215-218, 217, 218, 235, 79 and 120, wherein the positions are numbered as in SEQ ID NO: 1; preferably, the BZ transposase has a substitution mutation at one or more of the following positions: 134, 137, 138, 144 and 189.

[0136] In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has a substitution mutation at positions 189, 208, and 216, and simultaneously has a substitution mutation at one or more positions selected from 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, wherein the positions are numbered as in SEQ ID NO: 1; preferably, it simultaneously has a substitution mutation at one or more of the following positions: 134, 137, 138, 144, and 251.

[0137] In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has a substitution mutation at positions 189, 216, 208 and 251, and a substitution mutation at one or more positions selected from 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, wherein the positions are numbered as in SEQ ID NO: 1; preferably, the BZ transposase has a substitution mutation at one or more of the following positions: 134, 137, 138 and 144.

[0138] In some embodiments, compared with SEQ ID NO: 1, the BZ transposase of the present invention has a substitution mutation at position 138, 189, 216, 208 and 251, and a substitution mutation at one or more positions selected from 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; preferably, it has a substitution mutation at one or more of the following positions: 134, 137 and 144.

[0139] In some embodiments, the mutation at each position is a mutation to a positively charged amino acid. Preferably, when comprising a mutation at S38, Q71 or H110, the amino acid at position S38, Q71 or H110 is mutated to a positively charged amino acid, such as H, K or R.

[0140] In some embodiments, the substitution mutations at each position are selected from the substitution mutations shown in Table A and Table B. Preferably, the mutation at position 22 is from D to A, L, I, G or V. In some embodiments, the substitution mutations at each of the above positions are independently selected from the group consisting of: 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, wherein the numbers refer to the amino acid position numbers of SEQ ID NO: 1, and the letters after the numbers represent the amino acid residues after the substitution mutations.

[0141] Exemplary BZ transposases may comprise one or more amino acid substitutions from Table C. Sometimes, a BZ transposase may comprise 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. Preferably, the BZ transposase comprises one or more amino acid substitutions selected from the group consisting of N5S, F21K, S38R, Q71H, Q71R, Q79R, H110R, H110K, K134A, K137T, Q138K, V144E, G189A, G216A, and K251T. More preferably, the BZ transposase comprises one or more mutations selected from the group consisting of S38R, Q71H, Q71R, H110K, and H110R.

[0142] Exemplary BZ transposases comprise one or more amino acid substitutions or substitution combinations from Table D and Table E. Sometimes, a BZ transposase may comprise at least one amino acid substitution or substitution combination 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 substitution combinations from Table D and Table E. Preferably, the BZ transposase comprises one or more amino acid substitutions or substitution combinations selected from the group consisting of Q71R\H110R, Q71R\Q79R\H110R, G216A\Q71R\Q79R\H110R, H208V\Q71R\Q79R\H110R, H208V\G216A\Q71R\Q79R\H110R.

[0143] Highly active BZ transposase

[0144] Another aspect of the present disclosure provides a hyperactive BZ transposase. As used herein, a "hyperactive" BZ transposase may refer to any BZ transposase that has increased transposition efficiency compared to the wild-type ZB transposase having the amino acid sequence of SEQ ID NO: 1.

[0145] Transposition efficiency can be measured according to the percentage of the successful transposition event that occurs in the host cell colony, and this percentage is normalized with the amount of the transposon and transposase introduced into this host cell colony.In many cases, when comparing the transposition efficiency of two or more transposases, for the transfection of host cells under identical or similar transfection conditions, each of identical transposon construct and two or more transposases is paired.The amount of the transposition event in the host cell can be checked by various methods.For example, the transposon construct can be designed to contain the reporter gene between the terminal inverted repeat sequence, and the transfected cells that this reporter gene is positive can be counted as the cells in which the successful transposition event occurs, which can draw the estimated value of the amount of the transposition event.In some embodiments, when comparing the transposition efficiency of two or more different transposons, for the transfection of host cells under identical or similar transfection conditions, identical transposase and every kind of different transposons are paired.Similar methods can be used to measure transposition efficiency.Also methods well known to those skilled in the art can be implemented to compare transposition efficiency.

[0146] Also provided herein is a method for obtaining a highly active BZ transposase. An exemplary method may include systematically modifying the amino acids of the BZ transposase to increase the net charge of the amino acid sequence. The method may include systematically mutating the DNA binding and oligomerization domains to positively charged histidine (H), lysine (K), or arginine (R) residues. Without wishing to be bound by a particular theory, the increase in the net charge of the DNA binding and oligomerization domains at neutral pH can increase the stability of the transposase / transposon, transposase / transposase interaction complex, enhance the binding ability between the target DNA and the transposase protein and the transposase, thereby improving the integration efficiency of the transposase.

[0147] The hyperactive BZ transposase may comprise one or more amino acid substitutions. In many cases, the one or more amino acid substitutions may be substitutions of non-conservative amino acids with conservative amino acids in the wild-type ZB sequence (SEQ ID NO: 1). Non-limiting examples of 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.

[0148] In some cases, the BZ transposase may comprise amino acid substitutions S38R, Q71R, Q79R, H110R, G216A. The BZ transposase may also comprise amino acid substitutions H208V, K251T, G189A, Q138K, Q138R, K134A, K137T, V144E, N005S, F21K, K120S, N125L, or one or more.

[0149] The inventors have also discovered that ZB or BZ transposases can be fused with other functional polypeptides or protein domains without affecting the transposase or transposon gene integration function. Therefore, the transposase of the present invention can also be a fusion transposase comprising the ZB or BZ transposase, wherein the fusion transposase further comprises a functional polypeptide.

[0150] 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.

[0151] In some embodiments, the leucine zipper domain is derived from the Basic_leucine-zipper_C protein family of IPR020983, the bZIP protein family of IPR004827, and the bZIP_sf protein family of IPR046347 in the InterPro protein database (https: / / www.ebi.ac.uk / interpro / ). In some embodiments, the leucine zipper domain is selected from C / EBPα (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), and LZIP (whose amino acid sequence is shown in SEQ ID NO: 15), all of which can increase transposition efficiency.

[0152] In some embodiments, the DNA sequence-specific or non-specific binding domain can be located at the C-terminus or N-terminus of the transposase. In some embodiments, the nuclear localization signal domain can be located at the C-terminus or N-terminus of the transposase, preferably the N-terminus. In some embodiments, the leucine zipper domain is located at the N-segment of the transposase. In some embodiments, 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. When located at the same end of the transposase, from the N-terminus to the C-terminus, it can be a DNA sequence-specific or non-specific binding domain-nuclear localization signal domain, or it can be a nuclear localization signal domain-DNA sequence-specific or non-specific binding domain. When located at 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-terminus and C-terminus of the transposase, or at the C-terminus and N-terminus, respectively.

[0153] In the fusion transposase, the BZ transposase and the DNA sequence-specific binding domain can be directly connected or separated by a linker. Typically, the linker 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. Exemplarily, 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 sequence of the linker is GGGS.

[0154] The present invention also includes mutants of the BZ transposase or fusion transposase described in any embodiment. As used herein, "mutant" or "variant" includes mutants of the transposase, as long as the mutant retains the respective biological functions of the antibody, transmembrane region, and intracellular domain. For example, mutants of antibodies suitable for use in the present invention include those having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with the comparative antibody. Sequence identity between two aligned sequences can be calculated using, for example, NCBI's BLASTp. Alternatively, compared to the comparative sequence, the mutants described in the present invention have one or more (e.g., within 20, within 15, within 10, within 8, within 5, or within 3, such as 1-20, 1-10, etc.) amino acid residue insertions, substitutions, or deletions. For example, in the art, conservative substitutions with amino acids having similar or similar properties generally do not alter the function of the protein or polypeptide. "Amino acids with similar or similar properties" include, for example, families of amino acid residues having similar side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In one or more embodiments, the mutant retains the amino acid substitution at position 5, 21, 22, 35, 38, 56, 61, 71, 73, 79, 110, 125, 134, 137, 138, 144, 186, 188, 189, 204, 215-218, 215, 216, 217, 218, 235, 251, 289, 120, or 208 of the BZ transposase described herein.

[0155] The transposase sequence described herein may be a modified polypeptide sequence. Modifications (generally without altering the primary structure) include chemical derivatization of the polypeptide in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those resulting from glycosylation during polypeptide synthesis and processing or during further processing steps. Such modifications can be accomplished by exposing the polypeptide to a glycosylation enzyme (e.g., a mammalian glycosylase or deglycosylase). Modifications also include sequences containing phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, and phosphothreonine). Also included are polypeptides modified to enhance their resistance to proteolysis or optimize their solubility.

[0156] It should be understood that in gene cloning operations, it is often necessary to design suitable restriction sites, which inevitably introduce one or more irrelevant residues at the end of the expressed amino acid sequence, but this does not affect the activity of the target sequence. In order to construct a fusion protein, promote the expression of a recombinant protein, obtain a recombinant protein that is automatically secreted outside the host cell, or facilitate the purification of the recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus, or other suitable regions within the protein of the recombinant protein, for example, including but not limited to suitable linker peptides, signal peptides, leader peptides, terminal extensions, etc. Therefore, 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 can be used herein. For example, 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 the protein.

[0157] The transposases described herein can spontaneously penetrate cells by direct contact with the cells and can be delivered along with transposon DNA to genetically modify various cell lines, embryonic, hematopoietic, and induced pluripotent stem cells.

[0158] polynucleotide molecules

[0159] The present invention provides polynucleotide molecules encoding the transposases (BZ transposases or fusion transposases) described herein, and / or transposon DNA containing mutated or unmutated ITR sequences. The present invention also provides complementary sequences to the coding sequences of the transposases. The polynucleotides may be recombinant or synthetic; they may comprise DNA, RNA, and PNA (peptide nucleic acid) and may be hybrids thereof. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The present invention also encompasses degenerate variants of the polynucleotide sequences encoding fusion proteins, i.e., nucleotide sequences encoding the same amino acid sequence but with a different nucleotide sequence.

[0160] In some embodiments, the polynucleotide molecule comprises DNA encoding the BZ transposase or the fusion transposase. The polynucleotide may also comprise a nucleic acid sequence encoding a transposon that can be recognized by the BZ transposase or the fusion transposase. The polynucleotide is present in a DNA vector. The vector may be a cloning vector or an expression vector. The expression vector comprises an expression cassette for the transposase, which is a nucleic acid construct containing a promoter, a transposase coding sequence, and a PolyA tailing signal sequence. The expression vector also typically contains other elements commonly contained in vectors, such as multiple cloning sites, resistance genes, replication initiation 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 DNA forms such as minicircle plasmids, nanoplasmids, and doggybone that do not contain antibiotics and / or replicon DNA sequences.

[0161] In some embodiments, the polynucleotide comprises a messenger RNA (mRNA) encoding the BZ transposase or the fusion transposase. In some embodiments, the mRNA is chemically modified, including but not limited to pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine (m5C), 5-methoxyuridine (5moU).

[0162] The present invention also relates to polynucleotides that hybridize to the above-mentioned polynucleotide sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0163] The polynucleotide molecules can be prepared by conventional methods in the art, and the corresponding vectors can be constructed, usually by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequences by artificial synthesis, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments and then connecting them, a very long fragment of the sequence can be obtained. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein. Alternatively, the sequences of the various parts of the fusion transposase can be obtained as above and then connected to obtain the full length of the CAR.

[0164] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the host cells after propagation by conventional methods. Recombinant vectors can be constructed using methods well known to those skilled in the art, see, for example, Sambrook et al., Ausubel (1989) or other standard textbooks. Vectors containing the nucleic acid molecules of the present 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 commonly used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other cellular hosts, see Sambrook et al.

[0165] In addition, a DNA sequence encoding a protein of the invention (or a fragment thereof, or a derivative thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the invention by chemical synthesis.

[0166] The present invention also relates to nucleic acid constructs comprising 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 a variety of ways to ensure expression of the transposase. The nucleic acid constructs can be manipulated prior to insertion into a vector, depending on the specific expression vector or requirements. The regulatory sequences and manipulations required for expressing proteins via DNA or mRNA are known in the art. Techniques for altering polynucleotide sequences using recombinant DNA methods are also known in the art.

[0167] In certain embodiments, 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 generally 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 for integrating the target sequence into the cell genome. These vectors can be used to transform appropriate host cells to enable protein expression. Vectors generally contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. The sequences (collectively referred to as "flanking sequences" in certain embodiments) generally include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a multi-linker region for inserting a nucleic acid encoding an antibody to be expressed, and a selectable marker element.

[0168] The type of vector is not limited, for example, plasmids, phagemids, phage derivatives, animal viruses and cosmids, and can be changed according to the host cell to be introduced. Viral vector technology is well known in the art and is described in, for example, 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, herpes viruses and lentiviruses.

[0169] To assess transposase expression, the vector introduced into the cells may also contain either or both a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected by the viral vector.

[0170] transposon

[0171] A transposon comprises a combination of DNA elements positioned between two terminal inverted repeat sequences, which can be recognized by the transposase described in any embodiment of the present invention. The combination of DNA elements includes, but is not limited to, sequence elements well known to those skilled in the art, such as promoters, enhancers, expressed genes, 5-UTRs, and 3-UTRs. In some embodiments, the two terminal inverted repeat sequences each independently comprise a sequence that is 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% identical to SEQ ID NO: 2, 11, or 12.

[0172] In some embodiments, the DNA element combination includes an expressed gene. Accordingly, the DNA element combination may also include a promoter selected from CMV, EFS, MND, EF1α, CAGC, PGK, UBC, U6, H1 and Cumate. In some embodiments, the expressed gene encodes a protein selected from a cell receptor, an immune checkpoint protein, a cytokine and any combination thereof. In some embodiments, the expressed gene encodes a cell receptor selected from a T cell receptor (TCR), a B cell receptor (BCR), a chimeric antigen receptor (CAR) or any combination thereof.

[0173] CAR may comprise a polypeptide (such as scFv) that binds to a tumor cell membrane antigen, a hinge region, a transmembrane region, and an intracellular signaling region in sequence. The hinge region, transmembrane region, and intracellular signaling region known in the art for constructing CAR can be used to construct the CAR of the present invention. Typically, a polypeptide that binds to a tumor cell membrane antigen is able to bind to a membrane antigen widely expressed by tumor cells with moderate affinity. The polypeptide is usually inserted with an antigen epitope, and the insertion position is selected from any one, two, or three of the following three positions: the N-terminus of the polypeptide, between the polypeptide and the hinge region, and inside the polypeptide. The polypeptide that binds to a tumor cell membrane antigen is a natural polypeptide or an artificially synthesized polypeptide; preferably, the artificially synthesized polypeptide is a single-chain antibody or a Fab fragment. The chimeric antigen receptor of the present invention can be directed against 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, SCC, AFU, EBV-VCA, POA and PROGRP. In some embodiments, CARs suitable for use in the present invention may refer to CN202111681582.0, the entire contents of which are incorporated herein by reference.

[0174] In some embodiments, 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. In certain embodiments, the antibody is a chimeric antibody, a recombinant antibody, a humanized antibody, or a human antibody. In certain embodiments, the antibody is affinity-tuned. Non-limiting examples of antibodies of the present invention include single-chain variable fragments (scFv), VHHs, single-domain antibodies (sdABs), small modular immunopharmaceuticals (SMIP) molecules, or nanobodies. In certain embodiments, VHHs are camelids. Alternatively or in addition, in certain embodiments, VHHs are humanized. Non-limiting examples of antibody fragments of the present invention include complementary determining regions, variable regions, heavy chains, light chains, or any combination thereof. Non-limiting examples of antibody mimetics of the present invention include: affibodies, Afflilin molecules, affimers, Affitin molecules, alphabodies, anticalins, and Avimer molecules, DARPins, Fynomers, Kunitz domain peptides, or monobodies. Non-limiting examples of protein scaffolds of the present invention include Centyrin.

[0175] Transposons are typically provided in the form of nucleic acid constructs, particularly DNA vectors. In this regard, any DNA vector that facilitates the introduction of transposons into cells can be used, such as minicircle plasmids, nanoplasmids, doggybone plasmids, and the like. The DNA plasmids may or may not contain antibiotic and / or replicon sequences.

[0176] host cells

[0177] As used herein, "host cell" refers to a prokaryotic or eukaryotic cell capable of replicating a vector and / or expressing a heterologous gene encoded by a vector when expressing a heterologous nucleic acid sequence. A host cell can serve as a recipient for a vector or mRNA. A host cell can be "transfected" or "transformed," which refers to a process by which an exogenous nucleic acid is transfected or transduced into a host cell. Transformed cells include the primary subject cell and its progeny. As used herein, the terms "engineered" and "recombinant" cells or host cells often refer to cells into which an exogenous nucleic acid sequence, such as a vector or mRNA, has been introduced. Thus, recombinant cells can be distinguished from naturally occurring cells that do not contain the introduced recombinant nucleic acid.

[0178] Herein, host cells include cells that carry the polynucleotide molecules described herein and / or produce the transposases described herein. Specifically, the present invention provides cells that carry the transposases described herein and / or their coding sequences. The cells may also contain a nucleic acid sequence encoding a transposon that can be recognized by the transposase. The polynucleotide molecules may also contain a nucleic acid sequence encoding a transposon that can be recognized by the BZ transposase or the fusion transposase.

[0179] The type of cell is not limited, as long as it can express the transposase described herein or can use the transposase described herein to achieve transposition of the transposon. In some embodiments, the cell is a primary cell isolated from a subject. The subject is a healthy subject or has been diagnosed with a disease (e.g., cancer or tumor).

[0180] In some embodiments, the cells are isolated from the blood of the subject, such as PBMCs or cells derived therefrom. The cells may comprise primary immune cells, such as primary leukocytes. In some embodiments, the cells comprise primary T cells. The primary T cells comprise γδ T cells, helper T cells, memory T cells, natural killer T cells, effector T cells, or any combination thereof. In some embodiments, the primary immune cells comprise CD3+ cells.

[0181] In some embodiments, the cells comprise stem cells. The stem cells are selected from the group consisting of embryonic stem cells, hematopoietic stem cells, epidermal stem cells, epithelial stem cells, bronchial alveolar 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.

[0182] The nucleic acid constructs, vectors, and mRNAs of the present invention can be introduced into cells of interest. In this context, methods of introduction include transfection of the cells by electroporation, microinjection, calcium phosphate precipitation, cationic polymers, dendrimers, liposomes, microparticle bombardment, fugene, direct sonication, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, nucleofection, or any combination thereof. In certain embodiments, electroporation is used to deliver the nucleic acid construct or recombinant expression vector.

[0183] The obtained transformants can be cultured using conventional methods to express the BZ transposase or fusion transposase of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for host cell growth. In embodiments where 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 a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a further period of time.

[0184] The polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various separation methods using 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 renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0185] Transposition system

[0186] The present invention also relates to a system for genome editing (transposon system), comprising: (1) a BZ transposase or fusion transposase as described herein, or a polynucleotide encoding the same, and / or (2) a transposon DNA comprising a mutated or unmutated inverted terminal repeat sequence. The transposon is as described elsewhere herein. Exemplary wild-type inverted terminal repeat sequences are shown in SEQ ID NO: 2, 11, or 12.

[0187] The polynucleotide comprises DNA or messenger RNA (mRNA) encoding the BZ transposase or the fusion transposase. In some embodiments, the transposon is present in a DNA vector. In some embodiments, the polynucleotide and the transposon are present in the same plasmid.

[0188] The inventors have discovered that the terminal inverted repeat sequence of a transposon can be mutated to improve transposition efficiency. In some embodiments, the terminal inverted repeat sequence of a transposon does not contain a CpG motif compared to a wild-type terminal inverted repeat sequence (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, wherein the terminal inverted repeat sequence is a sequence in which the CpG in SEQ ID NO: 11 or 12 is mutated to ApG, GpG, TpG, CpA, or CpT.

[0189] Methods and uses

[0190] The present invention also relates to a method for preparing a cell, comprising the steps of introducing the BZ transposase or fusion transposase as described herein and a transposon that can be recognized by the BZ transposase or fusion transposase into the cell.

[0191] In some embodiments, the introducing comprises contacting the cell with a polynucleotide encoding the BZ transposase or the fusion transposase. The polynucleotide comprises DNA or messenger RNA (mRNA) encoding the BZ transposase or the fusion transposase.

[0192] In some embodiments, the introducing comprises contacting the cell with the BZ transposase or the fusion transposase, preferably by adding the BZ transposase or the fusion transposase directly to a culture medium containing the cell (preferably a cell culture medium containing cells of the target organism). In direct contacting of the BZ transposase or the fusion transposase with the target cell according to the present invention, no reagents, carriers or methods that alter protein penetration across the cell membrane may be used.

[0193] In some embodiments, the introducing comprises contacting the cell with a DNA vector containing the transposon. In some embodiments, the DNA vector comprises a minicircle plasmid. In some embodiments, the introducing comprises contacting the cell with a plasmid vector containing the transposon and a polynucleotide encoding the BZ transposase or the fusion transposase.

[0194] The present invention also provides a method for genetically engineering cells using a 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 the use of a protein transfection reagent or procedure to introduce the transposase protein into the cell. The method includes a process of directly contacting the transposase with the cell to be transposase-treated, for example, by incubating the cell with a cell culture medium containing the transposase. The method of the present invention includes a step of introducing the transposase protein without using any vector, reagent or method that alters the penetration of the protein across the cell membrane.

[0195] The term "protein transfection" in the context of the present invention should be understood broadly to refer to any method or reagent that is sufficient to introduce a protein that cannot effectively enter the target cell into the target cell. Common 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 methods such as protein electroporation.

[0196] 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 comprising a transgene introduced by the transposon; (b) administering the genetically modified cell to a patient in need of the treatment. In some embodiments, the patient has been diagnosed with cancer or a tumor. In some embodiments, the administration comprises infusing the genetically modified cell into a blood vessel of the patient.

[0197] The present invention also relates to the use of the BZ transposase or fusion transposase described herein, its coding sequence (DNA or RNA) or nucleic acid construct, cell, or genome editing system in the preparation of products, such as gene editing kits, engineered immune cells, or pharmaceutical compositions. The immune cells include γδ T cells, helper T cells, memory T cells, natural killer T cells, effector T cells, etc.

[0198] The scope of the present invention also encompasses kits comprising the BZ transposase or fusion transposase described herein, their coding sequences (DNA or RNA) or nucleic acid constructs, cells, or genome editing systems. The kits further comprise one or more of a nucleic acid encoding a transposon recognized by the BZ transposase or the fusion transposase, or a nucleic acid construct thereof (e.g., a DNA vector comprising the transposon), host cells, a cell culture medium suitable for the host cells, cytokines, and instructions for use.

[0199] Pharmaceutical compositions and administration

[0200] The transposases, nucleic acid molecules, and cells of the present invention can be administered alone or as pharmaceutical compositions in combination with a diluent and / or other components, such as related cytokines or cell populations. Thus, the present invention also provides pharmaceutical compositions comprising the transposases, nucleic acid constructs, gene editing systems, or cells prepared by the methods described herein and pharmaceutically acceptable excipients.

[0201] In the present invention, a "pharmaceutically acceptable excipient" is a pharmaceutically or food-acceptable carrier, solvent, suspending agent or excipient used to deliver the transposase, nucleic acid construct, gene editing system or cell of the present invention to an animal or human. Herein, a pharmaceutically acceptable excipient is non-toxic to the recipient of the composition at the dose and concentration used. It may include various types of carriers or excipients commonly used in the treatment of proteins, nucleic acids or cells known in the art. 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. In some embodiments, 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 used in combination with the pharmacological compound. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th ed., AR Genrmo, ed., 1990, Mack Publishing Company. The optimal pharmaceutical composition can be determined depending on the intended route of administration, mode of delivery, and desired dosage.

[0202] The pharmaceutical composition of the present invention can be selected for parenteral delivery, for inhalation or delivery through the digestive tract (such as oral), for example, for intravenous delivery. The preparation of the composition is within the technology of this area. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations that contain immune cells, particularly immune cells (such as T cells), in sustained or controlled release delivery formulations. The pharmaceutical composition of the present invention can also be used in a manner suitable for the disease to be treated (or prevented).

[0203] Pharmaceutical compositions for in vivo administration are typically provided in the form of sterile formulations. Sterilization is achieved by filtration through a sterile filtration membrane. Compositions for parenteral administration can be stored in lyophilized form or in solution (e.g., frozen preparations). Parenteral compositions are typically placed in containers with sterile access ports, such as intravenous solution strips or bottles with stoppers pierceable by hypodermic needles.

[0204] Once formulated, the pharmaceutical composition is stored in sterile vials in the form of a solution, suspension, gel, emulsion, solid, crystal, frozen product, or dehydrated or lyophilized powder. The pharmaceutical formulation (e.g., frozen preparation) can be stored in a ready-to-use form or in a form that is further formulated before administration. For example, suitable for delivery of the pharmaceutical composition described herein can be a frozen preparation that can withstand long-distance transportation without damaging the cells. In addition to the cells themselves, the frozen preparation typically also includes components such as cell freezing solution and human serum albumin (HSA). Before administration (e.g., intravenous infusion), the frozen pharmaceutical composition needs to be stored at low temperatures (e.g., placed in liquid nitrogen). After thawing, the frozen preparation can be infused into the patient directly or formulated as an infusion composition. Those skilled in the art are aware of the components and concentrations of conventional freezing solutions. For example, the freezing solution or infusion composition may also contain dimethyl sulfoxide, sodium chloride, glucose, sodium acetate, potassium chloride, or magnesium chloride, etc., and its concentration can be determined by those skilled in the art (e.g., experienced physicians) based on the conditions of the cells, disease, and patient.

[0205] In some embodiments of the invention, the genetically modified cells of the invention or compositions thereof can be combined with other therapies known in the art.

[0206] "Patient," "subject," "individual," and the like are used interchangeably herein to refer to a living organism, such as a mammal, in which an immune response can be elicited. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and transgenic species thereof.

[0207] In a specific embodiment, the present invention obtains a mutant BZ enzyme nucleic acid sequence by PCR, connects it to a plasmid and transforms Escherichia coli to obtain an engineered BZ transposase. Transfection of CHO cells and / or PBMC cells with an expression vector or mRNA for the engineered BZ transposase and a transposon containing eGFP or an expression gene results in transposon expression. Replacing the BZ transposase with a fusion transposase fused with other polypeptides does not affect the transposase or transposon gene integration function. Furthermore, cells are transfected with a transposon and incubated with a culture medium containing wild-type ZB transposase or engineered BZ transposase, and transposon expression is also observed.

[0208] The present invention is further described in detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should in no way be construed as being limited to the following examples, but rather should be construed to include any and all variations that become apparent as a result of the teachings provided herein. The methods and reagents used in the examples are, unless otherwise stated, conventional methods and reagents in the art.

[0209] Example

[0210] This article adopts the following method.

[0211] (1) Site-directed mutagenesis for preparation of BZ mutants

[0212] High-Fidelity DNA Polymerase (New England BioLabs) was used for all site-directed mutagenesis. For single-point mutations, rolling circle PCR was performed, followed by digestion with DpnI restriction enzyme, and 5 μL of the digestion reaction product was transformed into TOP10 E. coli competent cells. For combined mutations, multiple PCR reactions were used for mutagenesis, and the PCR products were purified using an agarose gel DNA recovery kit (Tianmo Bio) and purified using Hieff A 10 μL ligation reaction was performed on the purified PCR product using the Plus Multi One Step Cloning Kit (YEASEN). 5 μL of the ligation reaction product was then transformed into TOP10 competent E. coli cells. Three single clones were selected for each mutant and cultured. Half of each clone was sequenced and maintained. For mutants with correct sequences, plasmids for transfection were prepared using the NucleoBond Xtra MiDi EF Plasmid Preparation Kit (MACHEREY-NAGEL). Plasmid samples were randomly tested for supercoiling percentage and endotoxin content.

[0213] (2) Measurement of transfection efficiency in CHO-K1 cells

[0214] On the day of electroporation, CHO-K1 cells were digested with trypsin containing 0.25% EDTA, collected and centrifuged, and resuspended in 1× DPBS for counting. 1.5-2.5×10 6 After centrifugation, remove the supernatant. Following the instructions for the Lonza 2B electroporation reagent, take 100 μl of electroporation buffer, mix 4 μg of the transposase plasmid and 4 μg of the transposon plasmid with the electroporation reagent, and add it to the centrifuged cell pellet. Resuspend the cell pellet in an electroporation cuvette and place it in a Lonza 2B electroporator, setting the electroporation program to H-014. After electroporation, transfer the cells to a six-well plate in complete culture medium and culture. Fluorescence imaging was performed on days 5, 9, and 13 after electroporation, and the transposition efficiency of the transposase in CHO-K1 cells was determined by flow cytometry.

[0215] (3) Measurement of transfection efficiency in PBMC (peripheral blood mononuclear cells) cells

[0216] On the day of electroporation, the frozen PBMCs were thawed and counted, and 1×10 7After centrifugation, remove the supernatant. According to the instructions of Lonza 2B electroporation reagent, take 100ul of electroporation solution, mix 4ug of transposase plasmid and 4ug of transposon plasmid with electroporation reagent, and add it to the centrifuged cell pellet. After resuspending, add it to the electroporation cup, place it in the Lonza 2B electroporator, and set the electroporation program to U-014. After electroporation, transfer the cells to a six-well plate with complete culture medium (AIM-V + 2% FBS) and culture it, supplemented with 500U / ml of IL-2. Transfer the plate on the 5th day after electroporation, and take fluorescent photos, count, and flow cytometry to detect the transposition efficiency of transposase in PBMC cells on D5, D9, and D13.

[0217] For fluorescence photography analysis, GFP fluorescence was captured using an Olympus inverted fluorescence microscope on days 5, 9, and 13 with an exposure time of 500 ms and a magnification of 100×. The transposition efficiency of the transposase in the cells was evaluated by the number of GFP fluorescent cells and the intensity of the fluorescence.

[0218] For flow cytometric analysis, stable integration of the gene was assessed by measuring GFP fluorescence in cells grown without drug selection. Transfected cells were harvested at the indicated time points, washed once with 1× DPBS, and resuspended in 500 μl of DPBS before being transferred to flow cytometers or 96-well plates for analysis. Cells were analyzed using the Cytex SpectroFlo, and GFP expression was assessed using the FITC channel.

[0219] (4) Screening of transposase mutants in CHO-K1 cells

[0220] On the day of electroporation, CHO-K1 cells were digested with trypsin containing 0.25% EDTA, collected and centrifuged, and resuspended in 1× DPBS for counting. 1.5-2.5×10 6 After centrifugation, remove the supernatant. Following the instructions for the Lonza 2B electroporation reagent, take 100 μl of electroporation buffer, mix 4 μg of different transposase mutant plasmids and 4 μg of the transposon plasmid (pZB-dCG-eGFP) with the electroporation reagent, and add it to the centrifuged cell pellet. Resuspend the cells in an electroporation cuvette and place them in a Lonza 2B electroporator, setting the electroporation program to H-014. After electroporation, transfer the cells to a six-well plate in complete culture medium and culture. Fluorescence imaging is performed on days 5, 9, and 13 after electroporation. Transfected cells are harvested, washed once with 1× DPBS, and resuspended in 500 μl of DPBS before being transferred to a flow cytometer or 96-well plate for analysis. Cells are analyzed using the Cytex SpectroFlo, and GFP expression is assessed using the FITC channel. Different transposase mutants are screened and evaluated based on their final transposition efficiency.

[0221] (5) Screening of transposase mutants in PBMC cells

[0222] On the day of electroporation, the frozen PBMCs were thawed and counted, and 1×10 7 The cells were centrifuged and the supernatant removed. Following the instructions for the Lonza 2B electroporation reagent, 100 μl of electroporation buffer was added to the centrifuged cell pellet, 4 μg of the transposase plasmid and 4 μg of the transposon plasmid (pZB-dCG-eGFP) were mixed with the electroporation reagent, and the mixture was added to the centrifuged cell pellet. The cells were resuspended and placed in an electroporation cuvette. The cells were placed in a Lonza 2B electroporator and electroporation program was set to U-014. After electroporation, the cells were transferred to a six-well plate in complete medium (AIM-V + 2% FBS) supplemented with 500 U / ml of IL-2. The plates were transferred on day 5 after electroporation, and fluorescence images were taken on days 5, 9, and 13. The transfected cells were counted, harvested, washed once with 1× DPBS, and resuspended in 500 μl of DPBS before being transferred to a flow cytometer or 96-well plate for analysis. Cells were analyzed using the Cytex SpectroFlo, and GFP expression was assessed using the FITC channel. Different transposase mutants were screened and evaluated based on their final transposition efficiency.

[0223] Example 1: Screening for BZ transposase mutants with efficient gene integration in CHO and PBMC cells

[0224] Implementation purpose:

[0225] 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.

[0226] Implementation methods

[0227] The vector sequence of the transposon constructed in this example is SEQ ID NO: 3, which contains an eGFP fluorescent protein expression cassette. Engineered BZ transposases with amino acid substitutions at different positions were screened on CHO-K1 and / or PBMC cells to identify mutants with high enzymatic activity. The vector sequence containing the wild-type ZB transposase is shown in SEQ ID NO: 4. By replacing the codons for the amino acids at the corresponding positions according to the sequence of SEQ ID NO: 4, different vector sequences for engineered BZ transposases can be obtained. The amino acid sequence of the wild-type ZB transposase is shown below:

[0228] To test the transposition efficiency of different engineered BZ transposases, the engineered transposase or transposon constructed were transfected into CHO cells and / or PBMC cells at a certain mass ratio using electroporation. WT ZB transposase and PiggyBac (PB) transposase (its sequence is as described in CN105154473A) were used as control transposases and transfected into CHO and / or PBMC cells. Cells were grown in complete medium (without drug selection) and eGFP expression was assessed by fluorescence photography and flow cytometry at day 13-17 after transfection.

[0229] Implementation Results

[0230] A series of BZ transposase mutants with higher transposition efficiencies than the wild-type transposase were identified (Figure 2). Among these mutants, N5S, F21K, S38R, Q71H, Q71R, Q79R, H110R, H110K, K134A, K137T, Q138K, V144E, G189A, G216A, and K251T exhibited significantly enhanced transposition activity. In particular, mutations to amino acids outside the second helix of the BZ transposase HTH domain (e.g., S38R, Q71H, Q71R, H110K, and H110R) to positively charged amino acids such as histidine (H), lysine (K), or arginine (R) significantly increased transposition efficiency.

[0231] Table C:

[0232] Example 2: Screening of BZ transposase combination mutants with efficient gene integration in CHO and PBMC

[0233] Purpose of the experiment

[0234] This example tests the transposition efficiency of different engineered BZ transposase mutant combinations to identify BZ transposase mutant combinations with efficient gene integration.

[0235] Experimental steps:

[0236] Two batches of multiple 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 consisted of a single point mutant with moderate strength screened in Example 1 and two, three or more combination mutants of Q71R / H110R (Table D), and the other batch consisted of two, three or more combination mutants of a single point mutant with moderate strength screened in Example 1 (Table E).

[0237] Experimental results:

[0238] The results showed that compared with the wild-type ZB transposase, combinations of two, three or more single-point mutants with moderate strength showed better transposition efficiency (Figures 7 and 8); combinations of two, three or more Q71R / H110R single-point mutants with moderate strength also showed better transposition efficiency, among which Q7iR\H110R, Q71R\Q79R\H110R, G216A\Q71R\Q79R\H110R, H208V\Q71R\Q79R\H110R, and H208V\G216A\Q71R\Q79R\H110R showed significant improvements (Figures 5 and 6). Among the engineered transposase BZ combination mutants examined, the combination mutant numbered 206 (Q71R / H110R) was selected for comparison with PiggyBac (PB). The results are shown in Figures 3 and 4. 206 (Q71R / H110R) has comparable or better effects than PiggyBac (PB) in both CHO-K1 and PBMC cells.

[0239] Table D

[0240] Table E

[0241] Example 3: ZB or BZ transposase in fusion protein form has no effect on its gene integration ability

[0242] Experimental purpose: This example is used to demonstrate that ZB or BZ transposase can be fused with other functional polypeptides or protein domains without affecting the transposase or transposon gene integration function.

[0243] Experimental methods:

[0244] The present embodiment tests the transposition efficiency of the exemplary fusion transposase comprising NLS. Different NLS tags are connected to the N-terminus of the transposase to generate different types of transposases. Different NLS tags are added to the wild-type ZB transposase, and its amino acid sequence is shown in Table F. By means of electroporation, the above-mentioned exemplary fusion transposase comprising NLS is transfected into PBMC cells, and the cells are grown in complete medium (without drug selection). The transposition efficiency of the exemplary fusion transposase comprising NLS is assessed by flow cytometry on the 13th day after transfection by detecting the expression of fluorescent protein.

[0245] Table F

[0246] Experimental results:

[0247] As shown in the results ( FIG. 9 ), the transposition efficiency of the exemplary NLS-containing fusion transposases tested above was not affected by the NLS polypeptide of the fusion protein. These tests demonstrate that the N-terminus of the ZB or BZ transposase proteins can be coupled to different polypeptides or protein domains to generate different types of transposases without affecting their transposition integration function.

[0248] Example 4: BZ transposase can mediate transposon gene integration in protein form

[0249] Purpose of the experiment:

[0250] This example is used to demonstrate that the protein form of BZ transposase can mediate the gene integration of transposons.

[0251] Experimental methods:

[0252] CHO-K1 cells were revived and cultured until the cells were in good growth condition, digested and washed, and the transposon (carrying the eGFP reporter gene) was transfected into the cells using the Lonza 2B system (Lonza) by electroporation. After 4.5 hours of electroporation, the BZ transposase in protein form was added to the culture medium. Empty cells were used as a viability control. After 14 days of proliferation and culture of the electroporated cells after transfection, the cells were harvested, washed once with DPBS, and resuspended in 200 μL of DPBS buffer. Cells were analyzed using a Cytek Northern Lights (Cytek Biosciences) flow cytometer, and EGFP expression was assessed using the FITC channel.

[0253] Experimental results:

[0254] The results (Figure 10) show that the addition of engineered BZ transposase proteins (either wild-type ZB protein or a combined mutant designated 206) to the electroporation system mediates transposon expression. Twenty days after transfection, the wild-type ZB protein exhibited a transposition efficiency of approximately 6-7%. Compared to the wild-type ZB protein, mutant 206 exhibited approximately twice the transposition efficiency 14 days after transfection.

[0255] Example 5: BZ transposase in mRNA form can mediate genomic integration of CAR plasmid or antibody plasmid DNA and be used for cell gene therapy

[0256] Experimental purpose: This example is used to demonstrate that the highly efficient BZ transposase in the form of mRNA can be used for cell gene therapy, and its effect is comparable to that of the most efficient PiggyBac (PB) transposase currently in the art.

[0257] Experimental methods:

[0258] A donor DNA plasmid 257 containing messenger RNA (mRNA) encoding the BZ transposase (ZB protein with Q71R, H110R mutations) or the PB transposase and its transposon system is prepared, wherein the mRNA is pseudouridine chemically modified mRNA to engineer CD4+ / CD8+ T cells.

[0259] The CD4+ / CD8+T cells used in the embodiment were extracted from the peripheral blood of healthy people and separated using CD4+ / CD8+ magnetic beads. The cells were plated in six-well plates coated with anti-CD3 / anti-CD28 or anti-CD3 / 4-1BBL at a cell count of 1E7 and cultured in complete medium containing 100U / ml of IL-2 (without drug selection). The cells were incubated for two days for activation. The inventors used a transposon plasmid (257) carrying a CAR gene (CAR information is disclosed in CN202111681582.0, the entire contents of which are incorporated herein by reference. This embodiment uses the CAR shown in SEQ ID NO: 99 in CN202111681582.0) and an exemplary transposase BZ mRNA to electroporate activated CD4+ / CD8+T cells. Transgenic expression was monitored by flow cytometry for 9 days after electroporation. Under different activation modes, transposon plasmids (257) carrying CAR genes with different costimulatory domains 4-1BB and CD28 were co-transfected with PB mRNA transposase and exemplary transposase BZ mRNA, respectively (Table G).

[0260] Table G:

[0261] Experimental results:

[0262] As shown in the results (Figures 11 and 12), compared with the PB mRNA transposase, 9 days after transfection, the transposition efficiency of the exemplary BZ transposase mRNA was comparable to that of the PB mRNA transposase, with no significant difference, and there was no significant difference in the T cell proliferation efficiency and multiples. This shows that the BZ transposase in the form of mRNA can mediate the gene integration of CAR and / or antibody plasmid DNA used for gene therapy, and the effect is comparable to the most efficient PiggyBac (PB) transposase in the art. As shown in Figure 13, the anti-CD3 / 4-1BBL activation method was selected to prepare CAR-T for the killing experiment of tumor cells L363. The results also showed that there was no significant difference in the killing effect of CAR-T cells prepared by the new BZ transposase and CAR-T cells prepared by the PB transposase.

[0263] Example 6: CpG mutations in the BZ transposon ITR maintain the gene integration function of the transposition system

[0264] Experimental purpose: To eliminate CpG motifs in the BZ transposon ITR mutation, reduce immunogenicity, and maintain the gene integration function of the transposition system. The wild-type BZ transposon 5' ITR sequence and 3' ITR sequence (reverse complement) are shown in SEQ ID NOs: 11 and 12.

[0265] Experimental methods:

[0266] The only CpG motif in the ITR was mutated to ApG, GpG, TpG, CpA, or CpT. A transposon plasmid carrying the ITR mutation and the eGFP reporter gene was co-transfected with BZ mRNA into CHO-K1 cells. The cells were grown in complete medium (without drug selection). Flow cytometry was used to measure the expression of fluorescent protein on day 14 after transfection to assess the transposition efficiency of the ITR-mutated transposon.

[0267] Experimental results:

[0268] The results, shown in Figure 14, show that compared to the wild-type BZ ITR sequence, mutations of the ITR CpG on the BZ transposon plasmid to CpA or TpG did not affect the gene integration function of the transposition system. Mutations to ApG, GpG, and CpT decreased gene integration efficiency but still enabled effective transposition. A control plasmid containing CpG showed no gene integration efficiency in the absence of BZ mRNA.

[0269] Example 7: Fusion of a leucine zipper structure can enhance the transposition activity of ZB or BZ transposase

[0270] Experimental purpose: This example is used to demonstrate that ZB or BZ transposase can be fused with a leucine zipper structure and improve the gene integration function of ZB or BZ transposase.

[0271] Experimental methods:

[0272] This example tests the transposition efficiency of an exemplary fusion transposase containing three ZIPs. Different ZIP structures were attached to the N-terminus of the transposase to generate different types of transposases. Different ZIP structures were added to the mutant BZ transposase (ZB transposase with Q71R and H110R mutations), whose amino acid sequences are shown in Table H. HD transfection reagent (Promoga) was used to co-transfect the exemplary fusion transposase containing ZIP and the transposon donor carrying the screening gene NEO into Hela cells, with three replicate wells for each group. 2×10 4 The cells were cultured in a 10 cm dish and G418 selection solution (600 ng / ul) was added. After two weeks of selection, the cells were stained with Gimesa stain and positive clones were counted using ImageJ to assess the transposition efficiency of each transposase group.

[0273] Table H:

[0274] Experimental results:

[0275] As shown in the results ( FIG15 ), the transposition efficiency of the exemplary fusion transposases tested above was enhanced to varying degrees by different ZIP structures, with CEBPO exhibiting the best activity. These tests demonstrate that ZB or BZ transposase proteins can be coupled to different ZIP structures to generate transposases with higher activity.

[0276] Although specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details in light of all the teachings disclosed herein, and such modifications are within the scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

[0277] sequence of this article

Claims

1. A transposase, which is a mutant transposase or a fusion transposase, characterized in that: The mutant transposase comprises amino acid mutations compared to the wild-type ZB transposase as shown in SEQ ID NO: 1, wherein the amino acid mutations are one or more amino acid substitutions in the DNA binding and oligomerization domain, the DDE catalytic domain, or a combination thereof of the wild-type ZB transposase; and / or the amino acid mutations comprise one or more mutations selected from the group consisting of 5S, 21C, 21K, 35I, 38R, 56L, 61R, 71H, 71R, 73L, 79R, 110K, 110R, 125L, 134A, 137T, 138G, 138K, 144A, 144E, 186N, 188Y, 189A, 204V, 215-218DAVQ, 216A, 217V, 218K, 235R, 251T, or 289A; The fusion transposase is a wild-type ZB transposase or a mutant transposase fused with a functional polypeptide, wherein the functional polypeptide is a DNA sequence-specific or non-specific binding domain and / or a cell nucleus localization signal domain.

2. The transposase according to claim 1, wherein The mutant transposase is a transposase having any one or more of the following mutations compared to SEQ ID NO: 1: Q71R\H110R, Q71R\Q79R\H110R, G216A\Q71R\Q79R\H110R, H208V\Q71R\Q79R\H110R、 H208V\G216A\Q71R\Q79R\H110R、 F21K\D22A\Q71R\H110R, N005S\F21K / D22A\Q71R\Q79R\H110R、 K120S\N125L\Q71R\Q79R\H110R、 G216A\H208V\G189A\Q71R\Q79R\H110R、 G216A\H208V\K251T\Q71R\Q79R\H110R、 G216A\H208V\K251T\G189A\Q71R\Q79R\H110R、 G216A\H208V\K251T\G189A\Q138K\Q71R\Q79R\H110R, G216A\H208V\K251T\G189A\Q138R\Q71R\Q79R\H110R, G216A\H208V\K251T\G189A\K134A\Q71R\Q79R\H110R, G216A\H208V\K251T\G189A\Q138K\K134A\Q71R\Q79R\H110R, G216A\H208V\K251T\G189A\Q138R\K134A\Q71R\Q79R\H110R, G216A\H208V\K251T\G189A\Q138K\V144E\Q71R\Q79R\H110R, G216A\H208V\K251T\G189A\Q138K\K137T\Q71R\Q79R\H110R, G216A\Q71R\H110R, H208V\Q71R\H110R, H208V\G216A\Q71R\H110R, G216A\H208V\G189A\Q71R\H110R、 G216A\H208V\K251T\Q71R\H110R、 G216A\H208V\K251T\G189A\Q71R\H110R、 G216A\H208V\K251T\G189A\Q138K\Q71R\H110R、 G216A\H208V\K251T\G189A\Q138R\Q71R\H110R、 G216A\H208V\K251T\G189A\K134A\Q71R\H110R、 G216A\H208V\K251T\G189A\Q138K\K134A\Q71R\H110R, G216A\H208V\K251T\G189A\Q138R\K134A\Q71R\H110R, G216A\H208V\K251T\G189A\Q138K\V144E\Q71R\H110R, G216A\H208V\K251T\G189A\Q138K\Q71R\H110R, G216A\H208V\K251T\G189A\Q138R\Q71R\H110R、 G216A\H208V\K251T\G189A\K134A\Q71R\H110R, G216A\H208V\K251T\G189A\Q138K\K134A\Q71R\H110R, G216A\H208V\K251T\G189A\Q138R\K134A\Q71R\H110R, G216A\H208V\K251T\G189A\Q138K\V144E\Q71R\H110R, G216A\H208V\K251T\G189A\Q138K\K137T\Q71R\H110R, or N005S\F21K / D22A\Q71R\H110R.

3. The transposase according to claim 1, wherein The mutant transposase is a transposase having any one or more of the following mutations compared to SEQ ID NO: 1: G216A\H208V, G216A\H208V\G189A、 G216A\H208V\K251T, G216A\H208V\K251T\G189A、 G216A\H208V\K251T\G189A\Q138K、 G216A\H208V\K251T\G189A\Q138R、 G216A\H208V\K251T\G189A\K134A、 G216A\H208V\K251T\G189A\Q138K\K134A, G216A\H208V\K251T\G189A\Q138R\K134A、 G216A\H208V\K251T\G189A\Q138K\V144E, or G216A\H208V\K251T\G189A\Q138K\K137T.

4. The transposase according to any one of claims 1 to 3, wherein 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, preferably, the amino acid sequence of the leucine zipper domain is as shown in any one of SEQ ID NOs: 13-15, and / or The nuclear localization signal domain includes SV40 NLS, C-myc NLS, TAF1 NLS, TP53 NLS, STAT3 NLS or any combination thereof, and / or The functional polypeptide and the wild-type ZB transposase or its mutant may or may not contain a linker.

5. A polynucleotide encoding the transposase according to any one of claims 1 to 4; preferably, the polynucleotide is DNA or messenger RNA.

6. A transposon that can be recognized by the transposase according to any one of claims 1 to 4, wherein the transposon contains a mutant inverted terminal repeat sequence, preferably, the mutant inverted terminal repeat sequence does not contain a CpG motif, and more preferably, the CpG motif in the mutant inverted terminal repeat sequence is deleted or mutated compared with the wild-type inverted terminal repeat sequence SEQ ID NO: 11 or 12.

7. The transposon according to claim 6, wherein The mutation is to mutate the CpG motif into ApG, GpG, TpG, CpA or CpT, preferably into TpG or CpA.

8. The transposon according to claim 6, wherein The transposon comprises a target gene expression cassette located between terminal inverted repeat sequences. Preferably, the target gene encodes a protein selected from cell receptors, immune checkpoint proteins, cytokines, T cell receptors, B cell receptors, chimeric antigen receptors, and any combination thereof.

9. A transposon system, characterized in that The invention comprises the transposase according to any one of claims 1 to 4 or the polynucleotide encoding the transposase according to claim 5; and a transposon, wherein the transposon is a ZB transposon or the transposon according to any one of claims 6 to 8.

10. The transposon system according to claim 9, wherein The polynucleotide encoding the transposase is mRNA, preferably, the mRNA is chemically modified, including but not limited to pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine nucleoside (m5C), 5-methoxyuridine (5moU).

11. The transposon system according to claim 10 or 11, wherein: The polynucleotide encoding the transposase and / or the transposon are present in a DNA vector, preferably, the DNA vector is a plasmid vector or a minicircle DNA vector, preferably a non-antimicrobial plasmid vector; or The polynucleotide encoding the transposase and / or the transposon are present in a viral vector. Preferably, the viral vector is selected from an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a herpes simplex viral vector or a vaccinia viral vector.

12. The transposon system according to claim 11, wherein The polynucleotide encoding the transposase and the transposon are present in the same vector or in different vectors.

13. A host cell comprising or capable of producing the transposase according to any one of claims 1 to 4, the polynucleotide according to claim 5, the transposon according to any one of claims 6 to 8, or the transposon system according to any one of claims 9 to 12.

14. A method for preparing cells, characterized in that: include: a step of introducing the transposase according to any one of claims 1 to 4, the polynucleotide according to claim 5, the transposon according to any one of claims 6 to 8, or the transposon system according to any one of claims 9 to 12 into a cell, Preferably, The introducing comprises contacting the cell with the polynucleotide, and / or The introducing comprises contacting the cell with a DNA vector containing the transposon, and / or The introducing comprises contacting the cell with mRNA encoding the transposase and a DNA plasmid vector containing the transposon, and / or The introducing comprises contacting the cell with a DNA plasmid vector containing the polynucleotide and the transposon, and / or The introducing comprises contacting the cell with a plasmid vector containing the polynucleotide and a DNA plasmid vector containing the transposon, and / or The introducing comprises contacting the cell with the transposase.

15. The method according to claim 14, wherein The cells are from animals, such as vertebrates or invertebrates, preferably mammals, more preferably humans; and / or the cells are immune cells, preferably T cells.

16. The method according to claim 15, wherein The introducing comprises transfecting the cell by electroporation, microinjection, calcium phosphate precipitation, cationic polymers, dendrimers, liposomes, lipid nanoparticles, microparticle bombardment, fugene, direct sonic loading, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, nucleofection, virus-mediated transformation, or any combination thereof.

17. The method according to any one of claims 14 to 16, wherein: Said introducing comprises electroporating said cells.

18. The method according to claim 17, wherein The introduction comprises contacting cells with the mRNA of the transposase and a plasmid containing the transposon. Preferably, the plasmid is a microcarrier, and / or the plasmid is a micro-antibody-free plasmid.

19. A kit comprising: the transposase according to any one of claims 1 to 4, the polynucleotide according to claim 5, the transposon according to any one of claims 6 to 8, the transposon system according to any one of claims 9 to 12, or the cell according to claim 13.

20. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the cell according to claim 13 or the cell prepared by the method according to any one of claims 14 to 18.

21. Use of the transposase according to any one of claims 1 to 4, the polynucleotide according to claim 5, the transposon according to any one of claims 6 to 8, the transposon system according to any one of claims 9 to 12, the cell according to claim 13, or the pharmaceutical composition according to claim 20, which is selected from any one of the following (1) to (6): (1) Application in the preparation of drugs or reagents for integrating the target gene expression cassette into the host cell genome; (2) Application in the preparation of tools for integrating target gene expression cassettes into the host cell genome; (3) Application in the preparation of transgenic animals and transgenic cells; (4) Application in the preparation of drugs or preparations for genome research, gene therapy, cell therapy, or stem cell induction and post-induction differentiation; (5) Application in the preparation of tools for genomic research, gene therapy, cell therapy, or stem cell induction and post-induction differentiation; (6) Application in the preparation of kits, engineered immune cells or pharmaceutical compositions.

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