Transposase mutants with improved gene transfer efficiency

Abstract

The present invention relates to the field of transposases. In particular, the present invention provides a polypeptide comprising or consisting of a Tc1 / mariner superfamily transposase, e.g., Sleeping Beauty transposase, having a substitution at amino acid position 124. The transposase of the present invention has been found to have improved transposition efficiency. The present invention also provides nucleic acids encoding the transposases, cells expressing the nucleic acids, kits and pharmaceutical compositions comprising the transposases for use in, for example, gene therapy, e.g., adoptive T cell therapy, or uses and methods for gene delivery into the genome of cells.

Description

[Technical Field]

[0001] The present invention relates to the field of transposases. In particular, the present invention provides a polypeptide comprising or consisting of a Tc1 / mariner superfamily transposase, e.g., Sleeping Beauty transposase, having a substitution at amino acid position 124. The transposase of the present invention has been found to have improved transposition efficiency. The present invention also provides nucleic acids encoding the transposases, cells expressing the nucleic acids, kits and pharmaceutical compositions comprising the transposases for use in, for example, gene therapy, e.g., adoptive T cell therapy, or uses and methods for gene delivery into the genome of cells. [Background technology]

[0002] The ability of nucleic acids to integrate their sequences into new loci has evolved in diverse ways. The large family of transposons in maize, first described by Barbara McClintock [1], possesses the ability to move genetic information within the genome. Transposable elements (TEs) can be classified into two groups. Class I TEs follow a copy-and-paste mechanism and use an RNA intermediate for this process, so-called retrotransposons [2]. Class II transposons rely solely on a DNA intermediate for their transposition process. Within this category, subclass I transposons follow a cut-and-paste mechanism, during which the transposon is excised from one genomic location and reintegrated at a different location [2]. The Tc1 / mariner superfamily follows this canonical cut-and-paste mechanism. TEs in this superfamily are flanked by terminal inverted repeats (TIRs) and contain genes encoding transposases, the enzymes that catalyze the transposition reaction [2]. The transposase binds to the TIR, excises the TE from the donor locus, and reintegrates the TE adjacent to the TA target sequence, ultimately resulting in a TA target site duplication [2].

[0003] The minimal components required for the transposition reaction are a TIR and a transposase. TEs containing both of these components are therefore considered autonomous TEs [3]. Many autonomous TEs have been converted to non-autonomous derivatives by modification of the transposase coding region. These non-autonomous TEs can still function but require a functional transposase expressed by another element within the same cell [3]. This trans-complementation between the two functional components (the transposase and the specific TIR recognized and activated by the transposase) forms the basis for converting transposons into gene vector systems suitable for moving any gene of interest into the genome of a host cell (Figure 1).

[0004] One member of the Tc1 / mariner superfamily is the Sleeping Beauty (SB) transposon [3]. The SB transposon was reconstructed from a fossil DNA sequence in a fish genome and was the first DNA transposon shown to be active in vertebrates [3]. SB transposons have been widely used as genetic engineering tools in various preclinical studies and clinical trials [4]. The structural and biochemical characteristics of the SB transposase, which catalyzes the transposition reaction, are of particular interest because the efficiency of SB transposon integration into target cell genomes can be improved based on the enzymatic activity of the transposase. SB transposase consists of an N-terminal DNA-binding domain (aa 1–110) and a C-terminal catalytic domain (DDE) (aa 114–340) [3]. Both domains are connected by a flexible linker region containing a nuclear localization signal (NLS) (aa 97–123) [3]. The DNA-binding domain consists of two subdomains, the PAI and RED subdomains, which form a helix-turn-helix (HTH) motif important for recognition and binding to transposon DNA [3, 5]. The catalytic domain, which contains three conserved amino acids [D153, D244, and E279 (DDE)] in the catalytic center, catalyzes the DNA hydrolysis required for excision and transesterification reactions that occur during integration [5–7].

[0005] As shown in Figure 6, other transposases of the Tc1 / mariner superfamily, such as Frog Prince, have a similar structure.

[0006] Since the discovery of the SB transposon, several mutations have been identified that result in an overall higher integration efficiency. These mutations resulted in the current most active SB transposase variant, called SB100X [8]. Other mutations, such as the K248T mutation, which results in an integration-deficient SB transposase [9], or the K248R mutation, which results in a safer integration profile, have broadened the application of the SB transposase

[10] .

[0007] The WVPHEL (sequence number 25) motif (aa 119-124) of the Tc1 / mariner transposase Mos1, which forms a dimerization interface, plays a key role in downregulating transposition through an allosteric mechanism

[11] . Mutations in this region have been shown to result in hyperactive mariner transposase mutants

[11] .

[0008] However, this motif is not present in all Tc1 / mariner transposases. For example, the most widely used transposase, SB, contains KKPLL (SEQ ID NO: 26) at positions 119-123, which is homologous to the WVPHEL motif present in Mos1 (see alignment in Figure 6). Related transposases in this family, such as Frog Prince (FP), ZB, Tdr1, and Passport, also contain a motif similar to that of SB.

[0009] Ivics et al. previously showed that a deletion of aa 117-123, which contains the ARKKPLL (SEQ ID NO: 27) motif, in SB disrupts nuclear localization.

[26] Because this motif in SB aligns with the WVPHEL motif in the mariner transposase, mutations that confer hyperactivity in mariner are unlikely to function in SB because they would disrupt nuclear localization. Summary of the Invention [Problem to be solved by the invention]

[0010] In light of this, the inventors addressed the problem of providing further advantageous variants of transposases of the Tc1 / mariner superfamily, e.g. with improved transposition efficiency. [Means for solving the problem]

[0011] This problem is solved by the subject matter of the claims. In particular, the present invention provides a polypeptide having transposability, comprising or consisting of a Tc1 / mariner superfamily transposase having a substitution at amino acid position 124. The amino acid at position 124 is not Q. The transposase is preferably a Sleeping Beauty transposase having at least 80% amino acid identity, optionally at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to SEQ ID NO: 18.

[0012] We surprisingly identified a position amenable to substitution, amino acid 124, located immediately adjacent to but outside the region (aa 119–123) corresponding to the Mos1 WVPHEL motif in the SB100X coding sequence. Mutations, specifically a glutamine 124 to cysteine ​​124 substitution (Q124C), exhibited a two-fold increase in transposition activity and resistance to overproduction suppression described in various Tc1 / mariner transposases

[11] . Introduction of the Q124C mutation also increased the efficiency of other clinically relevant SB transposase mutants, including K248R. Overall, these findings demonstrate the utility of mutations at position 124, particularly the Q124C mutation, to enable improved speed of SB transposon-mediated genome engineering in preclinical and clinical applications. DETAILED DESCRIPTION OF THE INVENTION

[0013] The term "transposase" as used herein refers to an enzyme that can mediate a transposition reaction as a component of a functional nucleic acid-protein complex. "Transposition reaction" as used herein refers to the reaction in which a transposon is inserted into a target nucleic acid. The main components of the transposition reaction are a transposon and a transposase or integrase enzyme. The appropriate TIR sequence of a transposon is known in the art for each transposase. For example, pT2 or pT4 transposon, preferably pT4 transposon, can be transposed by SB.

[0014] A polypeptide having transposase activity may comprise or consist of a transposase. That is, the polypeptide may be a fusion protein of a transposase. The fusion protein may be fused at the N-terminus or C-terminus, although, as described in more detail below, the positions referred to herein are determined solely in relation to the transposase portion of the polypeptide. In particular, the polypeptide may consist of a transposase.

[0015] The transposase may lack 1 to 10 N-terminal and / or C-terminal amino acids of a naturally occurring full-length transposase. Preferably, the transposase may lack the methionine at position M1, for example, when the transposase is included in a fusion protein that includes another protein at the N-terminus of the transposase.

[0016] The term "transposition activity" as used herein refers to the activity of a given transposase, which can be assessed in a transposition reaction. "Transposition efficiency" is used interchangeably. A suitable experimental setup is described in the experimental section of this specification, or the classical binary transposition assay described in Ivics, 1997 Cell 91: 501-510 can be used.

[0017] The transposase of the present invention is a Tc1 / mariner superfamily transposase. Figure 6 shows an alignment of sequences of transposases in the Tc1 / mariner transposase superfamily. The alignment algorithm used to generate the alignment referenced was CLUSTAL Omega using the following settings: dealign input sequence: none, mbed-like clustering guide tree: yes, mbed-like clustering repeats: yes, combined repeat count: default, maximum guide tree repeats: default, maximum hmm repeats: default. Amino acid conservation is highlighted in MView. The alignment includes secondary structure information obtained for the catalytic domain of SB transposase highlighted in ESPript 3.0 (PDB entry code: 5CR4).

[0018] The transposase of the present invention is preferably active in human cells. Suitable transposases are, for example, Sleeping Beauty (SB), Frog Prince (FP), Minos, ZB, Tdrl, and Passport. SB is preferred.

[0019] Each transposase is classified by its amino acid identity to the respective "wild-type" transposase sequence in Figure 6. That is, if a transposase has at least 80% amino acid identity to any of the listed sequences and has the highest sequence identity to one of the sequences, it is classified as a transposon of that class.

[0020] Preferably, the transposase is an SB transposase having at least 80% amino acid identity, optionally at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to SEQ ID NO: 1 (SB100X), optionally with only one, two, or three amino acid differences from said sequence, one of which is a substitution at position 124.

[0021] In a preferred embodiment, the transposase is SB100X comprising a substitution at position 124, wherein the amino acid at position 124 is not Q. The sequence is provided as SEQ ID NO: 18. While SB100X is itself a hyperactive transposase, the inventors surprisingly showed that SB100X with a Q124C substitution (SEQ ID NO: 19) further enhances transposition activity. This also applies to mutant forms of the transposase that contain one additional substitution (e.g., a substitution at position 187, 247 or 248, e.g., SBlOOX Q124C H187V, SEQ ID NO:20; SBlOOX Q124C P247R, SEQ ID NO:21; SBlOOX Q124C K248R, SEQ ID NO:22), two additional substitutions (e.g., a substitution at positions 187 and 247 or 187 and 248, e.g., SBlOOX Q124C H187V P247R or Q124C H187V K248R), three additional substitutions (e.g., a substitution at positions 187, 247 and 248, e.g., SBlOOX Q124C H187V P247R K248R), or four additional substitutions. Transposases of the invention having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO: 18 are therefore preferred transposases of the invention. Preferred additional substitutions in SB are disclosed, for example, in PCT / EP2022 / 075007. Exemplary SB transposase variants of the invention include: a) SB100X Q124X1 (X1 is not Q, X1 is preferably C), H187X2 (X2 is not H, X2 is optionally V); b) SB100X Q124X1 (X1 is not Q, X1 is preferably C), P247X2 (X2 is not P, X2 is optionally R); c) SB100X Q124X1 (X1 is not Q, X1 is preferably C), K248X2 (X2 is not K, X2 is optionally R) is.

[0022] Alternatively, the transposase may be a Tdr1 transposase having at least 80% amino acid identity, optionally at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to SEQ ID NO: 2. Optionally, there are only one, two, or three amino acid differences from said sequence, one of which is a substitution at position 124.

[0023] The transposase may also be a ZB transposase having at least 80% amino acid identity, optionally at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to SEQ ID NO: 3. Optionally, there are only 1, 2, or 3 amino acid differences from the sequence, with one of the differences being a substitution at position 124.

[0024] In one embodiment, the transposase can be an FP (Frog Prince) transposase having at least 80% amino acid identity, optionally at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to SEQ ID NO: 4. Optionally, there are only one, two, or three amino acid differences from the sequence, one of which is a substitution at position 124.

[0025] The transposase can also be a Passport transposase having at least 80% amino acid identity, optionally at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to SEQ ID NO: 5. Optionally, there are only one, two, or three amino acid differences from the sequence, one of which is a substitution at position 124.

[0026] Alternatively, the transposase can be a Minos transposase having at least 80% amino acid identity, optionally at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to SEQ ID NO: 11. Optionally, there are only one, two, or three amino acid differences from said sequence, one of which is a substitution at position 124.

[0027] In the context of the present invention, the polypeptides of the present invention have enhanced transposition activity (i.e., greater than 100%, preferably greater than 110%, greater than 120%, greater than 150%, or about 200% or greater) compared to an otherwise identical polypeptide that does not contain the substitution at position 124. Transposases that do not contain the substitution at position 124 have an amino acid residue at that position in the respective original or "wild-type" transposase as set forth in the alignment of Figure 6. For example, for SB or Tdrl transposase, "wild-type" position 124 is Q; for ZB it is N; for FP it is S; and for Passport it is K. Enhanced transposition activity need not be present at all transposase concentrations, but is present at at least one concentration that is equal to or higher than the concentration at which overexpression inhibition is detected. Enhanced activity is preferably determined using the assay described below with reference to Figure 3B.

[0028] As used in the context of the present invention, a "position" in a transposase refers to an amino acid that aligns with the amino acid sequence of a "wild-type" full-length reference transposase, preferably the full-length amino acid sequence of SB of SEQ ID NO: 1, in an alignment of amino acid sequences. The position of the reference transposase is determined from the first N-terminal amino acid. Thus, position 124 of SB in the full-length amino acid sequence of SEQ ID NO: 1 is "Q." Thus, amino acid position 124 of another transposase is the amino acid that aligns with the "Q" of SB at position 124 of SEQ ID NO: 1 in the alignment shown in Figure 6.

[0029] The amino acid at position 124 may be selected from the group consisting of C, A, R, N, D, E, G, H, I, L, K, M, F, P, S, T, W, Y and V. In one embodiment, the amino acid at position 124 is C, A, R, D, E, G, H, I, L, M, F, P, S, T, W, Y and V. The amino acid at position 124 may also be, for example, C, A, D, E, G, H, I, L, M, F, P, W, Y and V.

[0030] The present inventors found that transposases of the present invention having a C or G at position 124 are particularly hyperactive. A significant increase in hyperactivity was also found for transposases having any of D, N, E, L, M, F, S, T, Y, or V at position 124. Transposases having an A, R, H, or I at position 124 still showed increased hyperactivity (see Figure 7). The transposase is preferably an SB transposase.

[0031] When the transposase is an SB transposase, as is preferred in the context of the present invention, the amino acid at position 124 can be C, A, R, N, D, E, G, H, I, L, K, M, F, P, S, T, W, Y and V. Preferred amino acids in said context are C, S or Met, most preferably C. The same applies to Tdr1 transposase.

[0032] When the transposase is a ZB transposase, the amino acid at position 124 can be C, A, R, D, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V. Preferred amino acids in this context are C, S, or Met, and most preferably C.

[0033] When the transposase is an FP transposase, the amino acid at position 124 can be C, A, R, N, D, E, G, H, I, L, K, M, F, P, T, W, Y, and V. A preferred amino acid in this context is C or Met, most preferably C.

[0034] When the transposase is a Passport transposase, the amino acid at position 124 can be C, A, R, N, D, E, G, H, I, L, M, F, P, S, T, W, Y, and V. A preferred amino acid in this context is C or Met, most preferably C.

[0035] When the transposase is a Minos transposase, the amino acid at position 124 can be C, A, R, N, D, E, G, H, I, L, K, M, F, P, S, W, Y, and V. A preferred amino acid in the above context is C or Met, most preferably C.

[0036] As shown herein, a significant increase in transposition activity was achieved when the amino acid at position 124 was C. Therefore, a substitution resulting in a C at position 124 is advantageous. Thus, a polypeptide consisting of or comprising an SB transposase having a Q124C substitution, preferably comprising the amino acid sequence of SEQ ID NO: 19 or containing one, two, three, or four amino acid differences compared to SEQ ID NO: 19, is a preferred embodiment of the present invention.

[0037] As described in PCT / EP2022 / 075007, transposases of the Tc1 / mariner superfamily containing a substitution at any of positions 187, 247, or 248, such as the K248R substitution, have improved integration patterns compared to transposases without substitutions at these positions. Such transposases have a better safety profile because they reduce transposon integration into exons and transcriptional regulatory regions of genes in the human genome. Thus, transposons frequently integrate into genetic safe harbors. These substitutions have been found to reduce transposition activity.

[0038] However, the present inventors have shown that a substitution at position 124 can rescue the transposition activity, resulting in a more active transposase. Therefore, a polypeptide of the present invention further comprising a substitution at any of positions 187, 247, and / or 248 is another preferred embodiment of the present invention. Therefore, the polypeptide of the present invention can also be a polypeptide as defined in PCT / EP2022 / 075007, particularly a polypeptide with increased specificity for integration into the genome. For example, a) the substituted amino acid at position 187 may be A, N, C, Q, G, I, L, M, S, V, W, K, R, E, P, T and S, preferably V; b) the substituted amino acid at position 247 may be R, C, A or S; preferably R; and / or c) The substituted amino acid at position 248 may be R, S, V, I or C, preferably R.

[0039] Preferred substitutions at position 187 of the SB transposase are H187A, H187N, H187C, H187Q, H187G, H187I, H187L, H187M, H187S, H187V, H187W, H187K, H187R, H187E, H187P, H187T and H187S, with the H187V substitution being most preferred.

[0040] Preferred substitutions at position 247 of the SB transposase are P247R, P247C, P247A and P247S substitutions, with the P247R substitution being most preferred.

[0041] As a substitution at position 248 of the SB transposase, K248R, K248S, K248V, K248I, or K248C substitutions are preferred, with K248R substitution being most preferred.

[0042] The transposase of the present invention into which a substitution at position 124 is introduced may be a hyperactive transposase. In a preferred embodiment, the polypeptide of the present invention comprises SEQ ID NO: 1 or a variant thereof that has transposition activity and has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1, wherein SEQ ID NO: 1 or its variant contains a substitution that increases its transposition activity, preferably one or more of the substitutions described below. The increased transposition activity may be compared to the activity of SB of SEQ ID NO: 1. As mentioned above, various variants of naturally occurring transposases have been described. For example, WO 2009 / 003671 describes variants of transposases (particularly SB) that are hyperactive, i.e., have increased transposition activity compared to wild-type transposase, particularly SB. The substitutions of the present invention are preferably introduced into a variant of a transposase that contains a substitution or substitutions that enhance one or more properties, particularly the transposition activity of the transposase. Thus, in one embodiment, the polypeptides of the invention contain the following substitution or groups of substitutions: (1) K14R, K13D, K13A, K30R, K33A, T83A, I100L, R115H, R143L, R147E, A205K / H207V / K208R / D210E, H207V / K208R / D210E, R214D / K215A / E216V / N217Q; M243Q, E267D, T314N, and / or G317E; (2) K14R / / R214D / K215A / E216V / N217Q; (3) K33A / R115H / R214D / K215A / E216V / N217Q / M243H; (4) K14R / K30R / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / M243H; (5) K13D / K33A / T83N / H207V / K208R / D210E / M243Q; (6) K13A / K33A / R214D / K215A / E216V / N217Q; <h2 style=";text-align:left;direction:ltr">(7) K33A / T83N / R214D / K215NE216V / N217Q / / G317E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (8) K14R / T83A / M243Q;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (9) K14R / T83A / I100L / M243Q;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (10) K14R / T83A / R143L / M243Q;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (11) K14R / T83A / R147E / M243Q;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (12) K14R / T83A / M243Q / E267D;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (13) K14R / T83A / M243Q / T314N;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (14) K14R / K30R / I110L / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / M243H;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (15) K14R / K30R / R143L / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / / M243H;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (16) K14R / K30R / R147E / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / M243H;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (17) K14R / K30R / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / / M243H / E267D;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (18) K14R / K30R / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / M243H / T314N;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (19) K14R / K30R / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / / M243H / G317E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (20) K14R / K33A / R115H / R214D / K215A / E216V / N217Q / M243H;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (21) K14R / K30R / R147E / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / / M243H / T314N;<h2 style=";text-align:left;direction:ltr"> (22) K14R / K30R / R143U / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / / M243H / E267D; (23) K14R / K30R / R143L / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / / M243H / T314N; (24) K14R / K30R / R143L / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / / M243H / G317E; (25) K14R / K33A / R115H / R143L / R214D / K215A / E216V / N217Q / M243H; (26) K14R / K33A / R115H / R147E / / R214D / K215A / E216V / N217Q / M243H; (27) K14R / K33A / R115H / R214D / K215A / E216V / N217Q / / M243H / E267D; (28) K14R / K33A / R115H / R214D / K215A / E216V / N217Q / / M243H / T314N; (29) K14R / K33A / R115H / R214D / K215A / E216V / N217Q / / M243H / G317E; (30) K14R / T83A / M243Q / G317E; or (31) K13A / K33A / T83N / R214D / K215A / E216V / N217Q Further comprising at least one of:

[0043] For example, the transposase of the present invention may be the SB100X transposase of SEQ ID NO:18 or, preferably, SEQ ID NO:19.

[0044] The transposase of the present invention may also be a transposase with improved aqueous solubility compared to SB of SEQ ID NO: 1, such as the highly soluble transposase described by Querques et al.

[19] or described in EP 3673053 A. To improve solubility, the transposase may further comprise, for example, at least one substitution at amino acid positions 176 and / or 212, where the amino acid at position 176 is not C and the amino acid at position 212 is not I, preferably the amino acid at positions 176 and / or 212 is S, and optionally both the amino acids at positions 176 and 212 are S.

[0045] Another object of the invention is a nucleic acid encoding a polypeptide of the invention.

[0046] The nucleic acids of the present invention may be any nucleic acid, e.g., ribonucleic acid, including mRNA, DNA, cDNA, chromosomal DNA, extrachromosomal DNA, plasmid DNA, and viral DNA (including recombinant viral vectors). All nucleic acid variants encoding the polypeptides of the present invention are provided, including nucleic acid variants with different nucleotide sequences due to the degeneracy of the genetic code. Nucleotide sequences of nucleic acid variants that result in improved expression of the encoded fusion protein in a selected host organism, e.g., human cells, are particularly preferred. Tables for appropriately adapting nucleic acid sequences to the specific transcription / translation machinery of host cells are known to those skilled in the art. Generally, it is preferable to adapt the G / C content of the nucleotide sequence to specific host cell conditions. For expression in human cells, it is preferable to increase the G / C content by at least 10%, more preferably at least 20%, 30%, 50%, 70%, or even more preferably 90% of the maximum G / C content (encoding each peptide variant of the present invention). Preparation and purification of such nucleic acids and / or derivatives are typically carried out by standard procedures.

[0047] Preferably, the nucleic acid is suitable for expressing the polypeptide of the present invention in mammalian cells, for example, human cells such as stem cells or lymphocytes (e.g., T lymphocytes). When the nucleic acid is DNA, it preferably contains at least a gene regulatory region. The regulatory region may be, for example, a transcriptional regulatory region selected from the group consisting of a promoter, an enhancer, a silencer, a locus control region, and a boundary element. A promoter or other expression control region may be operably linked to the nucleic acid encoding the polypeptide of the present invention, for example, to regulate the expression of the polypeptide / protein quantitatively or tissue-specifically. The promoter may be a constitutive or inducible promoter.

[0048] The nucleic acids of the present invention encoding the polypeptides of the present invention can be linear or circular isolated fragments, or can be inserted into a vector, preferably as a plasmid or recombinant viral DNA. The terms "vector" and "expression vector" are used interchangeably and refer to a polynucleotide or a mixture of polynucleotides and proteins that can be introduced into cells, preferably mammalian cells, or that can introduce the nucleic acids of the present invention into cells, preferably mammalian cells. Examples of vectors include, but are not limited to, minicircles, plasmids, cosmids, phages, viruses, or artificial chromosomes. In particular, vectors can be used to transfer the nucleic acids of the present invention into appropriate host cells. Once transferred into a host cell, the expression vector can replicate independently of or simultaneously with the host chromosomal DNA, producing several copies of the vector and its inserted DNA. When a replication-incompetent expression vector is used (as is often the case for safety reasons), the vector may not replicate but simply mediate expression of the nucleic acid. Depending on the type of expression vector, the expression vector may be lost from the cell, i.e., the neoantigen encoded by the nucleic acid may only be expressed transiently. The expression vector may also be stable within the cell. An expression vector typically contains an expression cassette, ie, the elements necessary to allow transcription of a nucleic acid into an mRNA molecule.

[0049] Preferably, nucleic acid is mRNA, minicircle or plasmid.It has been shown to be beneficial to use as little DNA as possible, that is, most preferably in the form of mRNA, or alternatively as minicircle, to introduce transposase into mammalian cells, particularly stem cells or primary cells (such as primary T cells).If nucleic acid is not mRNA, said nucleic acid sequence is operably linked to at least one transcription control unit, preferably a promoter active in human cells.

[0050] Another object of the present invention is a cell comprising a nucleic acid of the present invention and / or a polypeptide of the present invention. Typically, the cell will comprise both. The cell may be a bacterial cell, for example in the context of plasmid propagation, but is preferably a eukaryotic cell, in particular a mammalian cell. The cell may be, for example, a human cell, a mouse cell, a rabbit cell, or a rat cell. Human cells are preferred throughout the present invention. The cell may be a stem cell (e.g., a pluripotent stem cell, a hematopoietic stem cell) or a lymphocyte (e.g., a T lymphocyte). For example, the cell is preferably a human cell selected from the group comprising a pluripotent stem cell or a human lymphocyte (preferably a human T lymphocyte). The cell may also be a tumor cell.

[0051] Furthermore, the present invention provides a) any polypeptide of the invention, nucleic acid of the invention, and / or cell of the invention; and b) a nucleic acid comprising a cargo nucleic acid flanked by terminal inverted repeats (TIRs) of a transposon operable by said transposase; A kit comprising:

[0052] Such a kit may be used to deliver a cargo nucleic acid, e.g., a therapeutic amino acid, to the genome of a cell, e.g., a human cell. The cargo nucleic acid is typically an exogenous nucleic acid, i.e., a nucleic acid exogenous to the cell to which the nucleic acid is delivered. The therapeutic nucleic acid may, for example, encode a protein that is reduced or deficient in a metabolic disease, or may encode, for example, a tumor suppressor gene, an immunomodulator, e.g., a cytokine, an antigen, an antibody, a T cell receptor (TCR), or a chimeric antigen receptor (CAR), in the context of gene therapy for said disease. In a preferred embodiment, the therapeutic nucleic acid is useful for the treatment of cancer. The kit can be used for in vivo, ex vivo, or in vitro applications, for use in medicine, e.g., cancer treatment. Thus, the kit can be a medical kit. Alternatively, the cargo nucleic acid may encode a fluorescent protein and / or another selectable marker.

[0053] The present invention also provides pharmaceutical compositions comprising the polypeptides of the present invention, the nucleic acids of the present invention, the cells of the present invention, and / or components of the kits of the present invention. Pharmaceutical compositions typically further comprise a pharmaceutically acceptable carrier and / or excipient. The term "carrier" refers to a natural or synthetic organic or inorganic component with which an active ingredient is combined to facilitate, enhance, or enable application. According to the present invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents, excipients, or encapsulating substances suitable for administration to a subject. Possible carrier substances (e.g., diluents) include, for example, sterile water, Ringer's solution, lactated Ringer's solution, saline, bacteriostatic saline (e.g., saline containing 0.9% benzyl alcohol), phosphate-buffered saline (PBS), Hank's solution, fixed oils, polyalkylene glycols, hydrogenated naphthalenes, and biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers. In one embodiment, the carrier is PBS. The resulting solution or suspension is preferably isotonic with the blood of the recipient. Suitable carriers and their formulations are described in more detail in Remington's Pharmaceutical Sciences, 17th ed., 1985, Mack Publishing Co.

[0054] The pharmaceutical composition is preferably used in adoptive T cell therapy or gene therapy.Gene therapy can be used to treat metabolic disease or immune disease.In one embodiment, gene therapy includes but is not limited to autologous or heterologous T cell therapy, gene therapy that targets any cell type in blood, hematopoietic stem cell therapy, liver gene therapy, central nervous system gene therapy, eye gene therapy, muscle gene therapy, skin gene therapy and / or gene therapy for cancer treatment.

[0055] The pharmaceutical compositions of the present invention are also useful for vaccination therapy to incorporate antigens (e.g., specific tumor antigens, e.g., MAGE-1) into antigen-presenting cells (e.g., human professional antigen-presenting cells such as dendritic cells, macrophages, or B cells, or their precursor cells); tumor vaccination or the treatment of infectious diseases caused by pathogens, e.g., leprosy, tetanus, whooping cough, typhoid, paratyphoid fever, cholera, plague, tuberculosis, meningitis, bacterial pneumonia, anthrax, botulism, bacterial dysentery, Pathogen antigen vaccination for treating flu, diarrhea, food poisoning, syphilis, gastroenteritis, trench fever, influenza, scarlet fever, diphtheria, gonorrhea, toxic shock syndrome, Lyme disease, typhus, listeriosis, peptic ulcer, and legionellosis; e.g., acquired immunodeficiency syndrome, adenoviridae infections, alphavirus infections, arbovirus infections, vector-borne diseases, bunyaviridae infections, caliciviridae infections, chickenpox, genital warts, coronavirus infections, coxsackievirus infections, cytomegalovirus infections, deworming, flu, flu-like illnesses ... The subject method may be for the treatment of viral infections that result in, for example, leukemia, leukemia, leukemia-associated diseases, leukemia-associated diseases (e.g., leukemia-associated diseases), ..., leukemia-associated diseases, leukemia-associated diseases, leukemia-associated diseases, leukemia-associated diseases, leukemia-associated diseases, le

[0056] According to the present invention, the pharmaceutical composition contains an effective amount of an active substance, such as a polypeptide, nucleic acid, vector, or cell described herein, to produce a desired reaction or a desired effect. The pharmaceutical composition of the present invention is preferably sterile. The pharmaceutical composition may be provided in a uniform dosage form and may be prepared by a known method. The pharmaceutical composition of the present invention may be, for example, in the form of a solution or suspension.

[0057] Pharmaceutically acceptable carriers, adjuvants, or vehicles that may be used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, a partial glyceride mixture of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and wool fat. The pharmaceutical compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion. Preferably, the pharmaceutical composition is administered orally, intraperitoneally, or intravenously. The sterile injectable form of the pharmaceutical composition of the present invention may be an aqueous or oily suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a 1,3-butanediol solution. Acceptable vehicles and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally used as solvents or suspending media.

[0058] The pharmaceutical composition of the present invention is preferably used to treat diseases, in particular diseases caused by genetic defects, such as cystic fibrosis, hypercholesterolemia, hemophilia (e.g., A, B, C, or XIII), immunodeficiencies including HIV, Huntington's disease, α-antitrypsin deficiency, and tumors of the gastrointestinal tract, such as colon cancer, melanoma, kidney cancer, lymphoma, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), gastrointestinal tumors, lung cancer, glioma, thyroid cancer, breast cancer, prostate tumor, hepatocellular carcinoma, various virus-induced tumors (such as papillomavirus-induced cancers (e.g., cervical cancer)), adenocarcinoma, herpesvirus-induced tumors (e.g., Burkitt's lymphoma, EBV-induced B-cell lymphoma), hepatitis B-induced tumors (hepatocellular carcinoma), HTLV-1 and HTLV-2-induced lymphoma, acoustic neuroma, and the like. neurinoma, lung cancer, pharyngeal cancer, anal cancer, glioblastoma, lymphoma, rectal cancer, astrocytoma, brain tumor, stomach cancer, retinoblastoma, basal cell tumor, brain metastasis, medulloblastoma, vaginal cancer, pancreatic cancer, testicular cancer, melanoma, bladder cancer, Hodgkin's syndrome, meningioma, Schneeberger's disease, bronchial cancer, pituitary cancer, mycosis fungoides, esophageal cancer, breast cancer, schwannoma, spinal cell carcinoma, Burkitt's lymphoma, lyngeal cancer, thymoma, corpus carcinoma, bone cancer, non-Hodgkin's lymphoma, urethral cancer, CUP syndrome, oligodendroglioma, vulvar cancer, intestinal cancer, esophageal cancer The present invention is suitable for treating cancers selected from carcinoma, small intestine tumor, craniopharyngeoma, ovarian cancer, ovarian cancer, liver cancer, leukemia, or skin or eye cancer.

[0059] The pharmaceutical composition is preferably for the treatment of a human subject. The present invention also provides a method of treating a subject, such as a human subject in need of treatment, for any of the diseases mentioned herein, comprising administering to said subject an effective amount of the pharmaceutical composition of the present invention or an effective amount of a component of the pharmaceutical kit of the present invention.

[0060] The use of a polypeptide of the invention, a nucleic acid of the invention, a cell of the invention, or a kit of the invention for introducing an exogenous nucleic acid into the genome of a cell, eg in vitro, is also described.

[0061] Furthermore, the present invention provides a method for preparing a cell wherein an exogenous nucleic acid has been integrated into the genome of the cell, optionally an in vitro method, comprising the steps of: a) providing isolated cells; b) providing said cell with any of the polypeptides of the present invention; and c) providing the cell with a nucleic acid comprising an exogenous nucleic acid flanked by terminal inverted repeats (TIRs) of a transposon operable by the transposase; The present invention provides a method comprising:

[0062] In some cases, the polypeptide is provided to the cell by introducing the nucleic acid of the present invention into the cell. Alternatively, the polypeptide can be provided in the form of a polypeptide, for example, as the highly soluble transposase described herein. In one embodiment, the exogenous nucleic acid is included in the nucleic acid or vector of the present invention. The exogenous nucleic acid can also be administered separately.

[0063] In one embodiment, the nucleic acid of the present invention and / or the exogenous nucleic acid is provided to cells using a method selected from the group consisting of electroporation, microinjection, lipoprotein particles, and virus-like particles. Electroporation has been found to be particularly suitable for transfecting primary cells, such as T cells.

[0064] Another object of the invention is a method for preparing a polypeptide of the invention, comprising culturing a cell of the invention containing a nucleic acid of the invention and isolating the polypeptide.

[0065] The present invention is further illustrated in the accompanying figures and examples, which are not intended to limit the invention. All documents cited herein are incorporated by reference in their entirety. [Brief explanation of the drawings]

[0066] [Figure 1] Schematic diagram of gene delivery by Sleeping Beauty transposition. SB transposase is introduced into cells along with donor DNA, in the form of DNA (e.g., expression plasmid), mRNA, or recombinant protein, containing the transposon to be activated. The donor DNA can be vectorized as a plasmid or minicircle. After binding within the transposon's terminal inverted repeats (TIRs, box) that flank the gene of interest (GOI, box), SB transposase (circle) excises the transposon from the donor DNA (black strand) and integrates it into a site within the genomic target DNA (strand). [Figure 2] Validation of the Q124C mutation in the Sleeping Beauty transposase. (A) The Q124C mutant of SB100X exhibits a hyperactive phenotype as assayed by colony formation transposition assay in human HepG2 cells. (B) Venus expression in SB-transfected hiPSCs reached stable levels at day 14 (measured by flow cytometry). (C) The Q124C mutant results in an increased percentage of stably Venus-modified hiPSCs (measured by flow cytometry). [Figure 3] Mechanistic insights into the hyperactivity of the Q124C mutation in the Sleeping Beauty transposase. (A) Model of the SB transposase and Q124 aa side chain (highlighted) bound to the SB transposon and target DNA. (B) The Q124C mutant demonstrates its hyperactivity, especially at high doses (assayed by a dose-dependent colony formation transposition assay in human HepG2 cells). (C) In reactivated colony formation assays with puro selection (excision) and G418 and puro double selection (transposition), the Q124C mutant exhibits increased excision rates, resulting in an overall higher transposition rate compared to SB100X. [Figure 4]Validation of the Q124C mutation in mouse primary hematopoietic stem cells (HSCs). (A) The Q124C mutant of SB100X exhibits a hyperactive phenotype in HSCs at high doses, as assayed by Venus expression in SB-nucleofected HSCs. Venus expression reaches stable expression levels by day 9. (B) Viability of HSCs nucleofected with minicircle Venus and SB transposase, assayed over time by Zombie staining and flow cytometry analysis. [Figure 5-1] Combining the Q124C mutant with other Sleeping Beauty transposase variants rescues transposition activity. (A) Introduction of the Q124C mutant into the K248R mutant partially rescues its transposition activity (as assessed by colony-forming transposition assay in human HepG2 cells). (B) Insertion frequency into genomic safe harbors. (C) Insertion frequency into all genomic safe harbor subcategories. (D) Insertion frequency into exons. (E) Insertion frequency 10 kb upstream of the transcription start site (TSS). [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 6-1]Alignment of the amino acid sequences of the Sleeping Beauty transposase and the closely related Tc1 / mariner transposase (SEQ ID NOS: 1-17). Figure 6 allows those skilled in the art to determine the amino acid position in all other aligned transposases that corresponds to amino acid 124 or other specific amino acid positions. This amino acid position can be substituted with a different amino acid in the same manner as exemplarily outlined for SB. Additional transposases can be added to the alignment, allowing the identification of the amino acid corresponding to amino acid 124 of SB in these transposases. At the top of the sequence alignment, a secondary structure typical of transposases of the Tc1 / mariner superfamily is shown: α1 helix-α2 helix-β1 sheet-β2 sheet-β3 sheet-β4 sheet-β5 sheet-α3 helix-β6 sheet-η1-α4 helix-η2-α5 helix-α6 helix-α7 helix-α8 helix belonging to the corresponding amino acid stretch. The amino acid sequence of SB (SEQ ID NOS: 1) was used as the reference amino acid sequence in this alignment. All amino acids corresponding to amino acids 187, 247, and 248 of SB are highlighted in boxes. [Figure 6-2] Same as above. [Figure 7] Transposition efficiency of single amino acid substitution mutants at position 124 in SB100X transposase (measured by colony formation assay in human HeLa cells). Cells were seeded into 6-well plates at a density of 200,000 cells per well and transfected with 500 ng of each transposase expression plasmid and 500 ng of puromycin resistance gene-tagged SB transposon using Transit-LT1 transfection reagent. Some of the mutants exhibit a hyperactive phenotype compared to SB100X (n=3; significance assessed by t-test: *p<0.05, **p<0.01). [Example]

[0067] result Q124C mutation confers hyperactivity in human hepatocyte-derived HepG2 cells and human induced pluripotent stem cells (hiPSCs) To assay the transposition activity of the Q124C mutation in the SB100X coding sequence, we transfected expression plasmids (pcGlobin2) encoding either SB100X or the Q124C mutant into HepG2 cells along with a puromycin resistance (puro) gene-tagged SB vector (pT2 / Puro) and assessed the relative efficiency of transposition by counting puro-resistant cell colonies after antibiotic selection. The Q124C mutant results in a two-fold increase in transposition rate compared with SB100X, the currently most active SB transposase mutant (Figure 2A). This hyperactivity was also demonstrated in more clinically relevant human induced pluripotent stem cells (hiPSCs). Expression plasmids encoding the two active SB transposase mutants, SB100X and Q124C, and the catalytically inactive mutant DAE were transfected into hiPSCs along with a Venus fluorescent gene-tagged SB transposon minicircle. Fluorescence of hiPSCs was tracked over time, and hiPSCs reached stable expression by day 14 (Figure 2B). At day 14, the Q124C mutant showed a two-fold increase in the number of Venus-modified hiPSCs compared with the SB100X mutant (Figure 2C).

[0068] The Q124C mutation confers resistance to overproduction suppression and is manifested already at the excision stage To better understand the mechanistic reasons for the hyperactivity of the Q124C mutant in the SB100X coding sequence, we performed transposition assays with different doses of the SB transposase expression plasmid. It is a known phenomenon in the Tc1 / mariner TE that high levels of transposase result in overproduction suppression, and therefore a nonlinear saturation increase in the transposition rate

[11] . Because the Q124 mutation site is close to a protein-protein dimer interaction interface that tends to allosterically exert its overproduction suppression effect, we speculate that the hyperactivity of the Q124C mutant should be particularly evident at high transposase concentrations. The location of Q124, along with the SB transposon ends and DNA target site, is highlighted in a model of the SB transposase (Figure 3A). The model is based on the crystal structure of the catalytic domain

[12] and the NMR structures of the PAI

[13] and RED

[14] subdomains, and is superimposed with the full-length structure of the related mariner transposase, Mos1

[15] .

[0069] In a standard transposition assay comparing SB100X with Q124C, which yields puro-resistant colonies, transposition hyperactivity was demonstrated at higher doses (500 and 2500 ng of expression plasmid) (Figure 3B). These observations led us to conclude that the Q124C variant possesses a certain level of resistance to overproduction suppression. Thus, the Q124C variant expands the dose of SB transposase that can be used in preclinical and clinical settings, resulting in an overall higher transposition rate.

[0070] We established a transgenic human HepG2-derived reporter cell line harboring a single copy of a neomycin resistance gene (neo)-tagged SB transposon that disrupts the open reading frame (ORF) of the puro resistance gene. Therefore, these cells are G418-resistant and puro-sensitive. When SB transposase is expressed in these reporter cells, standard excision of the SB transposon and subsequent repair of the broken DNA ends by the non-homologous end joining (NHEJ) double-stranded DNA repair pathway reconstitutes the puro ORF, thereby resulting in a selectable puro-resistant phenotype. Selection with puro allows us to measure the excision rate of different transposase mutants, and dual selection with G418 and puro allows us to score the efficiency of the complete transposition reaction (excision + integration). Transfection of this reporter cell line with two mutants, SB100X and Q124C, demonstrates that hyperactivity is already evident at the excision stage, resulting in an overall increase in the transposition rate (Figure 3C). This is consistent with the described effect of resistance to overproduction suppression, since this effect primarily affects the initiation of the excision complex.

[0071] The Q124C mutation results in hyperactivity in hematopoietic stem cells (HSCs) To assay the transposition activity of the Q124C mutation in the SB100X coding sequence in primary hematopoietic stem cells (HSCs), lineage-negative bone marrow HSCs were isolated from mice and nucleofected with expression plasmids encoding two active SB transposase mutants, SB100X and Q124C, and a catalytically inactive mutant DAE, along with a Venus fluorescent gene-tagged SB minicircle. HSC fluorescence (Figure 4A) and viability (Figure 4B) were tracked over time, and HSCs reached stable expression by day 9. While no difference was observed in Venus-positive HSCs at a dose of 1 μg expression plasmid, at a higher dose of 2 μg expression plasmid, the Q124C mutation resulted in a two-fold increase in stable expression of Venus-positive HSCs (Figure 4A). HSC viability was only mildly affected at the higher dose of Q124C and rapidly recovered by day 9 (Figure 4B). This experimental data indicates that the hyperactivity of the Q124C mutant is also manifested in primary cells.

[0072] Introduction of the Q124C mutation into the K248R mutant rescues metastatic activity Other SB transposase variants, such as the K248R mutation in the SB100X coding sequence, offer certain advantages, such as a safer integration profile compared to SB100X

[10] . The K248R variant integrates less frequently into genes and promoter regions and more frequently into genomic locations considered safe harbors. However, the transposition activity of this variant is reduced compared to SB100X. Introducing the Q124C mutation into the K248R version can rescue the transposition activity of this variant to some extent. The transposition activity of various variants was assayed by standard colony formation assays, and puro-resistant colonies were counted (Figure 5A).

[0073] Integration of therapeutic gene constructs into safe sites in the human genome via SB transposase prevents insertional genomic toxicity and associated carcinogenic risks. Genomic "safe harbors" are regions of the human genome that can tolerate the integration of new DNA without adversely affecting the host cell. A chromosomal site or region can be bioinformatically designated as a GSH if it meets the following criteria: (i) no overlap with any transcription unit, (ii) at least 50 kb from the 5' end of any gene, (iii) at least 300 kb from cancer-related genes and (iv) from microRNA genes, and (v) outside of ultraconserved elements (UCEs) [16, 17]. The frequency of insertions into GSHs was increased by the introduction of the Q124C mutation into the coding sequence of the SB transposase (Figure 5B). In all subcategories of GSHs, the double mutant Q124C / K248R increased insertion frequency (Figure 5C). When the insertion frequency in the exon (Fig. 5D) and 10 kb upstream of the transcription start site (Fig. 5E) was assayed, the Q124C single mutation had a lower insertion frequency compared to SB100X, whereas the double mutant (Q124C / K248R) retained its lower insertion frequency compared to the single mutant (K248R).

[0074] Introducing the Q124C mutation into the K248R variant increases the value of this variant in clinical applications, where efficiency and safety play important roles, especially compared to viral vectors. The Q124 residue is located near the protein surface, which is involved in DNA interactions (Figure 3A). Therefore, combining the Q124C mutation with other SB transposase variants may also increase transposition activity.

[0075] material and method Site-directed mutagenesis of SB100X transposase All mutations were generated using Q5 polymerase (NEB, Ipswich, MA, USA) and the plasmid pcGlobin2-SB100X. 5'-phosphorylated primers for specific positions were designed with back-to-back annealing of the 5' ends. Primers were synthesized with a 5'-phosphate to enable downstream intramolecular ligation and were ordered from Eurofins (Eurofins / MWG, Luxembourg). Primer sequences were [SB100X-Q124_to_C-rev: 5'Phos-CTTCTTCCTTGCTGAGTGG-3' (SEQ ID NO: 23) and SB100X-Q124_to_C124-fwd: 5'Phos-CCACTGCTCTGCAACCGACATAAGAAAGCC-3' (SEQ ID NO: 24)]. PCR cycling conditions were set according to the manufacturer's instructions. The annealing temperature of the mutagenic primers was calculated using the "NEB Tm calculator™" software (https: / / www.neb.com / tools-and-resources / interactive-tools / tm-calculator). PCR products were purified with a QIAquick PCR purification kit (QIAGEN, Venlo, The Netherlands), eluted in 30 μl elution buffer, and digested with 2 μl DpnI (NEB, Ipswich, MA, USA) for 2 hours at 37°C, followed by heat inactivation at 80°C for 20 minutes. Linear double-stranded PCR products were circularized by ligation with T4 DNA ligase (NEB, Ipswich, MA, USA) overnight at 16°C. Circular PCR products were transformed into chemically competent Escherichia coli ( E. coli ) (Invitrogen / Life Technologies, Carlsbad, CA, USA), grown in Luria-Bertani (LB) medium for 1 h, and plated onto LB agar plates containing 100 μg / ml ampicillin to select for ampicillin resistance.To confirm the presence of the desired mutations and the absence of undesired mutations, plasmid DNA from several colonies was purified using a QIAprep spin miniprep kit (QIAGEN, Venlo, The Netherlands) and Sanger sequenced by Eurofins (Eurofins / MWG, Luxembourg).

[0076] Metastasis assay For the transfection assay in HepG2 cells, 3 × 10^5 cells were seeded into 6-well plates one day before transfection. Transfection was performed using TransIT-LT1 transfection reagent (Mirus Bio LLC, Madison, WI, USA) according to the manufacturer's protocol. Each transfection reaction was adjusted with the pmaxGFP plasmid to ensure the same total amount of transfected DNA. The transposon expression plasmid (pcGlobin2) was co-transfected with the transposon donor plasmid (pT2 / Puro). 48 h after transfection, cells were trypsinized, and 1–10% of the cells were replated onto 10 cm plates and selected for transposon integration using 1 μg / ml puromycin (InvivoGen, San Diego, CA, USA). After 2 weeks of selection, cell colonies were fixed with 10% (vol / vol) formaldehyde in phosphate-buffered saline (PBS), stained with methylene blue in PBS, and counted. For in vitro comparison of relative translocation efficiency, at least three independent experiments were performed.

[0077] Venus modification of hiPSCs by Sleeping Beauty hiPSCs were cultured and handled according to the protocol described by Skarnes et al.

[18] . Transfections were performed using TransIT-LT1 transfection reagent (Mirus Bio LLC, Madison, WI, USA) according to the manufacturer's protocol for hiPSCs. 2 μg of a transposon expression plasmid (pcGlobin2) and a transposon donor minicircle (mcVenus) were cotransfected into 2 × 10 hiPSCs. Venus expression was assessed every 3–4 days by cytofluorimetric analysis using a BD FACS-SORB (BD FACS™, East Rutherford, NJ, USA) and analyzed by FlowJo (FlowJo LLC, Ashland, OR, USA). At least three independent experiments were performed for in vitro comparison of relative transposition efficiencies.

[0078] Reactivation assay For reactivation assays with the modified HepG2 reporter cell line [HepG2-PB(SB2#23)], 4 × 10^5 cells were seeded into 6-well plates one day before transfection. Transfection was performed using Lipofectamine 3000 transfection reagent (Invitrogen AG, Waltham, MA, USA) according to the manufacturer's protocol. 1 μg of the transposon expression plasmid (pcGlobin2) was transfected into HepG2-PB(SB2#23) cells. 72 h after transfection, cells were trypsinized, and 100% of the cells were replated onto two 10 cm plates and selected for transposon excision using 1 μg / ml puromycin (InvivoGen, San Diego, CA, USA) or double-selected for transposition using 1 μg / ml puromycin and 1 mg / ml G418 (InvivoGen, San Diego, CA, USA). After 3 weeks of selection, cell colonies were fixed with 10% (vol / vol) formaldehyde in phosphate-buffered saline (PBS), stained with methylene blue in PBS, and counted. For in vitro comparison of relative metastatic efficiency, at least three independent experiments were performed.

[0079] Generation of SB insertion library To analyze the target site selection properties of SB100X transposase mutants, 4 x 10^5 HepG2 cells were seeded into 6-well plates one day before transfection. Transfections were performed with QIAGEN-purified plasmid DNA using TransIT-LT1 transfection reagent according to the manufacturer's protocol. 1 μl of TransIT-LT1 transfection reagent was used to transfect 550 ng of DNA containing 500 ng of pT2Bpuro and 50 ng of a helper plasmid expressing the mutant transposase or pcGlobin2-SB100X. 48 hours after transfection, cells were trypsinized, diluted into multiple 10 cm dishes containing DMEM supplemented with 1 μg / ml puromycin, and selected for growth over a 2-week period. At least 10,000 puromycin-resistant HepG2 cell colonies were trypsinized and centrifuged at 1,000 rpm for 5 minutes. The pellet was washed with PBS, and genomic DNA was extracted from the cells using the Qiagen DNeasy Blood & Tissue kit according to the manufacturer's protocol.

[0080] To generate the SB insertion site library, 2 μg of DNA was sheared to an average fragment size of 600 bp in a 50 μl Screw-Cap microtube using a Covaris M220 ultra-solicitor instrument with the following settings: 50 W peak incident power, 20% duty factor, 200 cycles per burst, and 28 s processing time. 1.2 μg of sheared DNA was blunted using the NEBNext End Repair Module (NEB) according to the manufacturer's recommendations, 5' phosphorylated, and 3' A-tailed using the NEBNext dA-Tailing Module (NEB). DNA was purified using a Clean and Concentrator kit (Zymo Research), eluted in 8 μl 10 mM Tris pH 8.0 (EB), and ligated with 50 pmol of T-linker (see below) using T4 ligase (NEB) in a 20 μl volume overnight at 16°C. T-linkers were generated by annealing 100 pmol each of the oligonucleotides Linker_TruSeq_T+ and Linker_TruSeq_T- in 10 mM Tris-Cl, pH 8, 50 mM NaCl, and 0.5 mM EDTA. After heat inactivation, the ligation product, including the unintegrated fragment of transposon donor plasmid DNA, was digested with 50 μl of DpnI (NEB) for 3 hours. The DNA was column-purified and eluted in 20 μl of EB. Six μl of the eluate was used for PCR I with 25 pmol each of the linker and transposon inverted repeat-specific primers: Linker and T-Bal-Long, under the following conditions: 98°C for 30 s; 10 cycles of 98°C for 10 s, 72°C for 30 s; and 15 cycles of 98°C for 10 s, a ramp to 62°C (1°C / s) for 30 s, 72°C for 30 s, and 72°C for 5 min. All PCR reactions were performed using NEBNext High-Fidelity 2x PCR Master Mix. PCRs were column purified and eluted in 20 μl EB. 10 μl was used for PCR II with primers Nested and LAM-SB-50 using the following program: 98°C for 30 s; 98°C for 10 s, ramp to 65°C (1°C / s) for 30 s, 72°C for 30 s, and 12 cycles of 72°C for 5 min.One-third of the column-purified PCR II was used for PCR III with primers PE-nest-ind-N and SB-20-bc-ill-N (where N is the number of Illumina TrueSeq indexes) to barcode the samples using the following PCR program: 98 °C for 30 s; 12 cycles of 98 °C for 10 s, a ramp to 64 °C (1 °C / s) for 30 s, 72 °C for 30 s, and 72 °C for 5 min. The final PCR products were separated on a 1% agarose gel, and a 200-500 bp smear was isolated and purified.

[0081] Sequencing and analysis of insertion sites Insertion site libraries were prepared as previously described

[19] and sequenced on an Illumina instrument using a 150-bp, single-end setting. After adapter and quality trimming (Phred score ≥ 20) using fastp

[20] , reads downstream of the transposon sequence specific primer were tested and filtered for the presence of remaining transposon inverted terminal repeats (ITRs) and a minimum length of 28 bases of genomic sequence for mapping using bowtie2

[21] in -sensitive and -end-to-end settings. Mapped loci were considered valid if the mapping quality of the supporting reads was ≥ 20. Every insertion site had to be supported by at least 10 independent reads in the TA target site of the human genome (hg38). When multiple insertions were detected within 10 bases, the insertion site supported by the greatest number of independent reads was considered valid. The distribution of insertion sites in various gene categories was investigated using the Genomation package

[22] . A computer-generated random set of 100,000 loci from the human hg38 genome assembly was used as a reference to investigate the distribution of insertion sites in different genomic bins. Genomic safe harbor coordinates for the hg38 assembly were generated according to previously defined criteria

[16] .

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Cancer 2011, 12, 51-58, doi:10.1038 / nrc3179. 17. Papapetrou, E.P.; Lee, G.; Malani, N.; Setty, M.; Riviere, I.; Tirunagari, L.M.S.; Kadota, K.; Roth, S.L.; Giardina, P.; Viale, A.; et al. Genomic safe harbors permit high β-globin transgene expression in thalassemia induced pluripotent stem cells. Nat. Biotechnol. 2011, 29, 73-78, doi:10.1038 / nbt.1717. 18. Skarnes, W.C.; Pellegrino, E.; McDonough, J.A. Improving homology-directed repair efficiency in human stem cells. Methods 2019, 164-165, 18-28, doi:10.1016 / j.ymeth.2019.06.016. 19. Querques, I.; Mades, A.; Zuliani, C.; Miskey, C.; Alb, M.; Grueso, E.; Machwirth, M.; Rausch, T.; Einsele, H.; Ivics, Z.; et al. A highly soluble Sleeping Beauty transposase improves control of gene insertion. Nat. Biotechnol. 2019, 37, 1502-1512, doi:10.1038 / s41587-019-0291-z. 20. Chen, S.; Zhou, Y.; Chen, Y.; Gu, J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 2018, 34, i884-i890, doi:10.1093 / bioinformatics / bty560. 21. 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Claims

1. A polypeptide having transposability, comprising a Sleeping Beauty (SB) transposase having at least 80% amino acid identity to SEQ ID NO: 18 and a substitution at amino acid position 124, wherein the amino acid at position 124 is not Q.

2. 10. A polypeptide according to any one of the preceding claims, having enhanced transposition activity compared to an otherwise identical transposase that does not contain the substitution at position 124.

3. 10. The polypeptide of claim 9, wherein the amino acid at position 124 is selected from the group consisting of C, A, R, N, D, E, G, H, I, L, M, F, S, T, Y, and V.

4. 10. The polypeptide of any one of the preceding claims, wherein the transposase comprises the amino acid sequence of SEQ ID NO: 19 or has 1, 2, 3 or 4 amino acid differences compared to SEQ ID NO: 19, preferably the transposase has a Q124C substitution.

5. 2. The polypeptide of claim 1, wherein the amino acid at position 124 is C.

6. The polypeptide of any one of claims 1 to 4, wherein the amino acid at position 124 is G.

7. The polypeptide of any one of claims 1 to 4, wherein the amino acid at position 124 is D.

8. The polypeptide of any one of claims 1 to 4, wherein the amino acid at position 124 is N.

9. The polypeptide of any one of claims 1 to 4, wherein the amino acid at position 124 is E.

10. The polypeptide according to any one of claims 1 to 4, wherein the amino acid at position 124 is L.

11. The polypeptide according to any one of claims 1 to 4, wherein the amino acid at position 124 is M.

12. The polypeptide of any one of claims 1 to 4, wherein the amino acid at position 124 is F.

13. The polypeptide of any one of claims 1 to 4, wherein the amino acid at position 124 is S.

14. The polypeptide of any one of claims 1 to 4, wherein the amino acid at position 124 is T.

15. The polypeptide of any one of claims 1 to 4, wherein the amino acid at position 124 is Y.

16. The polypeptide of any one of claims 1 to 4, wherein the amino acid at position 124 is V.

17. further comprising a substitution at any of positions 187, 247 and / or 248; Depending on the situation, a) the substituted amino acid at position 187 is A, N, C, Q, G, I, L, M, S, V, W, K, R, E, P, T and S, preferably V; b) the substituted amino acid at position 247 is R, C, A or S; preferably R; and / or c) the substituted amino acid at position 248 is R, S, V, I or C, preferably R; A polypeptide according to any one of the preceding claims.

18. The following substitutions or groups of substitutions: (1) K14R, K13D, K13A, K30R, K33A, T83A, I100L, R115H, R143L, R147E, A205K / H207V / K208R / D210E, H207V / K208R / D210E, R214D / K215A / E216V / N217Q; M243Q, E267D, T314N, and / or G317E; (2) K14R / / R214D / K215A / E216V / N217Q; (3) K33A / R115H / R214D / K215A / E216V / N217Q / M243H; (4) K14R / K30R / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / M243H; (5) K13D / K33A / T83N / H207V / K208R / D210E / M243Q; (6) K13A / K33A / R214D / K215A / E216V / N217Q; (7) K33A / T83N / R214D / K215NE216V / N217Q / / G317E; (8) K14R / T83A / M243Q; (9) K14R / T83A / I100L / M243Q; (10) K14R / T83A / R143L / M243Q; (11) K14R / T83A / R147E / M243Q; (12) K14R / T83A / M243Q / E267D; (13) K14R / T83A / M243Q / T314N; (14) K14R / K30R / I110L / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / M243H; (15) K14R / K30R / R143L / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / / M243H; (16) K14R / K30R / R147E / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / M243H; (17) K14R / K30R / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / / M243H / E267D; (18) K14R / K30R / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / M243H / T314N; (19) K14R / K30R / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / / M243H / G317E; (20) K14R / K33A / R115H / R214D / K215A / E216V / N217Q / M243H; (21) K14R / K30R / R147E / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / / M243H / T314N; (22) K14R / K30R / R143U / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / / M243H / E267D; (23) K14R / K30R / R143L / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / / M243H / T314N; (24) K14R / K30R / R143L / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / / M243H / G317E; (25) K14R / K33A / R115H / R143L / R214D / K215A / E216V / N217Q / M243H; (26) K14R / K33A / R115H / R147E / / R214D / K215A / E216V / N217Q / M243H; (27) K14R / K33A / R115H / R214D / K215A / E216V / N217Q / / M243H / E267D; (28) K14R / K33A / R115H / R214D / K215A / E216V / N217Q / / M243H / T314N; (29) K14R / K33A / R115H / R214D / K215A / E216V / N217Q / / M243H / G317E; (30) K14R / T83A / M243Q / G317E; or (31) K13A / K33A / T83N / R214D / K215A / E216V / N217Q 10. The polypeptide of claim 9, further comprising at least one of:

19. A nucleic acid encoding a polypeptide according to any one of the preceding claims.

20. 20. The nucleic acid of claim 19, wherein the nucleic acid is an mRNA, a minicircle, or a plasmid, and wherein if the nucleic acid is not an mRNA, the nucleic acid sequence may be operably linked to at least one transcriptional control unit, preferably a promoter active in human cells.

21. A cell comprising a nucleic acid according to claim 19 or 20 and / or a polypeptide according to any one of claims 1 to 18.

22. 22. The cell of claim 21, which is a human cell selected from the group comprising pluripotent stem cells or human lymphocytes, preferably human T lymphocytes.

23. a) a polypeptide according to any one of claims 1 to 18, a nucleic acid according to claim 19 or 20, and / or a cell according to claim 21 or 22; and b) a nucleic acid comprising a cargo nucleic acid flanked by terminal inverted repeats (TIRs) of a transposon operable by said transposase; Kit including:

24. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 18, a nucleic acid according to claim 19 or 20; a cell according to claim 21 or 22, and / or a kit according to claim 23, and optionally a pharmaceutically acceptable carrier and / or excipient, A pharmaceutical composition, preferably for use in adoptive T cell therapy or gene therapy.

25. 24. Use of a polypeptide according to any one of claims 1 to 18, a nucleic acid according to claim 19 or 20, a cell according to claim 21 or 22, or a kit according to claim 23, for introducing an exogenous nucleic acid into the genome of a cell, optionally in vitro.

26. 1. A method for preparing a cell wherein an exogenous nucleic acid has been integrated into the genome of the cell, optionally an in vitro method, comprising: a) providing isolated cells; b) providing said cells with a polypeptide according to any one of claims 1 to 18; and c) providing the cell with a nucleic acid comprising an exogenous nucleic acid flanked by terminal inverted repeats (TIRs) of a transposon operable by the transposase; Including, Optionally, the method wherein the polypeptide is provided to the cell by introducing into the cell a nucleic acid according to claim 19 or 20.

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