Novel transposase systems

Abstract

The present invention relates to a novel, functional transposase system. More specifically, the present invention relates to a DNA transposon comprising a heterologous polynucleotide flanked by transposon flanking regions and a recombinant transposase from Acyrthosiphon pisum (AP) or Aphis craccivora (AC) comprising an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 4, fused to a heterologous nuclear localization signal, and optionally containing at least one activity-improving mutation, as well as a polynucleotide or expression vector encoding the DNA transposon or transposase, and an expression system comprising the transposase / transposon pair. The present invention also relates to methods for stably integrating a polynucleotide into a cell or for stably expressing a protein of interest using a transposase / transposon pair.

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, the entire contents of which are incorporated by reference. A copy of said XML, created on September 18, 2023, is named "118877P1140PC_sequence listing" and is 103,000 bytes in size.

[0002] FIELD OF THE INVENTION The present invention relates to a novel, functional transposase system. More specifically, the present invention relates to a DNA transposon comprising a heterologous polynucleotide flanked by transposon flanking regions and a recombinant transposase from Acyrthosiphon pisum (AP) or Aphis craccivora (AC) comprising an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 4, fused to a heterologous nuclear localization signal, and optionally containing at least one activity-improving mutation, as well as a polynucleotide or expression vector encoding the DNA transposon or transposase, and an expression system comprising the transposase / transposon pair. The present invention also relates to methods for stably integrating a polynucleotide into a cell or for stably expressing a protein of interest using a transposase / transposon pair.

[0003] background Stable integration of transgenes into host cell genomes is of particular interest in the fields of gene therapy, cell and genome engineering, cell line development, and recombinant protein production. Transgene integration can be achieved, for example, by transfecting linearized DNA material into cells. In this case, integration events are based on random double-strand breaks in the genome, often resulting in random integration of DNA fragments or concatemers at various genomic loci. Transposase enzymes, on the other hand, are proteins that recognize specific DNA sequences (inverted terminal repeat (ITR) sequences within transposon flanking regions), cleave the transposon cargo together with the transposon flanking sequences, including the ITRs, and reversibly integrate it into the genomic locus at a predefined site containing the target sequence, preferably the short motif "TTAA." Transposases typically target transcriptionally active and / or accessible genomic loci, resulting in the defined integration of the transposon cargo / transgene without fragmentation, concatemer formation, rearrangement, or recombination. Therefore, transposase-mediated integration results in more stable recombinant cell lines with higher expression levels compared to random integration. By controlling the heterologous gene between the transposon flanking sequences, transposases can be modulated to integrate essentially any DNA sequence into various genomic loci in cells.

[0004] One of the first functionally active Piggy-Bac transposase / transposon pairs discovered was from the nettle looper, Trichoplusia ni, and was demonstrated to be active in a variety of cells and organisms. Another previously reported transposase is a Tc1 / mariner-type system, e.g., the highly active Sleeping Beauty SB100X from fish (Mates L et al., Nat Genet., 41(6):753-61 (2009)). Both are related to the DNA transposon class or class II transposons and use a "cut and paste" mechanism. In contrast, class I transposons, also known as retrotransposons, use a "copy and paste" mechanism and include, for example, non-LTR TRAS retrotransposons (Monti V. et al., Journal of Heredity, 104(4): 547-553 (2013)) and LTR retroelements (Bernet G. et al., Mobile Genetics Elements, 1(2): 97-102 (2011)). Most published genome sequences contain transposase / transposon-like sequences, but only a few have been demonstrated to be active. Mobile genetic elements, such as active transposase / transposon pairs, can cause genomic instability and, therefore, can be harmful to cells or organisms. As a result, most transposase / transposon elements have become inactive over the years due to, for example, mutations, deletions, insertions, or other genomic rearrangements. Since their previous publication, only a few active transposase / transposon pairs have been reported.Active transposases / transposons have been discovered in Trichoplusia ni, Xenopus tropicalis, Bombyx mori, and more recently in Oryzias latipes, Amyelois transitella, Heliothis virencens, Agrotis ipsilon, and Helicoverpa armigera (US 2015 / 291975, US 2017 / 101629, US 2020 / 318121, US 2020 / 318135, US 2020 / 318107; Hikosaka, Mol Biol Evol 24, 2648-2656 (2007)). These findings demonstrate that most experimentally tested transposase / transposon-like pairs from nature are no longer functional. For example, US 2020 / 031807 reports that PiggyBac-like transposases and transposons naturally occur in a wide range of organisms, but transposition activity has not been reported for most of them. Like other transposable elements, PiggyBac transposase / transposon-like sequences are prone to indels, recombination, and mutations that inactivate many copies, and are also prone to exhibiting large diversity, as described by Bouallegue and Rouault et al. (Genome Biol. Evol., 2017, 9(2): 323-339). Still, the authors focused only on the genome level and molecular evolution without providing activity data. Tc1 / mariner transposases represent an even more unrelated class of transposases; mariner-like elements in genome sequences have been examined in silico by Bouallegue and Filee et al. (BMC Genomics, 2017, 18(1): 1-12), but again, no activity data were presented. The presence of numerous transposase / transposon-like sequences does not provide any information regarding the presence of active transposase / transposon pairs and, conversely, increases the complexity of identifying novel active transposase / transposon pairs.

[0005] The availability of genome sequences from a variety of species and organisms offers the possibility of discovering unknown transposase / transposon-like sequences. However, identifying novel and functionally active transposase / transposon pairs remains extremely challenging. Transposase / transposon sequences are highly diverse and share little sequence homology with existing sequences, making sequence-based prediction of transposase activity and functionality virtually impossible.

[0006] Furthermore, only a few expression systems using functional transposase / transposon pairs are commercially available, including hyperactive piggy-Bac transposase (Transposagen / Lonza), hyperactive piggy-Bac transposase fused to a chromatin leader element (DirectedLuck™, ProBiogen), hyperactive transposase Leap-In® 1 and 2 (ATUM), or hyperactive sleeping beauty transposase SB100X (Max-Delbruck Center; Mates L et al., Nat Genet., 41(6):753-61 (2009)). Therefore, there is a need for additional functional transposase / transposon pairs, particularly those that are amenable to further improvement, e.g., in terms of stability and productivity of the cargo or transgene encoded thereby.

[0007] Summary of the Invention The present invention relates to novel transposase / transposon pairs, e.g., derived from Acyrthosiphon pisum (AP) or Aphis craccivora (AC). Each novel transposase / transposon pair represents an additional, highly efficient orthogonal transposase / transposon pair that is functionally active in eukaryotic cells and has transposition efficiencies at least equivalent to those of the commercially available transposase / transposon systems mentioned above. The transposases of the present invention specifically recognize defined transposon ends containing inverted terminal repeats (ITRs), which are substantially different from any published transposase / transposon system, and transposition results in the stable integration of any cargo polynucleotide (e.g., a polynucleotide encoding a protein of interest, a non-coding RNA, a viral genome, or a gene) into the genome of a host cell of interest.

[0008] In a first aspect, the present invention relates to a DNA transposon comprising a heterologous polynucleotide flanked by transposon flanking regions, wherein one transposon flanking region comprises, at one transposon end, the nucleotide sequence of SEQ ID NO: 38, and the other transposon flanking region comprises, at the other transposon end, the nucleotide sequence of SEQ ID NO: 39. The DNA transposon of the present invention is transposable by a transposase comprising the amino acid sequence of SEQ ID NO: 4.

[0009] In a specific embodiment, one transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 40 at one transposon end, and the other transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 41 at the other transposon end. Preferably, each of the one and other transposon flanking regions further comprises at least one inverted internal repeat (IIR) comprising (i) an internal repeat motif comprising the sequence tggtctac and (ii) its reverse complement (iii) separated by 6 to 3, preferably 4, nucleotides. More preferably, at least one inverted internal repeat (IIR) motif has the nucleotide sequence of SEQ ID NO: 43. In a preferred embodiment, (a) one transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 45 or 46 and further comprises an inverted internal repeat motif having the nucleotide sequence of SEQ ID NO: 43 separated by approximately 50-200 nucleotides, and / or the other transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 47 or a sequence having at least 95% sequence identity to SEQ ID NO: 47, wherein the sequence comprises at least the nucleotide sequence of SEQ ID NO: 41 and the nucleotides of SEQ ID NO: 43; or (b) one transposon flanking region and the other transposon flanking region are inverted and comprise the reverse complement of the sequence set forth in (a).

[0010] In particular embodiments, (a) one transposon flanking region comprises a sequence having at least 85% nucleotide sequence identity to SEQ ID NO: 2, 8, 10, or 58 and / or the other transposon flanking region comprises a sequence having at least 90% nucleotide sequence identity to SEQ ID NO: 3, 9, 11, or 59, or (b) one transposon flanking region and the other transposon flanking region are inverted and comprise the reverse complement of the sequence set forth in (a). Preferably, one transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 2, 8, 10, or 58 or the nucleotide sequence of SEQ ID NO: 29, 62, 63, or 64 and / or the other transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 3, 9, 11, 59, 48, or 49 or the nucleotide sequence of SEQ ID NO: 30, 65, 66, 67, 60, or 61. Alternatively, one transposon flanking region is inverted relative to the other transposon flanking region, with one transposon flanking region comprising the complement of SEQ ID NO: 3, 9, 11, 59, 48 or 49 or the complement of SEQ ID NO: 30, 65, 66, 67, 60 or 61 and / or the other transposon flanking region comprising the complement of SEQ ID NO: 2, 8, 10 or 58 or the complement of SEQ ID NO: 29, 62, 63 or 64, respectively.

[0011] In a preferred embodiment, the heterologous polynucleotide comprises at least one sequence selected from the group consisting of a sequence encoding a gene of interest, a complementary DNA (cDNA), a genome of interest, and another genetic element.

[0012] Additionally, an expression vector is provided that comprises a DNA transposon of the present invention.

[0013] In another aspect, the present invention relates to a recombinant transposase comprising at least one heterologous nuclear localization signal (NLS), preferably at least a heterologous C-terminal and / or N-terminal NLS, fused to the transposase, wherein the transposase is an Acyrthosiphon pisum transposase or an Aphis craccivora transposase, and wherein the transposase comprises an amino acid sequence having at least 90% sequence identity to amino acids 10 to 585 of SEQ ID NO:4.

[0014] In a related aspect, the invention relates to a recombinant transposase comprising an amino acid sequence having at least 90% sequence identity to amino acids 10-585 of SEQ ID NO: 4 and at least one heterologous NLS fused to the transposase. In a preferred embodiment, the transposase is an Acyrthosiphon pisum transposase or an Aphis craccivora transposase.

[0015] In another related aspect, the invention relates to a recombinant transposase comprising an amino acid sequence having at least 90% sequence identity to amino acids 10-585 of SEQ ID NO:4, and further comprising at least one mutation, preferably wherein at least one heterologous NLS is fused to the transposase. In a preferred embodiment, the recombinant transposase of the invention has increased activity compared to the transposase of SEQ ID NO:4. At least one mutation may be an amino acid substitution selected from the group consisting of K87Y, Q273V, V212I / I215L, I363V / K365S, K87Y / Q273V, K87Y / A264S / Q273V, A264S / Q273V, S270P / Q273V, K87Y / A264S, L583F, K576I, S372E, S277N and any combination thereof and / or at least one mutation is a deletion of N584 and / or E585, wherein the indicated amino acid positions of the substitutions and / or deletions correspond to the amino acid positions in the sequence of SEQ ID NO:4. Preferably, at least one mutation is an amino acid substitution selected from the group consisting of K87Y, Q273V, V212I / I215L, I363V / K365S, A264S / Q273V, K87Y / A264S or combinations thereof and / or at least one mutation is a deletion of N584 and / or E585.

[0016] In yet another aspect, the invention relates to a polynucleotide encoding a transposase comprising an amino acid sequence having at least 90% sequence identity to amino acids 10 to 585 of SEQ ID NO:4, operably linked to a eukaryotic promoter.

[0017] In a related aspect, polynucleotides are provided that encode the recombinant transposases of the invention.

[0018] Furthermore, the present invention relates to an expression vector encoding the recombinant transposase of the present invention or an expression vector comprising a polynucleotide encoding the transposase of the present invention.

[0019] In yet another aspect, the present invention relates to an isolated mRNA encoding a recombinant transposase of the present invention.

[0020] Also provided is an expression system or kit comprising: (a) a recombinant transposase source selected from the group consisting of (i) an expression vector encoding the recombinant transposase of the present invention, (ii) an isolated mRNA of the present invention, and (iii) a recombinant transposase of the present invention; and (b) a DNA transposon of the present invention or an expression vector comprising said DNA transposon of the present invention.

[0021] Also provided is a eukaryotic cell comprising a DNA transposon of the invention. In certain embodiments, the eukaryotic cell is a yeast or mammalian cell, preferably a mammalian cell, more preferably a rodent or human cell.

[0022] Also provided are non-human transgenic animals comprising the DNA transposons of the invention.

[0023] Also provided is a method for producing a cell comprising a stably integrated heterologous polynucleotide, the method comprising: (a) introducing into a eukaryotic cell a DNA molecule comprising a DNA transposon of the invention or an expression vector comprising a DNA transposon of the invention, wherein the heterologous polynucleotide comprises a sequence encoding a gene of interest, a complementary DNA (cDNA), a genome of interest, or another genetic element, and wherein the heterologous polynucleotide further comprises a sequence encoding a selectable marker; (b) introducing into the eukaryotic cell a recombinant transposase source, wherein the recombinant transposase source is selected from the group consisting of (i) an expression vector encoding a recombinant transposase of the invention, (ii) an isolated mRNA of the invention, and (iii) a recombinant transposase of the invention; and (c) culturing the eukaryotic cell in a medium under conditions to select for the selectable marker, wherein the DNA transposon is stably integrated into the genome of the eukaryotic cell.

[0024] Also disclosed is a method for producing a protein of interest, comprising: (a) introducing into a eukaryotic cell a DNA molecule comprising a DNA transposon of the present invention or an expression vector comprising a DNA transposon of the present invention, wherein the heterologous polynucleotide comprises a sequence encoding at least one protein of interest and further comprises a sequence encoding a selectable marker; and (b) introducing into the eukaryotic cell a recombinant transposase source, wherein the recombinant transposase source is (i) an expression vector encoding a recombinant transposase of the present invention, (ii) an isolated mRNA of the present invention, and (iii) a recombinant transposase of the present invention. (c) culturing eukaryotic cells in a medium under conditions to select for a selectable marker, wherein the heterologous polynucleotide comprises a sequence encoding at least one protein of interest, and the sequence encoding the selectable marker is integrated into the genome of the eukaryotic cell; (d) optionally isolating a single clone for clonal propagation to prepare a monoclonal cell line; (e) culturing the eukaryotic cells under conditions to produce the protein of interest; and (f) collecting and optionally purifying the protein of interest. [Brief explanation of the drawings]

[0025] [Figure 1] Figure 1: Plasmid map. Top: An exemplary plasmid encoding an AP transposase with a heterologous nuclear localization signal (NLS) under the control of a CMV promoter. Bottom: An exemplary transposon vector encoding the heavy and light chain genes of a therapeutic antibody under the control of a CMV promoter. A metabolic selection marker (glutamine synthase) is expressed under the control of an SV40 promoter. The transposon cargo is defined by the flanking sequences of SeqID2 left (SEQ ID NO: 2) and SeqID3 right (SEQ ID NO: 3). Both flanking sequences have the ITR sequences of SEQ ID NO: 1. [Figure 2]Figure 2: Selection experiment in which CHO cells lacking endogenous glutamine synthase (GS) (CHO K1 GS- / -) were transfected with Acyrthosiphon pisum transposase and a transposon, where the transposon provides the GS selection marker. As a control, CHO K1 GS- / - cells were transfected with the same transposon plasmid but not the transposase plasmid. Viability is monitored during the selection process for establishment of pools of recombinant CHO cells. % Viability is shown versus days post-transfection [days]. [Figure 3] Figure 3: Productivity of stable CHO pools transfected with transposons expressing AP transposase and therapeutic antibodies. Control cells were as described in Figure 2. Titers [mg / L] are shown against days after transfection [days]. [Figure 4] Figure 4: Determination of the copy number of heavy chain (HC) and light chain (LC) genes integrated into the genome by ddPCR of stable CHO pools transfected with AP transposase and transposons. [Figure 5] Figure 5: Selection experiment after transfection of CHO-K1 GS- / - cells with various Acyrthosiphon pisum (AP) transposase constructs with or without the indicated N- and / or C-terminal heterologous nuclear localization signals (NLSs) and transposons. Viability was monitored during the selection process of transfected CHO pools. % viability is shown versus days post-transfection [days]. AP: wild-type AP transposase (SEQ ID NO: 4), NLS_AP: wild-type AP transposase with a heterologous N-terminal NLS (SEQ ID NO: 6), and NLS_AP_NLS: wild-type AP transposase with heterologous N- and C-terminal NLSs (SEQ ID NO: 7). [Figure 6]Figure 6: Antibody titration of stable CHO pools 23 days after transfection with various Acyrthosiphon pisum transposase constructs with or without the indicated N- and / or C-terminal heterologous nuclear localization signal (NLS). Titers [mg / L] for the various constructs are shown. AP: wild-type AP transposase (SEQ ID NO: 4), NLS_AP: wild-type AP transposase with a heterologous N-terminal NLS (SEQ ID NO: 6), and NLS_AP_NLS: wild-type AP transposase with heterologous N- and C-terminal NLSs (flagged, SEQ ID NO: 7). [Figure 7] Figure 7: Selection experiment after transfection of CHO-K1 GS- / - cells with N-terminal Flag-tagged (Flag-NLS_AP_NLS, SEQ ID NO: 7) and N-terminal Flag-untagged (NLS_AP_NLS, SEQ ID NO: 32) Acyrthosiphon pisum transposase constructs and transposons. Viability was monitored during the selection process of transfected CHO pools. % Viability is shown versus days post-transfection [days]. [Figure 8] Figure 8: Titration of stable pools comparing Flag-tagged (SEQ ID NO: 7) and non-Flag-tagged (SEQ ID NO: 32) transposase constructs. [Figure 9] Figure 9: (A) Schematic representation of the left and right flanking sequences of the truncated versions tested. (B) Selection experiments after co-transfection of various Acyrthosiphon pisum transposon constructs with varying lengths of flanking sequences (right and left) as indicated and a plasmid encoding the AP transposase. Viability was monitored during selection of transfected CHO pools. % Viability is shown versus days post-transfection [days]. (C) Productivity of CHO pools transfected with the AP transposase and transposons with varying lengths of flanking sequences as indicated. Titer [mg / L] is shown versus days post-transfection [days]. [Figure 10]Figure 10: (A) Sequence alignment of tested left flanking sequences SEQ ID NO:2, SEQ ID NO:10, and SEQ ID NO:12, where the inverted terminal repeat (ITR) core motif (SEQ ID NO:1) is shown in bold, the binding motif (aggcgcg) is underlined, and the internal repeat (IR) motif (tggtctac) and its reverse complement are shown in italics and bold. (B) Sequence alignment of tested right flanking sequences SEQ ID NO:3, SEQ ID NO:11, and SEQ ID NO:13, where the ITR core motif (SEQ ID NO:1) is shown in bold, the binding motif (SEQ ID NO:50) is underlined, and the internal repeat (IR) motif (tggtctac) and its reverse complement are shown in italics and bold. [Figure 11]Figure 11: (A) Schematic representation of the left and right flanking sequences of the tested truncated versions. (B) Selection experiment applying a constant full-length left flanking sequence of the AP transposon (SEQ ID NO:2) and SEQ ID NO:3 (WT) or a truncated right flanking sequence (SEQ ID NO:9, SEQ ID NO:11, and SEQ ID NO:13, respectively). Viability [%] is monitored during selection of CHO pools. (C) Productivity of CHO pools transfected with a constant full-length left flanking sequence of the AP transposon (SEQ ID NO:2) and SEQ ID NO:3 (WT) or a truncated right flanking sequence (SEQ ID NO:9 and SEQ ID NO:11, respectively). Productivity (titer [mg / L]) is monitored at the indicated days post-transfection. (D) Selection experiment applying SEQ ID NO:2 (WT) or a truncated left flanking sequence of the AP transposon (SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12, respectively) and a constant full-length right flanking sequence (SEQ ID NO:3). Viability [%] is monitored during selection of CHO pools. (E) Productivity of CHO pools transfected with SEQ ID NO:2 (WT) or truncated left flanking sequences of the AP transposon (SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12, respectively) and a constant full-length right flanking sequence (SEQ ID NO:3). Productivity (titer [mg / L]) is monitored at the indicated days after transfection. (F) Selection experiment applying truncated left and right flanking sequences of the AP transposon (left SEQ ID NO:10 and right SEQ ID NO:13 or left SEQ ID NO:12 and right SEQ ID NO:11) compared to wild-type AP transposon (SEQ ID NO:2 and 3). Viability [%] is monitored during selection of CHO pools. [Figure 12] Figure 12: Selection experiments in which the left (SEQ ID NO: 10) and right (SEQ ID NO: 11) transposon sequences were swapped as shown schematically (top) were analyzed by monitoring viability (%) (middle) and productivity (titer [mg / L]) (bottom) at the indicated days post-transfection. [Figure 13]Figure 13: (A) Schematic representation of the left and right flanking sequences of the modified versions tested. Solid symbols represent native structures, while lightly filled symbols represent artificially introduced structures: inverted terminal repeats (ITRs), inverted internal repeats (IIRs), open circles (binding sites). (B) Selection experiment applying the indicated modified left and right flanking sequences of the AP transposon. Viability [%] is monitored during selection of CHO pools. (C) Productivity of CHO pools transfected with the indicated modified left and right flanking sequences of the AP transposon. Productivity (titer [mg / L]) is monitored at the indicated days post-transfection. [Figure 14] Figure 14: Selection experiment after transfection of two transposons encoding glutamine synthase selection markers together with the AP transposase. Viability is monitored during the selection process of stable CHO pools. % viability is shown against the number of days after transfection [days]. [Figure 15] Figure 15: Antibody titers measured 21 days post-transfection using two transposon-encoded glutamine synthase selectable markers together with the AP transposase. [Figure 16] Figure 16: Evaluation of fed-batch bioprocess performance of stable recombinant monoclonal antibody-expressing CHO cell pools generated using AP transposase. Three pools (AP cell pools 1–3) derived from different transfection reactions were cultured in biological duplicates (N=2). (A) Viable cell density [×10 viable cells / ml], (B) cell viability [%], and (C) recombinant monoclonal antibody titer [mg / L] were monitored daily for the indicated culture times [days] (antibody titer measurements were initiated on day 9). [Figure 17]Figure 17: Selection experiment applying functional mutants of the Acyrthosiphon pisum transposase. Viability is monitored during selection of the CHO pool. Compared to wild-type AP, a representative mutant, mutant AP K87Y, displays a hyperactivity phenotype, and mutant A264S displays a similar phenotype. % Viability is shown versus days after transfection [days]. [Figure 18] Figure 18: Expression of the transmembrane protein NRP1 for bioassay. (A) Determination of the gene copy number of the transfected NRP1 transgene in stable cell pools by ddPCR. Duplicates of three independent experiments (replicate 1: first bar, replicate 2: second bar) were analyzed and are shown as copy number / cell. (B) Determination of the relative gene expression of the transfected NRP1 transgene in stable cell pools by ddPCR. Duplicates of three independent experiments were analyzed. [Figure 19] Figure 19: Cell surface staining of expressed NRP1 in stably transfected cell pools analyzed by flow cytometry. Histograms show samples stained with anti-NRP1 antibody ("NRP1", top curve) or isotype control ("Iso2A", middle curve) and unstained samples (bottom curve). Bars indicate gates set for positive staining. [Figure 20]Figure 20: Activity of Aphis craccivora (AC) transposase and transposon and cross-reactivity of AC and Acyrthosiphon pisum (AP) transposase and transposon. CHO pools transfected with AP / AC transposase and transposon, AC / AP transposase and transposon, and AC / AC transposase and transposon were compared with AP / AP transposase and transposon as a control for selection behavior and productivity. (A) Selection experiment applying functionally distinct combinations of AC and AP transposons and transposases. Viability [%] is monitored during selection of CHO pools. (B) Productivity of CHO pools transfected with AP / AC transposase and transposon and AC / AC transposase and transposon compared with AP / AP transposase and transposon as a control. Productivity (titer [mg / L]) is monitored at the indicated days post-transfection. (C) Sequence alignment of AP transposase (SEQ ID NO: 4) and AC transposase (SEQ ID NO: 26), (D) sequence alignment of the left transposon flanking region of AP (SEQ ID NO: 2) and AC (SEQ ID NO: 29), and (E) sequence alignment of the right transposon flanking region of AP (SEQ ID NO: 3) and AC (SEQ ID NO: 30). [Figure 21]Figure 21: Evaluation of fed-batch bioprocess performance of stable recombinant monoclonal antibody-expressing CHO cell pools generated using AC transposon / AP transposase (left column, AC / AP), AP transposon / AP transposase (middle column, AP / AP), and AC transposon / AP transposase V212I+I215L+I363V+K365S+Q273V (right column, AC / AP V212I+I215L+I363V+K365S+Q273V). Three pools derived from different transfection reactions were cultured in biological duplicates (N=2). (A) Viable cell density [×10 viable cells / ml], (B) cell viability [%], (C) lactate [g / L], and (D) recombinant monoclonal antibody titer [mg / L] were monitored daily and are shown for the indicated incubation times [days] (antibody titer measurements were initiated on day 6). [Figure 22] Figure 22: Demonstration of AP transposase functionality in human suspension-adapted HEK293F cells using the fluorescent marker zsGreen as a transgene and measuring fluorescence by flow cytometry. (A) FACS measurement and quantification of the zsGreen-positive population in host cells alone (host) and with (+) or without (-) AP transposase at 8 days post-transfection. (B) FACS measurement and quantification of the zsGreen-positive population in host cells alone (host) and with (+) or without (-) AP transposase at 14 days post-transfection. (C) Comparison of % zsGreen-positive cells in + / -AP after 8 days (top) and 14 days (bottom) in separate samples. [Figure 23]Figure 23: Detection of infectious emGFP-encoding adeno-associated virus (AAV) particles produced by either HEK293 or HEK293_Cap_Tet-Rep producer cells transiently transfected with pHelper and pTransfer. Cell culture supernatant from either HEK293 or HEK293_Cap_Tet-Rep producer cells was transferred to pre-seeded HEK293 cells (96-well plate, 50 μL of cell culture supernatant per well). After 48 h, emerald green fluorescent protein (emGFP) expression was assessed by fluorescence microscopy using a Cytation 5. The upper panel shows a representative bright-field image of a cross-section of a cell layer of HEK293 or HEK293_Cap_Tet-Rep producer cells, and the lower panel shows GFP expression within the same cross-section. The white scale bar indicates 300 μm. Although AAV-mediated emGFP expression was not observed in cells treated with culture supernatant from HEK293 cells transfected with pHelper and pTransfer plasmids (lacking Rep and Cap gene expression), emGFP expression was detected in cells treated with cell culture supernatant from stable HEK293_Cap_Tet-Rep cells, indicating that the AAV Rep and Cap genes were successfully introduced into HEK293 cells using AP transposase with the AP transposon. [Figure 24] Figure 24: Selection experiment in which different transposase / transposon-like sequences (Aplysia californica, Onthophagus taurus (mutants 1, 2 and 3), Vanessa tameamea and Acyrthosiphon pisum) were tested for transposition activity in CHO cells deficient in endogenous glutamine synthase (GS) (CHO K1 GS- / -) with each transposon flanking region adjacent to the GS selectable marker. Viability is monitored during the selection process for establishment of pools of recombinant CHO cells. % viability is shown versus days post-transfection [days].

[0026] Detailed Description The term "comprises" or "comprising" means "including, but not limited to." This term is intended to be open-ended and to specify the presence of any stated feature, element, integer, step, or component, but is not intended to exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" includes the more restrictive terms "consisting of" and "consisting essentially of." With respect to sequences, the terms "having an amino acid sequence of" and "comprising amino acids of" are used interchangeably and include the embodiment "consisting of an amino acid sequence of." Similarly, the term "encoding" or "encodes" is intended to be open-ended, allowing the presence or addition of one or more other features, elements, or components. Furthermore, the singular and plural forms are not used in a restrictive manner. As used herein, the singular forms "a," "an," and "the" designate both the singular and the plural, unless expressly stated to designate only the singular.

[0027] As used herein, the terms "encoding" or "encodes" refer to a sequence or polynucleotide chain that codes for a gene, RNA, and / or protein, and particularly to encoding a transcribed and / or translated sequence product. This term encompasses the transcription of a DNA sequence into an RNA sequence, in the case of a protein, into an mRNA sequence, or in other cases, into non-coding RNA, such as siRNA, miRNA, etc. Furthermore, this term encompasses the translation of an mRNA sequence into an amino acid sequence, i.e., a protein. Thus, a gene or cDNA of interest may, for example, encode an mRNA or protein of interest or a non-coding RNA. A cDNA may also encode a viral genome, such as in the case of an RNA virus, e.g., VSV.

[0028] The term "protein" is used interchangeably with "amino acid sequence" or "polypeptide" and refers to a polymer of amino acids of any length. These terms also include proteins that have been post-translationally modified by reactions that include, but are not limited to, glycosylation, acetylation, phosphorylation, glycation, or proteolytic processing. Modifications and changes, such as fusion with other proteins, amino acid sequence substitutions, deletions, or insertions, can be made to the structure of a polypeptide while the molecule maintains its biologically functional activity. For example, specific amino acid sequence substitutions can be made in a polypeptide or its underlying nucleic acid coding sequence to obtain a protein with the same properties.

[0029] The term "nucleic acid sequence" is used interchangeably with "polynucleotide" and refers to DNA or RNA of any length. In the context of DNA transposons or expression vectors, particularly plasmids and integration into the genome of a cell or host cell, one of skill in the art will understand it to refer to a DNA sequence or molecule.

[0030] As used herein, the term "eukaryotic cell" refers to a cell having a nucleus within a nuclear membrane, and includes animal cells, human cells, plant cells, and yeast cells. In the present invention, "eukaryotic cell" particularly encompasses mammalian cells, such as cells derived from Chinese hamster ovary (CHO) cells or HEK293 cells. As used herein, mammalian cells refer to all cells or cell lines derived from mammals, such as human or rodent cells. The cells referred to herein are cells maintained in culture, such as cell lines or cell line-derived cells, i.e., immortalized cells or ex vivo primary cells. Primary cells are cells isolated from an organ tissue or organism and maintained in vitro for propagation and / or adoptive cell transfer into a patient. In the case of adoptive cell transfer into a patient (preferably a human patient), the primary cells are preferably autologous cells of the patient.

[0031] As used herein, the term "about" refers to a 10% variation of the specified value, for example, about 50% includes a 45-55% variation.

[0032] As used herein, the term "selection stringency" refers to the period after transfection until the cell culture viability reaches more than 70% and the doubling time reaches 48 hours or less.The longer the period, the more stringent the selection behavior.For example, attenuated glutamine synthase exhibits more stringent selection behavior compared to CHO wild-type glutamine synthase.

[0033] As used herein, the term "transposon flanking region" refers to a sequence that flanks a heterologous polynucleotide. The left and right transposon flanking regions, together with the heterologous polynucleotide, form a DNA transposon having one end (e.g., the left (5') end) and the other end (e.g., the right (3') end) indicated by the target sequence. Each transposon flanking region contains the target sequence (e.g., TTAA), an inverted terminal repeat (ITR), and typically further contains a binding motif and additional sequence between the ITR / binding motif and the heterologous polynucleotide that includes at least one inverted internal repeat (IIR), optionally and preferably at least one additional binding motif adjacent to the IIR. The at least one IIR may be directly adjacent to the ITR / binding motif and / or the IIR may be distant from the ITR / binding motif.

[0034] As used herein, the term "DNA transposon" refers to a heterologous polynucleotide flanked by left and right transposon flanks, where the transposon has a target sequence (e.g., TTAA) at each end followed by an ITR (left transposon flanking region) or preceded by an ITR (right transposon flanking region). Thus, one transposon end (e.g., the left or 3' end of the heterologous polynucleotide) contains the target sequence followed by an ITR, and the other transposon end (e.g., the right or 5' end of the heterologous polynucleotide) contains the ITR followed by the target sequence. Thus, sequences encoding the target sequence / ITR (e.g., SEQ ID NOs: 38 and 39) represent the two ends of the transposon. Herein, a DNA transposon may also be referred to as a transposon.

[0035] As used herein, the term "heterologous polynucleotide" refers to a sequence that is heterologous to the transposon flanking region, i.e., not derived from the same organism and / or the same genetic location as the transposon flanking region, and includes any recombinant polynucleotide. As used herein, the term "recombinant" refers to a DNA molecule created by methods of genetic recombination (e.g., cloning) that bring together genetic material from multiple sources or introduce mutations. A heterologous polynucleotide or recombinant polynucleotide may comprise a gene of interest, complementary DNA (cDNA, such as encoding a protein of interest or a viral genome of interest), a genome of interest (e.g., a viral genome of interest), or any other genetic element. A recombinant polynucleotide may also encode a recombinant transposase heterologous to the transposon flanking region, although in certain embodiments of the invention, the heterologous polynucleotide is not a polynucleotide encoding a transposase from Acyrthosiphon pisum or is not a polynucleotide encoding a transposase.

[0036] As used herein, the term "transposase" refers to a class of enzymes that can bind to the ends of a transposon and catalyze its movement to another part of the genome in cis (i.e., within the genome) and trans (e.g., from an expression vector, e.g., a plasmid, to the genome).

[0037] As used herein, the term "transposase / transposon-like sequence" refers to a sequence containing elements characteristic of transposases and transposons, such as a potential transposase sequence containing repetitive ITR-like elements and catalytic amino acid residues (DDE-motifs) 3' and 5' of the potential transposase sequence, without reference to its functional activity in cells. For example, US 2020 / 0318107 discloses that PiggyBac-like transposases and transposons naturally occur in a wide range of organisms, including Acyrthosiphon pisum (XP_001948139), and notes that for most of these, transposition activity has not been described. The transposases of the present invention are also PiggyBac-type, but are distinct from the sequence of the Acyrthosiphon pisum transposase (XP_001948139) mentioned in US 2020 / 0318107, with which the AP transposase gene of the present invention shares only about 21% sequence identity. In contrast, active transposase / transposon sequences are referred to herein as (active) transposase / (DNA) transposon pairs or systems.

[0038] As used herein, the term "nuclear localization signal (NLS)" refers to a short peptide that functions as a signal fragment mediating the transport of proteins from the cytoplasm to the nucleus. NLSs are reviewed in Lu et al. (Cell Commun Signal (2021) 19: 60), which is incorporated herein by reference. Briefly, classical nuclear localization signals (cNLSs) encompass two categories, termed "monopartite" (MP) and "bipartite" (BP). MP NLSs are generally single clusters of 4 to 8 basic amino acids containing four or more positively charged residues (arginine (R) or lysine (K)). The characteristic motif of MP NLSs is K(K / R)X(K / R), where X can be any residue. For example, the NLS of SV40 large T antigen is PKKKRKV (SEQ ID NO: 5), which has five consecutive positively charged amino acids. In contrast, BP NLSs are characterized by two clusters of 2–3 positively charged amino acids separated by a linker region of 9–12 amino acids containing several proline (P) residues. The consensus sequence can be represented as R / K(X)10-12KRXK. For example, the C-terminal NLS of nucleoplasmin is KRPAATKKAGQAKKKK (SEQ ID NO: 14). Non-classical NLSs (ncNLSs) do not resemble canonical signals and are not rich in arginine or lysine residues, such as the "proline-tyrosine" category termed PY-NLSs.

[0039] As used herein, the term "% sequence identity" refers to the number of exactly matching characters between two different sequences in a sequence alignment, or in the case of nucleotide sequences, exactly matching bases, or in the case of amino acid sequences, exactly matching amino acids.

[0040] As used herein, the term "fusion protein" refers to a protein produced by joining in frame two or more genes encoding originally separate proteins or protein fragments. Fusion proteins are also referred to as chimeric proteins. Fusion proteins include, but are not limited to, Fc-fusion proteins. The term "fusion" also refers to the joining, i.e., fusion, of two proteins or protein fragments of originally separate proteins to form a single protein. This particularly includes the introduction of a heterologous nuclear localization signal into the sequence of the transposase of the present invention. The term "introduction of a heterologous nuclear localization signal" refers to the introduction of a heterologous NLS in addition to and / or in place of the native putative nuclear localization signal. The heterologous NLS may be any NLS different from the native putative NLS of the transposase of the present invention (e.g., SEQ ID NO: 4 or SEQ ID NO: 26).

[0041] DNA transposons and polynucleotides or expression vectors encoding said DNA transposons The present invention provides novel transposase / transposon pairs, e.g., derived from Acyrthosiphon pisum (AP) or Aphis craccivora (AC), for transposing polynucleotides encoding various different proteins or non-coding RNAs into cells and stably expressing them through integration into the cellular genome. These novel transposase / transposon pairs are functionally active in eukaryotic cells and represent additional, highly efficient orthogonal transposase / transposon pairs with transposition efficiencies at least equivalent to commercially available transposase / transposon systems. The transposases of the present invention specifically recognize defined transposon ends containing inverted terminal repeats (ITRs), and, substantially different from all published transposase / transposon systems, transposition stably integrates any cargo polynucleotide into the cellular genome. The transposase / DNA transposon system of the present invention is a Piggy-Bac transposase / transposon pair.

[0042] More specifically, in one aspect, the present invention relates to a DNA transposon comprising a heterologous polynucleotide flanked by transposon flanking regions, wherein one transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 38 at one end of the transposon, and the other transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 39 at the other end of the transposon. The sequence of SEQ ID NO: 38 consists of the target sequence TTAA and an inverted terminal repeat (SEQ ID NO: 1), and the sequence of SEQ ID NO: 39 consists of the inverted terminal repeat (SEQ ID NO: 15) and the target sequence TTAA. The nucleotide sequences of SEQ ID NOs: 38 and 39 and the nucleotide sequences of SEQ ID NOs: 1 and 15 are reverse complementary to each other. However, the one and the other transposon flanking regions of the DNA transposon of the present invention are preferably not reverse complementary to each other over the entire length of the transposon flanking regions. The nucleotide sequences of SEQ ID NOs: 38 and 39 represent the ends of the one and the other transposon flanking regions. Thus, for example, the nucleotide sequence of SEQ ID NO:38 is at the left end (beginning) of the left transposon flanking region, and the nucleotide sequence of SEQ ID NO:39 is at the right end (end) of the right transposon flanking region, i.e., forming and representing the ends of a DNA transposon. The left and right transposon flanking regions may also be referred to as 3' and 5' transposon flanking regions, respectively, describing the upstream and downstream sequences of a heterologous polynucleotide. However, those skilled in the art will understand that the 3' and 5' ends may be reversed by inversion of the entire polynucleotide comprising the DNA transposon. Furthermore, those skilled in the art will understand that one and the other transposon flanking regions may be used as left and right transposon flanking regions, or alternatively, as right and left transposon flanking regions comprising reverse complement sequences. In particular, the transposon of the present invention is transposable by a transposase comprising the amino acid sequence of SEQ ID NO:4.

[0043] In a preferred embodiment, one transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 40 at one transposon end, and the other transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 41 at the other transposon end. The nucleotide sequence of SEQ ID NO: 40 comprises the nucleotide sequence of SEQ ID NO: 38 and further comprises the binding motif aggcgcg (SEQ ID NO: 50), and the nucleotide sequence of SEQ ID NO: 41 comprises the binding motif aggcgcg (SEQ ID NO: 50) and further comprises the nucleotide sequence of SEQ ID NO: 39, wherein the binding motif overlaps with the ITR sequence. Each of the one and the other transposon flanking regions preferably further comprises at least one inverted internal repeat (IIR) comprising (i) an internal repeat (IR) motif comprising the sequence tggtctac and (ii) its reverse complement, wherein (iii) the IR and its reverse complement are separated by 3 to 6, preferably 4, nucleotides. In a specific embodiment, the IR motif consists of the sequence tggtctac. Preferably, the four nucleotides separating IR tggtctac from its reverse complement are selected from the group consisting of AATT, AATC, AACT, AAGT, and AGGC. An internal repeat motif and its reverse complement, separated by 3 to 6, preferably 4, nucleotides, are referred to herein as an inverted internal repeat. In certain embodiments, at least one inverted internal repeat (IIR) motif has the nucleotide sequence of SEQ ID NO: 43 or 44. As used herein, the term "inverted internal repeat (IIR)" refers to an internal repeat (IR) motif followed downstream by its reverse complement, with an intervening nucleotide between the first internal repeat motif and the reverse complement. Here, the inverted internal repeat is located between the inverted terminal repeat and the heterologous polynucleotide. The IR motif of the transposon of the present invention has 3 to 6, preferably 4 to 5, and more preferably 4 intervening nucleotides between the nucleotide sequence tggtctac (SEQ ID NO: 42) and the first internal repeat motif and the reverse complement. The intervening nucleotides can be any nucleotide (ie, a, c, g, or t), and preferably the first intervening nucleotide is adenosine.Preferably, the intervening nucleotides are four selected from the group consisting of AATT, AATC, AACT, AAGT, and AGGC. More preferably, the four intervening nucleotides in at least one IIR in one transposon flanking region are different from those in at least one IIR in the other transposon flanking region. Even more preferably, the four intervening nucleotides in at least one IIR in one transposon flanking region are different from those in at least one IIR in the other transposon flanking region. When two or more IIRs are present in one or the other transposon flanking region, the four intervening nucleotides in IIRs within the same transposon flanking region are different. Thus, the IIR motif of the transposon of the present invention preferably has the nucleotide sequence of SEQ ID NO: 43, more specifically SEQ ID NO: 44. To increase sequence variation between one transposon flanking region and the other, the four intervening nucleotides in at least one IIR in one transposon flanking region are preferably different from those in the other transposon flanking region, and more preferably, the four intervening nucleotides in at least one IIR in one transposon flanking region are different from those in the other transposon flanking region, i.e., when two or more IIRs are present in one or the other transposon flanking region, the IIRs within the same transposon flanking region are also different from each other. The transposon may further comprise a second binding motif, cgcgcct, which may be adjacent to or overlapping the IIR, and preferably is adjacent to the IIR.

[0044] At least one IIR in one transposon flanking region and at least one IIR in the other transposon flanking region may independently be directly adjacent to the ITR binding motif or may be separated from the ITR binding motif by a nucleotide chain, preferably about 10 to 50 nucleotides, more preferably about 20 to 40 nucleotides, and even more preferably about 25 to 35 nucleotides. Preferably, at least one IIR in one transposon flanking region is directly adjacent to the ITR binding motif, and at least one IIR in the other transposon flanking region is separated from the ITR binding motif by a nucleotide chain. Alternatively, one and / or the other transposon flanking region may contain two or more IIRs, for example, two or three IIRs, preferably two IIRs. For example, one transposon flanking region may contain two IIRs, and the other transposon flanking region may contain one IIR (or vice versa). When there are two IIRs, the first IIR is preferably adjacent to the ITR and the binding region, and the second IIR is separated from the first IIR by about 25 to 250 nucleotides, preferably about 25 to 200 nucleotides, more preferably about 50 to 200 nucleotides, and even more preferably about 120 to 160 nucleotides. When there is one IIR, the IIR is preferably separated from the ITR binding motif (cgcgcct) by about 10 to 50 nucleotides, more preferably about 20 to 40 nucleotides, and even more preferably about 25 to 35 nucleotides. Here, the IIR preferably further comprises a binding motif (cgcgcct) or a binding motif-like motif (agcgcct). Thus, in a particular embodiment, the IIR binding motif has the sequence TGGTCTACAnnGTAGACCAmGCGCCT (SEQ ID NO: 68), preferably TGGTCTACAnnGTAGACCACGCGCCT (SEQ ID NO: 69) or TGGTCTACAnnGTAGACCAAGCGCCT (SEQ ID NO: 70), more preferably SEQ ID NO: 70.Those skilled in the art will also appreciate that one and the other transposon flanking regions can also be used in reverse orientation (e.g., in their respective reverse complement sequences).

[0045] In a preferred embodiment, one transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 45 or 46, and further comprises an inverted internal repeat (IIR) motif having the nucleotide sequence of SEQ ID NO: 43 separated by approximately 50 to 200 nucleotides, preferably wherein one transposon flanking region comprises SEQ ID NO: 10 or 63 or a sequence having 85% sequence identity to SEQ ID NO: 2 or 29, and / or the other transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 47 or a sequence having at least 95% sequence identity to SEQ ID NO: 47, wherein the nucleotide sequence comprises at least the nucleotide sequence of SEQ ID NO: 41 and the nucleotide sequence of SEQ ID NO: 43, preferably at least the nucleotide sequence of SEQ ID NO: 41 and the nucleotide sequence of SEQ ID NO: 68, 69 or 70. In more specific embodiments, the other transposon flanking region comprises a sequence having at least 97%, preferably 98%, more preferably 99% sequence identity to SEQ ID NO:47, wherein the nucleotide sequence comprises at least the nucleotide sequence of SEQ ID NO:41 and the nucleotide sequence of SEQ ID NO:43, preferably at least the nucleotide sequence of SEQ ID NO:41 and the nucleotide sequence of SEQ ID NO:68, 69, or 70. In particular embodiments, the other transposon flanking region comprises the nucleotide sequence of SEQ ID NO:48, 49, 60, or 61. The nucleotide sequence of SEQ ID NO:45 or 46 comprises the nucleotide sequence of SEQ ID NO:38 and further comprises the binding motif aggcgcg (SEQ ID NO:50) and a first IIR. Thus, the IIR further following the nucleotide sequence of SEQ ID NO:45 or 46 is a second IIR. Preferably, one transposon flanking region is an upstream (left or 5') transposon flanking region and the other transposon flanking region is a downstream (right or 3') transposon flanking region.Alternatively, one transposon flanking region can be inverted or swapped with the other (i.e., one transposon flanking region is the downstream transposon flanking region and the other is the upstream transposon flanking region), since both transposon flanking regions can be used as either the upstream or downstream transposon flanking region. Thus, in an alternative preferred embodiment, one transposon flanking region and the other transposon flanking region are inverted and comprise the reverse complement of the sequences set out above, i.e., for one transposon flanking region, they comprise the nucleotide sequence of SEQ ID NO: 45 or 46 and further comprise an inverted internal repeat (IIR) motif having the nucleotide sequence of SEQ ID NO: 43 separated by approximately 50 to 200 nucleotides, and for the other transposon flanking region, they comprise the nucleotide sequence of SEQ ID NO: 47 and / or the reverse complement of a sequence having at least 95% sequence identity to SEQ ID NO: 47, wherein this sequence comprises at least the nucleotide sequence of SEQ ID NO: 41 and the nucleotide sequence of SEQ ID NO: 43, preferably at least the nucleotide sequence of SEQ ID NO: 41 and the nucleotide sequence of SEQ ID NO: 68, 69 or 70.

[0046] In a more specific embodiment, (a) one transposon flanking region comprises a sequence having at least 85% nucleotide sequence identity to SEQ ID NO: 2, 8, 10 or 58 (preferably SEQ ID NO: 2 or 10, more preferably SEQ ID NO: 2) and / or the other transposon flanking region comprises a sequence having at least 90%, preferably at least 95%, nucleotide sequence identity to SEQ ID NO: 3, 9, 11 or 59 (preferably SEQ ID NO: 3 or 11, more preferably SEQ ID NO: 3); alternatively, (b) one transposon flanking region and the other transposon flanking region are inverted and comprise the reverse complement of the above-mentioned sequences, i.e., comprise the reverse complement of the sequence having at least 85% nucleotide sequence identity to SEQ ID NO: 2, 8, 10 or 58 and / or the reverse complement of the sequence having at least 90%, preferably at least 95%, nucleotide sequence identity to SEQ ID NO: 3, 9, 11, 59, 48 or 49. Preferably, one transposon flanking region comprises a sequence having at least 90%, preferably at least 95%, at least 97%, at least 98%, at least 99% or 100% nucleotide sequence identity to SEQ ID NO: 2, 8, 10 or 58 and / or the other transposon flanking region comprises a sequence having at least 95%, preferably at least 97%, at least 98%, at least 99% or 100% nucleotide sequence identity to SEQ ID NO: 3, 9, 11, 59, 48 or 49. Thus, in particular embodiments, one transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 2, 8, 10 or 58 and / or the other transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 3, 9, 11, 59, 48 or 49, or one transposon flanking region and the other transposon flanking region are inverted and comprise their reverse complementary sequences.

[0047] Furthermore, the left and right transposon flanking regions of Aphis craccivora (SEQ ID NOs: 29 and 30, respectively) share 85.7% and 95.8% sequence identity with the left and right transposon flanking regions of Acyrthosiphon pisum (SEQ ID NOs: 2 and 3, respectively) and were found to be similarly functional (selection behavior and productivity) when used in combination with the respective wild-type transposases (AC transposase: SEQ ID NO: 26 and AP transposase: SEQ ID NO: 4). Therefore, related transposase-transposon systems derived from AC are also transposase-transposon systems of the present invention. The left and right transposon flanking regions of AC are also functional when used in combination with the AP transposase. Therefore, the AP transposase / AC transposon system is also a transposase / DNA transposon system of the present invention.In a more specific embodiment, (a) one transposon flanking region comprises a sequence having at least 85% nucleotide sequence identity to SEQ ID NO:2, 8, 10 or 58 (preferably SEQ ID NO:2 or 10, more preferably SEQ ID NO:2) or SEQ ID NO:29, 62, 63 or 64 (preferably SEQ ID NO:29 or 63, more preferably SEQ ID NO:29) and / or the other transposon flanking region comprises a sequence having at least 85% nucleotide sequence identity to SEQ ID NO:3, 9, 11, 59, 48 or 49 (preferably SEQ ID NO:3 or 11, more preferably SEQ ID NO:3) or SEQ ID NO:30, 65, 66, 67, 60 or 61 (preferably SEQ ID NO:30 or 66, more preferably SEQ ID NO:30 or 66). or (b) a sequence having at least 85% nucleotide sequence identity with SEQ ID NO: 2, 8, 10 or 58 or SEQ ID NO: 29, 62, 63 or 64 and / or a reverse complement of a sequence having at least 90%, preferably at least 95% nucleotide sequence identity with SEQ ID NO: 3, 9, 11, 59, 48 or 49 or SEQ ID NO: 30, 65, 66, 67, 60 or 61, in which one transposon flanking region is inverted from the other transposon flanking region. Preferably, one transposon flanking region comprises a sequence having at least 90%, preferably at least 95%, at least 97%, at least 98%, at least 99% or 100% nucleotide sequence identity to SEQ ID NO: 29, 62, 63 or 64 and / or the other transposon flanking region comprises a sequence having at least 95%, preferably at least 97%, at least 98%, at least 99% or 100% nucleotide sequence identity to SEQ ID NO: 30, 65, 66, 67, 60 or 61.Thus, in certain embodiments, one transposon flanking region comprises the sequence of SEQ ID NO: 29, 62, 63 or 64 and / or the other transposon flanking region comprises the sequence of SEQ ID NO: 30, 65, 66, 67, 60 or 61, or one transposon flanking region and the other transposon flanking region are inverted and comprise their reverse complementary sequences.

[0048] Those skilled in the art will understand that the nucleotide sequence of the left transposon flanking region of the AP of SEQ ID NO: 2 is the full-length sequence, while the nucleotide sequences of SEQ ID NO: 8, 10 or 58 are truncated forms thereof containing the minimum essential elements, and that the nucleotide sequence of the left transposon flanking region of the AC of SEQ ID NO: 29 is the full-length sequence, while the nucleotide sequences of SEQ ID NO: 62, 63 or 64 are truncated forms thereof containing the minimum essential elements. Thus, in particular embodiments, (i) one transposon flanking region comprises a sequence having at least 85% (or at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100%) nucleotide sequence identity to at least SEQ ID NO:58, preferably at least SEQ ID NO:10, at least SEQ ID NO:8 or SEQ ID NO:2, or comprises a sequence having at least 85% (or at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100%) nucleotide sequence identity to at least SEQ ID NO:64, preferably SEQ ID NO:63, SEQ ID NO:62 or SEQ ID NO:29, and / or the other transposon flanking region comprises at least SEQ ID NO:48 or or (ii) a sequence having at least 90% (at least 95%, preferably at least 97%, at least 98%, at least 99% or 100%) sequence identity to SEQ ID NO: 49, preferably at least 90% (at least 95%, preferably at least 97%, at least 98%, at least 99% or 100%) sequence identity to SEQ ID NO: 60 or 61, preferably at least 90% (at least 95%, preferably at least 97%, at least 98%, at least 99% or 100%) sequence identity to SEQ ID NO: 67, at least 90% (at least 95%, preferably at least 97%, at least 98%, at least 99% or 100%) sequence identity to SEQ ID NO: 60 or 61, preferably at least 90% (at least 95%, preferably at least 90%>, ...

[0049] Preferably, (i) one transposon flanking region comprises a sequence having at least 85% (or at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100%) nucleotide sequence identity to at least SEQ ID NO:64, preferably at least SEQ ID NO:63, at least SEQ ID NO:62 or SEQ ID NO:29, and / or the other transposon flanking region comprises a sequence having at least 90% (at least 95%, preferably at least 97%, at least 98%, at least 99% or 100%) sequence identity to at least SEQ ID NO:60 or 61, preferably at least SEQ ID NO:67, at least SEQ ID NO:66, at least SEQ ID NO:65 or more preferably SEQ ID NO:30, or (ii) wherein one transposon flanking region and the other transposon flanking region are inverted and comprise the reverse complement of the sequence set out in (i). The full length and truncation sequences of the left and right transposon flanking regions of the AP and AC are summarized in Table A below.

[0050] [Table 1]

[0051] Those skilled in the art will understand that each of the one transposon flanking region and the other transposon flanking region may independently be an AP-derived sequence or an AC-derived sequence, preferably both the one transposon flanking region and the other transposon flanking region are AP-derived sequences or both are AC-derived sequences, and more preferably both the one transposon flanking region and the other transposon flanking region are AC-derived sequences (including their respective recited % sequence identities). For example, an AC-derived sequence is a sequence having at least 95% sequence identity with SEQ ID NO: 29, 62, 63, or 64 for one transposon flanking region and at least 97% sequence identity with SEQ ID NO: 30, 65, 66, 67, 60, or 61 for the other transposon flanking region (or its reverse complementary sequence); an AP-derived sequence is a sequence having at least 95% sequence identity with SEQ ID NO: 2, 8, 10, or 58 for one transposon flanking region and at least 97% sequence identity with SEQ ID NO: 3, 9, 11, 59, 48, or 49 for the other transposon flanking region (or its reverse complementary sequence), but is not limited to these. For the left transposon flanking region, at least SEQ ID NOs: 10, 8 and 2 (or SEQ ID NOs: 63, 62 and 29) are equally preferred, while for the right transposon flanking region, SEQ ID NOs: 9 or 3 (or SEQ ID NOs: 65 or 30) are preferred, with SEQ ID NO: 3 (or SEQ ID NO: 30) being more preferred, as function improves with length.

[0052] In certain preferred embodiments, (i) one transposon flanking region comprises a sequence having at least 95% (preferably, at least 97%, at least 98%, at least 99% or at least 100%) sequence identity to SEQ ID NO: 29, 62 or 63 and / or the other transposon flanking region comprises a sequence having at least 97% (preferably, at least 98%, at least 99% or at least 100%) sequence identity to SEQ ID NO: 30, 65 or 66, preferably SEQ ID NO: 30; or (ii) wherein one transposon flanking region and the other transposon flanking region are inverted and comprise the reverse complement of the sequence set forth in (i).

[0053] In the DNA transposons of the present invention, the transposon flanking regions flank a heterologous polynucleotide, which is a polynucleotide of interest. Those skilled in the art will understand that the heterologous polynucleotide of interest differs from the naturally occurring sequences flanking the transposon flanking regions. Thus, the polynucleotide of interest is not of the Aphididae or the Gastrorrhine order. In the DNA transposons of the present invention, the transposon flanking regions flank a heterologous polynucleotide comprising at least one sequence selected from the group consisting of a sequence encoding (or comprising) a gene of interest, a complementary DNA (cDNA), a genome of interest, and another genetic element. The gene of interest or cDNA may encode a protein of interest or a non-coding RNA (ncRNA). As used herein, the term "non-coding RNA" refers to an RNA molecule that is not translated into a protein. DNA from which functional non-coding RNA is transcribed may also be referred to as an RNA gene. Non-coding RNA (ncRNA) includes, but is not limited to, transfer RNA (tRNA), ribosomal RNA (rRNA), and small RNA (e.g., microRNA (miRNA), siRNA, shRNA, piRNA, snoRNA, snRNA), long non-coding RNA (lncRNA), antisense RNA, riboswitches, and ribozymes. The protein of interest is preferably a recombinant protein, including, but not limited to, therapeutic proteins, particularly secreted recombinant therapeutic proteins (e.g., antibodies, antibody-derived molecules or antibody mimetics, cytokines, hormones, fusion proteins, etc.), transmembrane receptors (e.g., chimeric antigen receptors (CARs) or cytokine receptors for overexpression in cells for use in in vitro assays or for engineering a patient's autologous cells, e.g., T cells or NK cells, for adoptive cell transfer), and enzymes (e.g., for genetic engineering of cells to produce proteins such as glycosylation-modifying enzymes). The transposons of the invention can also be used in virus production (eg, AAV or VSV) or in the generation of virus packaging cell lines.Thus, the protein of interest may be a viral protein, for example, preferably an AAV viral protein encoded by the AAV rep and / or cap genes.

[0054] The size of the transposon of the present invention is not limited and may include heterologous polynucleotides as large as 40 kb or 50 kb. Therefore, the heterologous polynucleotide may also include a sequence containing a genome of interest, for example, a viral genome of interest, such as the viral genome of adeno-associated virus (AAV) or vesicular stomatitis virus (VSV), or one or more viral genes, for example, AAV (rep and / or cap) or VSV. The AAV genome is flanked by two inverted terminal repeats (hereinafter referred to as viral inverted terminal repeats to distinguish them from the ITRs in the transposon of the present invention). The entire AAV genome, including the viral ITRs, comprises approximately 4.7 kb. In the context of the present invention, the AAV genome is typically a recombinant AAV (rAAV) genome and may encode a viral protein or a heterologous protein thereto, such as a therapeutic protein or a suicide gene product. Typically, in the case of an RNA virus, such as VSV, the viral genome is encoded by a genomic cDNA. Thus, the cDNA may encode the viral genome, a protein of interest, or a non-coding RNA. The transposons of the invention can further be used for the stable integration of other genetic elements, for example binding motifs or regulatory elements.

[0055] In certain embodiments, the heterologous polynucleotide comprises at least one sequence selected from the group consisting of a sequence encoding (or comprising) a gene of interest, a complementary DNA (cDNA), a genome of interest, and another genetic element, wherein the heterologous polynucleotide comprises at least one sequence under the control of a promoter and a transcription termination signal. The promoter can be any promoter compatible with the host cell. Preferably, the host cell is a eukaryotic host cell. In certain embodiments, the promoter is a eukaryotic promoter, preferably selected from the group consisting of the EF1a promoter, cytomegalovirus (CMV) promoter, GAPDH promoter, CAG promoter, herpes simplex virus thymidine kinase (HSV-TK) promoter, murine stem cell virus (MSCV) promoter, spleen-limited focus-forming virus (SFFV) promoter, SV40 promoter, and actin promoter, PGK promoter, and ubiquitin promoter. In certain or additional embodiments, the promoter is an inducible promoter, e.g., comprising a Tet regulatory element. The transcription termination signal is typically a polyadenylation site. Preferably, the heterologous polynucleotide comprises an expression cassette comprising a promoter, a sequence encoding a gene of interest, a complementary DNA (cDNA), a genome of interest, and at least one sequence selected from the group consisting of another genetic element, and a transcription termination signal. More preferably, the heterologous polynucleotide comprises an expression cassette comprising a promoter, at least one sequence encoding a gene of interest, a cDNA, or a genome of interest, and a transcription termination signal.

[0056] In certain embodiments, the heterologous polynucleotide comprises one or more sequences encoding an antibody heavy and / or light chain, a secreted therapeutic recombinant protein, a recombinant protein, a transmembrane receptor; a non-coding RNA mediating RNA interference (RNAi), preferably selected from the group consisting of siRNA, shRNA, lncRNA and miRNA; one or more viral proteins, a viral genome or viral genome cDNA, a ribozyme, a binding motif, a regulatory DNA element, another genetic element, and a combination of any of the above.

[0057] The protein of interest encoded by the gene of interest can be any protein, but is typically a therapeutic protein. As used herein, the term "therapeutic protein" refers to a protein that can be used for medical treatment of humans and / or animals. These include, but are not limited to, cytokines, growth factors, hormones, blood clotting factors, vaccines, interferons, fusion proteins, antibodies, antibody-derived molecules, and antibody mimetics. In certain embodiments, the therapeutic protein is selected from the group consisting of cytokines, hormones, fusion proteins, antibodies, antibody-derived molecules, and antibody mimetics.

[0058] In certain embodiments, the protein of interest is an antibody. When the protein of interest is an antibody, the DNA transposon or eukaryotic expression vector (particularly a mammalian expression vector) comprises a polynucleotide comprising a coding sequence for the heavy chain variable region and / or a coding sequence for the light chain variable region of the antibody. In certain embodiments, the DNA transposon comprises a polynucleotide comprising a coding sequence for the heavy chain and / or a coding sequence for the light chain variable region of the antibody. Thus, a polynucleotide comprising a coding sequence for the heavy chain variable region and a polynucleotide comprising a coding sequence for the light chain variable region can be expressed by the same DNA transposon or by separate DNA transposons. The DNA transposon may comprise a multicistronic expression cassette, e.g., a bicistronic expression cassette and / or multiple expression cassettes. A multicistronic expression cassette comprises two or more open reading frames separated by a sequence encoding an RNA element enabling translation initiation, e.g., an internal ribosome entry site (IRES). In a multicistronic expression cassette, two or more open reading frames are under the control of the same promoter. Thus, a polynucleotide encoding at least the variable region of the heavy chain and a polynucleotide encoding at least the variable region of the light chain can be expressed in the same expression cassette (e.g., separated by an IRES sequence) or in two separate expression cassettes. Furthermore, a selectable marker and a protein of interest and / or a non-coding RNA can be expressed in the same expression cassette or in separate expression cassettes. When the protein of interest is an antibody, a selectable marker (e.g., glutamine synthase) gene, a polynucleotide encoding at least the variable region of the heavy chain, and / or a polynucleotide encoding at least the variable region of the light chain can be expressed in the same expression cassette or in separate expression cassettes, or a mixture thereof.

[0059] In a preferred embodiment, the DNA transposon comprises a polynucleotide encoding a selectable marker (e.g., glutamine synthase) and at least one sequence encoding a gene of interest, a cDNA, a genome of interest, or another genetic element. Eukaryotic expression vectors, preferably mammalian expression vectors, comprising the DNA transposons of the present invention are preferably plasmids, bacterial artificial chromosomes (BACs), or non-integrating viral vectors. The plasmids, bacterial artificial chromosomes (BACs), or viral vectors can be introduced into eukaryotic host cells (e.g., mammalian host cells) via transfection or gene transfer, respectively.

[0060] A preferred protein of interest is an antibody, including fragments and derivatives thereof. Typically, antibodies are monospecific, but antibodies may also be multispecific. Thus, the present invention can be used to produce monospecific antibodies, multispecific antibodies or fragments thereof, preferably antibodies (monospecific), bispecific antibodies, trispecific antibodies or fragments thereof, preferably antigen-binding fragments thereof. Exemplary antibodies within the scope of the present invention include, but are not limited to, anti-CD2, anti-CD3, anti-CD20, anti-CD22, anti-CD30, anti-CD33, anti-CD37, anti-CD40, anti-CD44, anti-CD44v6, anti-CD49d, anti-CD52, anti-EGFR1 (HER1), anti-EGFR2 (HER2), anti-GD3, anti-IGF, anti-VEGF, anti-TNFalpha, anti-IL2, anti-IL-5R or anti-IgE antibodies, and are preferably selected from the group consisting of anti-CD20, anti-CD33, anti-CD37, anti-CD40, anti-CD44, anti-CD52, anti-HER2 / neu (erbB2), anti-EGFR, anti-IGF, anti-VEGF, anti-TNFalpha, anti-IL2 and anti-IgE antibodies.

[0061] The terms "antibody," "antibodies," or "immunoglobulin" are used herein in the broadest sense and encompass a variety of antibody structures, including (but not limited to) monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. There are various immunoglobulin classes: IgA, IgD, IgE, IgG, IgM, IgY, and IgW. Preferably, the antibody is an IgG antibody, more preferably an IgG1 antibody or an IgG4 antibody.

[0062] Antibodies can be of any species, including chimeric, humanized, and human antibodies. A "chimeric" antibody is a molecule in which domains or regions of the antibody are derived from different species. For example, the variable regions of the heavy and light chains may be derived from a rat or mouse antibody, while the constant region may be derived from a human antibody. In a "humanized" antibody, only minimal sequence is derived from a non-human species. In many cases, only the CDR amino acid residues of a human antibody are replaced with CDR amino acid residues from a non-human species, such as a mouse, rat, rabbit, or llama. Some key framework amino acid residues that affect antigen-binding specificity and affinity may also be replaced with non-human amino acid residues.

[0063] Typically, antibodies are tetrameric polypeptides composed of two pairs of heterodimers, each formed by a heavy chain and a light chain. Both the heterodimer and the tetrameric polypeptide structure are stabilized by interchain disulfide bridges. Each chain is composed of structural domains called "immunoglobulin domains" or "immunoglobulin regions." Here, the terms "domain" and "region" are used interchangeably. Each domain contains approximately 70 to 110 amino acids and forms a compact three-dimensional structure. Both heavy and light chains contain a "variable domain" or "variable region" at their N-terminus, which has few conserved sequences and is responsible for antigen recognition and binding. The light chain variable region is also called "VL," and the heavy chain variable region is also called "VH."

[0064] An "antibody fragment" or "antigen-binding fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments. Fab fragments consist of the variable regions of both chains held together by adjacent constant regions. These can be generated from conventional antibodies by protease digestion, e.g., papain, although Fab fragments can also be produced by genetic engineering. Additional antibody fragments include F(ab')2 fragments, which can be prepared by proteolytic cleavage with pepsin.

[0065] Using genetic engineering techniques, it is possible to produce truncated antibody fragments consisting only of the variable regions of the heavy chain (VH) and light chain (VL). These are called Fv fragments (Fragment variable = fragment of variable region). These Fv fragments lack the covalent bond between the two chains via the cysteines of the constant chains, and are therefore often stabilized. Advantageously, the variable region of the heavy chain and the variable region of the light chain are linked by a short peptide fragment of, for example, 10 to 30 amino acids, preferably 15 amino acids. In this way, a single peptide chain consisting of VH and VL linked by a peptide linker is obtained. This type of antibody protein is known as single-chain Fv (scFv). Examples of scFv antibody proteins are known to those skilled in the art. Therefore, antibody fragments and antigen-binding fragments also include Fv fragments, particularly scFv.

[0066] In recent years, various strategies have been developed to prepare multimeric scFv derivatives. These are intended, in particular, to produce recombinant antibodies with improved pharmacokinetic and biodistribution properties and enhanced binding activity. To achieve scFv multimerization, scFvs have been prepared as fusion proteins with multimerization domains. The multimerization domain can be, for example, the CH3 region of IgG or a coiled-coil structure (helical structure), such as a leucine zipper domain. On the other hand, there are also strategies in which interactions between the VH / VL regions of scFvs are used for multimerization (e.g., diabodies, tribodies, and pentabodies). Those skilled in the art will recognize that diabodies refer to bivalent homodimeric scFv derivatives. Shortening the linker of scFv molecules to 5-10 amino acids results in the formation of homodimers in which interchain VH / VL overlap occurs. Diabodies can be further stabilized by incorporating disulfide bridges. Examples of diabody-antibody proteins are known in the prior art.

[0067] Those skilled in the art will recognize that a minibody refers to a bivalent homodimeric scFv derivative, consisting of a fusion protein containing the CH3 region of an immunoglobulin, preferably an IgG, most preferably an IgG1, as a dimerization domain, linked to an scFv via a hinge region (e.g., also from IgG1) and a linker region. Examples of minibody-antibody proteins are known from the prior art.

[0068] Those skilled in the art will understand that triabody refers to a trivalent homotrimeric scFv derivative, in which VH and VL are directly fused without a linker sequence to form a trimer.

[0069] Those skilled in the art will also be familiar with so-called miniantibodies, which have a bivalent, trivalent or tetravalent structure and are derived from scFvs. Multimerization occurs via dimeric, trimeric or tetrameric coiled-coil structures. In a preferred embodiment of the present invention, the gene of interest encodes any of the desired polypeptides mentioned above, preferably a monoclonal antibody, derivative or fragment thereof.

[0070] Further included are single domain antibodies (sdAbs), also called nanobodies, which are antibody fragments of a single monomeric variable antibody domain, typically engineered from heavy chain antibodies found in camelids (VHH fragments) or cartilaginous fish (VNAR fragments).

[0071] Immunoglobulin fragments consisting of the CH2 and CH3 domains of an antibody heavy chain are called "Fc fragments," "Fc regions," or "Fc" because of their propensity to crystallize (Fc = fragment crystallizable). They can be formed from conventional antibodies by protease digestion, e.g., papain or pepsin, but can also be produced by genetic engineering. The N-terminal portion of an Fc fragment can vary depending on how many amino acids of the hinge region remain.

[0072] Antibodies comprising an antigen-binding fragment and an Fc region are sometimes referred to as full-length antibodies. Full-length antibodies can be monospecific or multispecific antibodies. Multispecific antibodies are antibodies that have at least two different antigen-binding sites, each binding to a different epitope. Multispecific antibodies include bispecific and trispecific antibodies. Bispecific antibodies have two different binding sites. Multispecific antibodies also include antibody formats other than full-length antibodies, such as antibody-derived molecules.

[0073] Bispecific antibodies typically combine antigen-binding specificities for target cells (e.g., malignant B cells) and effector cells (e.g., T cells, NK cells, or macrophages) in a single molecule. Exemplary bispecific antibodies include, but are not limited to, diabodies, BiTE (bispecific T cell engager) formats, and DART (dual affinity retargeting) formats. The diabody format separates the cognate variable domains of the heavy and light chains of two antigen-binding specificities onto two separate polypeptide chains, which are noncovalently associated. The DART format is based on the diabody format but provides additional stabilization through a C-terminal disulfide bridge. Trispecific antibodies are monoclonal antibodies that combine three antigen-binding specificities. Trispecific antibodies can be constructed based on bispecific antibody technology, which reconstitutes the antigen-recognition domains of two different antibodies into a single bispecific molecule. For example, a trispecific antibody has been generated that targets CD38 on cancer cells and CD3 and CD28 on T cells. Multispecific antibodies are particularly difficult to produce in high production quality.

[0074] As used herein, the term "antibody-derived molecule" refers to any molecule that contains at least an antigen-binding portion structurally related to an antibody. Antibody-derived molecules include modified full-length monospecific or bispecific antibodies or smaller antibody formats, including those described herein, that have been further modified with additional antigen-binding portions.

[0075] As used herein, the term "antibody mimetic" refers to a protein that binds to a specific antigen in a manner similar to an antibody, but is not structurally related to an antibody. Antibody mimetics include, but are not limited to, anticalins, affibodies, adnectins, monobodies, DARPins, affimers, and affitins.

[0076] Single domain antibodies (sdAbs) are also sometimes referred to as nanobodies. Those skilled in the art will appreciate that a protein may comprise more than one antigen-binding domain and therefore may be multivalent, preferably bivalent (e.g., a bivalent sdAb or a bivalent anticalin or any other bivalent antibody mimetic).

[0077] Another preferred therapeutic protein is a fusion protein, e.g., an Fc-fusion protein. Therefore, the present invention can be advantageously used to produce fusion proteins, e.g., Fc-fusion proteins. The effector portion of the fusion protein can be the complete sequence or any portion of the sequence of a native or modified heterologous protein. The immunoglobulin constant domain sequence can be derived from any immunoglobulin subtype, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 subtype or class, e.g., IgA, IgE, IgD, or IgM. Preferably, they are derived from human immunoglobulins, more preferably from human IgG, and even more preferably from human IgG1 and IgG2. Non-limiting examples of Fc-fusion proteins include MCP1-Fc, ICAM-Fc, EPO-Fc, and scFv fragments linked to the CH2 domain of a heavy chain immunoglobulin constant region containing an N-linked glycosylation site. Fc-fusion proteins can be constructed, for example, by genetic engineering approaches, by introducing a CH2 domain of a heavy chain immunoglobulin constant region containing an N-linked glycosylation site into another expression construct containing another immunoglobulin domain, an enzymatically active protein moiety, or an effector domain. Thus, Fc-fusion proteins of the invention also include, for example, a single-chain Fv fragment linked to a CH2 domain of a heavy chain immunoglobulin constant region containing an N-linked glycosylation site.

[0078] The term "cytokine" refers to small proteins released by cells that act as intercellular mediators, e.g., influencing the behavior of cells surrounding the secreting cell. Cytokines may be secreted by immune cells or other cells, e.g., T cells, B cells, NK cells, and macrophages. Cytokines may be involved in intercellular signaling events, e.g., autocrine signaling, paracrine signaling, and endocrine signaling. Cytokines may mediate a wide range of biological processes, including (but not limited to), immunity, inflammation, and hematopoiesis. Cytokines can be chemokines, interferons, interleukins, lymphokines, or tumor necrosis factors.

[0079] As used herein, "growth factor" refers to a protein or polypeptide capable of stimulating cell proliferation.

[0080] The heterologous polynucleotide flanked by the two transposon flanking regions may further comprise a sequence encoding a selectable marker. As used herein, the terms "selectable marker" and "selection marker" are used interchangeably herein and refer to a gene introduced into a cell that allows for selection in cell culture or single-cell clones. The selectable marker can be an antibiotic resistance gene, such as an ampicillin, chloramphenicol, tetracycline, or kanamycin resistance gene, pyromycin acetyltransferase, blasticidin acetyltransferase enzyme, hygromycin B phosphotransferase enzyme, or aminoglycoside 3' phosphotransferase enzyme. The selectable marker can also be a gene encoding a fluorescent marker, such as green fluorescent protein (GFP), eGFP, monomeric red fluorescent protein (mRFP) (e.g., mCherry), phycoerythrin, or a luminescent marker, such as luciferase. The selectable marker can also be a split selectable marker. Split selectable markers comprise split marker segments fused to inteins, i.e., protein splicing elements, expressed on separate vectors, which are recombined via protein trans-splicing to reconstitute the full-length marker protein in a host cell receiving the intended vector, as described, for example, in N. Jillette et al. (Nature Communications, 2019, 10:4968). Further suitable selectable markers are metabolic selectable markers, such as glutamine synthase (GS) or dihydrofolate reductase (DHFR), preferably a selectable marker gene encoding GS.

[0081] The DNA transposon of the present invention can be placed on any DNA molecule. In particular, the DNA transposon can be present on a vector, particularly an expression vector, such as a plasmid DNA, a plasmid without antibiotic resistance marker (pFAR), a minicircle (MC), a doggybone DNA (dbDNA), a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or a non-integrating viral vector. In a preferred embodiment, the DNA transposon is present on a plasmid DNA or a BAC.

[0082] In another aspect, the present invention relates to an expression vector comprising a DNA transposon of the present invention.

[0083] In yet another aspect, a eukaryotic cell is provided that contains a DNA transposon of the present invention. The eukaryotic cell can be any eukaryotic cell, for example, a yeast, insect, or mammalian cell. In the case of an insect cell, the insect cell is not an Aphididae or Gastrorrhinum insect cell. Preferably, the eukaryotic cell is a yeast or mammalian cell, more preferably a mammalian cell, even more preferably a rodent or human cell, for example, a Chinese hamster ovary (CHO) cell or a cell derived from HEK293 cells. These cells may be cells maintained in culture, for example, a cell line or a cell line-derived cell, i.e., an immortalized cell or an ex vivo primary cell. Suitable eukaryotic cells are further exemplified with respect to the methods of the present invention, and apply equally to the eukaryotic cells of the present invention.

[0084] In certain embodiments, the mammalian cell may contain, as heterologous polynucleotides flanked by transposon flanking regions, one or more viral genes, e.g., viral genes from AAV, preferably the AAV rep and / or cap genes, and optionally genes from a helper virus, e.g., genes from HSV or AdV. Such mammalian cells may be suitable as virus packaging cells. Therefore, also provided is the use of mammalian cells, wherein the heterologous genes are one or more viral genes for virus production, preferably AAV production.

[0085] In yet another aspect, a non-human transgenic animal is provided, wherein the transgenic animal comprises a DNA transposon of the invention. The non-human transgenic animal is preferably a mammal, such as a rodent, ruminant, or pig.

[0086] Recombinant Transposase The present invention further relates, in one aspect, to a recombinant transposase comprising at least one heterologous nuclear localization signal (NLS) fused to the transposase, wherein the transposase is an Acyrthosiphon pisum transposase. The at least one heterologous NLS may be fused to the N-terminus, C-terminus, or internal region. Those skilled in the art will understand that the heterologous NLS should be introduced without interfering with transposase activity. Thus, in a preferred embodiment, the recombinant transposase comprises at least a heterologous C-terminal and / or N-terminal NLS, i.e., at least a C-terminal or N-terminal, or a C-terminal and N-terminal heterologous NLS. In a more preferred embodiment, the recombinant transposase comprises at least a heterologous C-terminal and N-terminal NLS. Preferably, the recombinant transposase comprises an amino acid sequence having at least 90% sequence identity to amino acids 10 to 585 of SEQ ID NO: 4 or 26, preferably amino acids 10 to 585 of SEQ ID NO: 4. More preferably, the recombinant transposase comprises an amino acid sequence having at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 10 to 585 of SEQ ID NO: 4 or 26, preferably SEQ ID NO: 4, or having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4 or 26, preferably SEQ ID NO: 4. In certain embodiments, the recombinant transposase has about the same or better transposase activity compared to a transposase having the amino acid sequence of SEQ ID NO: 4, wherein the transposase activity is determined as enhanced recovery and / or productivity by selection using DNA transposons, preferably comprising transposon flanking regions having the nucleotide sequences of SEQ ID NOs: 2 and 3 or the nucleotide sequences of SEQ ID NOs: 29 and 30, encoding antibody heavy and light chains as proteins of interest.Determination of enhanced recovery and / or productivity by selection using a DNA transposon having the nucleotide sequences of SEQ ID NOs: 2 and 3 or the nucleotide sequences of SEQ ID NOs: 29 and 30 and including transposon flanking regions encoding antibody heavy and light chains as the protein of interest is exemplified, for example, in Example 9. Approximately the same or better transposase activity means at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 100%, compared to a transposase having the amino acid sequence of SEQ ID NO: 4. In a specific embodiment, the recombinant transposase of the present invention comprises the amino acid sequence of amino acids 10 to 585 of SEQ ID NO: 4 or SEQ ID NO: 26, preferably amino acids 10 to 585 of SEQ ID NO: 4. In a preferred embodiment, the recombinant transposase of the present invention comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 26, preferably SEQ ID NO: 4.

[0087] In a related aspect, the present invention relates to a recombinant transposase comprising an amino acid sequence having at least 90% sequence identity to amino acids 10-585 of SEQ ID NO:4. The recombinant transposase of the present invention may be an Acyrthosiphon pisum transposase, but may also be derived from a different species, e.g., Aphis craccivora. The Aphis craccivora transposase (SEQ ID NO:26) has 98.3% sequence identity to the amino acid sequence of SEQ ID NO:4. Thus, in certain embodiments, the recombinant transposase of the present invention is an Acyrthosiphon pisum transposase or an Aphis craccivora transposase. Preferably, at least one heterologous NLS is fused to the transposase. The at least one heterologous NLS may be fused to the N-terminus, C-terminus, or internal region. Those skilled in the art will appreciate that the heterologous NLS should be introduced without interfering with transposase activity. Thus, in a preferred embodiment, the recombinant transposase comprises at least a heterologous C-terminal and / or N-terminal NLS, i.e., at least a C-terminal or N-terminal or C-terminal and N-terminal heterologous NLS. In a more preferred embodiment, the recombinant transposase comprises at least a heterologous C-terminal and N-terminal NLS. Preferably, the recombinant transposase has at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 10 to 585 of SEQ ID NO:4, or comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:4. In certain embodiments, the recombinant transposase has at least 95%, preferably at least 97%, at least 98%, or at least 99% sequence identity to amino acids 10 to 585 of SEQ ID NO:26 or comprises an amino acid sequence having at least 95%, preferably at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:26.In certain embodiments, the recombinant transposase has approximately the same or better transposase activity as a transposase having the amino acid sequence of SEQ ID NO:4, where transposase activity is determined as enhanced recovery and / or productivity by selection using a DNA transposon comprising transposon flanking regions having the nucleotide sequences of SEQ ID NOs:2 and 3 or SEQ ID NOs:29 and 30 and encoding antibody heavy and light chains as the protein of interest. Determination of selection stringency and / or productivity using a DNA transposon comprising transposon flanking regions having the nucleotide sequences of SEQ ID NOs:2 and 3 or SEQ ID NOs:29 and 30 and encoding antibody heavy and light chains as the protein of interest is exemplified in Example 9. The same or better transposase activity means at least 90%, preferably at least 95%, more preferably at least 98%, and even more preferably at least 100%, as compared to a transposase having the amino acid sequence of SEQ ID NO:4. In a specific embodiment, the recombinant transposase of the present invention comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 26, preferably amino acids 10 to 585 of SEQ ID NO: 4, more preferably the amino acid sequence of SEQ ID NO: 4.In a specific embodiment, when the transposase comprises the amino acid sequence of amino acids 10 to 585 of SEQ ID NO: 26 or an amino acid sequence having at least 99% sequence identity with amino acids 10 to 585 of SEQ ID NO: 26, the DNA transposon used in combination preferably has: (i) one transposon flanking region having at least 90% (preferably at least 95%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity with at least SEQ ID NO: 64, preferably at least SEQ ID NO: 63, at least SEQ ID NO: 62, or SEQ ID NO: 29. or (ii) a DNA transposon in which one transposon flanking region and the other transposon flanking region comprise a sequence having at least 97% (preferably at least 98%, at least 99% or 100%) sequence identity to at least SEQ ID NO: 60 or 61, preferably at least SEQ ID NO: 67, at least SEQ ID NO: 66, at least SEQ ID NO: 65 or more preferably SEQ ID NO: 30; or (ii) wherein one transposon flanking region and the other transposon flanking region are inverted and comprise the reverse complement of the sequence described in (i).

[0088] In certain preferred embodiments, (i) one transposon flanking region comprises a sequence having at least 95% (preferably, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity to SEQ ID NO: 29, 62, or 63 and / or the other transposon flanking region comprises a sequence having at least 97% (preferably, at least 98%, at least 99%, or at least 100%) sequence identity to SEQ ID NO: 30, 65, or 66, preferably SEQ ID NO: 30; or (ii) wherein one transposon flanking region and the other transposon flanking region are inverted and comprise the reverse complement of the sequence described in (i), and the recombinant transposase is any transposase of the invention. In another preferred embodiment, the DNA transposon is any DNA transposon of the present invention (e.g., derived from AP or AC), and the recombinant transposase comprising an amino acid sequence having at least 99% sequence identity to amino acids 10 to 585 of SEQ ID NO: 4 optionally further comprises at least one mutation, wherein the at least one mutation is K87Y, Q273V, V212I / I215L, I363V / K365S, K87Y / Q and / or the at least one mutation is a deletion of N584 and / or E585, wherein the indicated amino acid positions of the substitutions and / or deletions correspond to the amino acid positions in the sequence of SEQ ID NO: 4. Preferably, the recombinant transposase comprises an amino acid sequence having at least 99% sequence identity with amino acids 10 to 585 of SEQ ID NO: 4 or further comprises at least one mutation, wherein the at least one mutation is a combination of K87Y or Q273V, V212 / I215L, and I363V / K365S.

[0089] The recombinant transposase of the present invention is capable of transposing a transposon comprising a heterologous polynucleotide adjacent to transposon flanking regions, wherein the left transposon flanking region has the nucleotide sequence of SEQ ID NO:2 and the right transposon flanking region has the nucleotide sequence of SEQ ID NO:3, or the left transposon flanking region has the nucleotide sequence of SEQ ID NO:29 and the right transposon flanking region has the nucleotide sequence of SEQ ID NO:30.

[0090] The heterologous NLS is preferably fused to the C-terminus and / or N-terminus of the recombinant transposase of the present invention. The heterologous NLS can be any NLS that mediates the transport of proteins from the cytoplasm to the nucleus. In mammalian cells, an NLS that mediates the transport of proteins from the cytoplasm to the nucleus is preferred. Typically, the NLS is derived from a eukaryotic protein, preferably a mammalian protein or a viral protein. The heterologous NLS can be a classical nuclear localization signal (cNLS), such as a monopartite NLS (MP NLS), including the amino acid sequence of SEQ ID NO: 5, and a bipartite NLS (BP NLS), including the amino acid sequence of SEQ ID NO: 14, a non-classical NLS (ncNLS), or any other type of NLS (also called atypical NLS). The heterologous NLS (e.g., SEQ ID NO: 5) can be fused to the recombinant transposase directly or via a peptide linker (e.g., SEQ ID NO: 22, where the linker has the sequence of SEQ ID NO: 52).

[0091] The recombinant transposase of the present invention may further comprise at least one mutation. Thus, in certain embodiments, the recombinant transposase comprises at least one mutation and has higher activity compared to the transposase of SEQ ID NO: 4. High activity may be determined for various reasons, such as half-life, stability, folding, activity, DNA binding, and dimerization. Exemplary mutations include, but are not limited to, amino acid substitutions selected from the group consisting of K87Y, Q273V, V212I / I215L, I363V / K365S, K87Y / Q273V, K87Y / A264S / Q273V, A264S / Q273V, S270P / Q273V, K87Y / A264S, L583F, K576I, S372E, S277N, and any combination thereof, and / or deletion of N584 and / or E585. wherein the indicated amino acid positions of the substitutions and / or deletions correspond to the amino acid positions in the sequence of SEQ ID NO:4. Preferably, at least one mutation is an amino acid substitution selected from the group consisting of K87Y, Q273V, V212 / I215L, I363V / K365S, A264S / Q273V, K87Y / A264S, L583F or a combination thereof, and / or at least one mutation is a deletion of N584 and / or E585. In a particular embodiment, at least one mutation is 1 to 7 amino acid substitutions, preferably 1 to 5 amino acid substitutions and / or a deletion of N584 and / or E585. In a more specific embodiment, at least one mutation is 1 to 7 mutations, preferably 1 to 5 mutations. More preferably, the at least one mutation is the substitution K87Y or a combination of Q273V, V212 / I215L, and I363V / K365S. In certain specific embodiments, the recombinant transposase comprises an amino acid sequence having at least 99% sequence identity with amino acids 10 to 585 of SEQ ID NO:4, and further comprising at least one mutation.wherein at least one mutation is one to five amino acid substitutions selected from the group consisting of K87Y, Q273V, V212 / I215L, I363V / K365S, A264S / Q273V, K87Y / A264S, and L583F, and optionally deletion of N584 and / or E585, preferably wherein at least one mutation is a combination of substitutions K87Y or Q273V, V212 / I215L, and I363V / K365S. Transposase activity is preferably determined as enhanced recovery and / or productivity by selection using a DNA transposon comprising transposon flanking regions having the nucleotide sequences of SEQ ID NOs: 2 and 3 or the nucleotide sequences of SEQ ID NOs: 29 and 30, and encoding antibody heavy and light chains as the protein of interest (e.g., as exemplified in Example 9). A highly active transposase means a transposase activity that is greater than 100%, preferably greater than about 120%, more preferably greater than about 150%, and even more preferably greater than about 200% of that of a transposase having the amino acid sequence of SEQ ID NO: 4. In a preferred embodiment, a recombinant transposase comprising at least one mutation of the present invention comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 10 to 585 of SEQ ID NO: 4 or 26, preferably SEQ ID NO: 4 or 26, more preferably SEQ ID NO: 4.

[0092] In yet another aspect, the present invention relates to a polynucleotide encoding a transposase, comprising an amino acid sequence having at least 90% sequence identity to at least amino acids 10-585 of SEQ ID NO:4 or SEQ ID NO:26, operably linked to a eukaryotic promoter. In certain embodiments, the eukaryotic promoter is not the native transposase promoter, i.e., a heterologous eukaryotic promoter. The promoter can be any promoter compatible with or active in a eukaryotic host cell. Suitable eukaryotic promoters include, but are not limited to, the EF1a promoter, cytomegalovirus (CMV) promoter, GAPDH promoter, CAG promoter, herpes simplex virus thymidine kinase (HSV-TK) promoter, murine stem cell virus (MSCV) promoter, spleen-limited focus-forming virus (SFFV) promoter, SV40 promoter, actin promoter, PGK promoter, and ubiquitin promoter. Additionally, the promoter may be an inducible promoter. The term "operably linked" is used synonymously with "under the control of" and means that an expression control sequence, e.g., a promoter (or transcription termination sequence), functionally controls the expression of one or more genes of interest, etc. Typically, an expression control sequence, e.g., a promoter (or transcription termination sequence), is contiguous with a gene of interest.

[0093] In yet another aspect, the present invention relates to a polynucleotide encoding the recombinant transposase of the present invention. The polynucleotide can be DNA or RNA. The transposase can be introduced into a host cell, for example, as a protein by microinjection or as DNA or mRNA by transduction or transfection. The DNA polynucleotide encoding the recombinant transposase can be part of an expression vector, such as a plasmid DNA, antibiotic resistance marker-free plasmid (pFAR), minicircle (MC), doggybone DNA (dbDNA), bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), or viral vector, preferably a non-integrating viral vector. Thus, the source of the recombinant transposase can be a protein, an expression vector, or mRNA. In a preferred embodiment, the recombinant transposase is introduced into a host cell by transfection of an expression vector, such as a plasmid DNA or mRNA. In a preferred embodiment, one or more codons of the polynucleotide encoding the transposase are selected for eukaryotic cell expression, preferably yeast, insect, or mammalian cell expression, more preferably mammalian cell expression, e.g., rodent or human cell expression, i.e., the polynucleotide encoding the transposase is codon-optimized, particularly for the expression host cell, e.g., a mammalian cell.

[0094] In yet another aspect of the present invention, there is provided an expression vector encoding a recombinant transposase of the present invention or comprising a polynucleotide encoding the recombinant transposase of the present invention. In a specific embodiment, the expression vector is a plasmid DNA, an antibiotic resistance marker-free plasmid (pFAR), a minicircle (MC), a doggie bone DNA (dbDNA), a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or a viral vector, preferably a plasmid DNA or a BAC.

[0095] In yet another aspect of the present invention, an isolated mRNA encoding the recombinant transposase of the present invention is provided. The mRNA can be modified to make it more stable. Those skilled in the art will be aware of mRNA modifications optimized for translation and stability, such as nucleotide substitutions using pseudouridine, 5-methyl-cytosine, or 5-methoxy-uridine, and / or capping of mRNA for translation, such as mCAP analog (m7G(5')ppp(5')G) or ARCA cap (Anti-Reverse Cap analog, 3'-O-Me-m7G(5')ppp(5')G).

[0096] In yet another aspect of the present invention, there is provided an expression system or kit comprising: (a) a recombinant transposase source selected from the group consisting of (i) an expression vector encoding a recombinant transposase of the present invention or comprising a polynucleotide encoding the recombinant transposase of the present invention, (ii) isolated mRNA encoding the recombinant transposase of the present invention, and (iii) a recombinant transposase of the present invention; and (b) a DNA transposon of the present invention or an expression vector comprising the DNA transposon of the present invention. The expression system or kit may further comprise (c) a transfection agent and / or (d) a eukaryotic cell.

[0097] Use of DNA transposons and / or recombinant transposases The present invention further relates to uses or methods of using the transposase / DNA transposon pair of the invention, i.e. the DNA transposon of the invention and the recombinant transposase of the invention.

[0098] In one aspect, a pharmaceutical composition is provided comprising (a) a DNA transposon of the invention and (i) a recombinant transposase of the invention or (ii) an isolated mRNA of the invention, or (b) a human cell comprising a DNA transposon of the invention, wherein the cell is preferably autologous to the patient.

[0099] In another aspect, the pharmaceutical composition of the present invention is for use in gene therapy or for use in the treatment of cancer or autoimmune disease. In a specific embodiment, the gene therapy can be replacement gene therapy for treating a genetic disease, such as a recessive genetic disease. In a specific embodiment, the cancer is a hematological cancer or solid cancer, preferably a hematological cancer. Preferably, the gene of interest encodes a chimeric antigen receptor (CAR), and the human cells are human T cells or human NK cells. In a preferred embodiment, the use comprises ex vivo transfection of the patient's autologous cells, followed by adoptive cell transfer of the autologous cells. Thus, in a specific embodiment, the pharmaceutical composition is for use in the treatment of cancer, particularly hematological cancer, where the autologous cells are T cells or NK cells, and the treatment comprises ex vivo transfection of autologous T cells or NK cells with a DNA transposon comprising a polynucleotide comprising a sequence encoding a CAR, and subsequent adoptive cell transfer of the autologous T cells or NK cells comprising the DNA transposon into the patient.

[0100] The present invention further relates to methods, in particular in vitro methods, for stably incorporating heterologous polynucleotides into cells, for example when preparing cell lines or for producing a product of interest, in particular a protein of interest, or for generating virus packaging cell lines.

[0101] In one aspect, the present invention relates to a method for producing a cell comprising a stably integrated heterologous polynucleotide, the method comprising: (a) introducing into a eukaryotic cell a DNA molecule comprising a DNA transposon of the present invention or an expression vector comprising said DNA transposon of the present invention, wherein the heterologous polynucleotide comprises a sequence encoding a gene of interest, a complementary DNA (cDNA), a genome of interest, or another genetic element, and wherein the heterologous polynucleotide further comprises a sequence encoding a selectable marker; (b) introducing into said eukaryotic cell a recombinant transposase source, wherein the recombinant transposase source is selected from the group consisting of (i) an expression vector encoding a recombinant transposase of the present invention, (ii) an isolated mRNA of the present invention, and (iii) a recombinant transposase of the present invention; and (c) culturing the eukaryotic cell in a medium under conditions for selecting for the selectable marker, wherein the DNA transposon is stably integrated into the genome of the eukaryotic cell. The method may further comprise the step of (d) isolating a single clone for clonal expansion to prepare a monoclonal cell line.

[0102] In another aspect, the present invention provides a method for producing a protein of interest, comprising: (a) introducing into a eukaryotic cell a DNA molecule comprising a DNA transposon of the present invention or an expression vector comprising said DNA transposon of the present invention, wherein the heterologous polynucleotide comprises a sequence encoding at least one protein of interest and further comprises a sequence encoding a selectable marker; (b) introducing into said eukaryotic cell a recombinant transposase source, wherein the recombinant transposase source comprises (i) an expression vector encoding a recombinant transposase of the present invention, (ii) an isolated mRNA of the present invention, and (iii) (b) culturing eukaryotic cells in a medium under conditions for selecting a selectable marker, wherein a heterologous polynucleotide comprising a sequence encoding at least one protein of interest and a sequence encoding a selectable marker is stably integrated into the genome of the eukaryotic cells; (c) optionally isolating a single clone for clonal propagation to prepare a monoclonal cell line; (d) culturing the eukaryotic cells under conditions for producing the protein of interest; and (d) collecting and optionally purifying the protein of interest.

[0103] In yet another aspect, the present invention relates to a method for producing a virus or virus-like particle of interest or a method for preparing a virus packaging cell line as specified herein in paragraphs 39 or 40, respectively.

[0104] In general, any DNA transposon of the present invention can be used in combination with any recombinant transposase of the present invention. However, those skilled in the art will understand that each DNA transposon needs to be tested in combination with a specific recombinant transposase, for example, as shown in the Examples. The identified recombinant transposases derived from AP (SEQ ID NO: 4) and AC (SEQ ID NO: 26) efficiently transpose DNA transposons containing AP (SEQ ID NOs: 2 and 3) and AC (SEQ ID NOs: 29 and 30) transposon flanking regions, respectively, resulting in comparable selection behavior and productivity, particularly when the same heterologous protein expression is used and / or detected. Surprisingly, it was found that recombinant AP transposases even more efficiently transpose DNA transposons containing AC (SEQ ID NOs: 29 and 30) transposon flanking regions. The same applies to truncated forms of AP (SEQ ID NOs: 2 and / or 3) or AC transposon flanking regions (SEQ ID NOs: 29 and / or 30), as described herein.

[0105] Certain preferred embodiments of the uses and methods of the present invention use (i) a DNA transposon comprising transposon flanking regions from an AC and an AC or AP transposase, or (ii) a DNA transposon comprising transposon flanking regions from an AP and an AP transposase, and in particularly preferred embodiments, a DNA transposon comprising transposon flanking regions from an AC and an AP transposase is used.

[0106] For example, in certain embodiments, the DNA molecule in step (a) comprises a DNA transposon comprising a heterologous polynucleotide flanked by transposon flanking regions, or an expression vector encoding said DNA transposon, wherein (i) one transposon flanking region comprises a sequence having at least 95% sequence identity to SEQ ID NO: 29, 62, 63, or 64 and / or the other transposon flanking region comprises a sequence having at least 97% sequence identity to SEQ ID NO: 30, 65, 66, 67, 60, or 61; or (ii) wherein one transposon the flanking region and the other transposon flanking region are inverted and comprise the reverse complement of the sequence described in (i), and the recombinant transposase source in step (b) encodes or is a recombinant transposase comprising an amino acid sequence having at least 90% sequence identity with amino acids 10 to 585 of SEQ ID NO: 4, optionally further comprising at least one mutation and / or at least one heterologous NLS fused to the transposase as defined for the recombinant transposase of the invention. Exemplary mutations include, but are not limited to, amino acid substitutions selected from the group consisting of K87Y, Q273V, V212I / I215L, I363V / K365S, K87Y / Q273V, K87Y / A264S / Q273V, A264S / Q273V, S270P / Q273V, K87Y / A264S, L583F, K576I, S372E, S277N, and any combination thereof, and / or deletions of N584 and / or E585, where the indicated amino acid positions of the substitutions and / or deletions correspond to the amino acid positions in the sequence of SEQ ID NO:4. Preferably, at least one mutation is an amino acid substitution selected from the group consisting of K87Y, Q273V, V212 / I215L, I363V / K365S, A264S / Q273V, K87Y / A264S, L583F or a combination thereof and / or at least one mutation is a deletion of N584 and / or E585.In a particular embodiment, the at least one mutation is 1 to 7 amino acid substitutions, preferably 1 to 5 amino acid substitutions and / or deletion of N584 and / or E585. In a more specific embodiment, the at least one mutation is 1 to 7 mutations, preferably 1 to 5 mutations. More preferably, the at least one mutation is the substitution K87Y or a combination of Q273V, V212 / I215L and I363V / K365S. In certain specific embodiments, the recombinant transposase comprises an amino acid sequence having at least 99% sequence identity to amino acids 10 to 585 of SEQ ID NO:4, and further comprises at least one mutation, wherein the at least one mutation is one to five amino acid substitutions selected from the group consisting of K87Y, Q273V, V212 / I215L, I363V / K365S, A264S / Q273V, K87Y / A264S, L583F and optionally a deletion of N584 and / or E585, preferably wherein the at least one mutation is the substitution K87Y or a combination of Q273V, V212 / I215L and I363V / K365S.

[0107] In a preferred embodiment, the DNA transposon comprises transposon flanking regions derived from AC and is used in combination with an AP transposase. In a specific embodiment, the transposase comprises the amino acid sequence of amino acids 10 to 585 of SEQ ID NO:26 or an amino acid sequence having at least 99% sequence identity to amino acids 10 to 585 of SEQ ID NO:26, and optionally further comprises at least one mutation described herein. The DNA transposons used in combination preferably have: (i) one transposon flanking region that is at least 90% (preferably at least 95%, at least 97%, at least 98%, at least 100%) identical to at least SEQ ID NO:64, preferably at least SEQ ID NO:63, at least SEQ ID NO:62, or SEQ ID NO:29; and / or the other transposon flanking region comprises a sequence having at least 97% (preferably at least 98%, at least 99% or 100%) sequence identity to at least SEQ ID NO: 60 or 61, preferably at least SEQ ID NO: 67, at least SEQ ID NO: 66, at least SEQ ID NO: 65 or more preferably SEQ ID NO: 30; or (ii) a DNA transposon in which one transposon flanking region and the other transposon flanking region are inverted and comprise the reverse complement of the sequence described in (i). The same as disclosed for the uses and methods of the invention apply equally to the expression system or kit of the invention.

[0108] The protein of interest is preferably a therapeutic protein, such as a therapeutic protein selected from the group consisting of cytokines, hormones, fusion proteins, antibodies, antibody-derived molecules and antibody mimetics, such therapeutic proteins being described in more detail herein above.

[0109] In a specific embodiment of the method of the present invention, a DNA molecule comprising a DNA transposon of the present invention or an expression vector encoding a transposase of the present invention can be introduced by transfection or transduction, preferably by transfection. Similarly, a recombinant transposase of the present invention or an mRNA encoding a recombinant transposase of the present invention is preferably introduced by transfection.

[0110] The production method of the present invention is an in vitro method. Eukaryotic host cells include, inter alia, yeast cells and mammalian cells, preferably mammalian cells. Yeast cells can be, but are not limited to, Saccharomyces cerevisiae, Pichia pastoris, Klyveromyces lactis, or marxianus. As used herein, mammalian cells refer to any cell or cell line derived from a mammal, e.g., human or rodent cells, and may also be referred to as "host cells" or "mammalian host cells." The cells referred to herein are cells maintained in culture, e.g., cell lines or cell line-derived cells, i.e., immortalized cells or ex vivo primary cells. Primary cells are cells isolated from an organ tissue or organism and maintained in vitro for propagation and / or adoptive cell transfer into a patient or subject. In the case of adoptive cell transfer, the primary cells are preferably autologous cells of the patient. Mammalian cells further include mammalian cell lines suitable for producing a product of interest, e.g., a heterologous protein and / or non-coding RNA of interest. Mammalian cells are preferably transformed and / or immortalized cell lines. Cell lines are adapted for continuous passage in cell culture, preferably serum-free cell culture and / or preferably suspension culture, and do not contain primary untransformed cells or cells that are part of an organ structure.

[0111] Preferably, the mammalian host cell is a human or rodent cell, more preferably a rodent cell, and even more preferably a CHO cell. Preferred mammalian cells for heterologous protein production are rodent or human cells. Preferred examples of mammalian cells or mammalian cell lines are CHO cells (e.g., DG44 and K1), NS0 cells, HEK293 cells (e.g., HEK293 cells and HEK293T cells), and BHK21 cells. Preferably, the mammalian cell or mammalian cell line is adapted to grow in suspension. In a preferred embodiment, the mammalian cell or mammalian cell line is a CHO cell. In a specific embodiment, the mammalian cell is an HEK293 cell or a CHO cell or a cell derived from an HEK293 cell or a CHO cell, and preferably the mammalian cell is a CHO cell or a CHO cell-derived cell.

[0112] Suitable rodent cells can be, for example, hamster cells, in particular BHK21, BHK TK-, CHO, CHO-K1, CHO-DXB11 (also called CHO-DUKX or DuxB11), CHO-S cells, and CHO-DG44 cells, or derivatives / progeny of any of such cell lines. Particularly preferred are CHO cells, such as CHO-DG44, CHO-K1, and BHK21, more preferably CHO-DG44 and CHO-K1 cells. Most preferred are CHO-DG44 cells. Also included are glutamine synthase (GS)-deficient derivatives of mammalian cells, in particular CHO-DG44 and CHO-K1 cells. In one embodiment of the invention, the mammalian cells are Chinese hamster ovary (CHO) cells, preferably CHO-DG44 cells, CHO-K1 cells, CHO DXB11 cells, CHO-S cells, CHO GS-deficient cells, or derivatives thereof. Suitable human cells are HEK293 cells or HEK293T cells. The host cells can also be murine cells, such as murine myeloma cells, e.g., NS0 cells and Sp2 / 0 cells, or derivatives / progeny of any of such cell lines.

[0113] Preferably, CHO cells, which enable an efficient cell line development process, are metabolically engineered, for example, by knocking out endogenous glutamine synthase (GS) to facilitate selection with methionine sulfoximine (MSX). As used herein, the term "GS gene knockout cells" refers to cells in which the endogenous GS gene has been knocked out, i.e., deleted or disrupted, resulting in disruption of GS enzyme function. Such cells may be referred to as GS- / - cells or GS- / + cells, depending on whether both or only one allele has been deleted or disrupted; preferably, GS- / - cells are used. Extracellular glutamine supplementation or the GS gene introduced by an expression vector is essential for cell survival of GS gene knockout cells. In a preferred embodiment, the mammalian host cells are CHO-K1 cells, more preferably CHO-K1-GS (GS- / -) cells. CHO cells commonly used in large-scale industrial production are often engineered to improve characteristics or facilitate selection of recombinant cells in the manufacturing process. Such manipulations include, but are not limited to, improving resistance to apoptosis, reducing autophagy, increasing cell proliferation, altering the expression of cell cycle regulatory proteins, chaperone manipulation, manipulation of the unfolded protein response (UPR), manipulation of the secretory pathway, and metabolic manipulation.

[0114] Non-limiting examples of mammalian cells that can be used within the context of the present invention are summarized in Table B. However, other mammalian cells, including (but not limited to) derivatives / progeny of these cells, human, mouse, rat, monkey and rodent cell lines, can also be used according to the present invention, particularly for the production of biopharmaceutical proteins.

[0115] [Table 2]

[0116] Most preferred are cells that are established, adapted, and cultured entirely under serum-free conditions, optionally in media that do not contain any animal-derived proteins / peptides. Commercially available media, such as Ham's F12 (Sigma, Deisenhofen, Germany), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium (DMEM; Sigma), Minimum Essential Medium (MEM; Sigma), Iscove's Modified Dulbecco's Medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, CA), serum-free CHO medium (Sigma), and protein-free CHO medium (Sigma), are exemplary suitable nutrient solutions. Any of these media can be supplemented with various compounds as needed, non-limiting examples of which include recombinant hormones and / or other recombinant growth factors (e.g., insulin, transferrin, epidermal growth factor, insulin-like growth factor), salts (e.g., sodium chloride, calcium, magnesium, phosphate), buffers (e.g., HEPES), nucleosides (e.g., adenosine, thymidine), glutamine, glucose or other equivalent energy sources, antibiotics, and trace elements. Any other necessary supplements can also be included at appropriate concentrations that would be known to those skilled in the art. For the growth and selection of genetically modified cells expressing a selectable gene, an appropriate selection agent is added to the culture medium.

[0117] The protein of interest encoded by a eukaryotic expression vector or produced by the methods of the invention is preferably produced in CHO cells in cell culture. After expression, the recombinant protein is harvested and further purified. The antibody can be recovered from the culture medium as a secreted protein in harvested cell culture fluid (HCCF) or from a cell lysate (i.e., a fluid containing the contents of cells lysed by any means, including, but not limited to, enzymatic, chemical, osmotic, mechanical, and / or physical disruption of the cell membrane and, optionally, the cell wall) and purified using the techniques described herein. According to the invention, this method involves providing, as a starting material, harvested cell culture fluid containing the protein of interest, e.g., an antibody, wherein the HCCF is derived from a CHO cell culture. Preferably, the protein of interest, e.g., an antibody, is recovered from the harvested cell culture fluid by cell separation, e.g., filtration and / or centrifugation. Thus, in certain embodiments, harvesting includes centrifugation, clarification, and / or filtration to produce a harvested cell culture fluid, preferably followed by one or more filtration steps, e.g., ultrafiltration and / or dialysis. The purified protein can optionally be formulated and optionally reformulated into a solid or liquid, preferably a liquid, composition designed for the intended use. Such formulations are, in principle, known to those skilled in the art and may include, for example, buffers, stabilizers, and / or other excipients for use in human or veterinary medical treatment.

[0118] In view of the above, it will be appreciated that the present invention also encompasses the following:

[0119] Item 1 provides a DNA transposon comprising a heterologous polynucleotide adjacent to transposon flanking regions, wherein one transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 38 at one end of the transposon, and the other transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 39 at the other end of the transposon.

[0120] Item 2 further defines the DNA transposon of Item 1, wherein one transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 40 at one transposon end, and the other transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 41 at the other transposon end.

[0121] Item 3 specifies the DNA transposon of Item 1 or 2, wherein each of the one and the other transposon flanking regions further comprises at least one inverted internal repeat (IIR) comprising (i) an internal repeat motif comprising the nucleotide sequence tggtctac and (b) its reverse complement (iii) separated by 3 to 6, preferably 4, nucleotides, and preferably wherein at least one inverted internal repeat (IIR) motif has the nucleotide sequence of SEQ ID NO:43.

[0122] Item 4 further includes (a) one transposon flanking region comprising the nucleotide sequence of SEQ ID NO: 45 or 46, and further comprising an inverted internal repeat (IIR) motif having the nucleotide sequence of SEQ ID NO: 43 separated by approximately 50 to 200 nucleotides, and / or the other transposon flanking region comprising the nucleotide sequence of SEQ ID NO: 47 or a sequence having at least 95% sequence identity to SEQ ID NO: 47, wherein the sequence comprises at least the nucleotide sequence of SEQ ID NO: 41 and the nucleotide sequence of SEQ ID NO: 43; or (b) wherein one transposon flanking region and the other transposon flanking region are inverted and comprise the reverse complement of the sequence described in (a), preferably wherein (a) one transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 45 or 46. and (b) wherein one transposon flanking region and the other transposon flanking region are inverted and comprise the reverse complement of the sequence described in (i).

[0123] Clause 5 further defines the DNA transposon of any one of clauses 1 to 4, wherein (a) one transposon flanking region comprises a sequence having at least 85% nucleotide sequence identity to SEQ ID NO: 2, 8, 10, or 58 and / or the other transposon flanking region comprises a sequence having at least 90% nucleotide sequence identity to SEQ ID NO: 3, 9, 11, or 59, or (b) wherein one transposon flanking region and the other transposon flanking region are inverted and comprise the reverse complement of the sequence set forth in (a). Paragraph 6 further provides: (1) (a) one transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 2, 8, 10, or 58, or the nucleotide sequence of SEQ ID NO: 29, 62, 63, or 64, and / or the other transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 3, 9, 11, 59, 48, or 49, or the nucleotide sequence of SEQ ID NO: 30, 65, 66, 67, 60, or 61; or (b) wherein one transposon flanking region and the other transposon flanking region are inverted and have the reverse of the sequence described in (a). or (2) (a) one transposon flanking region comprises a sequence having at least 95% sequence identity to SEQ ID NO: 29, 62, 63 or 64 and / or the other transposon flanking region comprises a sequence having at least 97% sequence identity to SEQ ID NO: 30, 65, 66, 67, 60 or 61; or (b) wherein one transposon flanking region and the other transposon flanking region are inverted and comprise the reverse complement of the sequence described in (a).

[0124] Paragraph 7 further defines the DNA transposon of any one of paragraphs 1 to 6, wherein the heterologous polynucleotide comprises at least one sequence selected from the group consisting of a sequence encoding a gene of interest, a complementary DNA (cDNA), a genome of interest, and another genetic element.

[0125] Paragraph 8 further provides the DNA transposon of paragraph 7, wherein the heterologous polynucleotide comprises at least one sequence under the control of a promoter and a transcription termination signal.

[0126] Paragraph 9 further provides the DNA transposon of paragraph 8, wherein the promoter is a eukaryotic promoter, preferably selected from the group consisting of (1) an EF1a promoter, a CMV promoter, a GAPDH promoter, a CAG promoter, a herpes simplex virus thymidine kinase (HSV-TK) promoter, an MSCV promoter, an SFFV promoter, an SV40 promoter, and an actin promoter, a PGK promoter, and a ubiquitin promoter, and / or (2) an inducible promoter.

[0127] Item 10 further comprises the heterologous polynucleotide comprising: (i) antibody heavy and / or light chains; (ii) secreted recombinant therapeutic proteins; (iii) recombinant proteins; (iv) transmembrane receptors, (v) a non-coding RNA that mediates RNA interference (RNAi), preferably a non-coding RNA selected from the group consisting of siRNA, shRNA, lncRNA, and miRNA; (vi) one or more viral proteins; (vii) a viral genome or viral genome cDNA; (viii) ribozymes, (ix) binding motifs, (x) Regulatory DNA elements (xi) other genetic elements and (xii) Any combination of (i) to (xi) The present invention provides a DNA transposon according to any one of Items 1 to 9, comprising at least one sequence selected from the group consisting of one or more sequences encoding:

[0128] Paragraph 11 further provides for the DNA transposon of any one of paragraphs 1 to 10, wherein the heterologous polynucleotide further comprises a sequence encoding a selectable marker, preferably a metabolic selectable marker, more preferably glutamine synthase (GS) or dihydrofolate reductase (DHFR), even more preferably GS.

[0129] Item 12 further defines the DNA transposon of any one of Items 1 to 11, wherein the transposon is transposable by a transposase comprising the amino acid sequence of SEQ ID NO:4.

[0130] Paragraph 13 further provides the DNA transposon of any one of paragraphs 1 to 12, wherein the DNA transposon is present on a plasmid DNA, a plasmid without an antibiotic resistance marker (pFAR), a minicircle (MC), a doggybone DNA (dbDNA), a bacterial artificial chromosome (BAC), a yeast artificial chromosome, or a non-integrating viral vector.

[0131] Item 14 provides an expression vector comprising the DNA transposon according to any one of Items 1 to 13.

[0132] Paragraph 15 provides a recombinant transposase comprising at least one heterologous nuclear localization signal (NLS), preferably at least a heterologous C-terminal and / or N-terminal NLS, fused to the transposase, wherein the transposase is an Acyrthosiphon pisum transposase.

[0133] Paragraph 16 further defines the recombinant transposase of Paragraph 15, wherein the transposase comprises an amino acid sequence having at least 90%, preferably 98%, sequence identity to amino acids 10 to 585 of SEQ ID NO:4.

[0134] Item 17 provides a recombinant transposase comprising an amino acid sequence having at least 90%, preferably 98%, sequence identity to amino acids 10 to 585 of SEQ ID NO:4.

[0135] Paragraph 18 further defines the recombinant transposase of Paragraph 17, wherein (a) the transposase is an Acyrthosiphon pisum transposase or an Aphis craccivora transposase, and / or (b) the transposase comprises at least one heterologous NLS fused to the transposase.

[0136] Item 19 further defines the recombinant transposase according to any one of Items 15 to 18, wherein the transposase comprises the amino acid sequence of amino acids 10 to 585 of SEQ ID NO: 4, preferably the amino acid sequence of SEQ ID NO: 4, or the amino acid sequence of amino acids 10 to 585 of SEQ ID NO: 26, preferably the amino acid sequence of SEQ ID NO: 26.

[0137] Paragraph 20 further provides the recombinant transposase of any one of paragraphs 15 to 19, wherein the transposase comprises at least one mutation and is more active than the transposase of SEQ ID NO:4.

[0138] Paragraph 21 further relates to a method for producing a transposase comprising the steps of: (a) a transposase comprising: a transposase having at least one mutation; (b) a transposase having at least one mutation; and (c) a transposase having at least one mutation; and (d) a transposase having at least one mutation; and 21. The recombinant transposase of any one of paragraphs 15 to 20, wherein the indicated amino acid positions of the substitutions and / or deletions correspond to the amino acid positions in the sequence of SEQ ID NO: 4, and preferably at least one mutation is an amino acid substitution selected from the group consisting of K87Y, Q273V, V212 / I215L, I363V / K365S, A264S / Q273V, K87Y / A264S, L583F or a combination thereof, and / or at least one mutation is a deletion of N584 and / or E585, optionally wherein at least one heterologous NLS is fused to the transposase.

[0139] Paragraph 22 provides a polynucleotide encoding a transposase, comprising an amino acid sequence having at least 90%, preferably 98%, sequence identity to amino acids 10 to 585 of SEQ ID NO:4, operably linked to a eukaryotic promoter.

[0140] Item 23 provides a polynucleotide encoding the recombinant transposase according to any one of Items 15 to 21.

[0141] Paragraph 24 further defines the polynucleotide of Paragraph 23, wherein the polynucleotide is DNA or RNA.

[0142] Paragraph 25 further provides for the polynucleotide of any one of paragraphs 22 to 24, wherein one or more codons of the polynucleotide encoding the transposase are selected for eukaryotic cell expression, preferably yeast or mammalian cell expression, more preferably mammalian cell expression (e.g., rodent or human cell expression).

[0143] Item 26 provides an expression vector comprising the polynucleotide according to any one of Items 22 to 25 or encoding the recombinant transposase according to any one of Items 15 to 21.

[0144] Item 27 provides an isolated mRNA encoding the recombinant transposase according to any one of Items 15 to 21.

[0145] Item 28 provides an expression system or kit comprising: (a) (i) the expression vector of Item 26; (ii) the isolated mRNA of Item 27; and (iii) the recombinant transposase of any one of Items 15 to 21; and (b) a recombinant transposase source selected from the group consisting of the DNA transposon of any one of Items 1 to 13 or the expression vector of Item 14.

[0146] Item 29 provides a eukaryotic cell comprising the DNA transposon of any one of Items 1 to 13.

[0147] Paragraph 30 further provides the eukaryotic cell of paragraph 29, wherein the eukaryotic cell is a yeast or mammalian cell, preferably a mammalian cell, more preferably a rodent or human cell.

[0148] Paragraph 31 further provides for the eukaryotic cell of paragraph 29 as a mammalian cell, wherein the heterologous polynucleotides flanking the transposon flanking regions are preferably one or more viral genes from AAV, more preferably the AAV rep and / or cap genes and optionally further at least one helper virus gene (e.g., from HSV or AdV).

[0149] Paragraph 32 further provides the eukaryotic cell of paragraph 29, wherein the cell is a viral packaging cell.

[0150] Paragraph 33 provides the use of the cell of paragraph 31 or 32 for virus production, preferably AAV production.

[0151] Item 34 provides a non-human transgenic animal comprising the DNA transposon according to any one of Items 1 to 13.

[0152] Item 35 provides a pharmaceutical composition comprising: (a) a DNA transposon according to any one of Items 1 to 13 and a recombinant transposase according to any one of Items 15 to 21 or an isolated mRNA according to Item 27; or (b) a human cell comprising a DNA transposon according to any one of Items 1 to 13, wherein the cell is preferably an autologous cell of the patient.

[0153] Paragraph 36 further provides for the pharmaceutical composition of paragraph 30 for use in gene therapy or cancer treatment.

[0154] Paragraph 37 provides a method for producing a cell comprising a stably integrated heterologous polynucleotide, the method comprising: (a) introducing into a eukaryotic cell a DNA molecule comprising the DNA transposon of any one of Paragraphs 1-13 or the expression vector of Paragraph 14, wherein the heterologous polynucleotide comprises a sequence encoding a gene of interest, a complementary DNA (cDNA), a genome of interest, or another genetic element, and wherein the DNA transposon further comprises a sequence encoding a selectable marker; (b) introducing into the eukaryotic cell a recombinant transposase source, wherein the recombinant transposase source is selected from the group consisting of (i) the expression vector of Paragraph 26, (ii) the isolated mRNA of Paragraph 27, and (iii) the recombinant transposase of any one of Paragraphs 15-21; and (c) culturing the eukaryotic cell in a medium under conditions to select for the selectable marker, wherein the DNA transposon is stably integrated into the genome of the eukaryotic cell.

[0155] Paragraph 38 is a method for producing a protein of interest, comprising: (a) introducing into a eukaryotic cell a DNA molecule comprising the DNA transposon of any one of paragraphs 1 to 13 or the expression vector of paragraph 14, wherein the heterologous polynucleotide comprises a sequence encoding at least one protein of interest and further comprises a sequence encoding a selectable marker; (b) introducing into the eukaryotic cell a recombinant transposase source, wherein the recombinant transposase source is selected from (i) the expression vector of paragraph 26, (ii) the isolated mRNA of paragraph 27, and (iii) the recombinant transposase of any one of paragraphs 15 to 21. (c) culturing eukaryotic cells in a medium under conditions to select for a selectable marker, wherein the heterologous polynucleotide comprises a sequence encoding at least one protein of interest, and the sequence encoding the selectable marker is integrated into the genome of the eukaryotic cell; (d) optionally isolating a single clone for clonal propagation to prepare a monoclonal cell line; (e) culturing the eukaryotic cells under conditions to produce the protein of interest; and (f) collecting and optionally purifying the protein of interest.

[0156] Paragraph 39 is a method for producing a virus or virus-like particle of interest, comprising: (a) introducing into a eukaryotic cell a DNA molecule comprising the DNA transposon of any one of Paragraphs 1 to 13 or the expression vector of Paragraph 14, wherein the heterologous polynucleotide comprises a sequence encoding at least one virus or virus-like particle of interest and further comprises a sequence encoding a selectable marker; (b) introducing into the eukaryotic cell a recombinant transposase source, wherein the recombinant transposase source is selected from (i) the expression vector of Paragraph 26, (ii) the isolated mRNA of Paragraph 27, and (iii) the recombinant transposase of any one of Paragraphs 15 to 21. (c) culturing eukaryotic cells in a medium under conditions to select for a selectable marker, wherein the heterologous polynucleotide comprises a sequence encoding at least one virus or virus-like particle of interest, and the sequence encoding the selectable marker is integrated into the genome of the eukaryotic cell; (d) optionally isolating a single clone for clonal propagation to prepare a monoclonal cell line; (e) culturing eukaryotic cells under conditions to produce the virus or virus-like particle; and (f) collecting and optionally purifying the virus or virus-like particle.

[0157] Paragraph 40 is a method for producing a viral packaging cell line, comprising: (a) introducing into a eukaryotic cell a DNA molecule comprising the DNA transposon of any one of paragraphs 1 to 13 or the expression vector of paragraph 14, wherein the heterologous polynucleotide comprises a sequence encoding at least one viral gene of interest and further comprises a sequence encoding a selectable marker; (b) introducing into the eukaryotic cell a recombinant transposase source, wherein the recombinant transposase source is selected from (i) the expression vector of paragraph 26, (ii) the isolated mRNA of paragraph 27, and (iii) the recombinant transposase source of paragraph 28. (c) culturing eukaryotic cells in a medium under conditions to select for a selectable marker, wherein the heterologous polynucleotide comprises a sequence encoding at least one viral gene, and the sequence encoding the selectable marker is integrated into the genome of the eukaryotic cell; (d) optionally isolating a single clone for clonal propagation to prepare a monoclonal cell line; and (e) culturing the eukaryotic cells under conditions to produce the at least one viral gene.

[0158] Paragraph 41 provides for the method of paragraph 40, further comprising, after step (c) or (d), respectively, step (c1) or (d1) of transiently transfecting the eukaryotic cell with at least one further viral gene and / or transducing the eukaryotic cell with a helper virus, and optionally, step (f) of harvesting and optionally purifying the virus or virus-like particle.

[0159] Paragraph 42 further provides the method of paragraph 40 or 41, wherein at least one viral gene is derived from AAV, preferably wherein the at least one viral gene is the AAV rep gene and / or cap gene.

[0160] Paragraph 43 further provides the method of paragraph 39, wherein the at least one virus or virus-like particle of interest is AAV or VSV, and wherein in the case of AAV, the method may further comprise (a) transducing the eukaryotic cell with a helper virus (e.g., from HSV or AdV) or (b) transfecting the eukaryotic cell with at least one helper virus gene (e.g., from HSV or AdV).

[0161] Paragraph 44 further provides the method of any one of paragraphs 39 to 43, wherein the eukaryotic cell is a mammalian cell.

[0162] Paragraph 45 further provides the method of paragraph 37 or 44, wherein the method is an in vitro method.

[0163] Example method Plasmid generation The transposon plasmids for stable transfection contain promoter-driven antibody expression cassettes, ampicillin resistance, and metabolic glutamine synthase selection markers. Plasmids encoding the transposases were cloned and co-transfected with each transposon. Circular plasmids were used for transfection.

[0164] Host cell culture CHO-K1 host cells (with a genomic knockout of the endogenous glutamine synthase gene) were cultured in host cell medium supplemented with L-glutamine. Host cell cultures were initiated at a seeding density of 3 x 10E05 cells / mL. Growth conditions were set at 36.5°C and 5% CO2 in a shaking incubator at 120 rpm in a shaker flask. Cell density and viability measurements were performed using a Cedex HiRe® cell count analyzer.

[0165] Transposase and transposon transfection into CHO The day before transfection, host cells were seeded into shake flasks at a cell density of 0.8 x 10E06 cells / mL. On the day of transfection, cell density and viability were measured, and the amount of cells required for transfection was calculated. The cells were centrifuged at 750 x g for 7 minutes, the supernatant was discarded, and 1-3 μg transposon and 1-2 μg transposase plasmid DNA were added per shot using the Neon® transfection system. An electroporation cuvette was filled with 3 mL of electroporation buffer E2, and cells (5 x 10E6 cells) were resuspended in approximately 90 μl of buffer R (total volume 100 μl) and mixed with 4 μg transposase and transposon plasmid. Transfection was performed at 1500 V, 10 mS, and two pulses. The transfected cells were transferred to 5 ml of pre-warmed host medium in a T25 ml flask and incubated at 8% CO2 and 37°C for at least 24 hours.

[0166] Alternatively, the transposase was transfected as mRNA (1-2 μg per transfection) using the same procedure.

[0167] Selection of stable CHO pools 24 hours after transfection, cells were transferred to selection conditions (medium without L-glutamine). 10 mL of selection medium was pre-warmed per pool in a T75 flask. Two stable pools were cultured for each transfection.

[0168] The cells were centrifuged (750 × G for 7 minutes) and the supernatant was discarded. The cells were resuspended in 20 mL of selection medium and incubated at 37°C with 8% CO2. Cells were monitored microscopically during selection. Additional medium (5 mL) was added after 3–5 days.

[0169] Passaging stable pools The selection step was considered successful when cell viability reached at least 70% and a doubling time of at least 48 hours. After selection, cells were passaged in 125 mL shake flasks in a total volume of 30 mL at 36.5°C and 5% CO2 with 120 rpm shaking, starting at 3 x 10E05 cells / mL. Samples for titer determination were collected periodically. Antibody titers were measured using a ForteBio Octet instrument with a Protein A biosensor.

[0170] Copy number measurement by ddPCR To measure the transgene copy number in stable pools, we applied the ddPCR (digital droplet PCR) method. Genomic DNA was isolated from washed and centrifuged stable pool cells (0.5 × 106 cells per sample) using QiaSymphonyn.

[0171] 300 ng of sample genomic DNA was digested with one or two restriction enzymes (according to the manufacturer's protocol) to cut the antibody cassette at both ends. Restriction enzyme digestion was performed at 37°C for 1 hour. The sample was then diluted 1:10 with nuclease-free water. To measure copy number, two reactions were prepared per sample: one containing the primer-probe set for the heavy chain and the other containing the primer-probe set for the light chain. The housekeeping gene Eif3i was used to normalize the DNA amount. Droplets were generated using an automated droplet generator (Biorad) followed by PCR. Analysis and evaluation were performed using a droplet reader (Biorad) and QuantaSoft software (Biorad).

[0172] Relative gene expression by ddPCR To measure relative transgene expression in stable pools, we applied ddPCR (digital droplet PCR). Total RNA was isolated from washed and centrifuged cells (0.5 × 10 cells per sample) of stable pools using QiaSymphonyn. The isolated RNA was diluted, and 0.5 ng was used to prepare a reaction mixture. This reaction mixture contained both components for reverse transcription of RNA and subsequent cDNA amplification. Both reactions were performed sequentially in the same tube using an optimized PCR protocol. The housekeeping gene Eif3i was used for DNA normalization. Droplets were generated using an automated droplet generator (Biorad), followed by RT-PCR followed by cDNA PCR in a single run. Analysis and evaluation were performed using a droplet reader (Biorad) and QuantaSoft software (Biorad).

[0173] Fed-batch bioprocess evaluation The bioprocess performance of a stable pool producing a monoclonal antibody (IgG1) was evaluated in an advanced microbioreactor system (ambr™15, Sartorius) under standard fed-batch culture conditions. The process duration was 14 days, with nutrient feeding initiated on day 2 after inoculation. Cells were seeded on day 0 at a density of 0.7 x 106 viable cells per mL, and the cell culture temperature was set at 34.5°C. Cell culture parameters were monitored daily (product titration began on day 9). The culture pH was set at 6.95 ± 0.25, and the glucose concentration was increased to 6 g / L if it fell below 4 g / L.

[0174] Cell surface staining and flow cytometry analysis NRP1 expression and cell surface presentation were assessed by cell surface staining with a fluorescent dye-labeled anti-NRP1 antibody and subsequent flow cytometric analysis. For cell staining, 0.5 × 10 cells per sample were washed and resuspended in a PBS-based buffer containing BSA, EDTA, and sodium azide, then transferred to a 96-well V-bottom plate. The plate containing the cells was centrifuged (300 × g, 5 min), and the PBS buffer was replaced with staining solution containing anti-NRP1 antibody or an isotype control. Host cell negative controls were incubated with PBS buffer. Samples were incubated at 4°C for 45 min in the dark. Stained cells were washed again with PBS buffer and then analyzed using a NovoCyte flow cytometer (Agilent). For analysis, the cell populations of interest were assessed using FSC-H vs. SSC-H scatter plots. The signal intensity of the bound antibody was determined by the coupled APC fluorescent dye in an APC-H histogram. The threshold for identifying APC-positive cells was set in a stained but untransfected host cell sample and kept constant for all subsequent measurements.

[0175] Example 1: Identification of a novel transposase / transposon pair from Acyrthosiphon pisum We describe the identification of a novel transposase / transposon pair from Acyrthosiphon pisum (AP). Based on the published genome sequence, 384 candidate transposase / transposon-like sequences were identified. These sequences were screened for repetitive ITR-like elements 3' and 5' of the candidate transposase sequences in the genomic vicinity. Furthermore, the candidate transposase sequences were evaluated for catalytic amino acid residues (DDE motifs). Fourteen transposase / transposon-like sequences were experimentally tested, and surprisingly, only one sequence pair (identified from the Acyrthosiphon pisum genome sequence) demonstrated active transposition in eukaryotic (Chinese hamster ovary, CHO) cells (see Figure 24 and Example 17 for selected results). Transposase activity was demonstrated in transfection experiments using a circular plasmid DNA vector and mRNA encoding the AP transposase.

[0176] To the best of our knowledge, this novel transposase / transposon pair from Acyrthosiphon pisum has not been reported previously. The novel transposase / transposon pair from Acyrthosiphon pisum itself represents an additional orthogonal transposase / transposon pair functionally active in eukaryotic cells. This transposase specifically recognizes the transposon ends discovered. This system is significantly different from any published transposase / transposon system, as transposition stably integrates any cargo gene into the genome of the host cell of interest.

[0177] Example 2: Identification and activity testing in CHO cells A novel transposase / transposon pair from Acyrthosiphon pisum (NCBI Reference Sequence: XP_029340918.1) was experimentally demonstrated to be active in CHO cells, resulting in the transfer of the transposon encoding flanking sequences and ITRs, as well as a monoclonal antibody expression cassette, into the host cell genome. CHO-K1 GS- / - cells (with a biallelic knockout of the endogenous glutamine synthase gene) were transfected with a transposon vector encoding a metabolic selectable marker (glutamine synthase) and an expression cassette for a therapeutic monoclonal antibody under the control of a CMV promoter. The transposon vector contained an ITR core sequence (SEQ ID NO: 1) within both the left flanking sequence of SEQ ID NO: 2 and the right flanking sequence of SEQ ID NO: 3. Additionally, a second plasmid encoding the Acyrthosiphon pisum transposase (SEQ ID NO: 4) with a heterologous N-terminal nuclear localization signal (SEQ ID NO: 5, full sequence SEQ ID NO: 6) under the control of the CMV promoter was transfected, and stable cells were selected in cell culture medium lacking L-glutamine (Figure 1).

[0178] As a control, CHO K1 GS- / - cells were transfected with the same transposon vector but without the transposase plasmid. The viability of the transfected cells was measured 18 days after transfection (Figure 2). CHO cells transfected with the transposon and transposase recovered from the selection process after approximately 12 days, with a viability of over 70%, indicating successful transposition, thus indicating stable integration of the transposon into the CHO cell genome. Viability further increased to a stable level of over 90%. In contrast, negative control cells (transfected with the transposon alone) did not recover from the selection experiment.

[0179] Stable transfection of antibody expression cassettes as transgene transposon cargo resulted in highly productive and stable recombinant CHO cell lines (Figure 3), which would be highly suitable for the production of therapeutic proteins. The established stable cell lines showed higher antibody titers and improved stability of antibody expression compared to pools established by random integration of the transgene. A negative control cell sample lacking the transposase plasmid did not yield highly productive stable cell pools.

[0180] Random integration of transgenes often leads to concatemerization, fragmentation, rearrangement, and recombination of the transgene, resulting in transgene copy imbalance and partial loss of transgene or selectable marker copies in the genomes of stable recombinant cell lines. In contrast, stable transgene integration mediated by transposases follows a cut-and-paste mechanism, leaving single intact copies of the transposon cargo at various genomic loci in the resulting stable recombinant cell lines. Genetic analysis of stable CHO pools cotransfected with AP transposase and transposons demonstrated that the copy numbers of the therapeutic monoclonal antibody heavy and light chain genes were highly balanced (Figure 4).

[0181] Example 3: Effect of heterologous nuclear localization signals on transposition efficiency The effects of adding additional heterologous nuclear localization signals to the C- and N-termini of the AP transposase gene were investigated to determine transposition efficiency and the generation of stable recombinant CHO cell pools expressing a therapeutic antibody. The AP transposase plasmid was cotransfected with a transposon plasmid encoding a therapeutic antibody under the control of a CMV promoter, SV40-controlled expression of the glutamine synthase gene as a metabolic selection marker, and flanking sequences of SEQ ID NO:2 and SEQ ID NO:3 (including the ITR sequences of SEQ ID NO:1). Initial sequence analysis showed that the naturally occurring transposase gene already exhibits an NLS-like sequence (SEQ ID NO:25) at its N-terminus. We investigated AP transposase genes without an additional N-terminal NLS (SEQ ID NO: 4), with one additional N-terminal NLS (SEQ ID NO: 6), or with two NLS motifs at both the N- and C-termini (transposase: SEQ ID NO: 7; NLS SEQ ID NO: 22 (N-terminus) and SEQ ID NO: 14 (C-terminus)). All constructs successfully survived the selection step, indicating that the antibody and selectable marker expression cassettes were stably integrated into the genome of CHO host cells through active transposition of the transposase / transposon combination. The construct with two NLS motifs (SEQ ID NO: 7) reached a viability of over 70% already after 7 days of selection, significantly faster than the other constructs with SEQ ID NO: 4 and SEQ ID NO: 6 (Figure 5). The other constructs reached a viability of over 70% only after 12 and 9 days, respectively.

[0182] These experiments demonstrated that increasing the number of NLS motifs and attaching these sequences to the N- and C-termini increased the transposition efficiency of the transposase / transposon system, as indicated by metabolic selection leading to faster growth of stably transfected cells. This may be driven by increased nuclear import and / or higher expression of the transposase enzyme. Furthermore, titration measurements confirmed that the AP transposase construct with two NLS motifs resulted in a higher productivity, approximately two-fold improved pool yield, compared to a transposase construct lacking an additional nuclear localization signal (Figure 6).

[0183] We further investigated whether the presence of the Flag tag used in the construct with the sequence of SEQ ID NO: 7 affects the transposase activity of the AP transposase. In a direct comparison, stable pools transfected with SEQ ID NO: 7 and SEQ ID NO: 32 (lacking the Flag tag) performed equally well, indicating that the Flag tag did not affect the performance and transposase activity of the AP transposase protein (Figures 7 and 8).

[0184] Example 4: Examination of flanking sequences The flanking sequences (SEQ ID NO: 2 and SEQ ID NO: 3) are essential for AP transposase to recognize the transposon and induce stable genomic integration via transposition. The flanking sequences were extracted from the genomic sequences 3' and 5' proximal to the transposase gene. To identify minimal functional flanking sequence motifs, a series of truncated flanking sequences (Seq IDs 2, 3, 10, 11, 12, and 13) were designed and tested for transposition in CHO cells. All truncated flanking sequences had the ITR sequence of SEQ ID NO: 1 in the left (5') flanking region or the reverse-complement ITR sequence of SEQ ID NO: 15 in the right (3') flanking region (Figure 9A).

[0185] CHO cells were transfected with a plasmid encoding the AP transposase and a transposon encoding an expression cassette for a therapeutic antibody with varying combinations of flanking sequences (see Figure 9A). Flanking sequence combinations SeqID2+3 (SEQ ID NO:2 and SEQ ID NO:3) and SeqID10+11 (SEQ ID NO:10 and SEQ ID NO:11) survived the selection process, indicating that the transposition event was active and the transposase / transposon combination was functional (Figures 9B and 9C). Flanking sequence combination SeqID12+13 (SEQ ID NO:12 and SEQ ID NO:13) remained non-functional (Figure 9B).

[0186] Stable and highly productive CHO pools could only be generated with functional transposons SeqID2+3 and SeqID10+11, where both transposon setups reached comparable titers (Figure 9C).

[0187] Sequence alignments of the left and right flanking sequences are shown in Figures 10A and 10B. The flanking sequences were further analyzed for internal repeat motifs. Functional flanking sequences further exhibit at least one internal repeat motif (IR) (tggtctac) and its reverse complement (together referred to as an inverted internal repeat (IIR)) separated by four nucleotides, where at least the first of these nucleotides is an adenosine (SEQ ID NO: 43). Conserved sequences of SEQ ID NO: 43 (or more specifically, SEQ ID NO: 44) were identified in both flanking arms: two in the left flanking arm and one in the right flanking arm.

[0188] Therefore, the right flanking region likely needs to contain at least one IIR (tggtctac) and its reverse complement separated by four nucleotides, i.e., at least nt 349 to nt 415 of SEQ ID NO: 3, or nt 88 to nt 154 of SEQ ID NO: 11, or SEQ ID NO: 47, 48, or 49. In the left transposon flanking arm, both IIRs are likely to be important. Therefore, the left flanking region is expected to require at least the sequence of SEQ ID NO: 40, 45, or 46, plus a variable stretch of nucleic acid, e.g., an inverted internal repeat motif having the sequence of SEQ ID NO: 43, separated by approximately 50 to 200 nucleotides.

[0189] Example 5: Further consideration of flanking sequences In further experiments, either the right or left flanking regions were truncated to better understand the individual effects and minimal motifs (Fig. 11A).

[0190] CHO cells were transfected as described in Example 4 with transposons encoding expression cassettes for therapeutic antibodies with a full-length AP left flanking sequence (SEQ ID NO:2) and a truncated AP right flanking sequence (SEQ ID NO:3 (WT), SEQ ID NO:9, SEQ ID NO:11, and SEQ ID NO:13, respectively) (Figure 11A, upper panel and Figures 11B and 11C) or a truncated left AP flanking sequence (SEQ ID NO:2 (WT), SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12, respectively) and a full-length right AP flanking sequence (SEQ ID NO:3) (Figures 11D and 11E). SEQ ID NO:8 is the left transposon flanking sequence truncated at 297 nucleotides, i.e., midway between SEQ ID NO:2 (340 nt) and SEQ ID NO:10 (214 nt). SEQ ID NO:9 is the right transposon flanking sequence truncated at 260 nucleotides, i.e., midway between SEQ ID NO:3 (415 nt) and SEQ ID NO:11 (145 nt).

[0191] In the first experiment, the left flanking sequence (SEQ ID NO: 2) was kept constant, while the right flanking sequence was systematically truncated (SEQ ID NOs: 9, 11, and 13). The selection behavior and productivity of stable pools were compared with the AP wild-type control with full-length flanking sequences (Seq ID 2 and Seq ID 3). When the right flanking sequence was systematically truncated to Seq ID 9 or Seq ID 11, the selection period was clearly longer compared to the wild-type control, but overall productivity remained unchanged (Figures 11B and 11C). Further shortening of the right flanking sequence to Seq ID 13 rendered the transposon dysfunctional, and cells did not recover from metabolic selection (Figure 11B). Alignment of the right flanking sequences (SEQ ID NOs: 3, 11, and 13 in Figure 10B) with annotated repeat and binding motifs suggests that deleting one of the internal repeats of the IIR motif in SEQ ID NO: 13 is detrimental to transposon function. Furthermore, the full-length right transposon flanking region (SEQ ID NO: 3) appears to be superior to all truncated right flanking sequences.

[0192] In a second experiment, the right flanking sequence (SEQ ID NO: 3) was kept constant, while the left flanking sequence was systematically truncated (SEQ ID NOs: 8, 10, and 12). The selection behavior and productivity of stable pools were compared to AP wild-type controls with full-length flanking sequences (SEQ ID NOs: 2 and 3). When the left transposon flanking sequence was systematically truncated to SeqID8 or SeqID10, the selection period and overall productivity remained unchanged. This suggests that transposon dysfunction is not expected as long as both IIR motifs remain intact (Figures 11D and 11E). Further shortening of the left flanking region to SEQ ID NO: 12 rendered the transposon dysfunctional, and cells did not recover from metabolic selection (Figure 11D). Figure 10A shows an alignment of the left transposon flanking sequence with annotated repeat and binding motifs. This suggests that deletion of one of the IIR motifs in SEQ ID NO: 12 abolishes the function of the transposon flanking region.

[0193] As expected, when the truncated left and right flanking sequences were combined (left: SeqIDs 10, 12; right: SeqIDs 13, 11), the transposon became dysfunctional and cells did not recover from metabolic selection (Figure 11F).

[0194] We further analyzed whether swapping the left and right transposon sequences affected selection and productivity. In this experiment, a transposon encoding an antibody expression cassette was transfected using SEQ ID NO:10 and SEQ ID NO:11 as the left and right transposon sequences, respectively, and a second transposon containing the reversed order was tested (Figure 12, top). Cell culture performance was assessed by monitoring the selection step and determining productivity. As can be seen in Figure 12 (middle and bottom), swapping the orientation of the flanking sequences can result in similar cell culture performance.

[0195] Example 6: Examination of motif changes in flanking regions Previous experiments have shown that the binding and repeat motifs found in the AP transposon flanking regions play an important role in mediating efficient transposition. Furthermore, the flanking regions were modulated by artificially introducing additional motifs, as shown in Figure 13A, to investigate the effects on selection behavior and productivity.

[0196] Based on the finding that removing the IIR motif from the left flanking region (see Figure 10A, SEQ ID NO: 12) impairs selection behavior (see Figure 11D), various modified left flanking regions were designed (Figure 13; SEQ ID NO: 53 (SeqID53), SEQ ID NO: 55 (SeqID55), and SEQ ID NO: 57 (SeqID57)). The first design (SeqID55) is a modified construct containing two additional binding motifs and a binding-IIR motif setup (SEQ ID NO: 54, corresponding to nt 1-54 of SEQ ID NO: 49 and nt 349-402 of SEQ ID NO: 3) taken from the right AP transposon flanking region, providing specific distances relative to the native motifs to avoid potential steric effects. In the second design (SeqID57), the construct was further complemented with an additional binding motif following the newly introduced IIR in an attempt to introduce further variation into the modular concept. The third design (SeqID53) takes the concept of the minimum length required for the flanking regions from previous experiments and knowledge of the significance of the second native IIR motif. This design (SeqID53) is a truncated variant containing only the ITRs of SeqID55, the first native binding and IIR motifs, and an artificially added binding and binding-IIR motif (SEQ ID NO: 54), which, like SeqID55, originate from the right flanking region but terminate immediately after this artificially introduced IIR. This setup will demonstrate whether the artificially added motif can rescue the selection behavior and productivity impairment caused by the removal of the second native IIR (SEQ ID NO: 12), as shown in Figure 11D.

[0197] All left flanking region designs were combined with a minimal right flanking region variant (SEQ ID NO: 49 or SeqID 49) with the intent of first, confirming that this minimal sequence was functional, and second, creating a baseline for subsequent changes in the right flanking region.

[0198] The results show that all of the introduced modifications significantly impaired metabolic selection behavior compared to the wild-type flanking region setups (SeqID2 and SeqID3) (Figure 13B). In contrast, overall productivity remained almost unchanged (Figure 13C). The effects of SeqID55 and SeqID57 were comparable, with a slight decrease for SeqID57 using the modular concept approach. Interestingly, the minimal right flanking region setup proved functional in all tested combinations. In particular, in combination with the minimal setup of SeqID53, productivity was almost equivalent to that of the wild-type. This also demonstrates that the artificially introduced motif of SeqID53 has a partial rescue effect on the removal of the second native repeat motif.

[0199] In a subsequent step, the above-tested left flanking region modifications (SeqID53, SeqID55, and SeqID57) were combined with a modified right flanking region (SeqID56) containing an additional copy of the native binding motif and binding-IIR motif unit (SEQ ID NO:54) to test the effect of the second IIR motif present in the right flanking sequence. Only the combination with SeqID55 resulted in similar metabolic selection behavior compared to the minimal sequence of SEQ ID NO:49, while the other combinations with SEQ ID NO:53 and SEQ ID NO:57 showed dramatically impaired recovery from selection (Figure 13B). Productivity of the modified right flanking region SEQ ID NO:56 remained lower than that of the wild-type and the combination with SEQ ID NO:49 (Figure 13C). In summary, the right flanking region showed high sensitivity to changes in the configuration of the binding and repeat motifs, which was reflected in the detrimental effects on selection behavior and productivity. However, the minimal right flanking region appears to be effective and sufficient for transfer.

[0200] Example 7: Combination with glutamate synthase from Providencia vermicola The AP transposase / transposon was tested on an improved transposon setup with an attenuated bacterial glutamine synthase selection marker from Providencia vermicola, as previously described in EP 22163849.7. Selection behavior was comparable between both metabolic selection markers tested (Figure 14).

[0201] Surprisingly, stable CHO pools transfected with transposons carrying the AP transposase and bacterial glutamine synthase selection marker showed an approximately 7-fold increase in recombinant antibody titer (Figure 15), demonstrating that the AP transposase and each transposon can also be used with different, more stringent metabolic selection markers.

[0202] Example 8: Bioprocess performance of stable pools generated by AP transposase To investigate the cell culture performance of stable CHO cells expressing recombinant proteins, stably selected pools were cultivated under fed-batch process conditions in a controlled bioreactor system.

[0203] To this end, CHO-K1 cells were cotransfected with transposons encoding the heavy and light chain genes of a monoclonal antibody in combination with the AP transposase and an attenuated bacterial glutamine synthase selectable marker from Providencia vermicola. To assess productivity in the bioreactor, three stable CHO cell pools expressing recombinant monoclonal antibodies were seeded into bioreactors (N=2 per pool) and cultured for 14 days. From day 2 onwards, nutrient feed solution was added to the cultures daily, and cell culture parameters, including cell growth, viability, and monoclonal antibody titer, were monitored up to day 14.

[0204] Stable CHO cell pools expressing monoclonal antibodies generated using AP transposase demonstrated very good and robust bioprocess performance, with final product titers reaching up to 5 g / L (Figure 16). In addition, all three stable pools showed similar culture behavior with respect to cell growth, viability, and product titer formation, indicating a high level of robustness and reproducibility.

[0205] Example 9: Bioprocess performance of stable pools generated with AP transposase mutants To further improve the AP transposase protein, novel protein variants were engineered and tested for improved activity based on wild-type AP transposase (nucleic acid sequence SEQ ID NO:31, amino acid sequence SEQ ID NO:32) with N- and C-terminal NLSs. CHO-K1 cells were transfected as described, and the selection process was monitored. Table 1 summarizes the protein variants tested. The variants were primarily designed based on a classical family shuffling approach.

[0206] [Table 3] TIFF2025536126000004.tif53161

[0207] Most mutants were dysfunctional, and no stable antibody-expressing CHO pools were obtained (--). Protein mutant A264S maintained functionality with comparable performance compared to wild-type AP (Table 1 and Figure 17). Surprisingly, protein mutant K87Y (nucleic acid sequence SEQ ID NO: 33, amino acid sequence SEQ ID NO: 34) showed improved selection and productivity behavior. Cells transfected with K87Y AP transposase grew faster from selection, reaching a viability of over 70% in 9 days compared to 12 days for wild-type AP transposase (Figure 17). This was accompanied by improved productivity (Table 1). Similar results were observed for I363V / K365S AP transposase (SEQ ID NO: 35) and K87Y / A264S AP transposase (SEQ ID NO: 37) compared to the wild-type (Table 1). Although not to the same extent, improved performance (selection and / or productivity) was also observed for AP transposases with the following mutations: Q273V, V212I / I215L, L583F, K576I, S372E, and S277E (Table 1). Combinations are expected to further improve performance and result in more active AP transposases. For example, the A264S / Q273V AP transposase (SEQ ID NO: 36) was shown to be more effective than either mutation alone, whereas the combination of K87Y and Q273V appeared to reduce the effectiveness of K87Y alone (K87Y / Q273V and K87Y / A264S / Q273V). Surprisingly, deletion of the last two amino acids (N584 / E585) also resulted in improved selection and productivity behavior.

[0208] The mutations L582F, K576I, I578F, and D577F, as well as the deletions N584 and E585, were predicted to improve the hydrophobicity of the C-terminus, thereby improving dimerization. At least L582F and the deletions N584 and E585 are expected to have a positive effect on selection behavior and productivity, and K576I is also expected to have a positive effect on selection behavior. Furthermore, S372E and Q492K are predicted to increase DNA binding, and S277N and T306K are predicted to optimize the catalytic domain. At least S372E and S277N are expected to have a positive effect on productivity without affecting selection behavior.

[0209] In this regard, it is noted that wild-type AP transposase exhibits transposition efficiency comparable to that of other commercially available transposases, which are typically highly active mutants (e.g., piggy-BAC or sleeping beauty transposase). Therefore, it is hypothesized that the highly active mutants of AP transposase described herein and further improved mutants of AP transposase, for example, obtained by combining mutations, have the potential to produce transposases with even higher transposition efficiency.

[0210] Example 10: Identification of additional AP transposase variants with improved activity profiles Further protein engineering and mutagenesis were performed to improve the activity profile of AP mutants using cellular assays. Primarily, selection behavior (the time it takes to recover from metabolic selection and reach at least 70% viability) and productivity during cell passage were investigated. Additional mutants and, in particular, combinations of mutations were found to improve either selection behavior or selection behavior and productivity. Both parameters are advantageous individually, and mutants with improvements in both parameters are particularly promising.

[0211] [Table 4]

[0212] With the exception of one mutant (K576I+L583F), further combinations of mutations resulted in improvements in at least one of selection behavior or productivity.

[0213] Example 11: Generation of bioassay cell lines stably expressing cell surface receptors The novel AP transposase system was further used to generate stable cell lines for cell-based bioassays using transmembrane receptor expression. This experiment aimed to provide deeper insight into the ability of the AP transposase system to enable the expression of various types of molecules other than common antibodies or secreted proteins. To generate bioassay cell lines, CHO K1 host cells were cotransfected with a vector containing the gene sequence for the cell surface receptor neuropilin-1 (NRP1) and a vector encoding the AP transposase. The transfected cells were then subjected to a selection process to generate stable cell pools. To evaluate the transgene integration efficiency, the average gene copy number of the cell pool was determined by isolating genomic DNA and performing droplet digital PCR (ddPCR). The same methodology concept was applied to the isolated RNA to determine transgene expression. In the final step, successful translation and cell surface presentation of the receptor protein was assessed by flow cytometry. Cell surface staining for flow cytometry analysis of NRP1 presentation was performed with a fluorochrome-conjugated anti-NRP1 antibody. Transfected cell pools were compared with untransfected host cell samples to determine cell surface NRP1 levels.

[0214] Duplicate transfected stable cell pools were generated in three unique experiments. Gene copy number analysis showed a highly homogenous pattern, both within and across experiments, with final copy numbers ranging from 3 to 8 copies per cell (Figure 18A). These results demonstrate a high level of robustness of AP transposase-associated gene integration in the context of generating homogenous stable cell pools.

[0215] The functionality and activity of the integrated transgene were confirmed at the transcriptional level. Relative gene expression of the NRP1 transgene could be observed in all three experiments (Figure 18B). Expression levels varied from a 2-fold increase to a maximum of 9-fold increase compared to the housekeeping gene. Again, duplicates within each experiment showed highly homogeneous gene expression levels. Across experiments, the homogeneity of gene expression was less pronounced. This effect was expected, as gene transcription is strongly influenced by downstream factors, such as the gene integration locus, epigenetic modifications, and metabolic constraints.

[0216] At the final stage of transgene expression, we analyzed the success of NRP1 receptor translation and localization. Based on the role of NRP1 receptor translocation to the extracellular membrane, we performed cell surface staining with a fluorochrome-labeled anti-NRP1 antibody on stably transfected cell pools. Stained cells were analyzed by flow cytometry to measure the signal intensity of the cell surface-bound fluorochrome antibody. This signal intensity correlates with NRP1 levels. To assess the effect of endogenous NRP1 levels, we included untransfected host cell samples in the analysis as a reference for determining the threshold for positive signal. Cell surface staining experiments revealed high levels of NRP1 signal in all transfected cell pools, with the majority of cell bodies exhibiting positive signals (Figure 19). Again, replicates within the experiment were highly comparable. Interestingly, samples from Experiment 2 showed at least two cell populations present in the cell pool with different signal intensity levels. This coincidence may explain the generally low relative gene expression levels observed in the previous analysis. In contrast, experiments 1 and 3 showed comparable signal intensities clearly derived from one major population. Overall, the histograms from the latter two experiments indicate a highly homogenous pool of cells.

[0217] Stable cell pools expressing the cell surface receptor NRP1 generated using the novel AP transposase system demonstrated successful results at the genetic, transcriptional, and translational levels. Transfected cells produced sufficient quantities of apparently structurally intact NRP1 receptors that were correctly localized to the cell surface. All generated cell pools showed high homology in gene copy number and a correlation between transcription and translation rates. The AP transposase system demonstrated its robustness and flexibility in generating stable pools expressing secreted proteins, e.g., molecules other than antibodies. Such cell pools may serve as a source of cell clones that can be utilized for various applications, including cell-based bioassays.

[0218] Example 12: Generation of stable cell lines for expression of recombinant proteins other than antibodies In another approach, we utilized a novel AP transposase system for the recombinant expression of alternative proteins, e.g., lipase. In this experiment, we investigated the ability of the AP transposase system to express tagged proteins with various molecular properties other than monoclonal antibodies.

[0219] Therefore, CHO-K1 cells were cotransfected with transposons encoding lipase and Providencia glutamine synthase and a vector encoding the AP transposase. After transfection, positively transfected cells were selected by culturing the cell culture in glutamine-free medium. After the selection process, the yield of recombinant lipase was measured by octet assay to capture the overexpressed lipases. Overall, five lipases were overexpressed, and two independent stable pools were obtained for each lipase.

[0220] Using the previously described combination of the novel AP transposase system and Providencia glutamine synthase, we generated recombinant pools expressing five cHCPs and were able to scale up to shake flasks within 7–10 days. Due to efficient integration of the transposon, only a 15–40% decrease in viability was observed for the various transfections during the selection process. Negative controls transfected with transposon alone did not recover after selection began. Interestingly, pools transfected with the same lipases exhibited nearly identical viability curves during the selection phase (data not shown). Differences in selection time and viability between recombinantly expressed lipases may correlate with the effects of overexpressed lipase on CHO cells.

[0221] After the selection process was completed, the resulting pool was inoculated into a shake flask fed-batch culture for 8 days to recombinantly express lipases with AP transposase. For all lipases, significant amounts of lipase protein were expressed, with protein yields ranging from 30 mg / L to a maximum of 1.5 g / L after 8 days, depending on the overexpressed lipase and its effect on the CHO expression host (data not shown).

[0222] Expression of recombinant lipases using the Acyrthosiphon pisum transposase system was successful for all five lipases. Interestingly, the final lipase protein yield strongly correlated with the selection period and the effect of the overexpressed lipase on CHO cell growth. In particular, overexpression of Lipase 3 limited CHO cell growth and was the least efficient lipase for recombinant overexpression. In contrast, overexpression of Lipase 1 showed little loss of viability during pool selection, and strong titers in the g / L range were obtained after only 8 days of fermentation. These data demonstrate the ability of the AP transposase system to recombinantly overexpress proteins other than therapeutic mAbs, provided the overexpressed enzyme does not adversely affect the expression host itself.

[0223] Thus, the identified transposase / transposon pair from Acyrthosiphon pisum provides a versatile system for generating stable cell lines expressing various recombinant proteins in CHO cells, and experiments are underway to confirm its applicability in human cells, such as HEK293 cells. It is anticipated that this system could also be used for the production of viruses, particularly VSV and adeno-associated viruses, or virus-like particles derived therefrom. Furthermore, it is anticipated that this system could be used for stable integration in gene therapy, particularly ex vivo integration into autologous cells followed by adoptive cell transfer into patients.

[0224] Example 13: Related Homologues A transposase from Aphis craccivora was discovered (nucleic acid sequence SEQ ID NO:27, amino acid sequence SEQ ID NO:26), which is closely related to the Acyrthosiphon pisum transposase. The amino acid sequences are highly homologous, sharing 98.3% sequence identity with the transposase protein (Table 3).

[0225] [Table 5]

[0226] Analysis of the context of nearby genomic sequences revealed that the ITR core sequence (SeqID2) was identical between both related strains, and the flanking sequences were highly homologous (Tables 4 and 5).

[0227] [Table 6]

[0228] [Table 7]

[0229] Given the high homology between the transposase and ITR / flanking sequences for Acyrthosiphon pisum and Aphis craccivora, the Aphis craccivora transposase / transposon combination may be more functional. To this end, we generated an Aphis craccivora transposase (SEQ ID NO: 28) containing N- and C-terminal NLSs and an N-terminal Flag tag.

[0230] Due to the high homology between the transposases and ITR / flanking sequences of Acyrthosiphon pisum and Aphis craccivora, transposase / transposon combinations were tested for cross-reactivity. The sequence alignment of the AP transposase (SEQ ID NO: 4) with the AC transposase (SEQ ID NO: 26) (Figure 20C), the sequence alignment of the AP left transposon flanking region (SEQ ID NO: 2) with the AC left transposon flanking region (SEQ ID NO: 29) (Figure 20D), and the sequence alignment of the AP right transposon flanking region (SEQ ID NO: 3) with the AC right transposon flanking region (SEQ ID NO: 30) (Figure 20E) are shown in Figure 20.

[0231] When glutamine synthase-deficient CHO cells were transfected with the Aphis craccivora transposase (SEQ ID NO:26) and an Acyrthosiphon pisum-based transposon (with flanking sequences of SEQ ID NO:2 and SEQ ID NO:3 and a glutamine synthase metabolic selection marker), the cells did not recover from selection (Figure 20A), indicating that the Aphis craccivora transposase does not function with the Acyrthosiphon pisum-based transposon.

[0232] In contrast, when the Aphis craccivora transposase (SEQ ID NO: 26) was combined with its native flanking sequences (SEQ ID NO: 29 and SEQ ID NO: 30), CHO cells recovered from selection and generated stable pools with antibody expression titers comparable to those of the AP transposase / transposon pair. This demonstrates that the discovery of additional functions is highly relevant to transposases / transposons in mammalian cells. Surprisingly, when the Aphis craccivora transposon with the flanking sequences of SEQ ID NO: 29 and SEQ ID NO: 30 was combined with the Acyrthosiphon pisum transposase (SEQ ID NO: 4), CHO cells recovered from selection significantly faster compared to either wild-type reference (Figure 20A). This combination resulted in a significant increase in overall growth and productivity, as determined by antibody titration (Figure 20B). This demonstrates an unexpectedly strong enhancement of cross-reactivity and overall system activity. Because the AP and AC transposases differ by only 10 amino acids, mutational analysis will indicate which amino acids account for the subtle differences in activity between the two transposases.

[0233] Thus, both the transposase / transposon pairs identified from Acyrthosiphon pisum and Aphis craccivora are excellent transposase / transposon pairs. Surprisingly, combining the Aphis craccivora transposon with the Acyrthosiphon pisum transposase was more effective, while the reverse combination was not functional. Furthermore, the AP transposase and / or AC transposase can be further improved by using mutants with improved activity, as described herein, e.g., in Examples 9, 10, and 14.

[0234] Example 14: Analysis of AP transposase mutants with improved activity profiles in combination with AC transposons As shown in Example 13, when the Aphis craccivora (AC) transposon with flanking sequences SEQ ID NO:29 and SEQ ID NO:30 was combined with the AP transposase (SEQ ID NO:4), CHO cells recovered from selection significantly faster compared to either wild-type control (AP / AP or AC / AC). This combination also resulted in significant improvements in overall growth and productivity. The superior performance of the AP transposase plus AC transposon was confirmed in a controlled bioreactor (Ambr15 system) and under representative controlled process conditions. Additionally, several combinations of the AP transposase mutants from Example 9 were further tested in parallel with the AC and AP transposons.

[0235] Cell culture performance was evaluated using a two-step approach. First, the selection period was evaluated and productivity in shake flasks was investigated over multiple passages. These experiments confirmed that selected combinations of AP transposase mutations outperformed the wild-type AP enzyme, confirming the superiority of the AC transposon in combination with the AP transposase. Overall selection behavior improved by up to 5 days, and productivity levels increased by up to 3-fold (see Table 6).

[0236] [Table 8]

[0237] The same stable pools were subjected to a controlled bioprocess in the Ambr15 system to investigate the cell culture process conditions in more detail. Cell growth (viable cell density) as well as titer and key metabolic parameters were studied in a 14-day fed-batch production process (Figure 21). Controlled bioreactor experiments confirmed the findings of the initial shake flask studies. The combination of wild-type AP transposase with AC transposon and the combination of AP mutant V212I+I215L+I363V+K365S+Q273V with AC or AP transposons showed excellent performance, with titers reaching over 6 g / L for the AC(transposon)-AP(transposase) and AC(transposon)-AP(transposase V212I+I215L+I363V+K365S+Q273V) sample pools on day 14. For comparison, the titer in the AP (transposon)-AP (transposase) sample pool reached approximately 3 g / L on day 14.

[0238] Thus, the data demonstrate that transposase performance can be improved by introducing one or more activity-improving mutations that have a positive effect on the generation of stable cell pools (selection behavior and productivity), which is also reflected in bioprocess parameters (cell viability and growth, as well as productivity) in controlled bioprocesses of stable cell pools. Furthermore, the data confirm the advantages of using AP transposases or AP transposase variants, which are particularly effective in combination with AC transposons.

[0239] Example 15: Application of AP transposase in human HEK293 cell line To demonstrate the functionality of the AP transposase in human suspension-adapted HEK293F cells, 5 × 10 cells were transfected with 5 μg transposon encoding the fluorescent protein zsGreen (using AP flanking regions, SEQ ID NOs: 2 and 3) and 4 μg AP transposase mRNA in a transfection volume of 50 μL using a MaxCyte® STx™ transfection device (MaxCyte, Rockville, MD, USA). Transfections were performed using the HEK2 Transfection Program and R-50x8™ Transfection Slides (MaxCyte®) as described by the supplier. Control cells were transfected with 5 μg transposon without transposase mRNA. After transfection, cells were transferred to 40 mL of fresh medium and cultured stationary at 37°C, 5% CO2, and 85% humidity for 5 days. Five days after transfection, cells were transferred to shake flasks at 37°C, 5% CO2, and 85% humidity, with 95 rpm shaking in a 25 mm orbital pattern, and passaged every 3–4 days. No selection for stable integration was applied during culture. At 8 and 14 days after transfection, zsGreen expression was measured using a NovoCyte flow cytometer system (Agilent, Santa Clara, CA, USA). Because HEK293F cells maintain transfected plasmids for several days, the measurement at day 8 represents expression from transiently transfected plasmids, whereas the measurement at day 14 represents expression after stable integration.

[0240] Figure 22 (A and C) shows that at day 8, slightly more than 40% of zsGreen-positive cells were observed in the presence and absence of AP transposase due to expression from a transiently transfected plasmid. While the percentage of zsGreen-expressing cells was negligible 14 days after transfection in the absence of AP transposase, approximately 23.68% of cells remained zsGreen-positive in the presence of AP transposase, demonstrating stable integration (Figures 22B and 22C). Therefore, this data demonstrates that the AP transposase system is compatible with cells from species other than hamsters.

[0241] Example 16: Generation of stable AAV HEK293 producer cell lines using AP transposase Generation of stable AAV producer cells: Stable adeno-associated virus (AAV) producer (packaging) cells containing stably integrated rep and cap genes were established by a sequential transfection process involving two plasmids in adherent human embryonic kidney 293 (HEK293) cells (ATTC #CRL-1573).

[0242] The first plasmid (pBIG-540-AP / Cap) contained the AAV2 cap gene controlled by its endogenous p40 promoter and the neomycin resistance gene controlled by the simian virus 40 (SV40) promoter, flanked by AP transposon flanking region sequences (SEQ ID NOs: 2 and 3).

[0243] The second plasmid contained the AAV2 rep gene, with one of its native promoters (p5) replaced by the inducible tetracycline (Tet) promoter. Additionally, this plasmid encoded two proteins essential for Tet promoter function, controlled by the mouse phosphoglycerate kinase (mpGK) promoter, and a hygromycin resistance gene controlled by the SV40 promoter. These sequences were also flanked by AP transposon flanking sequences (SEQ ID NOs: 2 and 3).

[0244] Both plasmids were sequentially transfected into HEK293 cells along with a plasmid encoding the AP transposase gene (SEQ ID NO: 18) with additional N- and C-terminal NLSs. First, HEK293 cells were transfected with pBIG-540-AP / Cap by electroporation using the Neon transfection system (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Twenty-four hours after transfection, the cells were subjected to antibiotic selection by treating them with 250 μg / μL Geneticin 418. After approximately four weeks of selection and cell recovery, a second plasmid (pBIG-560-AP / TET-Rep) was also transfected by electroporation into the HEK293 cell pool carrying the stable recombinant Cap. Twenty-four hours after transfection, the cells were subjected to a second round of antibiotic selection using both 250 μg / μL Geneticin 418 and 50 μg / μL Hygromycin. Once the cells recovered from double antibiotic selection (approximately 3–4 weeks), a stable Rep / Cap-harboring HEK293 cell pool (HEK293_Cap_Tet-Rep cells) was utilized for AAV production.

[0245] AAV production in unmodified HEK293 and stable HEK293_Cap_Tet-Rep producer cells: 7 x 10 total 5HEK293 or HEK293_Cap_Tet-Rep cells were seeded in 6-well plates using Dulbecco's modified Eagle's medium (DMEM) (Thermo Fisher Scientific, Waltham, Massachusetts, USA) supplemented with 10% fetal bovine serum (FBS). After 24 hours, cells were transfected with plasmids from the AAV-MAX Control Plasmid Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA, #A47672). For transfection, the pHelper and pAAV-CMV-EmGFP (pTransfer) plasmids were combined at a 1:1 molar ratio (1.4 μg DNA total per well) and complexed with FectoVIR (4.2 μL per well, Polyplus Transfection SA, Illkirch-Graffenstaden, France). During AAV production, the medium was supplemented with 500 ng / mL doxycycline to activate the Tet-inducible promoter. 72 hours after transfection, the cells were detached using a cell scraper. Both the cells and the cell culture supernatant were transferred to a tube and centrifuged at 300 × g for 7 minutes. The cell culture supernatant was then transferred to a new tube, while the cell pellet was resuspended in 300 μL of phosphate-buffered saline (PBS). Both tubes were stored at -80°C for subsequent analysis.

[0246] Transduction of HEK293 cells with AAV sample: 1 x 10 4HEK293 cells were seeded in 96-well microplates. After 24 hours, the cell culture medium was removed and the cells were treated with 0.5 μg mitomycin C (in DMEM medium) for 1 hour. The cells were then washed with PBS and refilled with 100 μL of fresh DMEM medium. For transduction, 50 μL of AAV sample (either various dilutions of AAV-containing lysate or undiluted AAV-containing cell culture supernatant) was transferred to adherently grown HEK293 cells per well. After 48 hours of culture, emerald green fluorescent protein (emGFP) fluorescence was analyzed by fluorescence microscopy using a Cytation 5 imaging system.

[0247] Although AAV-mediated emGFP expression was not observed in cells treated with culture supernatant from HEK293 cells transfected with pHelper and pTransfer plasmids (lacking Rep and Cap gene expression), emGFP expression was detected in cells treated with cell culture supernatant from stable HEK293_Cap_Tet-Rep cells, indicating that the AP transposase was successfully used in combination with the AP transposon to stably integrate the AAV Rep and Cap genes into HEK293 cells (Figure 23).

[0248] This data demonstrates that stable AAV-producing cell lines based on human HEK293 cells can be generated by stably integrating the required AAV genes via the AP transposase, expanding the applicability of the AP transposase system well beyond classical protein production to a variety of cell types.

[0249] Example 17: Activity testing of putative transposase / transposon-like sequences As described in Example 1, 384 candidate transposase / transposon-like sequences were identified based on characteristic motifs, such as the complete transposase open reading frame, catalytic DDE / DDD motif, and ITR flanking sequences, based on published genome sequences. Fourteen transposase / transposon-like sequences were experimentally tested, and transposase activity was demonstrated only for AP transposases with their respective transposon sequences. Transfection experiments using circular plasmid DNA vectors and mRNA encoding each transposase were performed as described in Example 2 for AP transposases. The results for the selected transposases and their selected transposon flanking sequences, as defined in Table 7 below, are shown in Figure 24.

[0250] [Table 9]

[0251] The results demonstrate that, unexpectedly, one transposase / DNA transposon pair was found to exhibit transposition activity. While predictions based on genetic elements in database sequences allow the identification of numerous transposase / transposon-like sequences, sequence-based predictions regarding the activity and functionality of these sequences are not possible, necessitating experimental testing rather than hoping that functional pairs exist within the sequences tested. Therefore, the identification of an active A. pisum transposase / DNA transposon pair was surprising.

[0252] [Table 10] TIFF2025536126000012.tif249161 TIFF2025536126000013.tif214161 TIFF2025536126000014.tif245161 TIFF2025536126000015.tif83161

Claims

1. A DNA transposon comprising a heterologous polynucleotide flanked by transposon flanking regions, One transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 38 at one transposon end, and the other transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 39 at the other transposon end. DNA transposons.

2. (a) one transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 40 at one transposon end, and the other transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 41 at the other transposon end; (b) each of the one and the other transposon flanking regions further comprises at least one inverted internal repeat (IIR) comprising (i) an internal repeat motif comprising the nucleotide sequence tggtctac and (ii) its reverse complement (iii) separated by 6 to 3, preferably 4, nucleotides, wherein preferably at least one inverted internal repeat (IIR) motif has the nucleotide sequence of SEQ ID NO: 43; and / or (c) The DNA transposon of claim 1, wherein (i) one transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 45 or 46 and further comprises an inverted internal repeat motif having the nucleotide sequence of SEQ ID NO: 43 separated by about 50-200 nucleotides, and / or the other transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 47 or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 47, wherein said sequence comprises at least the nucleotide sequence of SEQ ID NO: 41 and the nucleotide sequence of SEQ ID NO: 43; or (ii) wherein one transposon flanking region and the other transposon flanking region are inverted and comprise the reverse complement of the sequence described in (i).

3. (a) (i) one transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 45 or 46 and further comprises an inverted internal repeat motif having the nucleotide sequence of SEQ ID NO: 43 separated by about 50-200 nucleotides, wherein one transposon flanking region comprises a nucleotide sequence having 85% sequence identity to SEQ ID NO: 10 or 63 or SEQ ID NO: 2 or 29, and / or the other transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 47 or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 47, wherein the sequence comprises at least the nucleotide sequence of SEQ ID NO: 41 and the nucleotide sequence of SEQ ID NO: 43; or (ii) wherein one transposon flanking region and the other transposon flanking region are inverted and comprise the reverse complement of the sequence set forth in (i); and / or (b) (i) one transposon flanking region comprises a nucleotide sequence having at least 85% nucleotide sequence identity to SEQ ID NO: 2, 8, 10 or 58 and / or the other transposon flanking region comprises a nucleotide sequence having at least 90% nucleotide sequence identity to SEQ ID NO: 3, 9, 11 or 59; or (ii) one transposon flanking region; or (ii) wherein one transposon flanking region and the other transposon flanking region are inverted and comprise the reverse complement of the sequence set forth in (i); and / or (c) (i) one transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 2, 8, 10 or 58 or the sequence of SEQ ID NO: 29, 62, 63 or 64 and / or the other transposon flanking region comprises the nucleotide sequence of SEQ ID NO: 3, 9, 11, 59, 48 or 49 or the nucleotide sequence of SEQ ID NO: 30, 65, 66, 67, 60 or 61; or (ii) wherein one transposon flanking region and the other transposon flanking region are inverted and comprise the reverse complement of the sequence described in (i); or (d) The DNA transposon of claim 1 or 2, wherein (i) one transposon flanking region comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 29, 62, 63 or 64 and / or the other transposon flanking region comprises a nucleotide sequence having at least 97% sequence identity to SEQ ID NO: 30, 65, 66, 67, 60 or 61, or (ii) wherein one transposon flanking region and the other transposon flanking region are inverted and comprise the reverse complement of the sequence described in (i).

4. 4. The DNA transposon of any one of claims 1 to 3, wherein the heterologous polynucleotide comprises at least one sequence selected from the group consisting of a sequence encoding a gene of interest, a complementary DNA (cDNA), a genome of interest, and another genetic element.

5. The DNA transposon according to any one of claims 1 to 4, wherein the transposon can be transposed by a transposase comprising the amino acid sequence of SEQ ID NO:

4.

6. A DNA transposon according to any one of claims 1 to 5, Expression vector.

7. an amino acid sequence having at least 90% sequence identity to amino acids 10 to 585 of SEQ ID NO: 4, and at least one heterologous nuclear localization signal (NLS) fused to a transposase, preferably wherein the transposase is an Acyrthosiphon pisum transposase or an Aphis craccivora transposase; Recombinant transposase.

8. and a transposase comprising an amino acid sequence having at least 90% sequence identity to amino acids 10 to 585 of SEQ ID NO: 4, wherein the transposase comprises at least one mutation, preferably, wherein the at least one mutation is selected from the group consisting of K87Y, Q273V, V212I / I215L, I363V / K365S, K87Y / Q273V, K87Y / A264S / Q273V, A264S / Q273V, S270P ... and / or at least one mutation is a deletion of N584 and / or E585, wherein the indicated amino acid positions of the substitutions and / or deletions correspond to the amino acid positions in the sequence of SEQ ID NO: 4, and preferably wherein at least one heterologous NLS is fused to the transposase. Recombinant transposase.

9. A gene encoding a transposase comprising an amino acid sequence having at least 90% sequence identity with amino acids 10 to 585 of SEQ ID NO: 4, operably linked to a eukaryotic promoter; Polynucleotide.

10. A gene encoding the recombinant transposase according to claim 7 or 8. Polynucleotide.

11. 11. A method for producing a recombinant transposase comprising the steps of: Expression vector.

12. A gene encoding the recombinant transposase according to claim 7 or 8. Isolated mRNA.

13. (a) the following: (i) the expression vector according to claim 11; (ii) the isolated mRNA of claim 12; and (iii) The recombinant transposase according to claim 7 or 8. A recombinant transposase source selected from the group consisting of: (b) comprising the DNA transposon according to any one of claims 1 to 5 or the expression vector according to claim 6; Expression systems or kits.

14. The DNA transposon according to any one of claims 1 to 5 is contained therein, yeast or mammalian cells, preferably mammalian cells, more preferably rodent or human cells; Eukaryotic cells.

15. 1. A method for producing a cell containing a stably integrated heterologous polynucleotide, comprising: (a) introducing into a eukaryotic cell a DNA molecule comprising a DNA transposon according to any one of claims 1 to 5 or an expression vector according to claim 6, wherein the heterologous polynucleotide comprises a sequence encoding a gene of interest, a complementary DNA (cDNA), a genome of interest or another genetic element, and wherein the heterologous polynucleotide further comprises a sequence encoding a selectable marker; (b) introducing a recombinant transposase source into said eukaryotic cell, wherein the recombinant transposase source comprises: (i) the expression vector according to claim 11; (ii) the isolated mRNA of claim 12; and (iii) The recombinant transposase according to claim 7 or 8. selected from the group consisting of (c) culturing the eukaryotic cells in a medium under conditions for selecting the selectable marker, wherein the DNA transposon is stably integrated into the genome of the eukaryotic cells; Including, method.

16. 1. A method for producing a protein of interest, comprising: (a) introducing into a eukaryotic cell a DNA molecule comprising the DNA transposon of any one of claims 1 to 5 or an expression vector of claim 6, wherein the heterologous polynucleotide comprises a sequence encoding at least one protein of interest and further comprises a sequence encoding a selectable marker; (b) introducing a recombinant transposase source into said eukaryotic cell, wherein the recombinant transposase source comprises: (i) the expression vector according to claim 11; (ii) the isolated mRNA of claim 12; and (iii) The recombinant transposase according to claim 7 or 8. selected from the group consisting of (c) culturing the eukaryotic cells in a medium under conditions for selecting for the selectable marker, wherein the heterologous polynucleotide comprises a sequence encoding at least one protein of interest, and the sequence encoding the selectable marker is integrated into the genome of the eukaryotic cells; (d) optionally isolating a single clone for clonal propagation to prepare a monoclonal cell line; (e) culturing the eukaryotic cells under conditions for producing the protein of interest; (f) collecting and optionally purifying the protein of interest; Including, method.

17. 1. A method for producing a virus or virus-like particle of interest, comprising: (a) introducing into a eukaryotic cell a DNA molecule comprising the DNA transposon of any one of claims 1 to 5 or an expression vector of claim 6, wherein the heterologous polynucleotide comprises a sequence encoding at least one virus or virus-like particle of interest and further comprises a sequence encoding a selectable marker; (b) introducing a recombinant transposase source into said eukaryotic cell, wherein the recombinant transposase source comprises: (i) the expression vector according to claim 11; (ii) the isolated mRNA of claim 12; and (iii) The recombinant transposase according to claim 7 or 8. selected from the group consisting of (c) culturing the eukaryotic cells in a medium under conditions for selecting for the selectable marker, wherein the heterologous polynucleotide comprises a sequence encoding at least one virus or virus-like particle of interest, and the sequence encoding the selectable marker is integrated into the genome of the eukaryotic cells; (d) optionally isolating a single clone for clonal propagation to prepare a monoclonal cell line; (e) culturing the eukaryotic cells under conditions for producing the virus or virus-like particle of interest; (f) harvesting and optionally purifying the virus or virus-like particle; Including, method.

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