Transposase mutants with enhanced gene transfer efficiency

Abstract

The invention relates to the field of transposase. In particular, it provides a polypeptide comprising or consisting of a Tc1 / mariner superfamily transposase, such as a sleep beauty transposase, having a substitution at the 124 amino acid. The transposase of the invention was found to have enhanced transposon efficiency. The invention also provides nucleic acids encoding the transposase, cells expressing the same, kits and pharmaceutical compositions comprising the transposase (e.g., for gene therapy, e.g. Adoptive T cell therapy), or uses and methods of delivering a gene into the genome of a cell.

Description

[0001] The present invention relates to the field of transposases. In particular, it provides a polypeptide comprising or consisting of a Tc1 / mariner superfamily transposase, such as the Sleeping Beauty transposase, having a substitution at amino acid position 124. The transposase of the present invention was found to have enhanced transposition efficiency. The present invention also provides nucleic acids encoding the transposase, cells expressing it, kits and pharmaceutical compositions comprising the transposase (e.g., for gene therapy such as adoptive T cell therapy), or uses and methods for delivering genes into a cell genome.

[0002] The ability of nucleic acids to integrate their sequences into new sites has evolved in many ways. A large family of transposons, first described in maize by Barbara McClintock [1], has the ability to move their genetic information within the genome. Transposons (TEs) can be divided into two groups. Class I TEs are so-called retrotransposons that follow a copy-and-paste mechanism and use an RNA intermediate to carry out this process [2]. Class II transposons rely exclusively on DNA intermediates 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 another [2]. The Tc1 / mariner superfamily follows this typical cut-and-paste mechanism. TEs of this superfamily are flanked by inverted terminal repeats (TIRs) and contain genes encoding transposases, an enzyme factor that catalyzes the transposition reaction [2]. The transposase binds to the TIRs, excises the TE from the donor locus, and reintegrates it near the TA target sequence, ultimately resulting in duplication of the TA target site [2].

[0003] The minimum components required for a transposition reaction are a TIR and a transposase. Therefore, TEs containing these two components are considered autonomous TEs [3]. Many autonomous TEs have generated non-autonomous derivatives through modification of the transposase coding region. These non-autonomous TEs can still be mobilized, but require a functional transposase expressed by another element in the same cell [3]. This reverse complementarity between the two functional components (the transposase and the specific TIR recognized and mobilized by the transposase) is the basis for transforming transposons into a genetic vector system suitable for moving any target gene ( Figure 1 ).

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

[0005] Other transposases in the Tc1 / mariner superfamily, such as Frog Prince, have similar structures, such as Figure 6 shown.

[0006] Since the discovery of the SB transposon, several mutations have been found that increase the overall integration efficiency. These mutations ultimately led to the most active SB transposase variant currently, SB100X[8]. Other mutations have expanded the application spectrum of SB transposase, such as the K248T mutation that creates an integration-defective SB transposase[9] or the K248R mutation that creates a safer integration spectrum

[10] .

[0007] The WVPHEL (SEQ ID NO: 25) motif of the Tc1 / mariner transposase Mos1 (amino acids 119-124) forms a dimer interaction interface and plays an important role in downregulating the transposition reaction through an allosteric mechanism

[11] . Mutations in this region have been shown to result in the formation of hyperactive mariner transposase variants

[11] .

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

[0009] Ivics et al. have previously shown

[26] that deletion of amino acids 117-123, which contain the ARKKPLL (SEQ ID NO: 27) motif in SB, disrupts nuclear localization. Because this motif in SB matches the WVPHEL motif in mariner transposase, mutations that cause mariner hyperactivity are unlikely to play a role in SB, as these mutations would inhibit nuclear localization.

[0010] In view of this, the inventors addressed the problem of providing further advantageous variants of Tc1 / mariner superfamily transposases, for example with increased transposition efficiency.

[0011] This problem is solved by the subject matter of the claims. In particular, the present invention provides a polypeptide having transposition activity, which comprises or consists 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, which has at least 80% amino acid identity with SEQ ID NO: 18, optionally at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity or at least 99% sequence identity.

[0012] Outside the region corresponding to the WVPHEL motif of Mos1 (amino acids 119-123), next to amino acid 124 in the SB100X coding sequence, the inventors surprisingly discovered a position that can be substituted, where variants with mutations, specifically a variant in which glutamine at position 124 was substituted with cysteine ​​at position 124 (Q124C), had an approximately 2-fold increase in transposition activity and exhibited resistance to the overproduction inhibition described for various Tc1 / mariner transposases

[11] . Introduction of the Q124C mutation also improved the efficiency of other clinically relevant SB transposase variants, including K248R. Overall, these findings demonstrate the utility of mutations at position 124, and specifically the Q124C mutation, to increase the rate of application of SB transposon-mediated genome engineering in preclinical and clinical applications.

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

[0014] The polypeptide having transposition activity comprises a transposase or consists of a transposase, i.e. it can be a fusion protein of a transposase. The fusion protein can be fused to the N-terminus or the C-terminus, however, as described in more detail below, the nomenclature position is determined only relative to the transposase portion of the polypeptide. In particular, the polypeptide can consist of a transposase.

[0015] The transposase may lack 1 to 10 N- and / or C-terminal amino acid sequences of the full length of a naturally occurring transposase. Preferably, it may lack a methionine at position M1, for example, if the transposase is comprised in a fusion protein comprising 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 that can be assessed in a transposition reaction. "Transposition efficiency" is a synonym. Suitable experimental settings are described in the experimental section of this article, 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 An alignment of transposase sequences from the Tc1 / mariner transposase superfamily is shown. The alignment algorithm used to generate the alignment above was CLUSTAL Omega with the following settings: Dealign input sequences: No, mbed-like clustering guide tree: Yes, mbed-like clustering iterations: Yes, Combined iterations: Default, Max guide tree iterations: Default, Max hmm iterations: Default. Amino acid conservation is highlighted in MView. The alignment includes secondary structure information for the SB transposase catalytic domain (PDB entry code: 5CR4) highlighted with ESPript3.0.

[0018] The transposase of the invention is preferably active in human cells. Suitable transposases are, for example, Sleeping Beauty (SB), Frog Prince (FP), Minos, ZB, Tdr1 and Passport. SB is the first choice.

[0019] The corresponding transposase is based on its Figure 6That is, if the transposase has at least 80% amino acid identity with any of the sequences and has the highest sequence identity with one of the sequences, it is clustered as a transposon of the class.

[0020] Preferably, the transposase is a SB transposase having at least 80% amino acid identity to SEQ ID NO: 1 (SB100X), optionally having at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity or at least 99% sequence identity. Optionally, the sequence has only one, two or three amino acid differences, 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. SB100X itself is a highly active transposase, but the inventors surprisingly found that SB100X with the Q124C substitution (SEQ ID NO: 19) further enhanced the transposase activity. This also applies to variants of the transposase comprising one additional substitution (e.g. at position 187, 247 or 248, e.g. SB100X Q124C H187V, SEQ ID NO: 20, SB100X Q124C P247R, SEQ ID ID: 21; SB100X Q224CK248R, SEQ ID NO: 22), two additional substitutions (e.g. at positions 187 and 247 or 187 and 248, e.g. SB100X Q124C H187VP247R or Q124C H187V K248R), three additional substitutions (e.g. at positions 187, 247 and 248, e.g. SB100X Q124C H287V P247RK248R or four additional substitutions. Thus, variants of the transposase comprising one additional substitution (e.g. at position 187, 247 and 248, e.g. SB100X Q124C H287V P247RK248R) with SEQ ID NO: 23. NO:18 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 are 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 are

[0022] a)SB100X Q124X 1 , where X 1 Not Q, where X 1 Preferably C, H187X 2 , where X 2 Not H, where X 2 Optionally V;

[0023] b)SB100X Q124X 1 , where X 1 Not Q, where X 1 Preferably C, P247X 2 , where X 2 Not P, where X 2 Optionally R;

[0024] c)SB100X Q124X 1 , where X 1 Not Q, where X 1 Preferably C, K248X 2 , where X 2 Not K, where X 2 Optionally R.

[0025] Alternatively, the transposase may be a Tdr1 transposase having at least 80% amino acid identity to SEQ ID NO: 2, optionally at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity or at least 99% sequence identity. Optionally, it has only one, two or three amino acid differences from the sequence, one of which is a substitution at position 124.

[0026] The transposase may also be a ZB transposase having at least 80% amino acid identity to SEQ ID NO: 3, optionally at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity or at least 99% sequence identity. Optionally, it has only one, two or three amino acid differences from the sequence, one of which is a substitution at position 124.

[0027] In one embodiment, the transposase may be a FP (Frog Prince) transposase having at least 80% amino acid identity to SEQ ID NO: 4, optionally at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity, or at least 99% sequence identity. Optionally, it has only one, two, or three amino acid differences from the sequence, one of which is a substitution at position 124.

[0028] The transposase may also be a Passport transposase having at least 80% amino acid identity to SEQ ID NO: 5, optionally at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity or at least 99% sequence identity. Optionally, it has only one, two or three amino acid differences from the sequence, one of which is a substitution at position 124.

[0029] Optionally, the transposase may be a Minos transposase having at least 80% amino acid identity to SEQ ID NO: 11, optionally at least 90% sequence identity, at least 95% sequence identity, at least 98% sequence identity or at least 99% sequence identity. Optionally, it has only one, two or three amino acid differences from the sequence, one of which is a substitution at position 124.

[0030] In the context of the present invention, the polypeptide of the invention has an enhanced transposition activity (i.e., more than 100%, preferably more than 110%, more than 120%, more than 150% or about 200% or more) compared to an otherwise identical polypeptide not comprising the substitution at position 124. The transposase not comprising the substitution has at position 124 Figure 6 The amino acid residue at the position specified in the alignment for the corresponding native or "wild-type" transposase. For example, for SB or Tdr1 transposase, "wild-type" position 124 is Q. For ZB, it is N, for FP, it is S, and for Passport, it is K. The enhanced transposition activity is not necessarily present at all transposase concentrations, but at least one concentration is equal to or above the concentration at which overexpression inhibition is detected. The enhanced activity is best described below Figure 3 Determined by the assay shown in B.

[0031] As used in the context of the present invention, a "position" in a transposase refers to an amino acid that is aligned in an amino acid sequence alignment with the amino acid sequence of a "wild-type" full-length reference transposase, preferably with the full-length amino acid sequence of SB according to SEQ ID NO: 1. The position of the reference transposase is determined by the first N-terminal amino acid. Thus, according to SEQ ID NO: 1, position 124 of SB in the full-length amino acid sequence is "Q". Thus, amino acid position 124 in another transposase is an amino acid that is aligned with the "Q" of position 124 of SB in SEQ ID NO: 1, e.g. Figure 6 shown.

[0032] 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 be C, A, D, E, G, H, I, L, M, F, P, W, Y, and V.

[0033] The inventors have found that the transposases of the present invention having C or G at position 124 are particularly active. It has also been found that the activity of the transposases having any one of D, N, E, L, M, F, S, T, Y or V at position 124 is significantly increased. The transposases having A, R, H or I at position 124 still have enhanced activity (see Figure 7 ). The transposase is preferably SB transposase.

[0034] If the preferred transposase in the context of the present invention is SB transposase, the amino acid at position 124 may be C, A, R, N, D, E, G, H, I, L, K, M, F, P, S, T, W, Y and V. The preferred amino acid in said context is C, S or Met, most preferably C. The same applies to Tdr1 transposase.

[0035] If the transposase is a ZB transposase, the amino acid at position 124 may be C, A, R, D, E, G, H, I, L, K, M, F, P, S, T, W, Y and V. The preferred amino acid in this context is C, S or Met, most preferably C.

[0036] If the transposase is a FP transposase, the amino acid at position 124 may be C, A, R, N, D, E, G, H, I, L, K, M, F, P, T, W, Y and V. The preferred amino acid in this context is C or Met, most preferably C.

[0037] If the transposase is a Passport transposase, the amino acid at position 124 may be C, A, R, N, D, E, G, H, I, L, M, F, P, S, T, W, Y and V. The preferred amino acid in this context is C or Met, most preferably C.

[0038] If the transposase is a Minoan transposase, the amino acid at position 124 may be C, A, R, N, D, E, G, H, I, L, K, M, F, P, S, W, Y and V. The preferred amino acid in this context is C or Met, most preferably C.

[0039] As shown herein, a significant increase in transposition activity is achieved if the amino acid at position 124 is C. Therefore, substitutions leading to C at position 124 are advantageous. Therefore, a polypeptide consisting of or containing an SB transposase with a Q124C substitution is a preferred embodiment of the present invention, wherein, preferably, the transposase comprises the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having 1, 2, 3 or 4 amino acid differences compared to SEQ ID NO: 19.

[0040] As described in PCT / EP2022 / 075007, Tc1 / mariner superfamily transposases comprising a substitution (e.g., a K248R substitution) at any of positions 187, 247, or 248 have an improved integration pattern compared to transposases without substitutions at said positions. They have better safety because they reduce transposon integration into exons and transcriptional regulatory regions of genes in the human genome. Transposons are therefore integrated into genetic safe harbors at a higher frequency. These substitutions were found to reduce transposition activity.

[0041] However, the inventors have shown that substitution at position 124 can rescue transposition activity and result in a more active transposase. Therefore, the 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 may also be a polypeptide as defined in PCT / EP2022 / 075007, in particular a polypeptide having a specific gain of integration into the genome. For example,

[0042] 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;

[0043] b) the substituted amino acid at position 247 may be R, C, A or S; preferably, R, and / or

[0044] c) The substituted amino acid at position 248 may be R, S, V, I or C, preferably R.

[0045] For substitutions at position 187 in SB transposase, H187A, H187N, H187C, H187Q, H187G, H187I, H187L, H187M, H187S, H187V, H187W, H187K, H187R, H187E, H187P, H187T and H187S substitutions are preferred, with H187V substitution being most preferred.

[0046] For substitution at position 247 in SB transposase, P247R, P247C, P247A and P247S substitutions are preferred, and P247R substitution is most preferred.

[0047] For the substitution at position 248 in the SB transposase, K248R, K248S, K248V, K248I or K248C substitutions are preferred, with K248R substitution being most preferred.

[0048] The transposase of the present invention that introduces the substitution at position 124 may be a hyperactive transposase. In a preferred embodiment, the polypeptide according to the present invention comprises SEQ ID NO:1 or a variant thereof having transposition activity and having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:1, wherein SEQ ID NO:1 or a variant thereof comprises a substitution that increases its transposition activity, wherein the substitution is preferably one or more substitutions as described below. The increase in transposition activity is an increase in the activity of SB compared to 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, in particular variants of SB, which have hyperactivity, i.e., have increased transposition activity compared to wild-type transposases, in particular SB. Preferably, the substitution of the present invention is introduced into a transposase variant comprising a substitution or a substitution group to enhance one or more properties of the transposase, in particular the transposition activity. Therefore, in one embodiment, the polypeptide of the present invention further comprises at least one of the following substitutions or substitution groups:

[0049] (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;

[0050] (2)K14R / R214D / K215A / E216V / N217Q;

[0051] (3)K33A / R115H / R214D / K215A / E216V / N217Q / M243H;

[0052] (4)K14R / K30R / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / M243H;

[0053] (5)K13D / K33A / T83N / H207V / K208R / D210E / M243Q;

[0054] (6)K13A / K33A / R214D / K215A / E216V / N217Q;

[0055] (7)K33A / T83N / R214D / K215NE216V / N217Q / / G317E;

[0056] (8)K14R / T83A / M243Q;

[0057] (9)K14R / T83A / I100L / M243Q;

[0058] (10)K14R / T83A / R143L / M243Q;

[0059] (11)K14R / T83A / R147E / M243Q;

[0060] (12)K14R / T83A / M243Q / E267D;

[0061] (13)K14R / T83A / M243Q / T314N;

[0062] (14)K14R / K30R / I110L / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q /

[0063] M243H;

[0064] (15)K14R / K30R / R143L / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q /

[0065] M243H;

[0066] (16)K14R / K30R / R147E / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q /

[0067] M243H;

[0068] (17)K14R / K30R / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q /

[0069] M243H / E267D;

[0070] (18)K14R / K30R / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q /

[0071] M243H / T314N;

[0072] (19) K14R / K30R / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / /

[0073] M243H / G317E;

[0074] (20) K14R / K33A / R115H / R214D / K215A / E216V / N217Q / M243H;

[0075] (21) K14R / K30R / R147E / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / /

[0076] M243H / T314N;

[0077] (22) K14R / K30R / R143U / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / /

[0078] M243H / E267D;

[0079] (23) K14R / K30R / R143L / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / /

[0080] M243H / T314N;

[0081] (24) K14R / K30R / R143L / A205K / H207V / K208R / D210E / R214D / K215A / E216V / N217Q / /

[0082] M243H / G317E;

[0083] (25) K14R / K33A / R115H / R143L / R214D / K215A / E216V / N217Q / M243H;

[0084] (26) K14R / K33A / R115H / R147E / / R214D / K215A / E216V / N217Q / M243H;

[0085] (27) K14R / K33A / R115H / R214D / K215A / E216V / N217Q / / M243H / E267D;

[0086] (28)K14R / K33A / R115H / R214D / K215A / E216V / N217Q / M243H / T314N;

[0087] (29)K14R / K33A / R115H / R214D / K215A / E216V / N217Q / / M243H / G317E;

[0088] (30) K14R / T83A / M243Q / G317E; or

[0089] (31)K13A / K33A / T83N / R214D / K215A / E216V / N217Q.

[0090] For example, the transposase of the present invention may be the SB100X transposase of SEQ ID NO:18 or preferably SEQ ID NO:19.

[0091] The transposase of the present invention may also be a transposase having improved solubility in water compared to SB of SEQ ID NO: 1, such as the high solubility transposase described in Querques et al.

[19] or EP3673053A. In order to improve solubility, the transposase may, for example, further comprise at least one substitution in amino acid position 176 and / or 212, wherein the amino acid at position 176 is not C and the amino acid at position 212 is not I, preferably, wherein the amino acid at position 176 and / or 212 is S, wherein, optionally, both amino acids at positions 176 and 212 are S.

[0092] Another object of the invention is a nucleic acid encoding a polypeptide according to the invention.

[0093] Nucleic acid of the present invention can be any nucleic acid, such as ribonucleic acid, including mRNA, DNA, cDNA, chromosomal DNA, extrachromosomal DNA, plasmid DNA, viral DNA, and also recombinant viral vectors. All nucleic acid variants encoding the above-mentioned polypeptides of the present invention are provided, including nucleic acid variants with different nucleotide sequences due to the degradation of the genetic code. In particular, for example, in human cells, it is preferred to cause the nucleotide sequence of the nucleic acid variant of the encoded fusion protein to express improved in the selected host organism. Tables for appropriately regulating nucleic acid sequences according to the specific transcription / translation mechanism of the host cell are known to those skilled in the art. In general, it is preferred to adapt the G / C content of the nucleotide sequence to specific host cell conditions. For expression in human cells, it is preferred that the G / C content increases at least 10% of the maximum G / C content (encoding the corresponding peptide variant of the present invention), more preferably at least 20%, 30%, 50%, 70%, and even more preferably 90%. The preparation and purification of such nucleic acids and / or derivatives are usually carried out by standard procedures.

[0094] Preferably, the nucleic acid is suitable for expressing the polypeptide of the present invention in mammals, for example human cells, such as stem cells or lymphocytes, for example T lymphocytes. If the nucleic acid is DNA, it preferably comprises at least the regulatory region of the gene. The regulatory region can be a transcriptional regulatory region, for example selected from promoters, enhancers, silencers, locus control regions and boundary elements. Promoters or other expression control regions can be operably connected to the nucleic acid encoding the polypeptide of the present invention, for example, to regulate the expression of the polypeptide / protein in a quantitative or tissue-specific manner. The promoter can be a constitutive or inducible promoter.

[0095] The nucleic acid of the present invention encoding the polypeptide of the present invention can be a linear fragment or a circular isolated fragment, or inserted into a vector, preferably as a plasmid or recombinant viral DNA. The term "vector" or "expression vector" is used interchangeably and refers to a polynucleotide or a mixture of polynucleotides and proteins that can introduce the nucleic acid of the present invention into a cell, preferably a mammalian cell. Examples of vectors include, but are not limited to, minicircles, plasmids, cosmids, phages, viruses or artificial chromosomes. In particular, vectors can be used to transport the nucleic acid of the present invention into a suitable host cell. Once entering the host cell, the expression vector can replicate independently of the host chromosomal DNA or simultaneously with the host chromosomal DNA, and multiple copies of the vector and its inserted DNA can be produced. If an expression vector that cannot replicate is used, which often happens for safety reasons, the vector may not replicate, but simply express the nucleic acid directly. Depending on the type of expression vector, the expression vector may be lost from the cell, i.e., the new antigen encoded by the nucleic acid is only transiently expressed. It may also remain stable in the cell. The expression vector typically contains an expression cassette, i.e., an essential element that allows the nucleic acid to be transcribed into an mRNA molecule.

[0096] Preferably, the nucleic acid is an mRNA, a minicircle or a plasmid. It has been shown that it is beneficial to introduce the transposase into mammalian cells, in particular stem cells or primary cells, such as primary T cells, using as little DNA as possible, i.e. most preferably in the form of mRNA, optionally as a minicircle. If the nucleic acid is not an mRNA, the nucleic acid sequence is operably linked to at least one transcription control unit, preferably a promoter active in human cells.

[0097] 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 include both. The cell may be a bacterial cell, for example in the context of plasmid propagation, but preferably, it is a eukaryotic cell, in particular a mammalian cell. It may be, for example, a human cell, a mouse cell, a rabbit cell or a rat cell. Human cells are preferred throughout the invention. The cell may be a stem cell, for example a pluripotent stem cell, a hematopoietic stem cell or a lymphocyte, for example a T lymphocyte. For example, the cell is preferably a human cell selected from a pluripotent stem cell or a human lymphocyte, preferably a human T lymphocyte. The cell may also be a tumor cell.

[0098] In addition, the present invention provides a kit comprising

[0099] a) a polypeptide of the invention, a nucleic acid of the invention and / or a cell of the invention; and

[0100] b) a nucleic acid comprising a cargo nucleic acid flanked by inverted terminal repeats (TIR) ​​of a transposon capable of being mobilized by said transposase.

[0101] This kit can be used to deliver cargo nucleic acids (e.g., therapeutic amino acids) to the genome of cells (e.g., human cells). Cargo nucleic acids are typically exogenous nucleic acids, i.e., nucleic acids exogenous to the cells to be delivered. Therapeutic nucleic acids can, for example, encode proteins reduced or defective in metabolic diseases, such as in the context of gene therapy for the disease, encoding tumor suppressor genes, immunomodulators, such as cytokines, antigens, antibodies, T cell receptors (TCRs) or chimeric antigen receptors (CARs). In a preferred embodiment, therapeutic nucleic acids can be used to treat cancer. The kit can be used for in vivo, ex vivo or in vitro applications, such as for medicine, such as for cancer treatment. Therefore, it may be a drug kit. Cargo nucleic acids optionally encode fluorescent proteins and / or another selective marker.

[0102] The present invention also provides a pharmaceutical composition comprising a polypeptide of the present invention, a nucleic acid of the present invention; a cell of the present invention and / or a component of a kit of the present invention. The pharmaceutical composition generally further comprises a pharmaceutically acceptable carrier and / or excipient. The term "carrier" refers to an organic or inorganic component of natural or synthetic nature, in which the active ingredients are combined to promote, enhance or achieve application. According to the present invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents, excipients or encapsulating materials, which are suitable for administration to a subject. Possible carrier substances are (such as diluents) such as sterile water, Ringer's solution, lactated Ringer's solution, physiological saline, bacteriostatic saline (such as 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 formulation are described in detail in Remington's Pharmaceutical Sciences, 17th ed., 1985, Mack Publishing Co.

[0103] The pharmaceutical composition is preferably used for adoptive T cell therapy or gene therapy. Gene therapy may be the treatment of metabolic or immune diseases. In one embodiment, gene therapy includes but is not limited to autologous or allogeneic T cell therapy, gene therapy targeting any cell type in the 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 the treatment of cancer.

[0104] The pharmaceutical composition of the invention may also be used for vaccination therapy, for incorporating antigens into antigen presenting cells, such as human professional antigen presenting cells, such as dendritic cells, macrophages or B cells or their precursors, such as specific tumor antigens, such as MAGE-1, for tumor vaccination, or for treating pathological antigens of infectious diseases caused by pathogens, such as leprosy, tetanus, pertussis, typhoid, paratyphoid, cholera, plague, tuberculosis, meningitis, bacterial pneumonia, anthrax, botulism, bacterial dysentery, diarrhea, food poisoning, syphilis, gastroenteritis, trench fever, influenza, scarlet fever, diphtheria, gonorrhea, toxic shock syndrome, Lyme disease, typhus, listeriosis, peptic ulcer and Legionnaires' disease; for treating diseases causing, for example, acquired immunodeficiency syndrome, adenoviridae infections, alphavirus infections, arbovirus infections, Borna's disease , Bunyaviridae infection, calicivirus infection, varicella, condyloma acuminatum, coronavirus infection, coxsackievirus infection, cytomegalovirus infection, dengue fever, DNA virus infection, ecthyma, infectious diseases, encephalitis, arbovirus, Epstein-Barr virus infection, infectious erythema, Hantavirus infection, hemorrhagic fever, viral hepatitis, viral human herpes simplex, herpes zoster, herpes zoster oticus, herpes virus infection, infectious mononucleosis, avian influenza, human Lassa fever, measles, molluscum contagiosum, mumps, paramyxoviridae infection, phlebitis, polyomavirus infection, rabies, respiratory syncytial virus infection, Rift Valley fever, RNA virus infection, rubella, slow virus disease, smallpox, subacute sclerosing panencephalitis, tumor virus infection, warts, West Nile fever, viral diseases, yellow fever; for the treatment of protozoan infections that cause malaria, etc. The method can be used to deliver various therapeutic nucleic acids.

[0105] According to the present invention, the pharmaceutical composition comprises an effective amount of an active agent, such as a polypeptide, nucleic acid, vector or cell as described herein, to produce a desired reaction or desired effect. The pharmaceutical composition according to the present invention is preferably sterile. The pharmaceutical composition can be provided in a uniform dosage form and can be prepared in a manner known per se. The pharmaceutical composition according to the present invention can be in the form of, for example, a solution or a suspension.

[0106] Pharmaceutically acceptable carriers, adjuvants or solvents that can be used for 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, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene glycol-polyoxypropylene-block polymers, polyethylene glycol and lanolin. The pharmaceutical compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, orally, vaginally or by implanted reservoirs. The term parenteral injection as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the pharmaceutical composition is administered orally, intraperitoneally or intravenously. The sterile injection form of the pharmaceutical composition of the present invention can be an aqueous or oily suspension. These suspensions can be prepared using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. The sterile injection preparation can also be a sterile injection solution or suspension in a non-toxic parenteral acceptable diluent or solvent, such as a solution in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are generally used as solvents or suspension media.

[0107] The pharmaceutical composition of the present invention is preferably suitable for treating diseases, in particular diseases caused by genetic defects, such as cystic fibrosis, hypercholesterolemia, hemophilia, such as A, B, C or XIII, immunodeficiency including HIV, Huntington's disease, α-antitrypsin deficiency, and a variety of tumors selected from 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 tumors, liver cancer, various virus-induced tumors. Tumors, for example, papillomavirus-induced cancers (e.g., cervical cancer), adenocarcinomas, herpes virus-induced tumors (e.g., Burkitt's lymphoma, EBV-induced B-cell lymphoma), hepatitis B-induced tumors (hepatocellular carcinoma), HTLV-1 and HTLV-2-induced lymphomas, acoustic neuroma, lung cancer, pharyngeal cancer, anal cancer, glioblastoma, lymphoma, rectal cancer, astrocytoma, brain tumors, gastric cancer, retinoblastoma, basal cell tumor, brain metastasis, medulloblastoma, vaginal cancer, pancreatic cancer, testicular cancer, melanoma, bladder cancer, Hodgkin's syndrome, meningioma, Schneeberger's disease disease), bronchial cancer, pituitary cancer, mycosis fungoides, esophageal cancer, breast cancer, neurilemmoma, spinal tumor, Burkitt's lymphoma, laryngeal cancer, thymoma, somatic cell cancer, bone cancer, non-Hodgkin's lymphoma, urethral cancer, CUP-syndrome, oligodendroglioma, vulvar cancer, intestinal cancer, esophageal cancer, small intestine tumor, craniopharyngeal cancer, ovarial carcinoma, ovarian cancer, liver cancer, leukemia or skin cancer or eye cancer; etc.

[0108] The pharmaceutical composition is preferably used to treat a human subject. The present invention also provides a method of treating a subject, such as a human subject in need thereof, such as treating any disease described herein, comprising administering to the subject an effective amount of the pharmaceutical composition of the present invention or a component of the pharmaceutical kit of the present invention.

[0109] Also described are uses of the polypeptides of the invention, the nucleic acids of the invention, the cells of the invention or the kits of the invention for introducing exogenous nucleic acids into the genome of a cell, such as in vitro uses.

[0110] Furthermore, the present invention provides a method for preparing a cell having an exogenous nucleic acid integrated into the cell genome, optionally an in vitro method, comprising the following steps:

[0111] a) providing isolated cells;

[0112] b) providing a cell with a polypeptide according to any one of the present invention; and

[0113] c) providing a nucleic acid to a cell, said nucleic acid comprising an exogenous nucleic acid flanked by inverted terminal repeats (TIRs) of a transposon capable of being activated by said transposase.

[0114] Optionally, the polypeptide is provided to the cell by introducing the nucleic acid of the invention into the cell. Alternatively, the polypeptide can be provided in the form of a polypeptide, for example, as provided in the form of a high solubility transposase as described herein. In one embodiment, the exogenous nucleic acid is contained in a nucleic acid or vector of the invention. It can also be administered alone.

[0115] In one embodiment, the nucleic acid of the invention and / or exogenous nucleic acid is provided in a cell using a method selected from electroporation, microinjection, lipoprotein particles, virus-like particles. Electroporation has been found to be particularly suitable for transfecting primary cells, such as T cells.

[0116] Another object of the present invention is a method for preparing a polypeptide according to the present invention, comprising culturing a cell according to the present invention comprising a nucleic acid according to the present invention and isolating the polypeptide.

[0117] The present invention is further illustrated in the accompanying drawings and examples. These are not intended to limit the present invention. The cited documents are fully incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] Figure 1 : Schematic overview of gene delivery by Sleeping Beauty transposition. Schematic diagram of gene delivery by Sleeping Beauty transposition. SB transposase is introduced into the cell in the form of DNA (e.g., expression plasmid), mRNA, or recombinant protein together with donor DNA where the transposon to be moved is located. The donor DNA can be provided as a plasmid or minicircle vector. After binding to the terminal inverted repeats of the transposon (TIR, rectangle) flanking the gene of interest (GOI, rectangle), the SB transposase (circle) excises the transposon from the donor DNA (black strand) and integrates it into a site in the genomic target DNA (strand).

[0119] Figure 2 : Validation of the Q124C mutation in the Sleeping Beauty transposase. (A) The Q124C mutant of SB100X displays a hyperactive phenotype as determined by colony-forming transposition assays in human HepG2 cells. (B) Venus expression in SB-transfected hiPSCs measured by flow cytometry reaches a plateau at day 14. (C) The Q124C mutant results in an increased rate of stable Venus-modified hiPSCs as measured by flow cytometry.

[0120] Figure 3: Mechanistic insights into the hyperactivity of the Sleeping Beauty transposase Q124C mutant. (A) Model of SB transposase and the Q124 amino acid side chain (highlighted) bound to the SB transposon and target DNA. (B) The Q124C mutant was shown to be hyperactive at high doses by a dose-dependent colony formation transposition assay in human HepG2 cells. (C) In a reactivation colony formation assay with puromycin (puro) selection (excision) and G418 and puromycin dual selection (transposition), the Q124C mutant had an increased excision rate, resulting in an overall higher transposition rate than SB100X.

[0121] 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 detected by Venus expression in SB nuclear-infected HSCs. The expression of Venus reached a stable expression level at day 9. (B) The survival of nuclear-infected HSCs with minicircle Venus and SB transposase was detected over time by zombie staining and flow cytometric analysis.

[0122] Figure 5 : Combination of the Q124C mutant with other Sleeping Beauty transposase variants rescues transposition activity. (A) Introduction of the Q124C mutant into the K248R mutant can rescue its transposition activity to some extent, as determined by colony-forming transposition assays in human HepG2 cells. (B) Insertion frequency of genomic safe harbors. (C) Insertion frequency of all genomic safe harbor subcategories (D) Schematic representation of exonic insertions. (E) Schematic representation of insertions 10 kb upstream of the transcription start site (TSS).

[0123] Figure 6 : Amino acid sequence alignment of Tc1 / mariner transposases closely related to Sleeping Beauty transposase (SEQ ID NOs: 1-17). Figure 6This enables one skilled in the art to determine the amino acid position in all other aligned transposases corresponding to the amino acid at position 124 or other specified positions, which can be substituted with different amino acids in the same manner as the SB example. Other transposases can be added to the alignment to identify the amino acid in these transposases corresponding to the amino acid at position 124 of SB. In addition to the sequence alignment, the typical secondary structure of the Tc1 / mariner superfamily transposase is indicated: α1 helix-α2 helix-β1 fold-β2 fold-β3 fold-β4 fold-β5 fold-α3 helix-β6 fold-η1-α4 helix-η2-α5 helix-α6 helix-α7 helix-α8 helix belonging to the corresponding amino acid fragment. The amino acid sequence of SB (SEQ ID NO: 1) is 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 with boxes.

[0124] 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 plated on 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 SB transposon marked with a puromycin resistance gene using Transit-LT1 transfection reagent. Several mutants exhibited a hyperactive phenotype compared to SB100X (n=3; significance assessed by t-test: *p<0.05 **p<0.01). DETAILED DESCRIPTION

[0125] Example

[0126] result

[0127] The Q124C mutation causes hyperactivity in human hepatocyte-derived HepG2 cells and human induced pluripotent stem cells (hiPSCs)

[0128] To examine the transposition activity of the Q124C mutation in the SB100X coding sequence, we transfected expression plasmids (pcGlobin2) encoding SB100X or the Q124C mutant into HepG2 cells together with a puromycin resistance (puro) gene-tagged SB vector (pT2 / puromycin) and assessed the relative efficiency of transposition by counting puromycin-resistant cell colonies after antibiotic selection. The Q124C mutant resulted in a two-fold increase in transposition efficiency compared to the most active SB transposase variant, SB100X ( Figure 2A). This hyperactivity was also demonstrated in more clinically relevant human induced pluripotent stem cells (hiPSCs). hiPSCs were transfected with expression plasmids encoding two active SB transposase variants, SB100X and Q124C, and catalytically inactive variant DAE, along with SB transposon minicircles labeled with the Venus fluorescent gene. The fluorescence of the hiPSCs was tracked over time, and stable expression was achieved at day 14 ( Figure 2 B). At day 14, the Q124C variant increased the number of Venus-modified hiPSCs by two-fold compared to the SB100X variant ( Figure 2 C).

[0129] The Q124C mutation leads to resistance to overproduction inhibition and is already manifested during the excision step

[0130] To better understand the mechanistic reasons for the hyperactivity of the Q124C mutant in the SB100X coding sequence, transposition experiments were performed at different doses of the SB transposase expression plasmid. A known phenomenon in Tc1 / mariner TE is that high levels of transposase can lead to overproduction inhibition, resulting in a nonlinear saturation increase in transposition rate

[11] . Since the mutation site of Q124 is close to the protein-protein dimer interaction interface that is prone to allosteric overproduction inhibition, we speculated that the hyperactivity of the Q124C mutant should be manifested particularly at high transposase concentrations. In the SB transposase model, the position of Q124 is highlighted together with the SB transposon end and the DNA target site ( Figure 3 A) 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, superimposed with the full-length structure of the related mariner transposase Mos1

[15] .

[0131] In a typical transposition assay resulting in puromycin-resistant colonies, SB100X was compared to Q124C and showed hyperactive transposition at higher doses (500 and 2500 ng expression plasmid) ( Figure 3 B). These observations lead us to conclude that the Q124C variant confers a degree of resistance to overproduction inhibition. Therefore, 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.

[0132] We have established a transgenic, human, HepG2-derived reporter cell line that contains a single copy of the neomycin resistance gene (neo)-tagged SB transposon that disrupts the open reading frame (ORF) of the puromycin resistance gene. These cells are therefore G418-resistant and puromycin-sensitive. If the SB transposase is expressed in these reporter cells, canonical excision of the SB transposon and subsequent repair of the broken DNA ends by the non-homologous end joining (NHEJ) double-strand DNA repair pathway reconstitute the puromycin ORF, resulting in a selectable, puromycin-resistant phenotype. Selection with puromycin allows the excision rates of different transposase variants to be measured, while dual selection with G418 and puromycin allows the efficiency of the complete transposition reaction (excision + integration) to be scored. Transfection of this reporter cell line with two variants, SB100X and Q124C, showed that hyperactivity was already present in the excision step, resulting in an increased overall transposition rate ( Figure 3 C) This is consistent with the described effect of resisting overproduction inhibition, as this effect primarily affects the initiation of the excision complex.

[0133] Q124C mutation causes hematopoietic stem cell (HSC) overactivity

[0134] To examine 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 variants, SB100X and Q124C, and the catalytically inactive variant DAE, as well as SB minicircles labeled with the Venus fluorescent gene. The fluorescence of HSCs was tracked over time. Figure 4 A) and survival rate ( Figure 4 B), and reached stable expression on day 9. Although no difference was observed in Venus-positive HSCs at a dose of 1 μg of expression plasmid, at a high dose of 2 μg, the Q124C mutation of the expression plasmid resulted in a two-fold increase in stably expressing Venus-positive HSCs ( Figure 4 A). Under high-dose Q124C, the survival rate of HSCs was only slightly affected and recovered rapidly on day 9 ( Figure 4 B) This experimental data shows that the hyperactivity of the Q124C mutant is also manifested in primary cells.

[0135] Introduction of the Q124C mutation into the K248R mutation rescues transposition activity

[0136] Other SB transposase variants, such as the K248R mutation in the SB100X coding sequence, have certain advantages, such as safer integration characteristics compared to SB100X

[10] . The K248R variant has reduced integration rates into genes and promoter regions and increased integration rates into genomic locations considered safe havens. However, the transposition activity of this variant is reduced compared to SB100X. The transposition activity of the K248R variant could be rescued to some extent by introducing the Q124C mutation into the K248R variant. The transposition activity of the different variants has been determined in a standard colony formation assay, where puromycin-resistant colonies were counted ( Figure 5 A).

[0137] Integration of SB transposition-mediated therapeutic gene constructs into safe sites in the human genome can prevent insertional genomic toxicity and the associated risk of tumorigenesis. Genomic "safe harbors" are regions in 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 a 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) microRNA genes; and (v) regions outside ultra-conserved elements (UCEs) [16, 17]. By introducing the Q124C mutation into the coding sequence of the SB transposase, the insertion frequency of GSH was increased ( Figure 5 B). The insertion frequency of the double mutant Q124C / K248R was increased in all GSH subclasses ( Figure 5 C). By analyzing the inserted exon ( Figure 5 D) and 10 kb upstream of the transcription start site ( Figure 5 E) Representative, Q124C single mutation has lower insertion frequency compared to SB100X, while the double mutation (Q124C / K248R) maintains the lower insertion frequency compared to the single mutation (K248R).

[0138] The introduction of the Q124C mutation into the K248R variant led to an increased value of this variant in clinical applications, where efficiency and safety, especially compared with viral vectors, play an important role. It is clear that the Q124 residue is more located on the protein surface involved in DNA interaction ( Figure 3 A). Therefore, it is plausible that other SB transposase variants could also combine with the Q124C mutant to increase its transposition activity.

[0139] Materials and methods

[0140] Site-directed mutagenesis of SB100X transposase

[0141] All mutations were generated using Q5 polymerase (NEB, Ipswich, MA, USA) and plasmid pcGlobin2-SB100X. 5' phosphorylated primers were designed for specific positions and annealed back-to-back at the 5' ends. Primers were synthesized with 5'-phosphate to enable downstream intramolecular ligation reactions and ordered from Eurofins (Eurofins / MWG, Luxembourg). Primer sequences (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 TM Software calculation ( https: / / www.neb.com / tools-and-resources / interactive-tools / tm-calculator ). PCR products were purified using the QIAquick PCR purification kit (QIAGEN, Venlo, The Netherlands), eluted in 30 μl elution buffer, digested with 2 μl DpnI (NEB, Ipswich, MA, USA) at 37°C for 2 h, and then heat inactivated at 80°C for 20 min. Linear double-stranded PCR products were ligated overnight at 16°C with T4 DNA ligase (NEB, Ipswich, MA, USA) for circularization. The circularized PCR products were transformed into chemically competent Escherichia coli (Invitrogen / Life Technologies, Carlsbad, CA, USA), grown for 1 h in Luria-Bertani (LB) medium, and screened for ampicillin resistance by plating on LB agar plates containing 100 μg / ml ampicillin. To confirm the presence of the desired mutations and the presence of undesired mutations, plasmid DNA from several colonies was purified using the QIAprep spin miniprep kit (QIAGEN, Venlo, The Netherlands) and subjected to Sanger sequencing by Eurofins (Eurofins / MWG, Luxembourg).

[0142] Transposition assay

[0143] When performing transposition experiments in HepG2 cells, 3 × 10 5Cells were seeded onto six-well plates. Transfection was performed using TransIT-LT1 transfection reagent (Mirus Bio LLC, Madison, Wisconsin, USA) according to the manufacturer's protocol. Each transfection reaction was filled to the same total DNA amount transfected with plasmid pmaxGFP. Transposon expression plasmid (pcGlobin2) was co-transfected with transposon donor plasmid (pT2 / puromycin). 48 hours after transfection, cells were trypsinized, 1-10% of cells were re-seeded onto 10 cm plates, and 1 μg / ml puromycin was selected for transposon integration (InvivoGen, San Diago, California, USA). After 2 weeks of selection, cell colonies were fixed with phosphate buffered saline (PBS) containing 10% (volume / volume) formaldehyde, stained with PBS containing methylene blue, and counted. At least three independent experiments were performed to compare relative transposition efficiency in vitro.

[0144] Venus modification of hiPSCs with Sleeping Beauty

[0145] hiPSCs were cultured and processed according to the protocol of Skarnes et al.

[18] . Transfection was performed using TransIT-LT1 transfection reagent (Mirus Bio LLC, Madison, WI, USA) according to the protocol of the hiPSC manufacturer. 2 μg of transposon expression plasmid (pcGlobin2) was co-transfected with transposon donor minicircle (mcVenus) into 2 × 10 6 hiPSCs were analyzed by BD FACS-SORB (BD FACS) every three to four days. TM Venus expression was assessed once by cytofluorimetry (ELISA, East Rutherford, NJ, USA) and analyzed by FlowJo (FlowJo LLC, Ashland, OR, USA). For in vitro comparisons of relative transposition efficiencies, at least three independent experiments were performed.

[0146] Reactivation test

[0147] For reactivation assays in a modified HepG2 reporter cell line (HepG2 PB (SB2#23)), 4 × 10 5Cells were seeded into six-well plates. Transfection was performed using Lipofectamine 3000 transfection reagent (Invitrogen AG, Waltham, MA, USA) according to the manufacturer's protocol. 1 μg of transposon expression plasmid (pcGlobin2) was transfected into HepG2-PB (SB2#23) cells. 72 hours after transfection, cells were trypsinized and 100% of the cells were replated on two 10 cm plates and either 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 phosphate-buffered saline (PBS) containing 10% (vol / vol) formaldehyde, stained with PBS containing methylene blue, and counted. At least three independent experiments were performed for in vitro comparison of relative transposition efficiency.

[0148] Generation of SB insert library

[0149] To analyze the target site selection properties of the SB100X transposase mutant, 4 × 10 5 HepG2 cells were seeded onto 6-well plates. Transfection was performed with QIAGEN purified plasmid DNA using TransIT-LT1 transfection reagent according to the manufacturer’s protocol. 550 ng of DNA including 500 ng of pT2Bpuro, 50 ng of a helper plasmid expressing a mutant transposase or pcGlobin2-SB100X was transfected using 1 μl of TransIT-LT1 transfection reagent. 48 h after transfection, cells were trypsinized, diluted into multiple 10 cm dishes containing DMEM supplemented with 1 μg / ml puromycin, and selected for growth for 2 weeks. At least 10,000 puromycin-resistant HepG2 cell colonies were trypsinized and centrifuged at 1000 rpm for 5 min. The pellet was washed with PBS, and genomic DNA was extracted from the cells using the Qiagen DNeasy Blood and Tissue Kit according to the manufacturer’s protocol.

[0150] To generate the SB insertion site library, 2 μg of DNA was sheared to an average fragment size of 600 bp using a Covaris M220 sonicator in a 50 μl screw-cap microtube using the following settings: peak incident power 50 W, duty cycle 20%, 200 cycles per pulse, 28 seconds. 1.2 μg of sheared DNA was blunted and 5'-phosphorylated using the NEBNext End Repair Module (NEB) and the 3'-A-tail was blunted using the NEBNext dA Tail Addition Module (NEB) as recommended by the manufacturer. DNA was purified using a clean and concentrate kit (Zymo Research) and eluted in 8 μl 10 mM Tris pH 8.0 (EB) for ligation with 50 pmol T-adapters (see below) and T4 ligase (NEB) in a 20 μl volume overnight at 16°C. T-adapters were created by annealing 100 pmol of each oligonucleotide Linker_TruSeq_T+ and Linker_TruSeq_T- in 10 mM Tris-Cl pH 8, 50 mM NaCl, 0.5 mM EDTA. After heat inactivation, the ligation products encapsulating the non-integrated transposon donor plasmid DNA fragment were digested with 50 μl DpnI (NEB) for 3 hours, and then the DNA was column purified and eluted in 20 μl EB. 6 μl of the eluate was used for PCR I with 25 pmol of primers specific for the adaptor and transposon inverted repeats: adaptor and T-Bal-Long, respectively, under the following conditions: 98°C for 30 s; 98°C for 10 s, 72°C for 30 s, 10 cycles; 98°C for 10 s, increased to 62°C (1°C / s) for 30 s, 72°C for 30 s, 15 cycles; 75°C for 5 min. All PCR reactions were performed using NEBNext Ultra Fidelity 2× PCR Master Mix. PCR was purified and eluted with 20 μl EB columns, and 10 μl primers Nested and LAM-SB-50 were used for PCR II with the following program: 98°C for 30 s; 12 cycles of 98°C for 10 s, ramped to 65°C (1°C / sec) for 30 s, 72°C for 30 s, and 75°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 indices) for barcoding samples using the following PCR program: 98°C for 30 s; 98°C for 10 s, ramped to 64°C (1°C / sec) for 30 s, 72°C for 30 s, 12 cycles; 75°C for 5 min. The final PCR product was separated on 1% agarose gel, and a smear of 200-500 bp was isolated and purified.

[0151] Sequencing and analysis of insertion sites

[0152] Insertion site libraries were prepared as described previously

[19] and sequenced on an Illumina instrument using a 150 bp single-end setting. Reads of transposon sequences downstream of the SB-specific primer were tested after adapter and quality trimming (Phred score ≥ 20) using fastp

[20] and filtering for the remainder of the transposon inverted terminal repeats (ITRs) and a minimum length of 28 bases of genomic sequence for alignment using bowtie2

[21] in sensitive and end-to-end settings. Aligned loci were considered valid if the alignment quality of the reads supporting it was ≥ 20. Any insertion site needed to be supported by at least 10 independent reads from the human genome TA target site (hg38). If multiple insertions were detected within 10 bases, the insertion site with the highest number of independent reads was considered valid. The images of insertion sites in different gene classes were investigated using the Genomation software package

[22] . A random set of 100,000 loci from the computationally generated human hg38 genome assembly was used as a reference to investigate the representation of insertion sites in various genomic intervals. The genomic safe harbor coordinates for the hg38 assembly were created according to previously defined criteria

[16] .

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[0176] 23.PCT / EP2022 / 075007

[0177] 24.WO 2009 / 003671

[0178] 25.EP3673053A

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Claims

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

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

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

5. The polypeptide according to any one of the preceding claims, wherein the amino acid at position 124 is C.

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

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

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

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

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

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

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

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

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

17. A polypeptide according to any one of the preceding claims, further comprising a substitution at any of positions 187, 247 and / or 248, in, Optionally, 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.

18. The polypeptide according to any one of the preceding claims, further comprising at least one of the following substitutions or substitution groups: (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.

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

20. The nucleic acid according to claim 19, in, Optionally, if the nucleic acid is not mRNA, the nucleic acid sequence is operably linked to at least one transcription control unit, preferably a promoter active in human cells.

21. A cell comprising the nucleic acid of any one of claims 19-20 and / or the polypeptide of any one of claims 1-18.

22. The cell according to claim 21, wherein the cell is a human cell selected from a pluripotent stem cell or a human lymphocyte, preferably a human T lymphocyte.

23. A kit comprising a) a polypeptide according to any one of claims 1 to 18, a nucleic acid according to any one of claims 19 to 20 and / or a cell according to any one of claims 21 to 22; and b) a nucleic acid comprising a cargo nucleic acid flanked by inverted terminal repeats (TIR) of a transposon capable of being activated by said transposase.

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

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

26. A method for preparing a cell having an exogenous nucleic acid integrated into the cell genome, optionally an in vitro method, comprising the steps of: a) providing isolated cells; b) providing the cell with the polypeptide of any one of claims 1 to 18; and c) providing to the cell a nucleic acid comprising an exogenous nucleic acid flanked by inverted terminal repeats (TIRs) of a transposon capable of being mobilized by the transposase; Wherein, optionally, the polypeptide is provided to the cell by introducing the nucleic acid of any one of claims 19-20 into the cell.

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