Hyperactive transposons and transposases

Abstract

The present invention relates to a polypeptide comprising a piggyBac transposase containing at least one amino acid substitution, or a fragment or derivative thereof having transposase function. The present invention also relates to a transposable element comprising a piggyBac or piggyBac-like left repeat and a left internal repeat, wherein the left internal repeat contains at least one nucleotide alteration. The present invention also relates to a kit comprising the above-described transposase and / or transposable element. The present invention also relates to a targeting system comprising the above-described transposase and / or transposable element.

Description

[Technical Field]

[0001] The present invention relates to a polypeptide comprising a piggyBac transposase containing at least one amino acid substitution, or a fragment or derivative thereof having transposase function. The present invention also relates to a transposable element comprising a piggyBac or piggyBac-like left repeat and a left internal repeat, wherein the left internal repeat contains at least one nucleotide alteration. The present invention also relates to a kit comprising the above-described transposase and / or transposable element. The present invention also relates to a targeting system comprising the above-described transposase and / or transposable element. [Background technology]

[0002] Transposons have recently been developed as a powerful non-viral gene delivery tool. In particular, when integration of plasmid DNA is assisted using transposons, the performance of the resulting producer cell line can be improved. For example, transposons allow for the integration of larger heterologous DNA fragments and the integration of more copies of heterologous DNA into each genome. Furthermore, transposon-mediated integration provides an efficient method for reducing integration of the plasmid backbone and / or reducing concatemer formation.

[0003] Transposable elements, or transposons, are DNA segments that can move from one locus in the genome to another. Two classes of transposable elements have been distinguished: retrotransposons (class 1), which replicate via an RNA intermediate, and "cut-and-paste" DNA transposons (class 2). Class 2 transposons are characterized by short inverted terminal repeats (ITRs) and an element-encoded transposase, an enzyme with excision and insertion activity. In their natural configuration, the transposase gene is located between the inverted repeats. Some transposons of class 2, such as those of the moth Trichoplusia nii (Trichoplusia Transposons such as the PiggyBac transposon from the bat Myotis lucifugus (PiggyBat), a reconstructed transposon from a salmon species (Sleeping Beauty), and the Tol2 transposon from the medaka fish Oryzias latipes have been shown to facilitate the insertion of heterologous DNA into eukaryotic genomes. These transposons have numerous applications in the genetic manipulation of host genomes, including transgene delivery and insertional mutagenesis. For example, the piggyBac (PB) DNA transposon (formerly known as IFP2) has been used technically and commercially in genetic engineering due to its ability to efficiently transfer between vectors and chromosomes (U.S. Patent No. 6,218,185). In these applications, the DNA to be integrated is flanked on either side by two PB ITRs within the PB vector. By co-delivery of the PB transposase, the flanked DNA is precisely excised from the PB vector and integrated into the target genome at the TTAA-specific site.

[0004] To increase transformation efficiency, more active transposases have been developed. These hyperactive transposases result in a greater proportion of cells that integrate a given transposon and a greater number of transposon integrations per cell compared to wild-type transposases. Various strategies have been described in the art: for example, EP2160461 describes a hyperactive Sleeping Beauty transposase generated via side-directed mutagenesis. U.S. Pat. No. 9,534,234 (US9534234) describes a transposase that is highly active and can be used to transform a variety of cells. 4234) provides PB-like transposases from the silkworm Bombyx mori and the frog Xenopus tropicalis fused to heterologous nuclear localization sequences (NLSs). EP1546322 (EP1546322) discloses chimeric integrases containing a binding domain that recognizes a DNA landing pad and attracts the transposon-transposase complex to the landing pad, promoting integration in its vicinity. EP1594972 (EP1594972) claims a transposase, or a fragment or derivative thereof with transposase function, fused to a polypeptide binding domain capable of associating with a cellular or engineered polypeptide containing a DNA targeting domain.

[0005] Transformation efficiency can also be increased by using a more active transposon.

[0006] One application area of ​​transposases is the development of pharmaceutical cell lines. Chinese hamster ovary (CHO) cells are the most popular mammalian cell factories for producing therapeutic biologics due to their ability to grow in suspension culture, their capacity for complex post-translational modifications, and their low susceptibility to human viral infection. One of the main limitations of industrial production of recombinant therapeutic proteins is the time-consuming and labor-intensive cell line production and characterization process. The majority of available methods rely on random transgene integration. Multiple cassettes are often integrated in tandem, often at more or less active sites. Active chromosomal loci are rare, and thousands of clones must be screened to obtain high-producer cells. To reduce the scope of recombinant cell line screening and increase the productivity and stability of recombinant CHO cell lines, PB-mediated gene delivery was used (M. Matasci et al., The PiggyBac transposon enhances the frequency of CHO stable cell line generation and yields recombinant lines with superior productivity and stability. Biotechnology and Bioengineering, Vol. 108, No. 9, (2011)).

[0007] However, results regarding more active transposons and transposases (e.g., more active PB and PB-like transposons and transposases) are not (yet) satisfactory.

[0008] It would be highly desirable to develop transposons and transposases (eg, PB and PB-like transposons and transposases) that would result in a greater number of transposon integrations per cell compared to the current state of the art.

[0009] Recently, Morellet et al. reported that the C-terminal cysteine-rich domain (CRD) of PB transposase binds to specific DNA sequences at the left and right transposon ends, and also binds to an unexpected internal site at the left end (Nucleic Acids Research, 2018, Vol. 46, No. 5 2018 doi:10.1093 / nar / gky044).

[0010] The present inventors have surprisingly found that artificially inserted modifications within the left internal repeat of a transposable element shorten the time it takes for transfectants to recover viability during the selection stage. Artificial piggyBac (PB) and PB-like transfectants with at least one modification within the left internal repeat that increases the homology of the left internal repeat to the left repeat are shown to be useful. Transposable elements have not been described or suggested in the art, and it was unlikely that such a mutation would have any effect.

[0011] Furthermore, the present inventors have surprisingly established for the first time a targeting system / gene delivery system comprising a transposable element containing a piggyBac (PB) or PB-like artificial left internal repeat sequence for the improved generation of producer cell lines for the production of therapeutic proteins or for the high-yield production of viral particle-based biopharmaceuticals.

[0012] Furthermore, the inventors have surprisingly discovered a hyperactive piggyBac (PB) transposase that can move a transposon, e.g., a transposable element described herein, from one genomic location to another with greater efficiency than piggyBac (PB) transposases described in the art. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] U.S. Patent No. 6,218,185 [Patent Document 2] European Patent No. 2160461 [Patent Document 3] U.S. Patent No. 9,534,234 [Patent Document 4] European Patent No. 1546322 [Patent Document 5] European Patent No. 1594972 [Non-patent literature]

[0014] [Non-Patent Document 1] M. Matasci et al., The PiggyBac transposon enhances the frequency of CHO stable cell line generation and yields recombinant lines with superior productivity and stability. Biotechnology and Bioengineering, Vol. 108, No. 9, (2011) [Non-patent document 2] Nucleic Acids Research,2018,Vol.46,No.5 2018 doi:10.1093 / nar / gky044 Summary of the Invention [Means for solving the problem]

[0015] In a first aspect, the present invention relates to a polypeptide comprising a piggyBac transposase, or a fragment or derivative thereof having transposase function, comprising at least one amino acid substitution selected from the group consisting of: a substitution of isoleucine (I) at amino acid position 30 or a corresponding amino acid position with alanine (A) (I30A); a substitution of glutamine (Q) at amino acid position 118 or the amino acid position corresponding thereto with proline (P) (Q118P); a substitution of methionine (M) at amino acid position 185 or a corresponding amino acid position with valine (V) (M185V); a substitution of methionine (M) at amino acid position 282 or the amino acid position corresponding thereto with leucine (L) (M282L); and An asparagine (N) at amino acid position 538 or a corresponding amino acid position is substituted with an arginine (R) (N538R).

[0016] In a second aspect, the present invention provides a polynucleotide encoding a polypeptide according to the first aspect. Regarding leotide.

[0017] In a third aspect, the present invention relates to a vector comprising a polynucleotide according to the second aspect.

[0018] In a fourth aspect, the present invention provides a piggyBac or piggyBac-like Left repeat and including the left internal repeat sequence, the left internal repeat sequence comprises at least one nucleotide alteration; At least one nucleotide modification relates to the transposable element that increases the homology of the left internal repeat sequence to the left repeat sequence.

[0019] In a fifth aspect, the present invention provides a method for producing a transgenic cell, comprising: (i) providing cells; (ii) (iia) a transposable element, and a polypeptide according to the first aspect, or a polynucleotide according to the second aspect, or a vector according to the third aspect, or (iib) a transposable element according to the fourth aspect, and a transposase or a fragment or derivative thereof having transposase function, or a polynucleotide encoding a transposase or a fragment or derivative thereof having transposase function; a vector comprising a polynucleotide encoding a transposase or a fragment or derivative thereof having transposase function; (iic) a transposable element according to the fourth aspect, and a polypeptide according to the first aspect, or a polynucleotide according to the second aspect, or a vector according to the third aspect, into said cells, thereby producing transgenic cells. The present invention relates to a method, including:

[0020] In a sixth aspect, the present invention relates to a transgenic cell obtainable by a method according to the fifth aspect.

[0021] In a seventh aspect, the present invention relates to the use of a transgenic cell according to the sixth aspect for the production of a protein or a virus.

[0022] In an eighth aspect, the present invention provides a method for producing a composition comprising: (i) a transposable element, and a polypeptide according to the first aspect, or a polynucleotide according to the second aspect, or a vector according to the third aspect; or (ii) a transposable element according to the fourth aspect, and a transposase or a fragment or derivative thereof having transposase function, or a polynucleotide encoding a transposase or a fragment or derivative thereof having transposase function; a vector comprising a polynucleotide encoding a transposase or a fragment or derivative thereof having transposase function; or (iii) a transposable element according to the fourth aspect, and a polypeptide according to the first aspect, or a polynucleotide according to the second aspect, or a vector according to the third aspect, The present invention relates to a kit comprising:

[0023] In a ninth aspect, the present invention provides a method for producing a composition comprising: (i) a transposable element, and a polypeptide according to the first aspect, or a polynucleotide according to the second aspect, or a vector according to the third aspect; or (ii) a transposable element according to the fourth aspect, and a transposase or a fragment or derivative thereof having transposase function, or a polynucleotide encoding a transposase or a fragment or derivative thereof having transposase function; a vector comprising a polynucleotide encoding a transposase or a fragment or derivative thereof having transposase function; or (iii) a transposable element according to the fourth aspect, and a polypeptide according to the first aspect, or a polynucleotide according to the second aspect, or Vector according to the third aspect The present invention relates to a targeting system including:

[0024] This summary of the present invention does not necessarily describe all features of the present invention, and other embodiments will become apparent from review of the detailed description that follows.

[0025] The following drawings are merely illustrative of the present invention and should not be construed in any way as limiting the scope of the invention as set forth by the appended claims. [Brief explanation of the drawings]

[0026] [Figure 1]Figure 1 is a diagram showing the tested mutants of hyperactive PiggyBac transposase. PBw: wild-type (wt) PiggyBac transposase, Trichoplusia ni, GenBank accession number AAA87375.2; haPB1: transposase mutated at I30A, Q118P, M185V, M282L, and N538R compared to wt PiggyBac transposase; haPB2: transposase mutated at I30A, M282L, and N538R compared to wt PiggyBac transposase. The nucleotide and corresponding amino acid sequences are listed in SEQ ID NOs: 17 and 18 for PBw, 19 and 20 for haPB1, and 21 and 22 for haPB2. [Figure 2] Figure 2 is a diagram showing the tested mutants of the transposon end sequence (TES): Figure 2a is a schematic diagram of the transposon; Figure 2b is a diagram showing the tested transposon mutants. [Figure 3] Figure 3 is a diagram showing a map of the transposon expression vector. The promoter region is shown as a black block, and the polyadenylation signal (polyA) is shown as a white box. The coding regions of the antibiotic resistance gene, the selectable marker gene, and the light or heavy chain gene are shown as arrows. pac = puromycin-N-acetyltransferase; dhfr = dihydrofolate reductase; aph = kanamycin resistance. The positions of the tested transposon end sequences (TES) are shown as triangles. [Figure 4]Figure 4a shows the recovery of viability during the selection phase. In Figure 4a, circles represent the PB minimal wild-type TES transposable element and PBw; triangles represent the PB wild-type TES transposable element (5'TES 247 bp) and PBw; squares represent the PB artificial TES transposable element (5'TES 247 bp) and PBw; and asterisks represent the PB artificial TES (5'TES 247 bp) and haPB2. Figure 4b shows the viability of the PB wild-type TES transposable element, the PB artificial TES transposable element, and PBw five days after the start of selection. [Figure 5] FIG. 5 shows fed batch IgG antibody titer concentrations of CHO-DG44 clone pools after simple selection, showing the relative titers at day 14 from PBw and hyperactive transposase mutants. [Figure 6] FIG. 6 shows preferred combinations of single substitutions and substitutions in piggyBac transposase, or a fragment or derivative thereof, in which at least one of the following substitutions is present: I30A, Q118P, M185V, M282L, and / or N538R. DETAILED DESCRIPTION OF THE INVENTION

[0027] [Definition] Before describing the present invention in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0028] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0029] Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank accession numbers, sequence submissions, etc.), whether supra or infra, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. In the event of a conflict between a definition or teaching of such incorporated reference and a definition or teaching set forth herein, the text of this specification shall control.

[0030] The term "comprise" or variations thereof, such as "comprises" or "comprising," according to the present invention, is meant to include a stated integer or group of integers, but not to exclude any other integer or group of integers. "Of" means inclusive of the stated integer or group of integers, but excluding any modifications or other integers that materially affect or alter the stated integer. In accordance with the present invention, the term "consisting of" or variations such as "consists of" means inclusive of the stated integer or group of integers, but excluding any other integer or group of integers.

[0031] As used in the context of describing the present invention (especially in the context of the claims below), The terms "a," "an," and "the" and similar referents are to be construed as including both the singular and the plural unless otherwise indicated herein or clearly contradicted by context.

[0032] The terms "polypeptide" and "protein" are used interchangeably in the context of the present invention and refer to a long peptide-bonded chain of amino acids.

[0033] The term "polypeptide fragment" as used in the context of the present invention refers to a polypeptide that has a deletion, for example an amino-terminal deletion, and / or a carboxy-terminal deletion, and / or an internal deletion, compared to the full-length polypeptide.

[0034] As used herein, the term "transposase" refers to any enzyme that can bind to the ends of a transposable element and catalyze its transfer to another part of the genome by a cut-and-paste mechanism or a replicative transposition mechanism. The ends of the transposable element are preferably terminal repeats, such as terminal inverted repeats (TIRs). Thus, a transposase can not only recognize the terminal repeats surrounding the transfer element, but also recognize, for example, a target sequence on a new host DNA.

[0035] As used herein, the term "fragment" of a transposase "having transposase function" refers to a fragment derived from a naturally occurring transposase that lacks one or more amino acids compared to the naturally occurring transposase and has transposase function. For example, a fragment of a naturally occurring transposase still has transposase function, and in particular still mediates the excision and / or insertion of nucleotide sequences, e.g., DNA. Generally, a fragment of an amino acid sequence contains fewer amino acids than the corresponding full-length sequence, and the amino acid sequence present is in the same sequential order as the full-length sequence. Thus, a fragment does not contain any internal insertions or deletions in the portion of the full-length sequence represented by the fragment.

[0036] As used herein, the term "derivative" of a transposase "having transposase function" refers to a derivative of a naturally occurring transposase in which one or more amino acids have been substituted, deleted, inserted, and / or added compared to the naturally occurring transposase, and which still has transposase function. For example, a derivative of a naturally occurring transposase still has transposase function, and in particular still mediates the excision and / or insertion of nucleotide sequences, e.g., DNA. In contrast to a fragment, a derivative may contain internal insertions or deletions within the amino acids corresponding to the full-length sequence and may have similarity to the full-length coding sequence.

[0037] The above modifications are preferably achieved by recombinant DNA techniques. Further modifications may also be achieved by applying chemical modifications to the transposase.

[0038] Transposases and fragments or derivatives thereof can be recombinantly produced but still retain the same or essentially the same characteristics as naturally occurring transposases, particularly with respect to nucleotide sequences, e.g., DNA excision and / or insertion. For example, a transposase fragment or derivative referred to herein preferably retains at least 50%, more preferably at least 75%, and even more preferably at least 95% of the activity of the native protein. Such biological activity is readily determined by several assays known in the art, e.g., enzyme activity assays.

[0039] The (highly active / artificial) transposase or its derivatives having transposase function according to the present invention A fragment or derivative of contains at least one amino acid substitution selected from the group consisting of: a substitution of isoleucine (I) at amino acid position 30 or a corresponding amino acid position with alanine (A) (I30A); a substitution of glutamine (Q) at amino acid position 118 or the amino acid position corresponding thereto with proline (P) (Q118P); a substitution of methionine (M) at amino acid position 185 or a corresponding amino acid position with valine (V) (M185V); a substitution of methionine (M) at amino acid position 282 or the amino acid position corresponding thereto with leucine (L) (M282L); and An asparagine (N) at amino acid position 538 or a corresponding amino acid position is substituted with an arginine (R) (N538R).

[0040] The transposase, or a fragment or derivative thereof, has improved transposase function, particularly improved activity / ability to mediate the excision and / or insertion of nucleotide sequences, such as DNA, compared to transposases described in the art (e.g., wild-type / naturally occurring transposases). In particular, the transposase, or a fragment or derivative thereof, is capable of mobilizing a transposon, such as a transposable element described herein, from one genomic location to another genomic location with greater efficiency than transposases described in the art (e.g., wild-type / naturally occurring transposases).

[0041] The transposase or a fragment or derivative thereof may be recombinantly produced but still have improved characteristics compared to transposases described in the art (e.g., wild-type / naturally occurring transposases), particularly with respect to the excision and / or insertion of nucleotide sequences, e.g., DNA. For example, the transposase fragment or derivative referred to herein preferably has at least 10% more activity than the activity of the native protein, more preferably at least 20% more activity than the activity of the native protein, even more preferably at least 50% more activity than the activity of the native protein, and most preferably at least 75% more activity than the activity of the native protein. Such biological activity is readily determined by several assays known in the art, e.g., enzyme activity assays.

[0042] This transposase or a fragment or derivative thereof may be referred to as a recombinant, artificial, and / or heterologous transposase or a fragment or derivative thereof.

[0043] The transposase of the present invention or a fragment or derivative thereof having transposase function can be referred to as a hyperactive transposase or a fragment or derivative thereof. As used herein, the term "hyperactive transposase or a fragment or derivative thereof" refers to a transposase or a fragment or derivative thereof that has improved transposase function, particularly improved activity / ability to mediate the excision and / or insertion of nucleotide sequences, e.g., DNA, compared to transposases described in the art (e.g., wild-type transposases). In particular, as used herein, "hyperactive transposase or a fragment or derivative thereof" refers to a transposase or a fragment or derivative thereof that can mobilize a transposon, e.g., a transposable element described herein, from one genomic location to another genomic location with greater efficiency than transposases described in the art (e.g., wild-type transposases).

[0044] Histones are the building blocks of chromatin. Histone post-translational modifications form a signature that describes the chromatin state of a given locus. Euchromatin generally contains high levels of histones. Histone acetylation and / or methylation, particularly mono-methylation, are associated with histone modification. In particular, acetylation of lysine residues, for example, reduces the positive charge of histones, thereby weakening their interaction with negatively charged DNA and increasing the fluidity of nucleosomes (complexes of DNA and histones). Amino acid acetylation can also reduce the level of compaction of nucleosome arrays. The chromatin state of a given locus depends, for example, on molecules capable of post-translationally modifying histones, such as methylating and / or acetylating them (so-called "writers"), molecules capable of removing post-translational modifications, such as methylated and / or acetylated histones (so-called "erasers"), and molecules capable of readily identifying post-translational modifications of histones, such as methylation and / or acetylation (so-called "readers"). "Reader" molecules are recruited to such histone modifications and bind via specific domains, such as plant homeodomains (PHDs), zinc fingers, bromodomains, or chromodomains. The triple actions of "writing," "reading," and "erasing" establish a favorable local environment for transcriptional regulation, DNA damage repair, and so on.

[0045] As used herein, the term "chromatin reader element (CRE)" refers to any structure that accommodates modified histone residues and provides an accessible surface (such as a cavity or surface groove) that determines the type (e.g., acetylation or methylation, and acetylation vs. methylation) or state specificity (e.g., mono-, di-, or tri-methylation of lysine or arginine, etc.) of post-translational histone modification. A "chromatin reader element" also interacts with the adjacent sequence of the modified amino acid to distinguish sequence configurations. In particular, a "chromatin reader element" binds to a histone tail and recognizes specific post-translational modifications (PTMs), such as methylation on histones, e.g., methylation of lysine or arginine, and / or acetylation. As a result, the chromatin reader element recruits chromatin remodeling complexes and components of the transcription machinery to the binding site. A "chromatin reader element" is preferably an element that recognizes the degree of histone methylation, particularly the degree of histone mono-, di-, or tri-methylation, e.g., of lysine and / or arginine residues. Alternatively, a "chromatin reader element" is an element that recognizes the acetylation state of histones. As mentioned above, transcriptionally active euchromatin is generally associated with histone acetylation and / or methylation, particularly histone monomethylation. Preferably, the chromatin reader element is a "chromatin reader domain (CRD)." The chromatin reader domain may be a bromodomain, a chromodomain, a plant homeodomain (PHD) zinc finger, a WD40 domain, a Tudor domain, a double / tandem Tudor domain, an MBT domain, an ankyrin repeat domain, a zf-CW domain, or a PWWP domain. For example, bromodomains are found in chromatin-associated proteins such as histone acetyltransferases, which specifically recognize acetylated lysine residues. PHDs (especially PHD fingers) are also found in chromatin-associated proteins such as plant homeodomain proteins, such as transcription initiation factors. These can also recognize acetylated lysine residues. Chromatin reader domains that recognize histone methylation include PHD domains, chromodomains, WD40 domains, tudor domains, double / tandem tudor domains, MBT domains, ankyrin repeat domains, zf-CW domains, and PWWP domains. More preferably, the chromatin reader domain is a bromodomain or a plant homeodomain (PHD) zinc finger. Alternatively, the chromatin reader element is preferably an artificial chromatin reader element. The artificial chromatin reader element may be a microantibody, a single-chain antibody, an antibody fragment, an affibody, an affilin, an anticalin, an atrimer, a DARPin, an FN2 scaffold, a fynomer, or a Kunitz domain.

[0046] Chromatin reader elements, particularly chromatin reader domains, may be associated with transposases or fragments or derivatives thereof with transposase function. Chromatin reader elements, particularly transposases or fragments or derivatives thereof with transposase function linked to chromatin reader domains, can recognize specific histone post-translational modifications, such as methylation and / or acetylation, and thus can recognize active euchromatin.

[0047] As used herein, the term "DNA binding / targeting domain" refers to a moiety that is capable of specifically binding to a DNA region (including highly structured chromosomal regions such as repetitive regions in the nucleus) and is directly or indirectly involved in mediating the integration of a transposable element into said DNA region. A DNA region is preferably defined by a nucleotide sequence that is unique within each genome.

[0048] As used herein, the term "nuclear localization sequence / signal (NLS)" refers to a structure that tags a polypeptide for import into the cell nucleus by nuclear transport. Typically, this sequence / signal consists of one or more short sequences of positively charged lysines or arginines exposed on the surface of the polypeptide.

[0049] As used herein, the term "heterologous" refers to an element that is derived from another natural source, e.g., another organism, or that has been removed from its natural context, e.g., fused to, attached to, or linked to another molecule, or that is not normally found in nature. In particular, as used in the context of the present invention, the term "heterologous polypeptide" refers to a polypeptide that is not normally found in nature. As used in the context of the present invention, the term "heterologous nucleotide sequence" refers to a nucleotide sequence that is not normally found in nature. This term encompasses nucleic acids that meet at least one of the following criteria: (a) a nucleic acid that has been exogenously introduced into a given cell (thus, an "exogenous sequence" even if the sequence is foreign or native to the recipient cell); (b) the nucleic acid comprises a nucleotide sequence that is naturally found in a given cell (e.g., the nucleic acid comprises a nucleotide sequence that is endogenous to the cell), but is produced in the cell in unnatural amounts (e.g., greater than expected or greater than found in nature), or the nucleotide sequence differs from the endogenous nucleotide sequence such that the same encoded protein (a protein having the same or substantially the same amino acid sequence) as that found endogenously is produced in unnatural amounts (e.g., greater than expected or greater than found in nature); or (c) the nucleic acid comprises two or more nucleotide sequences or segments that are not found in the same relationship to each other in nature (e.g., the nucleic acid is recombinant).

[0050] The term "heterologous chromatin reader element (CRE)", in particular "chromatin reader domain (CRD)", "heterologous DNA-binding domain" or "heterologous nuclear localization sequence (NLS)", as used herein in reference to a transposase or a fragment or derivative thereof with transposase function, refers to an amino acid sequence that is not normally found closely associated with a transposase or a fragment or derivative thereof with transposase function in nature.

[0051] As used herein, the term "transposable element (also referred to as "transposon" or "jumping gene")" refers to a polynucleotide molecule that can change its position within a genome. Typically, a transposable element comprises a polynucleotide that encodes a functional transposase that catalyzes excision and insertion. However, the transposable element described in the context of the present invention lacks a polynucleotide that encodes a functional transposase. The transposon-based polynucleotide molecule no longer contains the complete sequence encoding a functional transposase, preferably a naturally occurring functional transposase. Preferably, the complete sequence encoding a functional transposase, preferably a naturally occurring functional transposase or a portion thereof, has been deleted from the transposable element. Alternatively, the gene encoding the transposase has been mutated such that it no longer contains the naturally occurring transposase or a fragment or derivative thereof that has transposase function, i.e., the function of mediating excision and / or insertion of the transposon into the target site. The transposable elements described herein possess sequences necessary for mobilization by a transposase provided in trans. These are repeated sequences at each end of the transposable element that contain binding sites for the transposase, allowing for excision and integration. The repeated sequences are also called terminal repeats. Preferably, the terminal repeats are terminal inverted repeats (TIRs). In particular, the terminal repeats are piggyBac terminal repeats or piggyBac-like terminal repeats. Herein, the terminal sequences of the transposable element are also referred to as "5'-transposon terminal sequence" and "3'-transposon terminal sequence." Instead of a polynucleotide sequence encoding a functional transposase, an exogenous polynucleotide sequence, e.g., a polynucleotide sequence of interest / heterologous polynucleotide sequence, such as a functional gene and regulatory elements that drive expression, is preferably part of a transposable element as described herein. In particular, these sequences are located between the terminal repeats, in other words, between the "5'-transposon end sequence" and the "3'-transposon end sequence." Preferably, the transposase recognizes the TA dinucleotide at each end of the transposable element, particularly in the repeat sequence of the transposable element, and excises the transposable element, for example, from a vector. Usually, two transposase monomers are involved in the excision of the transposable element, with one transposase monomer at each end of the transposable element. Finally, the transposase dimer complexed with the excised transposable element recognizes the TTAA site in the target sequence, thereby reintegrating the transposable element into the DNA of the host organism, for example, the host cell.

[0052] As used herein, the terms "5'-transposon end sequence" and "3'-transposon end sequence" refer to the portions of the 5' and 3' non-coding regions of the transposable elements described herein that are involved in the recognition of the transposable element by the transposase. They can form a functional complex with the transposase to carry out the transposition reaction. Other functional elements, such as enhancers and / or promoters, may be embedded within the transposon end sequence. For the piggyBac transposable element, the most commonly used vector configuration today is a 5'-transposon end sequence with a length of 311 bp and a 3'-transposon end sequence with a length of 235 bp.

[0053] The 5'-transposon end sequence of the transposable elements described herein comprises a left repeat sequence and a left internal repeat sequence.

[0054] As used herein, the term "left repeat" refers to a nucleotide sequence located between the 5' end of a transposon and the left internal repeat. In particular, the left repeat is located within the first 80 nucleotides of the 5'-transposon end sequence and is linked to the PB cysteine-rich domain or the PB-like cysteine-rich domain. For example, the left repeat of the original PB is a 19-bp DNA region separated by a 3-bp spacer from the 13-bp terminal inverted repeat. It is highly homologous to the right repeat and (highly homologous to) the left internal repeat. ) are homologous.

[0055] As used herein, the term "left internal repeat" refers to a nucleotide sequence located downstream of the left internal repeat within the 5'-transposon end sequence of a transposon. It is (highly) homologous to the left internal repeat. It is protected in DNA footprinting assays, for example, by the cross-brace zinc finger motif of piggyBac or piggyBac-like transposases. Recently, Morellet et al. reported that the C-terminal cysteine-rich domain (CRD) of PB transposase binds to specific DNA sequences at the left and right transposon ends, as well as to an unexpected internal site at the left end (Nucleic Acids Research, 2018, Vol. 46, No. 5 2018 doi:10.1093 / nar / gky044).

[0056] The left repeat sequence and the left internal repeat sequence contained in / part of the 5'-transposon end sequence may be linked to each other by a naturally occurring transposable element sequence or by a non-naturally occurring transposable element sequence, e.g. a (heterologous) linker sequence.

[0057] As used herein, the term "piggyBac (PB) transposon" refers to a transposon derived from the cabbage looper moth (Trichoplusia ni). The transposable segments were first discovered in a mutant baculovirus strain, hence their name "PB." The original PB element is approximately 2.4 kb and has identical 13-bp terminal inverted repeats and an additional asymmetric 19-bp internal repeat. These asymmetric 19-bp internal repeats are also referred to as the "left repeat" and "right repeat." As used herein, the term "piggyBac (PB) transposase" refers to the transposase derived from the cabbage looper moth (Trichoplusia ni) (GenBank accession number AAA87375.2; SEQ ID NO: 18 [Virology 172(1)156-169 1989]).

[0058] As used herein, the term "piggyBac-(PB-)-like transposon" refers to a transposon that is distinct from but has the same genetic structure as the transposon derived from the cabbage looper moth (Trichoplusia ni). PiggyBac-(PB-)-like transposon contains a left repeat sequence of approximately 12 to 17 bases, flanked by four base sequences corresponding to the integration target sequence that is duplicated upon transposon integration. For example, piggyBac-(PB-)-like transposons may be derived from Xenopus tropicalis, Bombyx mori (silkworm), Mus musculus, Homo sapiens, or Myotis lucifugus. As used herein, the term "piggyBac-(PB-)-like transposase" refers to a transposase other than the transposase from the cabbage looper moth (Trichoplusia ni). It is characterized by a DDE-like DDD motif with aspartic acid residues at positions corresponding to D268, D346, and D447 of the Trichoplusia ni PB transposase (SEQ ID NO: 18). PiggyBac-(PB-)-like transposons and transposases occur naturally in a wide range of organisms (Sakar, A. et al., (2003). Mol. Gen. Genomics 270:173-180). For example, piggyBac- (PB-)-like transposases are expressed in Xenopus tropicalis, Bombyx mori (silkworm), Mus musculus, Homo sapiens, and It may be derived from Myotis lucifugus or Myotis lucifugus.

[0059] The (highly active / artificial) transposable element of the present invention is a piggyBac or piggyBac-like Left repeat and including the left internal repeat sequence, The left internal repeat sequence comprises at least one nucleotide alteration, wherein the at least one nucleotide alteration increases the homology of the left internal repeat sequence to the left repeat sequence. The at least one nucleotide modification is preferably selected from the group consisting of a nucleotide substitution, a nucleotide deletion, a nucleotide addition, and a nucleotide insertion, or a combination thereof. This transposable element shortens the time required for viability recovery during the transfectant selection stage compared to state-of-the-art transposable elements (e.g., wild-type transposable elements). The transposable element may be referred to as a recombinant, artificial, and / or heterologous transposable element.

[0060] As used herein, the term "polynucleotide" refers to a polymer of deoxyribonucleotide or ribonucleotide bases, including both sense and antisense strands of DNA and RNA molecules. Specifically, a polynucleotide may be DNA, RNA, mRNA, cRNA, or a hybrid, both cDNA and genomic DNA, and the polynucleotide sequence may contain a combination of deoxyribonucleotide or ribonucleotide bases, as well as a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Polynucleotides can be obtained by chemical synthesis or recombinant methods. Preferably, the polynucleotide is a DNA or mRNA molecule.

[0061] As used herein, the term "polynucleotide of interest" relates to a nucleotide sequence. The nucleotide sequence may be an RNA sequence or a DNA sequence, preferably a DNA sequence. According to different aspects of the present invention, the polynucleotide of interest may encode a product of interest. The product of interest may be a polypeptide of interest, e.g., a protein, or an RNA of interest, e.g., mRNA, or a functional RNA, e.g., double-stranded RNA, microRNA, or siRNA. Functional RNA is frequently used to silence the corresponding target gene. Preferably, the polynucleotide of interest is operably linked to a suitable regulatory sequence (e.g., a promoter), which is well known and well described in the art and can affect the transcription of the polynucleotide of interest. The expression level of a desired product in a host organism, e.g., a host cell, can be determined based on either the amount of corresponding mRNA present in the cell or the amount of the desired product encoded by the polynucleotide of interest. For example, mRNA transcribed from a selected sequence can be quantified by PCR or Northern hybridization. Polypeptides can be quantified by various methods, such as by assaying for the biological activity of the polypeptide (e.g., by enzyme assay) or by using an antibody that recognizes and binds to the protein and employs an assay independent of such activity, such as Western blotting, ELISA, or radioimmunoassay. The polynucleotide of interest is preferably a polynucleotide encoding a polypeptide, a non-coding polynucleotide, a polynucleotide comprising a promoter sequence, a polynucleotide encoding an mRNA, a polynucleotide encoding a tag, and a viral polynucleotide. The polynucleotide of interest is preferably a heterologous / exogenous polynucleotide.

[0062] As used herein, the term "expression control sequence" refers to a nucleotide sequence that affects the expression of a coding sequence to which it is operably linked in a host organism, e.g., a host cell. Expression control sequences are sequences that control transcription, such as promoters, TATA boxes, enhancers, UCOE or MAR elements, polyadenylation signals, post-transcriptionally active elements such as RNA stabilization elements, RNA transport elements, and translation enhancers.

[0063] As used herein, the term "operably linked" means that one nucleotide sequence is linked to a second nucleotide sequence such that in-frame expression of the corresponding fusion or hybrid protein can be affected by avoiding frameshift or stop codons. The term also refers to the ligation of an expression control sequence to a coding nucleotide sequence of interest (e.g., a sequence encoding a protein) to effectively control the expression of said sequence. The term further refers to the ligation of a nucleotide sequence encoding an affinity tag or marker tag to a coding nucleotide sequence of interest (e.g., a sequence encoding a protein).

[0064] As used herein, the term "linker" refers to a stretch of amino acids, e.g., at least 2, 3, 4, or 5 amino acids, or a stretch of nucleotides, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, that serves no biological function in a host organism, such as a cell. The function of a linker is to connect or combine different polypeptides or polynucleotides, allowing these polypeptides or polynucleotides to perform their biological function that they would perform without being bound to the linker.

[0065] As used herein, the term "minicircle" refers to a DNA vector that is produced as a circular expression cassette that lacks any bacterial plasmid DNA backbone.

[0066] As used herein, a "variant" can be characterized by a degree of sequence identity to the parent amino acid sequence or nucleotide sequence from which it is derived. More precisely, an amino acid sequence variant in the context of the present invention can exhibit at least 80% sequence identity to its parent amino acid sequence. A nucleotide sequence variant in the context of the present invention can exhibit at least 80% sequence identity to its parent nucleotide sequence. As used herein, the term "at least 80% identical" refers to at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the respective parent / reference amino acid sequence or the respective parent / reference nucleotide sequence. Preferably, the amino acid sequence in question and the parent / reference amino acid sequence exhibit the indicated sequence identity over the entire length of the parent / reference amino acid sequence. Preferably, the nucleotide sequence in question and the parent / reference nucleotide sequence show the indicated sequence identity over the entire length of the parent / reference nucleotide sequence.

[0067] The "(highly active / artificial) transposable element" of the present invention shortens the time required for transfectants to recover viability during the selection step, compared to state-of-the-art transposable elements (e.g., wild-type transposable elements). The "(highly active) transposable element mutant" of the present invention has the above-mentioned advantageous effects. Furthermore, they still serve as substrates for the transposase, and therefore these mutants are functionally active mutants. The transposable elements or transposable element variants of the present invention can be referred to as recombinant, heterologous, artificial, and / or modified transposable elements or transposable element variants.

[0068] The "(hyperactive / artificial) transposases or fragments or derivatives thereof" of the present invention have improved transposase function, in particular improved activity / ability to mediate the excision and / or insertion of nucleotide sequences, e.g., DNA. The "transposases or fragments or derivatives thereof" of the present invention can also mobilize transposons, e.g., transposable elements described herein, from one genomic location to another with greater efficiency than transposases described in the art (e.g., wild-type transposases). The "(hyperactive) transposase mutants" of the present invention retain / have the above-mentioned advantageous effects. Moreover, they still recognize transposable elements as substrates. Therefore, these mutants are functionally active mutants. The transposases or transposase variants of the present invention can be referred to as recombinant, heterologous, artificial, and / or modified transposases or transposase variants.

[0069] Nucleotide and amino acid sequence similarity, i.e., percentage sequence identity, can be determined through sequence alignment. Such alignments can be performed using several art-known algorithms, preferably the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), hmmalign (HMMER package, http: / / hmmer.wustl.edu / ) or the CLUSTAL algorithm (Thompson JDet al. Nucleic Acids Res. 1994, 22:4673-80) (available, for example, at http: / / www.ebi.ac.uk / Tools / clustalw / or http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html or http: / / npsa-pbil.ibcp.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_clustalw.html). The preferred parameters used are the default parameters set out at http: / / www.ebi.ac.uk / Tools / clustalw / or http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html. The degree of sequence identity (sequence matching) can be calculated using, for example, BLAST, BLAT or BlastZ (or BlastX). A similar algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al. J. Mol. Biol. 1990, 215:403-410. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described by Altschul et al. Nucleic Acids Res. 1997, 25:3389-3402. When utilizing the BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence identity analysis is performed using the Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:I54-I62) or Markov Random Fields.

[0070] Residues in two or more polypeptides or polynucleotides are said to "correspond" to one another if the residues occupy analogous positions in the polypeptide or nucleotide structures. Analogous positions in two or more polypeptides may be related by amino acid sequence or It is well known in the art that similar positions in two or more polynucleotides can be determined by aligning polypeptide sequences based on structural similarity. It is also well known in the art that similar positions in two or more polynucleotides can be determined by aligning polynucleotide sequences based on nucleotide sequence or structural similarity. Such alignment tools are well known to those skilled in the art and can be obtained, for example, on the World Wide Web, for example, ClustalW (www.ebi.ac.uk / ClustalW) or Align (http: / / www.ebi.ac.uk / emboss / align / index.html), using standard settings, preferably Align EMBOSS: needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0071] As used herein, the term "host cell" refers to any cell that can be used for the production of proteins and / or viruses. This term also refers to any cell that can host the polypeptides, polynucleotides, and / or transposable elements described herein. The cell may be a prokaryotic or eukaryotic cell. Preferably, the cell is a eukaryotic cell. More preferably, the eukaryotic cell is a vertebrate cell, a yeast cell, a fungal cell, or an insect cell. The vertebrate cell may be a mammalian cell, a fish cell, an amphibian cell, a reptile cell, or an avian cell. The avian cell may be a chicken cell, a quail cell, a goose cell, or a duck cell, such as a duck retina cell or a duck somite cell. Even more preferably, the vertebrate cell is a mammalian cell. Most preferably, the mammalian cell is selected from the group consisting of Chinese hamster ovary (CHO) cells (e.g., CHO-K1 / CHO-S / CHO-DUXB11 / CHO-DG44 cells), human embryonic kidney (HEK293) cells, HeLa cells, A549 cells, MRC5 cells, WI38 cells, AGE1.CR cells, BHK cells, and Vero cells. The cell may also be contained in / part of an organism. The organism may be a prokaryotic or eukaryotic organism. Preferably, the organism is a eukaryotic organism. More preferably, the organism may be a fungus, an insect, or a vertebrate. The vertebrate may be a bird (e.g., a chicken, quail, goose, or duck), dog, weasel, rodent (e.g., a mouse, rat, or hamster), sheep, goat, pig, bat (e.g., a fruit bat or little bat), or human / non-human primate (e.g., a monkey or great ape). Most preferably, the organism is a mammal, such as a mouse, rat, pig, or human / non-human primate. Most preferably, the organism is a mammal, such as a mouse, rat, pig, or human / non-human primate.

[0072] [Embodiments of the present invention] The present inventors have surprisingly discovered a hyperactive piggyBac (PB) transposase that can mobilize a transposon, e.g., a transposable element described herein, from one genomic location to another with greater efficiency than piggyBac (PB) transposases described in the art (e.g., wild-type transposases). Furthermore, the present inventors have surprisingly discovered that artificially inserted modifications within the left internal repeat of a transposable element shorten the time to recover viability during the transfectant selection stage. Artificial piggyBac (PB) and PB-like transposable elements with at least one modification within the left internal repeat that increases the homology of the left internal repeat to the left repeat have not been described or suggested in the art. It was unlikely that such modifications would have any effect. Furthermore, the present inventors have surprisingly established for the first time a targeting system / gene delivery system comprising a transposable element containing a piggyBac (PB) or PB-like artificial left internal repeat sequence for the improved generation of producer cell lines for the production of therapeutic proteins or for the high-yield production of viral particle-based biopharmaceuticals. Ta.

[0073] Thus, in a first aspect, the present invention relates to a (recombinant / artificial) polypeptide comprising, consisting essentially of, or consisting of a (hyperactive) piggyBac transposase, or a fragment or derivative thereof with transposase function, comprising at least one amino acid substitution (e.g. at least 1, 2, 3, 4 or 5 amino acid substitutions) selected from the group consisting of: a substitution of isoleucine (I) at amino acid position 30 or a corresponding amino acid position with alanine (A) (I30A); a substitution of glutamine (Q) at amino acid position 118 or the amino acid position corresponding thereto with proline (P) (Q118P); a substitution of methionine (M) at amino acid position 185 or a corresponding amino acid position with valine (V) (M185V); a substitution of methionine (M) at amino acid position 282 or the amino acid position corresponding thereto with leucine (L) (M282L); and An asparagine (N) at amino acid position 538 or a corresponding amino acid position is substituted with an arginine (R) (N538R).

[0074] Single and combination substitutions in piggyBac transposase or a fragment or derivative thereof are listed in FIG.

[0075] In a preferred embodiment, the piggyBac transposase or a fragment or derivative thereof having transposase function comprises the following amino acid substitutions: a substitution of isoleucine (I) at amino acid position 30 or a corresponding amino acid position with alanine (A) (I30A); a substitution of glutamine (Q) at amino acid position 118 or the amino acid position corresponding thereto with proline (P) (Q118P); a substitution of methionine (M) at amino acid position 185 or a corresponding amino acid position with valine (V) (M185V); a substitution of methionine (M) at amino acid position 282 or the amino acid position corresponding thereto with leucine (L) (M282L); and An asparagine (N) at amino acid position 538 or a corresponding amino acid position is substituted with an arginine (R) (N538R).

[0076] In a more preferred embodiment, the piggyBac transposase or a fragment or derivative thereof having transposase function comprises the following amino acid substitutions: a substitution of isoleucine (I) at amino acid position 30 or a corresponding amino acid position with alanine (A) (I30A); a substitution of methionine (M) at amino acid position 282 or the amino acid position corresponding thereto with leucine (L) (M282L); and An asparagine (N) at amino acid position 538 or a corresponding amino acid position is substituted with an arginine (R) (N538R).

[0077] The wild-type piggyBac transposase preferably has the nucleotide sequence set forth in SEQ ID NO: 17 and the amino acid sequence set forth in SEQ ID NO: 18. Thus, in an even more preferred embodiment, the piggyBac transposase is having the amino acid sequence set forth in SEQ ID NO: 18, Substitution of isoleucine (I) at amino acid position 30 with alanine (A) (I30A), Substitution of glutamine (Q) at amino acid position 118 with proline (P) (Q118P), a substitution of methionine (M) at amino acid position 185 with valine (V) (M185V); a substitution of methionine (M) at amino acid position 282 with leucine (L) (M282L); and a substitution of asparagine (N) at amino acid position 538 with arginine (R) (N538R); or The piggyBac transposase is a variant thereof that is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of the piggyBac transposase, a substitution of isoleucine (I) at amino acid position 30 or a corresponding amino acid position with alanine (A) (I30A); a substitution of glutamine (Q) at amino acid position 118 or the amino acid position corresponding thereto with proline (P) (Q118P); a substitution of methionine (M) at amino acid position 185 or a corresponding amino acid position with valine (V) (M185V); a substitution of methionine (M) at amino acid position 282 or the amino acid position corresponding thereto with leucine (L) (M282L); and Substitution of asparagine (N) at amino acid position 538 or a corresponding amino acid position with arginine (R) (N538R) and at least one amino acid substitution (e.g., at least one, two, three, four, or five amino acid substitutions) selected from the group consisting of:

[0078] In an even more preferred embodiment, the piggyBac transposase having the amino acid sequence set forth in SEQ ID NO: 18 and the following amino acid substitutions: Substitution of isoleucine (I) at amino acid position 30 with alanine (A) (I30A), Substitution of glutamine (Q) at amino acid position 118 with proline (P) (Q118P), a substitution of methionine (M) at amino acid position 185 with valine (V) (M185V); a substitution of methionine (M) at amino acid position 282 with leucine (L) (M282L); and containing a substitution of asparagine (N) at amino acid position 538 with arginine (R) (N538R); or The piggyBac transposase may be a variant thereof that is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of the piggyBac transposase, wherein the variant comprises the following amino acid substitutions: a substitution of isoleucine (I) at amino acid position 30 or a corresponding amino acid position with alanine (A) (I30A); a substitution of glutamine (Q) at amino acid position 118 or the amino acid position corresponding thereto with proline (P) (Q118P); a substitution of methionine (M) at amino acid position 185 or a corresponding amino acid position with valine (V) (M185V); a substitution of methionine (M) at amino acid position 282 or the amino acid position corresponding thereto with leucine (L) (M282L); and Substitution of asparagine (N) at amino acid position 538 or a corresponding amino acid position with arginine (R) (N538R) Includes:

[0079] Therefore, piggyBac transposase specifically: having the amino acid sequence set forth in SEQ ID NO: 20, which comprises an alanine (A) at amino acid position 30, a proline (P) at amino acid position 118, a valine (V) at amino acid position 185, a leucine (L) at amino acid position 282, and an arginine (R) at amino acid position 538; or The piggyBac transposase is a variant thereof that is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, wherein the variant comprises an alanine (A) at or corresponding to amino acid position 30, a proline (P) at or corresponding to amino acid position 118, a valine (V) at or corresponding to amino acid position 185, a leucine (L) at or corresponding to amino acid position 282, and an arginine (R) at or corresponding to amino acid position 538. This piggyBac transposase has the nucleotide sequence set forth in SEQ ID NO:19.

[0080] In a most preferred embodiment, the piggyBac transposase has the amino acid sequence set forth in SEQ ID NO: 18, and the following amino acid substitutions: Substitution of isoleucine (I) at amino acid position 30 with alanine (A) (I30A), a substitution of methionine (M) at amino acid position 282 with leucine (L) (M282L); and containing a substitution of asparagine (N) at amino acid position 538 with arginine (R) (N538R); or The piggyBac transposase may be a variant thereof that is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of the piggyBac transposase, wherein the variant comprises the following amino acid substitutions: a substitution of isoleucine (I) at amino acid position 30 or a corresponding amino acid position with alanine (A) (I30A); a substitution of methionine (M) at amino acid position 282 or the amino acid position corresponding thereto with leucine (L) (M282L); and Substitution of asparagine (N) at amino acid position 538 or a corresponding amino acid position with arginine (R) (N538R) Includes:

[0081] Therefore, piggyBac transposase specifically: having the amino acid sequence set forth in SEQ ID NO: 22, which comprises an alanine (A) at amino acid position 30, a leucine (L) at amino acid position 282, and an arginine (R) at amino acid position 538; or The piggyBac transposase is a variant thereof that is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, wherein the variant comprises an alanine (A) at or corresponding to amino acid position 30, a leucine (L) at or corresponding to amino acid position 282, and an arginine (R) at or corresponding to amino acid position 538. The piggyBac transposase has the nucleotide sequence set forth in SEQ ID NO: 21. The piggyBac transposase, or a fragment or derivative thereof, has improved transposase function, in particular improved activity / ability to mediate the excision and / or insertion of nucleotide sequences, e.g., DNA, compared to transposases described in the art (e.g., wild-type / naturally occurring transposases). In particular, the piggyBac transposase described above, or a fragment or derivative thereof, is capable of mobilizing a transposon, e.g., a transposable element described herein, from one genomic location to another genomic location with greater efficiency than transposases described in the art (e.g., wild-type / naturally occurring transposases). Thus, the piggyBac transposase according to the first aspect, or a fragment or derivative thereof, may also be referred to as a hyperactive piggyBac transposase, or a fragment or derivative thereof.

[0082] In a further preferred embodiment, the polypeptide comprises at least one heterologous chromatin leader element (CRE). A polypeptide comprising a hyperactive transposase or a fragment or derivative thereof and at least one chromatin leader element (CRE) enables the targeting of transposable elements, particularly transposable elements of the present invention, to random locations within the genome with high transcriptional activity. In other words, a polypeptide comprising a transposase or a fragment or derivative thereof and at least one chromatin leader element enables the targeting of active chromatin. The result of this targeting process is the integration of a transposable element containing a polynucleotide of interest (e.g., encoding a protein or viral particle), particularly a transposable element of the present invention, into transcriptionally active chromatin via the transposase or a fragment or derivative thereof. This, in turn, enables the generation of high-producer cell lines for the production of biopharmaceuticals based on proteins (e.g., therapeutic proteins) or viral particles. At least one heterologous chromatin reader element (CRE) may be linked to the transposase or a fragment or derivative thereof, preferably via a linker. In particular, at least one heterologous CRE may be linked to the N-terminus and / or C-terminus of the transposase, preferably via a linker.

[0083] In a more preferred embodiment, the at least one heterologous chromatin reader element (CRE) is at least one heterologous chromatin reader domain (CRD). The at least one heterologous chromatin reader domain (CRD) may be linked to the transposase or a fragment or derivative thereof, preferably via a linker. In particular, the at least one heterologous CRD may be linked to the N-terminus and / or C-terminus of the transposase, preferably via a linker.

[0084] Preferably, the CRD recognizes histone methylation level and / or histone acetylation status. More preferably, the CRD is a plant homeodomain (PHD)-type zinc finger. Even more preferably, the PHD-type zinc finger is transcription initiation factor TFIID subunit 3PHD.

[0085] In an alternatively more preferred embodiment, at least one heterologous chromatin reader element (CRE) is an artificial CRE. Preferably, the artificial CRE recognizes a histone tail having a specific methylation site and / or acetylation site. More preferably, the artificial CRE is selected from the group consisting of a microantibody, a single-chain antibody, an antibody fragment, an affibody, an affilin, an anticalin, an atrimer, a DARPin, an FN2 scaffold, a fynomer, and a Kunitz domain.

[0086] Also (alternatively or additionally) in a preferred embodiment, the polypeptide comprises at least Each of the DNA-binding domains further comprises a heterologous DNA-binding domain (eg, at least one or two DNA-binding domains).

[0087] Also (alternatively or additionally), in a preferred embodiment, the polypeptide further comprises a heterologous nuclear localization signal (NLS), which may form the N-terminus of the transposase / polypeptide or the C-terminus of the transposase / polypeptide.

[0088] In a more preferred embodiment, the polypeptide comprises a transposase as described above or a fragment or derivative thereof, at least one heterologous chromatin reader element (CRE), and at least one heterologous DNA binding domain.

[0089] In an even more preferred embodiment, the polypeptide comprises the above-mentioned transposase or a fragment or derivative thereof, at least one heterologous chromatin reader element (CRE), at least one heterologous DNA-binding domain, and a heterologous nuclear localization signal (NLS).

[0090] In a second aspect, the present invention relates to a polynucleotide encoding a polypeptide according to the first aspect, said polynucleotide being preferably DNA or RNA such as mRNA.

[0091] In a third aspect, the present invention relates to a vector comprising a polynucleotide according to the second aspect. The terms "vector" and "plasmid" can be used interchangeably herein. The vector may be a viral vector or a non-viral vector. Preferably, the vector is an expression vector. In some embodiments, the vector is a small circle. Expression of a polynucleotide encoding a polypeptide according to the first aspect is preferably controlled by an expression control sequence. The expression control sequence may be a sequence that controls transcription, such as a promoter, an enhancer, a UCOE or MAR element, a polyadenylation signal, a post-transcriptional active element, such as an RNA stabilizing element, an RNA transport element, and a translation enhancer. Such expression control sequences are known to those skilled in the art. For example, a CMV promoter or a PGK promoter can be used as the promoter.

[0092] In a fourth aspect, the present invention provides a piggyBac or piggyBac-like Left repeat and including the left internal repeat sequence, the left internal repeat sequence comprises at least one nucleotide alteration; At least one nucleotide modification relates to the transposable element that increases the homology of the left internal repeat sequence to the left repeat sequence.

[0093] Preferably, the at least one nucleotide modification is selected from the group consisting of a nucleotide substitution (e.g., at least one, two, three, or four nucleotide substitution), a nucleotide deletion (e.g., at least one, two, three, or four nucleotide deletion), a nucleotide addition (e.g., at least one, two, three, or four nucleotide addition), and a nucleotide insertion (e.g., at least one, two, three, or four nucleotide insertion), or a combination thereof.

[0094] The piggyBac left repeat and / or left internal repeat are preferably derived from Trichoplusia ni. The piggyBac-like left repeat and / or left internal repeat are preferably derived from Xenopus tropicalis. ropicalis, Bombyx mori (silkworm), and Myotis lucifugus.

[0095] In a preferred embodiment, the piggyBac left internal repeat sequence is having the nucleotide sequence set forth in SEQ ID NO: 1, and Substitution of adenosine (A) at nucleotide position 3 with cytidine (C) (A3C), Substitution of adenosine (A) at nucleotide position 9 with thymidine (T) (A9T), Substitution of adenosine (A) at nucleotide position 10 with thymidine (T) (A10T), and Substitution of guanosine (G) at nucleotide position 12 with thymidine (T) (G12T) or The piggyBac left repeat sequence is a variant thereof that is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence, wherein the variant is a substitution of an adenosine (A) at nucleotide position 3 or a corresponding nucleotide position with a cytidine (C) (A3C); a substitution of adenosine (A) at nucleotide position 9 or the corresponding nucleotide position with thymidine (T) (A9T); a substitution of adenosine (A) at nucleotide position 10 or the corresponding nucleotide position with thymidine (T) (A10T), and Substitution of guanosine (G) at nucleotide position 12 or the corresponding nucleotide position with thymidine (T) (G12T) and at least one nucleotide substitution (e.g., at least one, two, three, or four substitutions) selected from the group consisting of:

[0096] In an alternatively preferred embodiment, the piggyBac-like left internal repeat sequence is having the nucleotide sequence set forth in SEQ ID NO:2 and containing a nucleotide insertion; an adenosine (A) is introduced between nucleotide positions 7 and 8, or A variant thereof wherein the piggyBac-like left internal repeat sequence is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, identical to the nucleotide sequence, wherein the variant comprises a nucleotide insertion wherein an adenosine (A) is introduced between nucleotide positions 7 and 8 or between the nucleotide positions corresponding thereto.

[0097] In an alternatively preferred embodiment, the piggyBac-like left internal repeat sequence is having the nucleotide sequence set forth in SEQ ID NO:3, and A substitution of guanosine (G) at nucleotide position 7 with cytidine (C) (G7C) and a substitution of thymidine (T) at nucleotide position 9 with cytidine (C) (T9C). or The piggyBac-like left internal repeat sequence is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99% identical to the nucleotide sequence, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110, 111, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 6%, 97%, 98%, or 99% identical variants thereof, wherein the variants are a substitution of guanosine (G) at nucleotide position 7 or the corresponding nucleotide position with cytidine (C) (G7C); and Substitution of thymidine (T) at nucleotide position 9 or the corresponding nucleotide position with cytidine (C) (T9C) The amino acid sequence of the present invention comprises at least one nucleotide substitution (e.g., at least one or two substitutions) selected from the group consisting of:

[0098] In an alternatively preferred embodiment, the piggyBac-like left internal repeat sequence is having the nucleotide sequence set forth in SEQ ID NO:4, and contains a nucleotide substitution in which thymidine (T) at nucleotide position 7 is replaced with adenosine (A) (T7A); or the piggyBac-like left internal repeat sequence is a variant thereof which is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e. 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to said nucleotide sequence, said variant being It contains a nucleotide substitution in which a thymidine (T) at nucleotide position 7 or the corresponding nucleotide position is replaced with an adenosine (A) (T7A).

[0099] In an alternatively preferred embodiment, the piggyBac-like left internal repeat sequence is having the nucleotide sequence set forth in SEQ ID NO:5, and A substitution of guanosine (G) at nucleotide position 6 with thymidine (T) (G6T), and a substitution of thymidine (T) at nucleotide position 14 with guanosine (G) (T14G). or a variant thereof, wherein the piggyBac-like left internal repeat sequence is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence, wherein the variant is a substitution of guanosine (G) at nucleotide position 6 or the corresponding nucleotide position with thymidine (T) (G6T); and Substitution of thymidine (T) at nucleotide position 14 or the corresponding nucleotide position with guanosine (G) (T14G) The amino acid sequence of the present invention comprises at least one nucleotide substitution (e.g., at least one or two substitutions) selected from the group consisting of:

[0100] In a further preferred embodiment, the piggyBac or piggyBac-like left repeat has a nucleotide sequence selected from the group consisting of SEQ ID NO: 23 to SEQ ID NO: 27, or a variant thereof that is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said nucleotide sequence.

[0101] It should be noted that the piggyBac or piggyBac-like left repeat sequence and the piggyBac or piggyBac-like left internal repeat sequence are preferably included in / are part of the 5'-transposon end sequence. Preferably, the piggyBac left repeat and the piggyBac left internal repeat are included together / combined. Also preferred is the piggyBac-like left repeat and the piggyBac-like left internal repeat together / combined. Thus, in a more preferred embodiment, the 5'-transposon end sequence comprises: (i) a piggyBac left repeat having the nucleotide sequence set forth in SEQ ID NO: 23, or a variant thereof that is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to said nucleotide sequence; and a piggyBac left internal repeat sequence having the nucleotide sequence set forth in SEQ ID NO: 1 and comprising at least one nucleotide substitution (e.g. at least one, two, three, or four substitutions) selected from the group consisting of A3C, A9T, A10T, and G12T, or a variant thereof which is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to said nucleotide sequence, said variant comprising at least one nucleotide substitution (e.g., at least one, two, three, or four substitutions) selected from the group consisting of A3C, A9T, A10T, and G12T (or at a position corresponding thereto); (ii) a piggyBac-like left repeat having the nucleotide sequence set forth in SEQ ID NO: 24, or a variant thereof that is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to said nucleotide sequence; and a piggyBac-like left internal repeat having the nucleotide sequence set forth in SEQ ID NO:2 and comprising an adenosine insertion between nucleotide positions 7 and 8, or a variant thereof which is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to said nucleotide sequence, said variant comprising an adenosine insertion between nucleotide positions 7 and 8 (or between nucleotide positions corresponding thereto); (iii) a piggyBac-like left repeat having the nucleotide sequence set forth in SEQ ID NO: 25, or a variant thereof that is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to said nucleotide sequence; and 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, wherein the variant has the nucleotide sequence set forth in SEQ ID NO: 3 and contains at least one nucleotide substitution (e.g., at least one or two substitutions) selected from the group consisting of G7C and T9C, or a variant thereof that is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, wherein the variant contains at least one nucleotide substitution (e.g., at least one or two substitutions) selected from the group consisting of G7C and T9C (or a position corresponding thereto). a piggyBac-like left internal repeat containing one or two substitutions, (iv) a piggyBac-like left repeat having a nucleotide sequence as set forth in SEQ ID NO: 26, or a variant thereof that is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to said nucleotide sequence; and a piggyBac-like left internal repeat sequence having the nucleotide sequence set forth in SEQ ID NO: 4 and comprising a nucleotide substitution in T7A, or a variant thereof which is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to said nucleotide sequence, said variant comprising a nucleotide substitution in T7A (or at a position corresponding thereto); (v) a piggyBac-like left repeat having the nucleotide sequence set forth in SEQ ID NO: 27, or a variant thereof that is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to said nucleotide sequence; and 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, wherein the variant comprises at least one nucleotide substitution (e.g., at least one or two substitutions) selected from the group consisting of G6T and T14G (or a position corresponding thereto).

[0102] The piggyBac or piggyBac-like left repeat sequence and the piggyBac or piggyBac-like left internal repeat sequence may be combined / linked to each other by a naturally occurring transposable element sequence or by a non-naturally occurring transposable element sequence, e.g., by a (heterologous) linker sequence.

[0103] In an alternatively more preferred embodiment, the 5'-transposon end sequence is (i) having the nucleotide sequence set forth in SEQ ID NO:6, or a variant thereof that is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to said nucleotide sequence, wherein said nucleotide sequence or variant thereof comprises the nucleotide sequence set forth in SEQ ID NO:1 containing at least one nucleotide substitution (e.g., at least one, two, three, or four substitutions) selected from the group consisting of A3C, A9T, A10T, and G12T; (ii) a nucleotide sequence as set forth in SEQ ID NO: 7, or a variant thereof that is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to said nucleotide sequence, wherein said nucleotide sequence or variant thereof comprises an adenosine insertion between nucleotide positions 7 and 8. 2, or (iii) a nucleotide sequence set forth in SEQ ID NO: 8, or a variant thereof that is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence, wherein the nucleotide sequence or variant thereof comprises the nucleotide sequence set forth in SEQ ID NO: 3 containing at least one nucleotide substitution (e.g., at least one or two substitutions) selected from the group consisting of G7C and T9C; (iv) a nucleotide sequence as set forth in SEQ ID NO: 9, or a variant thereof that is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to said nucleotide sequence, wherein said nucleotide sequence or variant thereof comprises the nucleotide sequence as set forth in SEQ ID NO: 4 containing a T7A nucleotide substitution; or (v) The nucleotide sequence set forth in SEQ ID NO: 10, or a variant thereof that is at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% or 99%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence, wherein the nucleotide sequence or variant thereof includes the nucleotide sequence set forth in SEQ ID NO: 5 that contains at least one nucleotide substitution (e.g., at least one or two substitutions) selected from the group consisting of G6T and T14G.

[0104] A left internal repeat sequence containing the above-described nucleotide modification may also be referred to as an artificial left internal repeat sequence / modified left internal repeat sequence. A particularly preferred artificial left internal repeat sequence / modified left internal repeat sequence has the nucleotide sequence set forth in SEQ ID NO: 28. Furthermore, a 5'-transposon end sequence containing the above-described nucleotide modification may also be referred to as an artificial 5'-transposon end sequence / modified 5'-transposon end sequence. A particularly preferred artificial 5'-transposon end sequence / modified 5'-transposon end sequence has the nucleotide sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 16.

[0105] It should be noted that the transposable element preferably comprises a piggyBac or piggyBac-like 3'-transposon end sequence. This is preferably a wild-type sequence. A preferred 3'-transposon end sequence has the nucleotide sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 14. Thus, in an even more preferred embodiment, the transposable element comprises a piggyBac or piggyBac-like 5'-transposon end sequence comprising the above-mentioned piggyBac or piggyBac-like left internal repeat sequence and the above-mentioned piggyBac or piggyBac-like left repeat sequence, as well as a piggyBac or piggyBac-like 3'-transposon end sequence.

[0106] The transposable element preferably comprises: at least one polynucleotide of interest, or At least one cloning site for inserting at least one polynucleotide of interest It should be noted that this includes especially, At least one polynucleotide of interest comprises a piggyBac or piggyBac-like 5'-transposon end sequence and a piggyBac or piggyBac-like 3'-transposon end sequence. '-operably linked to a transposon end sequence, or At least one cloning site for inserting at least one polynucleotide of interest is located between the piggyBac or piggyBac-like 5'-transposon end sequence and the piggyBac or piggyBac-like 3'-transposon end sequence. The at least one polynucleotide of interest may be operably linked to the piggyBac or piggyBac-like 5'-transposon end sequence and the piggyBac or piggyBac-like 3'-transposon end sequence by a native transposable element sequence or by a non-native transposable element sequence, for example by a (heterologous) linker sequence.

[0107] The at least one polynucleotide of interest is preferably selected from the group consisting of a polynucleotide encoding a polypeptide, a non-coding polynucleotide, a polynucleotide comprising a promoter sequence, a polynucleotide encoding an mRNA, a polynucleotide encoding a tag, and a viral polynucleotide.

[0108] The polynucleotide of interest may encode a therapeutic polypeptide, such as an antibody, antibody fragment, monoclonal antibody, viral protein, viral protein fragment, antigen, or hormone. Polypeptides may be used for gene therapy, e.g., treatment of monogenic diseases. In this case, the polynucleotide encoding the polypeptide is operably linked to a tissue-specific promoter. Polypeptides may also be used for cell therapy, particularly ex vivo. The cells may be pluripotent stem cells (iPSCs), human embryonic stem (hES) cells, human hematopoietic stem cells (HSCs), or human T lymphocytes. Non-coding polynucleotides may be useful in targeted gene disruption. Polynucleotides containing promoter sequences may enable activation of gene expression when a transposon is inserted adjacent to an endogenous gene. Polynucleotides may be transcribed into mRNA or functional non-coding RNA, such as shRNA or gRNA. Polynucleotides may include sequence tags for identifying the insertion site of the transposable element. Viral polynucleotides may be used for the production of viral particle-based biopharmaceuticals.

[0109] The expression of the polynucleotide of interest is preferably controlled by an expression control sequence. The expression control sequence may be a transcription control sequence, such as a promoter, an enhancer, a UCOE or MAR element, a polyadenylation signal, a post-transcriptional active element, such as an RNA stabilizing element, an RNA transport element, and a translation enhancer. Such expression control sequences are known to those skilled in the art. For example, a CMV promoter or a PGK promoter may be used as the promoter. The preferred structure of the transposable element containing the polynucleotide of interest (GOI = gene of interest) is shown in Figure 2a.

[0110] Thus, in an even more preferred embodiment, the transposable element comprises a piggyBac or piggyBac-like 5'-transposon end sequence comprising the above-mentioned piggyBac or piggyBac-like left internal repeat sequence and the above-mentioned piggyBac or piggyBac-like left repeat sequence, at least one polynucleotide of interest, and a piggyBac or piggyBac-like 3'-transposon end sequence. A preferred structure of a transposable element containing a polynucleotide of interest (GOI = gene of interest) is shown in Figure 2b. Alternatively, the transposable element may comprise a piggyBac or piggyBac-like 5'-transposon end sequence comprising the piggyBac or piggyBac-like left internal repeat sequence described above and the piggyBac or piggyBac-like left repeat sequence described above, and at least one cloning site for inserting at least one polynucleotide of interest. and a piggyBac or piggyBac-like 3'-transposon end sequence.

[0111] Preferably, the transposable element is circular and is contained in / is part of a plasmid vector or contained in / is part of a small circular DNA vector.

[0112] The transposable element and / or vector comprising the transposable element may further comprise elements that enhance expression (e.g., nuclear export signals, promoters, introns, terminators, enhancers, elements that affect chromatin structure, RNA export elements, IRES elements, CHYSEL elements, and / or Kozak sequences), selectable markers (e.g., DHFR, puromycin, hygromycin, zeocin, blasticidin, and / or neomycin), markers for in vivo monitoring (e.g., GFP or β-galactosidase), restriction endonuclease recognition sites (e.g., sites for insertion of exogenous nucleotide sequences such as multiple cloning sites), recombinase recognition sites (e.g., LoxP (recognized by Cre), FRT (recognized by Flp), or AttB / AttP (recognized by PhiC31)), insulators (e.g., MAR or UCOE), viral replication sequences (e.g., SV40 ori), and / or sequences compatible with DNA-binding domains.

[0113] It is also preferred that the transposable element is a piggyBac or piggyBac-like transposable element. More preferably, the piggyBac transposable element is derived from Trichoplusia ni. Alternatively, it is more preferred that the piggyBac-like transposable element is selected from the group consisting of Xenopus tropicalis, Bombyx mori (the silkworm), and Myotis lucifugus.

[0114] In a fifth aspect, the present invention provides a method (in vitro or in vivo) for producing a transgenic cell, comprising: (i) providing cells; (ii) (iia) a transposable element, and a polypeptide according to the first aspect, or a polynucleotide according to the second aspect, or a vector according to the third aspect, or (iib) a transposable element according to the fourth aspect, and a transposase or a fragment or derivative thereof having transposase function, or a polynucleotide encoding a transposase or a fragment or derivative thereof having transposase function; a vector comprising a polynucleotide encoding a transposase or a fragment or derivative thereof having transposase function; (iic) a transposable element according to the fourth aspect, and a polypeptide according to the first aspect, or a polynucleotide according to the second aspect, or a vector according to the third aspect, into said cells, thereby producing transgenic cells. The present invention relates to a method, including:

[0115] The method may be an in vitro method or an in vivo method. Preferably, the method is an in vitro method.

[0116] In one embodiment, the introduction is via electroporation, transfection, injection, lipofection, and / or (viral) infection.

[0117] Naturally, transposable elements contain a polynucleotide encoding a functional transposase that catalyzes excision and insertion. However, the transposable elements described in methods (iia), (iib), and (iic) above lack a polynucleotide encoding a functional transposase. This transposable element does not contain a complete sequence encoding a functional transposase, particularly a naturally occurring functional transposase. Preferably, the complete sequence encoding a functional transposase, particularly a naturally occurring functional transposase, or a portion thereof, is deleted from this transposable element. It can be replaced by at least one polynucleotide of interest (see below).

[0118] The transposase or a fragment or derivative thereof having transposase function is provided in trans in the above methods, for example as a polypeptide, such as a polypeptide according to the first aspect, as a polynucleotide, such as a polynucleotide according to the second aspect, or contained in a vector, such as a vector according to the third aspect.

[0119] The introduction of the transposable element described in the above methods (iia), (iib) and (iic) can be carried out via electroporation, transfection, injection, lipofection or (viral) infection. The transposable element can be transiently or stably introduced into the cell. In the first case, the transposable element is introduced as an extrachromosomal element, for example, as a linear DNA molecule, plasmid DNA, episomal DNA, small circular DNA, viral DNA or viral RNA. In the second case, the transposable element is stably introduced / inserted into the genome of the cell. Preferably, the transposable element is transiently introduced into the cell. More preferably, the transposable element is contained in a vector. Those skilled in the art are familiar with molecular biology techniques for introducing transposable elements into cells, such as microinjection, electroporation or lipofection, and know how to perform these techniques. It is particularly preferred that the transposable element is the transposable element according to the fourth aspect.

[0120] Introduction of the polypeptides, polynucleotides or vectors described in methods (iia), (iib) and (iic) above can also be carried out via electroporation, transfection, injection, lipofection and / or (viral) infection.

[0121] When a polynucleotide is introduced into a cell, the polynucleotide is subsequently transcribed and translated into a polypeptide within the cell. When a vector containing a polynucleotide is introduced into a cell, the polynucleotide is subsequently transcribed from the vector and translated into a polypeptide within the cell. The polynucleotide may be DNA or RNA, such as mRNA. Viral DNA or RNA may also be introduced. The polynucleotide may be transiently or stably introduced into the cell. In the first case, the polynucleotide is introduced as an extrachromosomal polynucleotide, for example, as a linear DNA molecule, a circular DNA molecule, a plasmid DNA, a viral DNA, an in vitro synthesized / transcribed RNA, or a viral RNA. In the second case, the polynucleotide is stably introduced / inserted into the genome of the cell. Preferably, the polynucleotide is transiently introduced into the cell. More preferably, the polynucleotide is comprised in a vector, in particular an expression vector. Viral DNA or RNA sequences may be introduced as part of a vector or in the form of a vector. It is particularly preferred that the polynucleotide is operably linked to a heterologous promoter allowing transcription of the transposase, or a fragment or derivative thereof with transposase function, in the cell or from a vector, e.g., an expression vector contained in the cell or a vector used for in vitro transcription.

[0122] Those skilled in the art are familiar with molecular biology techniques for introducing polypeptides or nucleic acid sequences encoding polypeptides into cells, such as techniques such as microinjection, electroporation, or lipofection, and know how to perform these techniques.

[0123] It is particularly preferred that the polypeptide is a polypeptide according to the first aspect, the polynucleotide is a polynucleotide according to the second aspect, or the vector is a vector according to the third aspect.

[0124] In a preferred embodiment, the transposable elements according to methods (iia), (iib), and (iic) are comprised in / are part of a polynucleotide molecule, preferably a vector. In this case, the polynucleotides according to methods (iia), (iib), and (iic) are also comprised in / are part of a polynucleotide molecule, preferably a vector. Thus, it is preferred that the polynucleotides according to methods (iia), (iib), and (iic) and the transposable elements according to methods (iia), (iib), and (iic) are in separate (different) polynucleotide molecules, preferably in (different) vectors. Particularly preferably, the transposable element of (iia) is comprised in / is part of a polynucleotide molecule, preferably a vector.

[0125] In another preferred embodiment, the transposable elements according to methods (iia), (iib) and (iic) and the polynucleotides according to methods (iia), (iib) and (iic) are comprised in / are part of the same polynucleotide molecule, preferably a vector. Particularly preferably, the transposable element of (iia) and the polynucleotide according to the second aspect are comprised in / are part of a polynucleotide molecule, preferably a vector.

[0126] The transposable elements described in methods (iia), (iib), and (iic) above carry sequences necessary for mobilization by a transposase provided in trans. These are repeat sequences at each end of the transposable element that contain binding sites for the transposase, allowing excision from the genome. Thus, in a further preferred embodiment, the transposable elements described in methods (iia), (iib), and (iic) above comprise terminal repeats (TRs). It is particularly preferred that the transposable element of (iia) comprises terminal repeats (TRs). The terminal repeats are preferably terminal inverted repeats (TIRs). In this regard, it should be noted that the transposase provided in trans is specific to the transposable element. In other words, the transposable element is specifically recognized by the transposase. .

[0127] In a further (alternatively or additionally) preferred embodiment, the transposable elements according to (iia), (iib) and (iic) of the above method comprise at least one polynucleotide of interest. It is particularly preferred that the transposable element of (iia) comprises at least one polynucleotide of interest. Preferably, at least one polynucleotide of interest is flanked by terminal repeats (TRs). More preferably, the terminal repeat is a terminal inverted repeat (TIR). For example, the transposable element described in (iia), (iib), and (iic) of the above method comprises a first transposable element-specific terminal repeat and a second transposable element-specific terminal repeat downstream of the first transposable element-specific terminal repeat. At least one polynucleotide of interest is located between the first transposable element-specific terminal repeat and the second transposable element-specific terminal repeat.

[0128] Even more preferably, the at least one polynucleotide of interest is selected from the group consisting of a polynucleotide encoding a polypeptide, a non-coding polynucleotide, a polynucleotide comprising a promoter sequence, a polynucleotide encoding an mRNA, a polynucleotide encoding a tag, and a viral polynucleotide.

[0129] The polynucleotide of interest may encode a therapeutic polypeptide, such as an antibody, antibody fragment, monoclonal antibody, viral protein, viral protein fragment, antigen, or hormone. Polypeptides may be used for gene therapy, e.g., treatment of monogenic diseases. In this case, the polynucleotide encoding the polypeptide is operably linked to a tissue-specific promoter. Polypeptides may also be used for cell therapy, particularly ex vivo. The cells may be pluripotent stem cells (iPSCs), human embryonic stem (hES) cells, human hematopoietic stem cells (HSCs), or human T lymphocytes. Non-coding polynucleotides may be useful in targeted gene disruption. Polynucleotides containing promoter sequences may enable activation of gene expression when a transposon is inserted adjacent to an endogenous gene. Polynucleotides may be transcribed into mRNA or functional non-coding RNA, such as shRNA or gRNA. Polynucleotides may include sequence tags for identifying the insertion site of the transposable element. Viral polynucleotides may be used for the production of viral particle-based biopharmaceuticals.

[0130] The expression of the polynucleotide of interest is preferably controlled by an expression control sequence. The expression control sequence may be a transcription control sequence, such as a promoter, an enhancer, a UCOE or MAR element, a polyadenylation signal, a post-transcriptional active element, such as an RNA stabilizing element, an RNA transport element, and a translation enhancer. Such expression control sequences are known to those skilled in the art. For example, a CMV promoter or a PGK promoter may be used as the promoter.

[0131] The transposable element and / or vector comprising the transposable element according to (iia), (iib) and (iic) of the above method may further comprise any of a number of elements, including elements that enhance expression (e.g., nuclear export signals, promoters, introns, terminators, enhancers, elements that affect chromatin structure, RNA export elements, IRES elements, CHYSEL elements, and / or Kozak sequences), selectable markers (e.g., DHFR, puromycin, hygromycin, zeocin, blasticidin, and / or neomycin), markers for in vivo monitoring (e.g., GFP or β-galactosidase), restriction endonuclease recognition sites (e.g., sites for insertion of exogenous nucleotide sequences such as multiple cloning sites), recombinase recognition sites, and the like. The nucleic acid sequence may further comprise a recognition site (e.g., LoxP (recognized by Cre), FRT (recognized by Flp), or AttB / AttP (recognized by PhiC31)), an insulator (e.g., MAR or UCOE), a viral replication sequence (e.g., SV40 on), and / or a sequence compatible with the DNA-binding domain for targeting and DNA-binding domain properties ("bridging") via an additional binding molecule, particularly one with a chromatin reader domain.

[0132] In the above method, not only one transposable element may be inserted into a cell, but two or more transposable elements may be inserted into a cell. The transposable elements may be different from each other, for example, because they contain different polynucleotides of interest. This is particularly desirable when two ORFs encoding antibody heavy chains (HC) or antibody light chains (LC) must be introduced into a cell. In this case, the two or more ORFs may be contained in the same transposable element or in separate transposable elements, preferably in separate transposable elements.

[0133] Also (alternatively or additionally), in a preferred embodiment, the transposable element according to (iia), (iib), and (iic) of the above method is a DNA transposable element. The DNA transposable element preferably comprises a terminal inverted repeat (TIR). It is particularly preferred that the transposable element of (iia) is a DNA transposable element, and that this DNA transposable element preferably comprises a terminal inverted repeat (TIR). The DNA transposable element may be a piggyBac or piggyBac-like transposable element.

[0134] It is particularly preferred that the transposable element in (iia) of the above method is selected from the group consisting of a wild-type piggyBac transposable element, a hyperactive piggyBac transposable element, a wild-type piggyBac-like transposable element, and a hyperactive piggyBac-like transposable element. It is particularly more preferred that the piggyBac-like transposable element is selected from the group consisting of a piggyBat transposable element, a piggyBac-like transposable element derived from Xenopus tropicalis, a piggyBac-like transposable element derived from Bombyx mori, and a piggyBac-like transposable element derived from Myotis lucifugus. It is also particularly more preferred that the piggyBac transposable element is derived from Trichoplusia ni.

[0135] Conservative DNA-based transposable elements are transferred by a cut-and-paste mechanism. This requires a transposase, the inverted terminal repeats of the transposable element, and a target sequence on the new host DNA molecule. The transposase is provided in trans in the above-described method. The transposase catalyzes the excision of the transposable element from its current location and the integration of the excised transposable element into the genome of the cell. In the cut-and-paste mechanism, the transposase specifically binds to the inverted terminal repeats of the transposable element and excises the transposable element from its current location, e.g., from a vector. The transposase then positions the transposable element, cuts the target DNA backbone, and then inserts the transposable element. Typically, two transposase monomers are involved in the excision of the transposable element, one at each end of the transposable element. Finally, the excised transposable element is inserted into the target DNA backbone. The transposase dimer complexed with the transposable element reintegrates the transposable element into the cellular DNA.

[0136] It is particularly preferred that the transposase or a fragment or derivative thereof in (iib) of the above method is selected from the group consisting of wild-type piggyBac transposase or a fragment or derivative thereof, hyperactive piggyBac transposase or a fragment or derivative thereof, wild-type piggyBac-like transposase or a fragment or derivative thereof, and hyperactive piggyBac-like transposase or a fragment or derivative thereof.It is even more preferred that the piggyBac-like transposase or a fragment or derivative thereof is selected from the group consisting of piggyBat transposase or a fragment or derivative thereof, piggyBac-like transposase from Xenopus tropicalis or a fragment or derivative thereof, piggyBac-like transposase from Bombyx mori or a fragment or derivative thereof, and piggyBac-like transposase from Myotis lucifugus or a fragment or derivative thereof. It is also particularly preferred that the piggyBac transposase or a fragment or derivative thereof is derived from Trichoplusia ni.

[0137] It is further particularly preferred that the transposase or a fragment or derivative thereof of the above method (iib) is bound / fused to at least one heterologous chromatin leader element (CRE). The at least one heterologous chromatin leader element (CRE) may be linked to the transposase or a fragment or derivative thereof, preferably via a linker. In particular, the at least one heterologous CRE may be linked to the N-terminus and / or C-terminus of the transposase, preferably via a linker.

[0138] It is particularly preferred that the at least one heterologous chromatin reader element (CRE) is at least one heterologous chromatin reader domain (CRD). The at least one heterologous chromatin reader domain (CRD) can be linked to the transposase or a fragment or derivative thereof, preferably via a linker. In particular, the at least one heterologous CRD can be linked to the N-terminus and / or C-terminus of the transposase, preferably via a linker.

[0139] Preferably, the CRD recognizes histone methylation level and / or histone acetylation status. More preferably, the CRD is a plant homeodomain (PHD)-type zinc finger. Even more preferably, the PHD-type zinc finger is transcription initiation factor TFIID subunit 3 PHD.

[0140] Alternatively, it is especially more preferred that at least one heterologous chromatin reader element (CRE) is an artificial CRE. Preferably, the artificial CRE recognizes histone tails having specific methylation and / or acetylation sites, and more preferably, the artificial CRE is selected from the group consisting of a microantibody, a single-chain antibody, an antibody fragment, an affibody, an affilin, an anticalin, an atrimer, a DARPin, an FN2 scaffold, a fynomer, and a Kunitz domain.

[0141] The cell may be a prokaryotic or eukaryotic cell. Preferably, the cell is a eukaryotic cell. More preferably, the eukaryotic cell is a vertebrate cell, a yeast cell, a fungal cell, or an insect cell. The vertebrate cell may be a mammalian cell, a fish cell, an amphibian cell, a reptile cell, or an avian cell. The avian cell may be a chicken cell, a quail cell, a goose cell, or an avian cell. The vertebrate cell may be a duck cell, such as a duck retina cell or a duck somite cell. Even more preferably, the vertebrate cell is a mammalian cell. Most preferably, the mammalian cell is selected from the group consisting of Chinese hamster ovary (CHO) cells (e.g., CHO-K1 / CHO-S / CHO-DUXB11 / CHO-DG44 cells), human embryonic kidney (HEK293) cells, HeLa cells, A549 cells, MRC5 cells, WI38 cells, AGE1.CR cells, BHK cells, and Vero cells.

[0142] The cells may be isolated cells (e.g., in a cell culture or cell line, e.g., in a stable cell line). The cells may also be cells of tissues outside of an organism. However, transgenic cells may then be inserted into an organism. Insertion of transgenic cells into an organism may be by infusion or injection, or by further means known to those skilled in the art.

[0143] A cell may also be part of / contained in an organism, for example a eukaryotic multicellular organism. In this case, the insertion of the transposable element, polypeptide, polynucleotide, or vector referred to in methods (iia), (iib), and (iic) above is carried out in vivo. In vivo delivery can be achieved by injection (either local or systemic). The polynucleotide / transposable element can be, for example, in the form of naked DNA, or DNA complexed with liposomes, PEI, or other condensing agents, or can be incorporated into infectious particles (virus particles or virus-like particles). Polynucleotide / transposable element delivery can also be performed using electroporation, a gene gun, or an aerosol.

[0144] The organism may be a prokaryote or a eukaryote. Preferably, the organism is a eukaryote. More preferably, the organism may be a fungus, an insect, or a vertebrate. The vertebrate may be a bird (e.g., a chicken, a quail, a goose, or a duck), a dog, a weasel, a rodent (e.g., a mouse, a rat, or a hamster), a sheep, a goat, a pig, a bat (e.g., a fruit bat or a small bat), or a human / non-human primate (e.g., a monkey or a great ape). Most preferably, the organism is a mammal, such as a mouse, a rat, a pig, or a human / non-human primate.

[0145] In a sixth aspect, the present invention relates to cells, in particular transgenic cells, obtainable / producible by a method according to the fifth aspect.

[0146] In a seventh aspect, the present invention relates to the use of a cell, in particular a transgenic cell, according to the sixth aspect for the production of a protein or a virus. The protein may be a therapeutic protein. The virus may be a vector (viral vector).

[0147] In an eighth aspect, the present invention provides a method for producing a composition comprising: (i) a transposable element, and a polypeptide according to the first aspect, or a polynucleotide according to the second aspect, or a vector according to the third aspect; or (ii) a transposable element according to the fourth aspect, and a transposase or a fragment or derivative thereof having transposase function, or a polynucleotide encoding a transposase or a fragment or derivative thereof having transposase function; a vector comprising a polynucleotide encoding a transposase or a fragment or derivative thereof having transposase function; or (iii) a transposable element according to the fourth aspect, and a polypeptide according to the first aspect, or a polynucleotide according to the second aspect, or a vector according to the third aspect, The present invention relates to a kit comprising:

[0148] The transposable elements according to (i), (ii), and (iii) are independent or separate components of the kit. The transposable elements according to (i), (ii), and (iii) can be provided / included in the kit as linear DNA molecules, plasmid DNA, episomal DNA, small circular DNA, viral DNA, or viral RNA. Preferably, the transposable element according to the fourth aspect is provided / included in the kit.

[0149] The transposable elements according to (i), (ii), and (iii) provided with / included in the kit lack a polynucleotide encoding a functional transposase. The transposable elements do not contain a complete sequence encoding a functional transposase, preferably a naturally occurring functional transposase. Preferably, the complete sequence encoding a functional transposase, particularly a naturally occurring functional transposase or a portion thereof, is deleted from the transposable elements.

[0150] The transposase is a separate or individual component of the kit. The transposase is provided with / included in the kit as a polypeptide, a polynucleotide, or contained in a vector. Preferably, the polypeptide is a polypeptide according to the first aspect, the polynucleotide is a polynucleotide according to the second aspect, or the vector comprising the polynucleotide is a vector according to the third aspect.

[0151] In a preferred embodiment, the transposable elements described in (i), (ii), and (iii) of the kit are comprised in / are part of a polynucleotide molecule, preferably a vector. In this case, the polynucleotides described in (i), (ii), and (iii) are also comprised in / are part of a polynucleotide molecule, preferably a vector. Therefore, it is preferred that the polynucleotides described in (i), (ii), and (iii) of the kit and the transposable elements described in (i), (ii), and (iii) of the kit are in separate (different) polynucleotide molecules, preferably in (different) vectors. Particularly preferably, the transposable element in (i) is comprised in / is part of a polynucleotide molecule, preferably a vector.

[0152] In another preferred embodiment, the transposable elements described in (i), (ii), and (iii) of the kit and the polynucleotides described in (i), (ii), and (iii) of the kit are contained in / are part of the same polynucleotide molecule, preferably a vector. Particularly preferably, the transposable element of (i) and the polynucleotide according to the second aspect are comprised in / are part of a polynucleotide molecule, preferably a vector.

[0153] The transposable elements described in (i), (ii), and (iii) of the kit The kit contains sequences necessary for mobilization by the transposase provided in trans. These are repeat sequences at each end of the transposable element that contain binding sites for the transposase, allowing excision from the genome. Therefore, in a further preferred embodiment, the transposable elements described in (i), (ii), and (iii) of the above kit comprise terminal repeats (TRs). It is particularly preferred that the transposable element of (i) comprises terminal repeats (TRs). The terminal repeats are preferably terminal inverted repeats (TIRs). In this regard, it should be noted that the transposase provided in trans is specific to the transposable element. In other words, the transposable element is specifically recognized by the transposase.

[0154] In a further (alternatively or additionally) preferred embodiment, the transposable elements according to (i), (ii), and (iii) of the kit are: at least one polynucleotide of interest, or It contains at least one cloning site for inserting at least one polynucleotide of interest.

[0155] The transposable element (i) is at least one polynucleotide of interest, or It is particularly preferred to include at least one cloning site for inserting at least one polynucleotide of interest.

[0156] Preferably, At least one polynucleotide of interest is adjacent to a TR, or At least one cloning site for inserting at least one polynucleotide of interest is located between the TRs.

[0157] More preferably, the terminal repeat is a terminal inverted repeat (TIR). For example, the transposable element described in (i), (ii), and (iii) of the above kit comprises a first transposable element-specific terminal repeat and a second transposable element-specific terminal repeat downstream of the first transposable element-specific terminal repeat. At least one polynucleotide of interest is located between the first transposable element-specific terminal repeat and the second transposable element-specific terminal repeat.

[0158] Even more preferably, the at least one polynucleotide of interest is selected from the group consisting of a polynucleotide encoding a polypeptide, a non-coding polynucleotide, a polynucleotide comprising a promoter sequence, a polynucleotide encoding an mRNA, a polynucleotide encoding a tag, and a viral polynucleotide.

[0159] The polynucleotide of interest may encode a therapeutic polypeptide, such as an antibody, antibody fragment, monoclonal antibody, viral protein, viral protein fragment, antigen, or hormone. The polypeptide may be used for gene therapy, e.g., treatment of a single-gene disorder. In this case, the polynucleotide encoding the polypeptide is operably linked to a tissue-specific promoter. The polypeptide may also be used for cell therapy, particularly ex vivo. The cell may be a pluripotent stem cell (iPSC), human embryonic stem (hES) cell, human hematopoietic stem cell (HSC), or human T lymphocyte. Non-coding polynucleotides may be useful in targeted gene disruption. A polynucleotide containing a promoter sequence may enable activation of gene expression when a transposon is inserted adjacent to an endogenous gene. The polynucleotide may be transcribed into mRNA or a functional non-coding RNA, e.g., shRNA or gRNA. The polynucleotide may include a sequence tag for identifying the insertion site of the transposable element. Viral polynucleotides may also be used for targeted gene disruption. The nucleotides can be used for the production of viral particle-based biopharmaceuticals.

[0160] The expression of the polynucleotide of interest is preferably controlled by an expression control sequence. The expression control sequence may be a transcription control sequence, such as a promoter, an enhancer, a UCOE or MAR element, a polyadenylation signal, a post-transcriptional active element, such as an RNA stabilizing element, an RNA transport element, and a translation enhancer. Such expression control sequences are known to those skilled in the art. For example, a CMV promoter or a PGK promoter may be used as the promoter.

[0161] The transposable element and / or the vector comprising the transposable element described in (i), (ii), and (iii) of the above kit may contain elements that enhance expression (e.g., nuclear export signals, promoters, introns, terminators, enhancers, elements that affect chromatin structure, RNA export elements, IRES elements, CHYSEL elements, and / or Kozak sequences), selectable markers (e.g., DHFR, puromycin, hygromycin, zeocin, blasticidin, and / or neomycin), in It may further comprise a marker for in vivo monitoring (e.g., GFP or β-galactosidase), a restriction endonuclease recognition site (e.g., a site for insertion of an exogenous nucleotide sequence, such as a multiple cloning site), a recombinase recognition site (e.g., LoxP (recognized by Cre), FRT (recognized by Flp), or AttB / AttP (recognized by PhiC31)), an insulator (e.g., MAR or UCOE), a viral replication sequence (e.g., SV40 ori), and / or a sequence compatible with the DNA-binding domain for targeting via an additional binding molecule, particularly one with a chromatin leader domain, and DNA-binding domain properties ("bridging").

[0162] The kit may contain not only one transposable element, but also two or more transposable elements. The transposable elements may be different from each other, for example, because they contain different target polynucleotides. This is particularly desirable when two ORFs encoding antibody heavy chains (HC) or antibody light chains (LC) must be introduced into cells. In this case, the two or more ORFs are contained in the same transposable element or in separate transposable elements, preferably separate transposable elements.

[0163] Also (alternatively or additionally), in a preferred embodiment, the transposable elements described in (i), (ii), and (iii) of the above kit are DNA transposable elements. The DNA transposable elements preferably contain terminal inverted repeats (TIRs). It is particularly preferred that the transposable element (i) is a DNA transposable element, and that this DNA transposable element preferably contains terminal inverted repeats (TIRs). The DNA transposable element may be a piggyBac or piggyBac-like transposable element.

[0164] It is particularly preferred that the transposable element (i) in the above kit is selected from the group consisting of a wild-type piggyBac transposable element, a hyperactive piggyBac transposable element, a wild-type piggyBac-like transposable element, and a hyperactive piggyBac-like transposable element. The piggyBac-like transposable element includes a piggyBat transposable element, a piggyBac-like transposable element derived from Xenopus tropicalis, a piggyBac-like transposable element derived from Bombyx mori, and a piggyBac-like transposable element derived from Myotypica. It is particularly preferred that the piggyBac-like transposable element is selected from the group consisting of piggyBac-like transposable elements derived from Myotis lucifugus, and it is particularly preferred that the piggyBac transposable element is derived from Trichoplusia ni.

[0165] Conservative DNA-based transposable elements are transferred via a cut-and-paste mechanism. This requires a transposase, the inverted terminal repeats of the transposable element, and a target sequence on the new host DNA molecule. The transposase is provided in trans in the above-described method. The transposase catalyzes the excision of the transposable element from its current location and the integration of the excised transposable element into the genome of the cell. In the cut-and-paste mechanism, the transposase specifically binds to the inverted terminal repeats of the transposable element and excises the transposable element from its current location, e.g., from a vector. The transposase then positions the transposable element, cuts the target DNA backbone, and then inserts the transposable element. Typically, two transposase monomers are involved in the excision of the transposable element, one at each end of the transposable element. Finally, the transposase dimer complexed with the excised transposable element reintegrates the transposable element into the cellular DNA.

[0166] It is particularly preferred that the transposase or a fragment or derivative thereof in (ii) of the above kit is selected from the group consisting of wild-type piggyBac transposase or a fragment or derivative thereof, hyperactive piggyBac transposase or a fragment or derivative thereof, wild-type piggyBac-like transposase or a fragment or derivative thereof, and hyperactive piggyBac-like transposase or a fragment or derivative thereof.It is particularly more preferred that the piggyBac-like transposase or a fragment or derivative thereof is selected from the group consisting of piggyBat transposase or a fragment or derivative thereof, piggyBac-like transposase from Xenopus tropicalis or a fragment or derivative thereof, piggyBac-like transposase from Bombyx mori or a fragment or derivative thereof, and piggyBac-like transposase from Myotis lucifugus or a fragment or derivative thereof. It is also particularly preferred that the piggyBac transposase or a fragment or derivative thereof is derived from Trichoplusia ni.

[0167] It is further particularly preferred that the transposase or a fragment or derivative thereof of (ii) in the above kit is bound to / fused to at least one heterologous chromatin leader element (CRE). The at least one heterologous chromatin leader element (CRE) can be linked to the transposase or a fragment or derivative thereof, preferably via a linker. In particular, the at least one heterologous CRE can be linked to the N-terminus and / or C-terminus of the transposase, preferably via a linker.

[0168] It is particularly preferred that the at least one heterologous chromatin reader element (CRE) is at least one heterologous chromatin reader domain (CRD). The at least one heterologous chromatin reader domain (CRD) can be linked to the transposase or a fragment or derivative thereof, preferably via a linker. In particular, the at least one heterologous CRD can be linked to the N-terminus and / or C-terminus of the transposase, preferably via a linker.

[0169] Preferably, the CRD is a histone methylation level and / or a histone acetylation state. More preferably, the CRD is a plant homeodomain (PHD)-type zinc finger. Even more preferably, the PHD-type zinc finger is transcription initiation factor TFIID subunit 3 PHD.

[0170] Alternatively, it is especially more preferred that at least one heterologous chromatin reader element (CRE) is an artificial CRE. Preferably, the artificial CRE recognizes histone tails having specific methylation and / or acetylation sites, and more preferably, the artificial CRE is selected from the group consisting of a microantibody, a single-chain antibody, an antibody fragment, an affibody, an affilin, an anticalin, an atrimer, a DARPin, an FN2 scaffold, a fynomer, and a Kunitz domain.

[0171] In one embodiment, the kit is a kit for the generation of cells, particularly transgenic cells.

[0172] In another embodiment, the kit further comprises instructions on how to generate the cells, in particular transgenic cells. With regard to preferred cells, reference is made to the fifth aspect of the invention.

[0173] The kit may further comprise a container containing a single component of the kit. The kit may also include materials such as buffers, reagents, and / or diluents that are desirable from a commercial and user standpoint.

[0174] In a ninth aspect, the present invention provides a method for producing a composition comprising: (i) a transposable element, and a polypeptide according to the first aspect, or a polynucleotide according to the second aspect, or a vector according to the third aspect; or (ii) a transposable element according to the fourth aspect, and a transposase or a fragment or derivative thereof having transposase function, or a polynucleotide encoding a transposase or a fragment or derivative thereof having transposase function; a vector comprising a polynucleotide encoding a transposase or a fragment or derivative thereof having transposase function; or (iii) a transposable element according to the fourth aspect, and a polypeptide according to the first aspect, or a polynucleotide according to the second aspect, or Vector according to the third aspect The present invention relates to a targeting system including:

[0175] The targeting system may be contained in / part of a cell or may be introduced into a cell. Introduction of the targeting system into a cell may be via electroporation, transfection, injection, lipofection, or (viral) infection.

[0176] The cell may be an isolated cell (e.g., in a cell culture or cell line, e.g., in a stable cell line). The cell may also be a cell of a tissue outside of an organism. The cell may also be part of / contained in an organism, e.g., a eukaryotic multicellular organism. In this case, insertion of the targeting system is in v It is done in ivo.

[0177] In a preferred embodiment, the transposable elements described in (i), (ii), and (iii) of the targeting system are contained in / are part of a polynucleotide molecule, preferably a vector. In this case, the polynucleotides described in (i), (ii), and (iii) are also contained in / are part of a polynucleotide molecule, preferably a vector. Therefore, it is preferred that the polynucleotides described in (i), (ii), and (iii) of the targeting system and the transposable elements described in (i), (ii), and (iii) of the targeting system are in separate (different) polynucleotide molecules, preferably (different) vectors. Particularly preferably, the transposable element in (i) is comprised in / is part of a polynucleotide molecule, preferably a vector.

[0178] In another preferred embodiment, the transposable elements described in (i), (ii), and (iii) of the targeting system and the polynucleotides described in (i), (ii), and (iii) of the targeting system are contained in / are part of the same polynucleotide molecule, preferably a vector. Particularly preferably, the transposable element of (i) and the polynucleotide according to the second aspect are comprised in / are part of a polynucleotide molecule, preferably a vector.

[0179] The transposable elements described in (i), (ii), and (iii) of the above targeting system possess sequences necessary for mobilization by the transposase provided in trans. These are repeat sequences at each end of the transposable element that contain binding sites for the transposase, allowing excision from the genome. Therefore, in a further preferred embodiment, the transposable elements described in (i), (ii), and (iii) of the above targeting system comprise terminal repeats (TRs). It is particularly preferred that the transposable element in (i) comprises terminal repeats (TRs). The terminal repeats are preferably terminal inverted repeats (TIRs). In this regard, it should be noted that the transposase provided in trans is specific to the transposable element. In other words, the transposable element is specifically recognized by the transposase.

[0180] In a further (alternatively or additionally) preferred embodiment, the transposable elements according to (i), (ii) and (iii) of the targeting system comprise at least one polynucleotide of interest. It is particularly preferred that the transposable element (i) comprises at least one polynucleotide of interest. Preferably, at least one polynucleotide of interest is flanked by TRs. More preferably, the terminal repeat is a terminal inverted repeat (TIR). For example, the transposable element described in (i), (ii), and (iii) of the above kit comprises a first transposable element-specific terminal repeat and a second transposable element-specific terminal repeat downstream of the first transposable element-specific terminal repeat. At least one polynucleotide of interest is located between the first transposable element-specific terminal repeat and the second transposable element-specific terminal repeat.

[0181] Even more preferably, the at least one polynucleotide of interest is a polynucleotide encoding a polypeptide, a non-coding polynucleotide, a polynucleotide comprising a promoter sequence, The polynucleotide is selected from the group consisting of a polynucleotide encoding a nucleotide, a polynucleotide encoding an mRNA, a polynucleotide encoding a tag, and a viral polynucleotide.

[0182] The polynucleotide of interest may encode a therapeutic polypeptide, such as an antibody, antibody fragment, monoclonal antibody, viral protein, viral protein fragment, antigen, or hormone. Polypeptides may be used for gene therapy, e.g., treatment of monogenic diseases. In this case, the polynucleotide encoding the polypeptide is operably linked to a tissue-specific promoter. Polypeptides may also be used for cell therapy, particularly ex vivo. The cells may be pluripotent stem cells (iPSCs), human embryonic stem (hES) cells, human hematopoietic stem cells (HSCs), or human T lymphocytes. Non-coding polynucleotides may be useful in targeted gene disruption. Polynucleotides containing promoter sequences may enable activation of gene expression when a transposon is inserted adjacent to an endogenous gene. Polynucleotides may be transcribed into mRNA or functional non-coding RNA, such as shRNA or gRNA. Polynucleotides may include sequence tags for identifying the insertion site of the transposable element. Viral polynucleotides may be used for the production of viral particle-based biopharmaceuticals.

[0183] The expression of the polynucleotide of interest is preferably controlled by an expression control sequence. The expression control sequence may be a transcription control sequence, such as a promoter, an enhancer, a UCOE or MAR element, a polyadenylation signal, a post-transcriptional active element, such as an RNA stabilizing element, an RNA transport element, and a translation enhancer. Such expression control sequences are known to those skilled in the art. For example, a CMV promoter or a PGK promoter may be used as the promoter.

[0184] The transposable element and / or the vector comprising the transposable element according to (i), (ii), and (iii) of the above targeting system may further comprise an element that enhances expression (e.g., a nuclear export signal, a promoter, an intron, a terminator, an enhancer, an element that affects chromatin structure, an RNA export element, an IRES element, a CHYSEL element, and / or a Kozak sequence), a selection marker (e.g., DHFR, puromycin, hygromycin, zeocin, blasticidin, and / or neomycin), an in It may further comprise a marker for in vivo monitoring (e.g., GFP or β-galactosidase), a restriction endonuclease recognition site (e.g., a site for insertion of an exogenous nucleotide sequence, such as a multiple cloning site), a recombinase recognition site (e.g., LoxP (recognized by Cre), FRT (recognized by Flp), or AttB / AttP (recognized by PhiC31)), an insulator (e.g., MAR or UCOE), a viral replication sequence (e.g., SV40 ori), and / or a sequence compatible with the DNA-binding domain for targeting via an additional binding molecule, particularly one with a chromatin leader domain, and DNA-binding domain properties ("bridging").

[0185] In the above-mentioned targeting system, not only one transposable element is included, but also two or more transposable elements can be included.The transposable elements can be different from each other, for example, because they contain different target polynucleotides.This is particularly desirable when two ORFs encoding antibody heavy chain (HC) or antibody light chain (LC) must be introduced into cells.In this case, the two or more ORFs can be included in the same transposable element or in different transposable elements, preferably in different transposable elements.

[0186] Also (alternatively or additionally) in a preferred embodiment, the targeting system The transposable elements described in (i), (ii), and (iii) of the system are DNA transposable elements. The DNA transposable elements preferably contain terminal inverted repeats (TIRs). It is particularly preferred that the transposable element of (i) is a DNA transposable element, and that the DNA transposable element preferably contains terminal inverted repeats (TIRs). The DNA transposable element may be a piggyBac or piggyBac-like transposable element.

[0187] It is particularly preferred that the transposable element (i) in the above-mentioned targeting system is selected from the group consisting of a wild-type piggyBac transposable element, a hyperactive piggyBac transposable element, a wild-type piggyBac-like transposable element, and a hyperactive piggyBac-like transposable element. It is particularly more preferred that the piggyBac-like transposable element is selected from the group consisting of a piggyBat transposable element, a piggyBac-like transposable element derived from Xenopus tropicalis, a piggyBac-like transposable element derived from Bombyx mori, and a piggyBac-like transposable element derived from Myotis lucifugus. It is also particularly more preferred that the piggyBac transposable element is derived from Trichoplusia ni.

[0188] It is particularly preferred that the transposase or fragment or derivative thereof in (ii) of the above-mentioned targeting system is selected from the group consisting of wild-type piggyBac transposase or a fragment or derivative thereof, hyperactive piggyBac transposase or a fragment or derivative thereof, wild-type piggyBac-like transposase or a fragment or derivative thereof, and hyperactive piggyBac-like transposase or a fragment or derivative thereof. It is particularly more preferred that the piggyBac-like transposase or a fragment or derivative thereof is selected from the group consisting of piggyBat transposase or a fragment or derivative thereof, piggyBac-like transposase from Xenopus tropicalis or a fragment or derivative thereof, piggyBac-like transposase from Bombyx mori or a fragment or derivative thereof, and piggyBac-like transposase from Myotis lucifugus or a fragment or derivative thereof. It is also particularly preferred that the piggyBac transposase or a fragment or derivative thereof is derived from Trichoplusia ni.

[0189] It is further particularly preferred that the transposase or a fragment or derivative thereof of (ii) in the above targeting system is bound to / fused to at least one heterologous chromatin leader element (CRE). The at least one heterologous chromatin leader element (CRE) can be linked to the transposase or a fragment or derivative thereof, preferably via a linker. In particular, the at least one heterologous CRE can be linked to the N-terminus and / or C-terminus of the transposase, preferably via a linker.

[0190] It is particularly preferred that the at least one heterologous chromatin reader element (CRE) is at least one heterologous chromatin reader domain (CRD). The at least one heterologous chromatin reader domain (CRD) can be linked to the transposase or a fragment or derivative thereof, preferably via a linker. In particular, the at least one heterologous CRD can be linked to the N-terminus and / or C-terminus of the transposase, preferably via a linker.

[0191] Preferably, the CRD recognizes histone methylation level and / or histone acetylation status. More preferably, the CRD is a plant homeodomain (PHD)-type zinc finger. Even more preferably, the PHD-type zinc finger is transcription initiation factor TFIID subunit 3 PHD.

[0192] Alternatively, it is especially more preferred that at least one heterologous chromatin reader element (CRE) is an artificial CRE. Preferably, the artificial CRE recognizes histone tails having specific methylation and / or acetylation sites, and more preferably, the artificial CRE is selected from the group consisting of a microantibody, a single-chain antibody, an antibody fragment, an affibody, an affilin, an anticalin, an atrimer, a DARPin, an FN2 scaffold, a fynomer, and a Kunitz domain.

[0193] In a further aspect, the present invention provides a method for producing a pharmaceutical composition comprising: at least one polynucleotide of interest according to the fourth aspect, and a polypeptide according to the first aspect, a polynucleotide according to the second aspect, or Vector according to the third aspect The present invention relates to a (transgenic) cell containing a transposable element, comprising:

[0194] With regard to further preferred embodiments of the cells and transposable elements, reference is made to the fifth aspect of the invention.

[0195] Various modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope of the invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be covered by the present invention.

[0196] The following examples are for illustrative purposes only and are not intended to limit the above-described invention in any way. [Example]

[0197] Example 1 Gene optimization, synthesis, and construction of transposase expression plasmids The amino acid sequences of piggyBac wt transposase (Trichoplusia ni; GenBank accession number AAA87375.2; SEQ ID NO: 18 [Virology 172(1)156-169 1989]) and its hyperactive mutants (SEQ ID NO: 20 and SEQ ID NO: 22) were reverse translated. The nucleotide sequences were optimized by knockout of cryptic splice sites and RNA destabilizing sequence elements, optimized for increased RNA stability, and further adapted in terms of codon usage to match the requirements of CHO cells (Cricetulus griseus). The nucleotide sequences were submitted to GeneArt Gene Synthesis (Life Sciences, 2014). The PBw coding sequence (CDS) was synthesized by Biosciences Technologies and used using standard cloning procedures to generate the constructs shown in Figure 1. The coding sequence (CDS) of PBw is shown in SEQ ID NO: 17, the coding sequence (CDS) of haPB1 is shown in SEQ ID NO: 19, and the coding sequence (CDS) of haPB2 is shown in SEQ ID NO: 21. The constructs were ligated into an expression vector, allowing transient expression of the transposase mutants under the control of the CMV promoter. In order: Sambrook, J., EF Fritsch and T. Maniatis: Cloning I / II / III, A Laboratory Manual New York / Cold Spring Harbor Laboratory Press, 1989, Second Edition.

[0198] Example 2 Construction of transposon plasmids Transposons containing variants of the natural and artificial PB transposon end sequences were generated and tested for their ability to be recognized by the PB transposase. The constructs tested are shown in Figure 2b. The nucleotide sequences of the transposon end sequences are set forth herein as follows: SEQ ID NO: 13 (piggyBac, Trichoplusia ni, 5'-transposon end sequence (wt) (357 bp)), SEQ ID NO: 14 (piggyBac, Trichoplusia ni, 3'-transposon end sequence (wt)), SEQ ID NO: 6 (piggyBac, Trichoplusia ni, 5'-transposon end sequence (wt) (248 bp)), SEQ ID NO: 11 (piggyBac, Trichoplusia ni, minimal 5'-transposon end sequence (wt)), SEQ ID NO: 12 (piggyBac, Trichoplusia ni), The following sequences are listed: SEQ ID NO: 16 (piggyBac, artificial 5'-transposon end sequence (357 bp), and SEQ ID NO: 15 (piggyBac, artificial 5'-transposon end sequence (248 bp)). The transposon end sequences were incorporated at the 5' and 3' positions relative to the bacterial backbone sequence carrying the bacterial replication origin and antibiotic resistance gene in the empty expression vectors PBGGPEx2.0m and PBGGPEx2.0p. Synthetic heavy or light chain fragments of monoclonal antibodies assembled with the signal peptide were ligated into the empty expression vector containing the transposon to generate the plasmids shown in Figure 3. The general procedure for constructing expression plasmids is described in Sambrook, J., E.F. Fritsch and T. Maniatis: Cloning I / II / III, A Laboratory Manual, New York / Cold Spring Harbor Laboratory Press, 1989, Second Edition.

[0199] Example 3 Generation and analysis of clone pools The dihydrofolate reductase-deficient CHO cell line CHO / DG44 [Urlaub et al., 1986, Proc Natl Acad Sci USA. 83(2):337-341] was used as the starter cell line. The cell line was maintained in serum-free medium. A plasmid containing one of the transposon mutants and a plasmid containing a transient expression vector for the expression of one of the transposase mutants were transfected by electroporation according to the manufacturer's instructions (Neon Transfection System, Thermo Fisher Scientific). 1.5 μg of circular HC and LC transposon vector DNA and 1.2 μg of circular transposase DNA were used for each transfection. Transfectants were subjected to selection with puromycin and methotrexate to eliminate untransfected cells and non- and low-producer cells. Two consecutive rounds of transfection and selection were performed using the same vector combinations, DNA amounts, and selection conditions. After a two-week selection period, the selection pressure was removed and the resulting clonal pool was subjected to a fed-batch culture process under typical conditions with a defined seeding cell density. The fed-batch culture process was carried out in shake flasks (SF125, Corning) with a working volume of 30 mL in a chemically defined medium. A chemically defined diet was applied every two days according to the typical feeding regimen. Viability was measured using a Vi-CELL viability analyzer. (Beckman Coulter). Antibody concentrations of cell culture supernatant samples were determined by the Octet® RED96 system (Fortebio) against purified expressed antibodies as a standard curve. Figure 4 shows the recovery of viability during the selection stage of transfectants generated using wild-type PB transposase or hyperactive transposase haPB2 and the transposon of Example 2. Compared to the wild-type transposon, faster recovery of viability was observed. Faster recovery of viability was observed when a transposon containing an artificial TES was used in combination with a hyperactive transposase. Figure 5 shows the results of fed-batch culture of clone pools derived from wtPB transposase and hyperactive transposase mutants on day 14 using the PB minimal wild-type TES transposon. Increased titers (5- to 6-fold) were observed for the hyperactive transposase mutants compared to the wild-type transposase.

[0200] Sequence Listing Overview: SEQ ID NO: 1: piggyBac, Trichoplusia ni, left internal repeat wild type (wt) SEQ ID NO: 2: piggyBac-like, Xenopus tropicalis, left internal repeat (wt) SEQ ID NO: 3: piggyBac-like, Bombyx mori, left internal repeat (wt) SEQ ID NO: 4: piggyBac-like, Myotis lucifugus#1, left internal repeat (wt) SEQ ID NO: 5: piggyBac-like, Myotis lucifugus#2, left internal repeat (wt) SEQ ID NO: 6: piggyBac, Trichoplusia ni, 5'-transposon end sequence (wt), containing SEQ ID NO: 1 (wt) (248 bp) SEQ ID NO: 7: piggyBac-like, Xenopus tropicalis, 5'-transposon end sequence (wt), containing SEQ ID NO: 2 (wt) SEQ ID NO:8: piggyBac-like, Bombyx mori, 5'-transposon end sequence (wt), contains SEQ ID NO:3 (wt) SEQ ID NO: 9: piggyBac-like, Myotis lucifugus#1, 5'-transposon end sequence (wt), containing SEQ ID NO: 4 (wt) SEQ ID NO: 10: piggyBac-like, Myotis lucifugus#2, 5'-transposon end sequence (wt), containing SEQ ID NO: 5 (wt) SEQ ID NO: 11: piggyBac, Trichoplusia ni, minimal 5'-transposon end sequence (wt) SEQ ID NO: 12: piggyBac, Trichoplusia ni, minimal 3'-transposon end sequence (wt) SEQ ID NO: 13: piggyBac, Trichoplusia ni, 5'-transposon end sequence (wt) (357 bp) SEQ ID NO: 14: piggyBac, Trichoplusia ni, 3'-transposon end sequence (wt) SEQ ID NO: 15: piggyBac, artificial 5'-transposon end sequence (248 bp) SEQ ID NO: 16: piggyBac, artificial 5'-transposon end sequence (357 bp) SEQ ID NO: 17: wt piggyBac transposase, Trichoplusia ni, DNA SEQ ID NO: 18: wt piggyBac transposase, Trichoplusia ni, protein SEQ ID NO: 19: piggyBac transposase mutant, haPB1, DNA SEQ ID NO: 20: piggyBac transposase mutant, haPB1, protein SEQ ID NO: 21: piggyBac transposase mutant, haPB2, DNA SEQ ID NO: 22: piggyBac transposase mutant, haPB2, protein SEQ ID NO: 23: piggyBac, Trichoplusia ni, left repeat (wt) SEQ ID NO: 24: piggyBac-like, Xenopus tropicalis, left repeat (wt) SEQ ID NO: 25: piggyBac-like, Bombyx mori, left repeat (wt) SEQ ID NO: 26: piggyBac-like, Myotis lucifugus#1, left repeat (wt) SEQ ID NO: 27: piggyBac-like, Myotis lucifugus#2, left repeat (wt) SEQ ID NO: 28: piggyBac, artificial left internal repeat

Claims

1. A polypeptide comprising a piggyBac transposase comprising at least one amino acid substitution selected from the group consisting of: The piggyBac transposase (i) having the amino acid sequence set forth in SEQ ID NO: 22, which contains an alanine (A) at amino acid position 30, a leucine (L) at amino acid position 282, and an arginine (R) at amino acid position 538; or the piggyBac transposase is a variant thereof that is at least 90% identical to the amino acid sequence, wherein the variant comprises an alanine (A) at or corresponding to amino acid position 30, a leucine (L) at or corresponding to amino acid position 282, and an arginine (R) at or corresponding to amino acid position 538; (ii) the polypeptide having the amino acid sequence set forth in SEQ ID NO: 20, comprising an alanine (A) at amino acid position 30, a proline (P) at amino acid position 118, a valine (V) at amino acid position 185, a leucine (L) at amino acid position 282, and an arginine (R) at amino acid position 538, or wherein the piggyBac transposase is a variant thereof that is at least 90% identical to the amino acid sequence, wherein the variant comprises an alanine (A) at or corresponding to amino acid position 30, a proline (P) at or corresponding to amino acid position 118, a valine (V) at or corresponding to amino acid position 185, a leucine (L) at or corresponding to amino acid position 282, and an arginine (R) at or corresponding to amino acid position 538.

2. The polypeptide of claim 1 , wherein the polypeptide comprises at least one heterologous chromatin reader domain (CRD).

3. The polypeptide of claim 2, wherein the CRD is a plant homeodomain (PHD)-type zinc finger.

4. The polypeptide of claim 3 , wherein the PHD-type zinc finger is a transcription initiation factor TFIID subunit 3 PHD.

5. A polynucleotide encoding the polypeptide of any one of claims 1 to 4.

6. A vector comprising the polynucleotide of claim 5.

7. An artificial transposable element comprising a transposon end sequence described in sequence number 16.

8. The transposable element is at least one polynucleotide of interest, or The transposable element of claim 7, comprising at least one cloning site for inserting at least one polynucleotide of interest.

9. The transposable element of claim 7 or claim 8, wherein the transposable element is a piggyBac transposable element.

10. 1. A method for producing a transgenic cell, comprising: (i) providing cells; (ii) (iia) a transposable element, and A polypeptide according to any one of claims 1 to 4, or A polynucleotide according to claim 5, or A vector according to claim 6, or (iib) a transposable element according to any one of claims 7 to 9, and a transposase or a fragment or derivative thereof having transposase function, or a polynucleotide encoding a transposase or a fragment or derivative thereof having transposase function; a vector comprising a polynucleotide encoding a transposase or a fragment or derivative thereof having transposase function; (iic) a transposable element according to any one of claims 7 to 9, and A polypeptide according to any one of claims 1 to 4, or A polynucleotide according to claim 5, or The vector according to claim 6 into said cells, thereby producing transgenic cells. A method comprising:

11. 11. The method of claim 10, wherein the transposable element of (iia) comprises at least one polynucleotide of interest.

12. 12. The method of claim 10 or claim 11, wherein the transposase of (iib) is selected from the group consisting of a wild-type piggyBac transposase, a hyperactive piggyBac transposase, a wild-type piggyBac-like transposase, and a hyperactive piggyBac-like transposase.

13. 13. The method according to claim 10, wherein the cell is a mammalian cell. How to do it.

14. A transgenic cell obtainable by the method according to any one of claims 10 to 13.

15. 15. Use of the transgenic cell of claim 14 for the production of a protein or a virus.

16. (i) a transposable element, and A polypeptide according to any one of claims 1 to 4, or A polynucleotide according to claim 5, or The vector of claim 6; or (ii) a transposable element according to any one of claims 7 to 9, and a transposase or a fragment or derivative thereof having transposase function, or a polynucleotide encoding a transposase or a fragment or derivative thereof having transposase function; a vector comprising a polynucleotide encoding a transposase or a fragment or derivative thereof having transposase function; or (iii) a transposable element according to any one of claims 7 to 9, and A polypeptide according to any one of claims 1 to 4, or A polynucleotide according to claim 5, or The vector according to claim 6 Kit including:

17. (i) The transposable element at least one polynucleotide of interest, or 17. The kit of claim 16, comprising at least one cloning site for inserting at least one polynucleotide of interest.

18. 18. The kit of claim 16 or claim 17, wherein the transposase (ii) is selected from the group consisting of a wild-type piggyBac transposase, a hyperactive piggyBac transposase, a wild-type piggyBac-like transposase, and a hyperactive piggyBac-like transposase.

19. (i) a transposable element, and A polypeptide according to any one of claims 1 to 4, or A polynucleotide according to claim 5, or The vector of claim 6; or (ii) a transposable element according to any one of claims 7 to 9, and a transposase or a fragment or derivative thereof having transposase function, or a polynucleotide encoding a transposase or a fragment or derivative thereof having transposase function; a vector comprising a polynucleotide encoding a transposase or a fragment or derivative thereof having transposase function; or (iii) a transposable element according to any one of claims 7 to 9, and A polypeptide according to any one of claims 1 to 4, or A polynucleotide according to claim 5, or The vector according to claim 6 ; Targeting system including.

20. The targeting system of claim 19, wherein the transposable element of (i) comprises at least one polynucleotide of interest.

21. 21. The targeting system of claim 19 or claim 20, wherein the transposase (ii) is selected from the group consisting of a wild-type piggyBac transposase, a hyperactive piggyBac transposase, a wild-type piggyBac-like transposase, and a hyperactive piggyBac-like transposase.

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