Mammalian cell lines for transient protein production

Abstract

The invention provides cells that have integrated a nucleic acid encoding a transposase into their genomes and are capable of stably maintaining the integrated nucleic acid through multiple rounds of cell propagation while expressing the transposase. Such cells are then transfected with at least one transposon encoding a polypeptide or polypeptides to be expressed. The transposase can then integrate the transposon into the genome of cells of the mammalian cell population. Cells transformed in this manner expressed higher yields of a desired protein and / or expressed over longer periods over conventional transient expression methods without integration.

Description

Mammalian Cell Lines for Transient Protein ProducƟon CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This applicaƟon claims the benefit of US63 / 683,648filed August 15, 2024 and US63 / 600,335filed November 17, 2023, each incorporated by reference in its enƟrety for all purposes. SEQUENCE LISTING

[0002] The applicaƟon includes sequences in an XMLfile named 621874SEQLIST.xml of 367,600 bytes created November 5, 2024, which is incorporated by reference. BACKGROUND

[0003] Mammalian cell lines such as HEK293 (and derivaƟves), cell lines derived from Chinese hamster ovary (CHO), and cell lines derived from mammalian amniocytes can be used to transiently express mammalian proteins, as well as proteins from other vertebrates and non- natural proteins. Transient expression is generally accomplished by introducing into the host cell line a nucleic acid encoding the protein to be expressed, culturing the transfected cells for a period of days or weeks, then harvesƟng the protein either from the media (for a secreted protein) or from the lysed cells (for a cytoplasmic protein) and discarding the cultured cells.

[0004] As the host cells divide and grow during culturing, nucleic acids introduced into the cells tend to be lost through a variety of mechanisms including simple diluƟon as cells divide, and degradaƟon by nucleases. As the encoding nucleic acid is lost from the cells, expression of the encoded protein is also reduced. One strategy to reduce this expression loss with culture Ɵme is to increase the persistence of introduced plasmids by incorporaƟng into them sequences from viruses that can support episomal plasmid replicaƟon and maintenance. One example is the OriP sequence from Epstein-Barr virus which enables EBNA (Epstein-Barr Nuclear AnƟgen)-dependent plasmid replicaƟon; the EBNA may be supplied by a gene also encoded on the plasmid, or may be supplied in trans. A second example is the SV40 origin of replicaƟon, which enables SV40 large T-anƟgen-dependent plasmid replicaƟon, again the SV40 T-anƟgen may be supplied by a gene also encoded on the plasmid, or may be supplied in trans.Both of these viral replicaƟon sequences extend the duraƟon of plasmid-encoded protein expression, and thus increase the amount of protein that can be transiently produced. However, the plasmids are not protected from nucleases indefinitely, so protein expression by transient methods yield substanƟally lower amounts of protein than can be achieved with stable cell lines.

[0005] Stable cell lines for expression of a heterologous protein may be prepared by integraƟng into the genome of the host cell line DNA sequences comprising open reading frame(s) encoding the protein to be expressed, operably linked to regulatory sequences (such as promoter, polyadenylaƟon signal and opƟonally introns) that make the open reading frame expressible in the host cell. One method for performing this integraƟon is to incorporate the DNA sequences into a transposon and to introduce the transposon into the host cell line together with a corresponding transposase. The transposase recognizes the ends of the transposon, excises the transposon from one DNA molecule and inserts it into a second DNA molecule. Once the transposon is integrated into a host cell chromosome, the transposon is replicated and protected from nuclease degradaƟon like the rest of the host chromosome.

[0006] The typical Ɵming for transient-expression, between iniƟal introducƟon of protein-encoding DNA sequences into a host cell and harvesƟng of culture supernatant or cell lysate for protein purificaƟon is 5-7 days for HEK cells and 1-2 weeks for CHO cells. The typical Ɵming for preparaƟon of a transposon-derived stable cell line is 1-4 weeks from introducƟon of the transposon comprising the protein-encoding DNA sequences plus the transposase, through the selecƟon process in which cells not containing transposons are killed. ParƟcularly for metabolic selecƟons such as those for glutamine synthetase or dihydrofolate reductase, the selecƟon process is not performed with high cell numbers or densiƟes to reduce the effects of cross-feeding: cells that have integrated the selecƟve genes, along with transposons, into their genomes, produce enough glutamine or tetrahydrofolate to feed those cells that did not integrate any transposons. Thus at the end of the selecƟon period, it is generally necessary to scale-up the cell pool before producing the target protein, so that it might take about 8 weeks post-transfecƟon to begin expressing protein from a stable pool.

[0007] There is therefore a need for cell lines and methods for transient protein producƟon that increase the yields above those obtainable using viral replicaƟon sequences, but that avoid the significantly longer Ɵmelines of stable cell line development, even with transposases and transposons. SUMMARY OF THE CLAIMED INVENTION

[0008] The invenƟon provides a method for expressing a protein, the method comprising (i) transfecƟng a mammalian cell populaƟon, the genomes of which comprise a nucleic acid encoding a transposase operably linked to regulatory sequences including a promoter and a polyadenylaƟon signal such that the transposase is expressible in the mammalian cell populaƟon, with a corresponding transposon transposable by the transposase, wherein the corresponding transposon comprises an open reading frame encoding an expressible polypepƟde operably linked to regulatory sequences including a promoter and a polyadenylaƟon signal such that the expressible protein is expressible in cells of the populaƟon into which the transposon has bene introduced; and (ii) culturing the transfected cell populaƟon under condiƟons such that the expressible polypepƟde is expressed.

[0009] OpƟonally, the transposase is a piggyBac-like transposase, an hAT transposase, a Mariner transposase or a Helitron transposase. OpƟonally, the transposase is a piggyBac-like transposase selected from a transposase derived from a naturally occurring transposase from the looper moth Trichoplusia ni, a transposase derived from a naturally occurring transposase from a Xenopus species, a transposase derived from a naturally occurring transposase from a Bombyx species, a transposase derived from a naturally occurring transposase from a Heliothis species, a transposase derived from a naturally occurring transposase from a Helicoverpaspecies, a transposase derived from a naturally occurring transposase from an Agro s species, atransposase derived from a naturally occurring transposase from an Amyelois species, atransposase derived from a naturally occurring transposase from a Myo s species and atransposase from an Oryzias species. OpƟonally the transposase is a Sleeping Beauty transposase, a Tol2 transposase, a Tc Buster transposase or a Helraiser transposase. OpƟonally, the promoter operably linked to the open reading frame encoding the transposase is aconsƟtuƟve promoter. OpƟonally, the promoter operably linked to the open reading frame encoding the transposase is an inducible promoter, and the method further comprises inducing the promoter before transfecƟng the mammalian cell populaƟon with the corresponding transposon. OpƟonally, the culturing step is performed with selecƟon for cells comprising the transposon over cells lacking the transposon. OpƟonally, the corresponding transposon further comprises a second open reading frame encoding a selecƟve marker that allows cells comprising the transposon to overcome a selecƟve agent (e.g., puromycin, hygromycin, blasƟcidin, zeocin, neomycin) and the culturing step further comprises supplying to culture medium the selecƟve agent (to inhibit growth of cells that do not comprise the corresponding transposon. OpƟonally, the corresponding transposon comprises a second open reading frame encoding an enzyme required to provide the nutrient (glutamine synthetase, dihydrofolate reductase) and the culturing step is performed in media lacking the nutrient (e.g., glutamine, tetrahydrofolate) thereby inhibiƟng growth of cells that do not comprise the corresponding transposon. OpƟonally, the open reading frame encoding the transposase in the genomes of the mammalian cell populaƟon is within a second transposon not transposable by the transposase. OpƟonally, the open reading frame encoding the transposase in the genomes of the mammalian cell populaƟon is within a retroviral vector, such as a lenƟviral vector. OpƟonally, the open reading frame encoding the transposase in the genomes of the mammalian cell populaƟon was introduced into the genomes by random integraƟon. OpƟonally, the mammalian cell populaƟon is a CHO cell or a HEK cell populaƟon.

[0010] OpƟonally, the expressible polypepƟde is an anƟbody chain, or an Fc fusion protein or a chain of a 3-chain or 4-chain mulƟ-specific anƟbody. OpƟonally, the expressible polypepƟde comprises a plurality of anƟbody chains separated by CHYSEL elements. OpƟonally, the corresponding transposon comprises a plurality of open reading frames encoding a plurality of expressible polypepƟdes, each operably linked to regulatory sequences. OpƟonally, the plurality of expressible polypepƟdes are chains of a mulƟ-chain anƟbody. OpƟonally, the plurality of open reading frames are in the same transcripƟonal unit separated by IRES elements. OpƟonally, the method further comprises transfecƟng the mammalian cell populaƟon with a second corresponding transposon transposable by the transposase, whereinthe second corresponding transposon comprises a second open reading frame encoding a second expressible polypepƟde operably linked to regulatory sequences including a promoter and a polyadenylaƟon signal such that the second expressible polypepƟde is expressible in cells of the populaƟon into which the second corresponding transposon has been introduced; wherein in the culturing step the second expressible polypepƟde is expressed. OpƟonally the expressible polypepƟde and second expressible polypepƟde assemble into a mulƟmeric polypepƟde.

[0011] OpƟonally, the culturing comprises feeding the cells. OpƟonally, the method further comprises changing the incubaƟon temperature of the cells during the culturing step to bias the cells to producƟon of the polypepƟde over propagaƟon. OpƟonally, the culturing of the cells results in propagaƟon of the cells.

[0012] OpƟonally, the method is performed without clonal selecƟon of a cell line from the cultured cells. OpƟonally, the cultured cells and their progeny integrate the transposon at different locaƟons in their genomes. OpƟonally, the locaƟons of the transposon in the genomes of the cultured cells and their progeny change with Ɵme. OpƟonally, the method further comprises purifying the expressible polypepƟde from the cultured transfected cells. OpƟonally, the purifying step is performed within 7-30 days of the transfecƟng step. OpƟonally, the purifying step is performed when the concentraƟon of the expressible polypepƟde is within + / - 20% of its maximum concentraƟon OpƟonally, the purified expressible polypepƟde includes polypepƟde expressed during selecƟon of the cultured cells for presence of a selecƟon marker encoded by the transposon.

[0013] OpƟonally, the method further comprises freezing the cultured transfected cells.

[0014] OpƟonally, the maximum yield of the expressible protein is 50-1500 mg / L. OpƟonally, the culturing is performed from 7-30 days. OpƟonally, the expressible protein is used for non-clinical research.

[0015] OpƟonally, the corresponding transposon is a component of an episome. OpƟonally, the corresponding transposon is not a component of an episome.

[0016] The invenƟon further provides a cell line having a genome comprising an open reading frame encoding a polypepƟde with at least 95% and opƟonally 100% sequence idenƟtyto an amino acid sequence selected from SEQ ID NOS:9-41, 46-69, 74, 158, 84, 89, 96, 101, 106, 157, 116, 130 or 141, operably linked to a promoter acƟve in the cell line. OpƟonally, the promoter has a sequence selected from SEQ ID NOS:1 and 2, or 42 and 43, or 75 and 76, or 80 and 81, or 85 and 86, or 90 and 91, or 97 and 98, or 102 and 103, or 107 and 108, or 112 and 113, or 128 and 129, or 137 and 138. The invenƟon further provides a CHO K1 cell line having a genome comprising a nucleic acid encoding a transposase of SEQ ID NO:16 fused at its N- terminus to a nuclear localizaƟon signal, operably linked to the heterologous promoter. OpƟonally, the CHO K1 cell line comprises six copies of the transposase at six locaƟons of the genome, such as the six locaƟons are as shown in Table 7. OpƟonally, the nucleic acid encoding the transposase comprises SEQ ID NO:152. DEFINITIONS

[0017] The singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a nucleic acid” includes a plurality of nucleic acids, reference to “a substrate” includes a plurality of such substrates, reference to “a variant” includes a plurality of variants, and the like.

[0018] Terms such as “connected,” “aƩached,” “linked,” and “conjugated” are used interchangeably to encompass direct as well as indirect connecƟon, aƩachment, linkage or conjugaƟon unless the context indicates otherwise.

[0019] When a range of values is recited, it is to be understood that each intervening integer value, and each fracƟon thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the invenƟon. When a value being discussed has inherent limits, for example where a component can be present at a concentraƟon of from 0 to 100%, or where the pH of an aqueous soluƟon can range from 1 to 14, those inherent limits are specifically disclosed. When a value is explicitly recited, it is to be understood that values which are about the same quanƟty or amount as the recited value are also within the scope of the invenƟon. When a combinaƟon isdisclosed, each sub combinaƟon of the elements of that combinaƟon is also specifically disclosed and is within the scope of the invenƟon. Conversely, when different elements or groups of elements are individually disclosed, combinaƟons thereof are also disclosed. When any element of an invenƟon is disclosed as having a plurality of alternaƟves, examples of that invenƟon in which each alternaƟve is excluded singly or in any combinaƟon with the other alternaƟves are also hereby disclosed; more than one element of an invenƟon can have such exclusions, and all combinaƟons of elements having such exclusions are hereby disclosed.

[0020] Unless defined otherwise, all technical and scienƟfic terms used have their ordinary meaning. Singleton, et. al., DicƟonary of Microbiology and Molecular Biology, 2nd Ed., John Wiley and Sons, New York (1994), and Hale & Marham, The Harper Collins DicƟonary of Biology, Harper Perennial, NY, 1991 provide guidance as to ordinary meaning.

[0021] Unless otherwise indicated, nucleic acids are wriƩen leŌ to right in 5’ to 3’ orientaƟon; amino acid sequences are wriƩen leŌ to right in amino to carboxy orientaƟon, respecƟvely.

[0022] If a DNA sequence is provided, the specificaƟon should be understood as addiƟonal disclosing the sequence of the RNA, which will be the same with the excepƟon that thymine (T) is replaced with uracil (U), and vice versa.

[0023] Nucleic acids are preferably provided with codon preferences for a cell in which expression is intended. The term “codon usage” or “codon bias” refers to the relaƟve frequencies with which different synonymous codons are used to encode an amino acid within an open reading frame. A nucleic acid sequence having codon preferences for a parƟcular target cell has a balance of synonymous codon choices that result in efficient translaƟon in that cell type. This balance is oŌen not calculable from observed genomic codon frequencies, but must be empirically determined, for example as described in US patents 7,561,972 and 7,561,973 and 8,401,798 and in Welch et. al. (2009) “Design Parameters to Control SyntheƟc Gene Expression in Escherichia coli”. PLoS ONE 4(9): e7002.hƩps: / / doi.org / 10.1371 / journal.pone.0007002. A nucleic acid originally isolated from one cell type to be introduced into a target cell of another type can undergo selecƟon of codon preferences for the target site cell such that at least 1 and someƟmes, 5, 20, 15, 20, 50, 100 ormore choices among synonymous codons differ between the nucleic acid introduced into the target cell from the original nucleic acid.

[0024] Two nucleic acids are “complementary” if the bases of one hydrogen bond to the bases of the other. For perfect complementarity, adenine (A) in thefirst nucleic acid must correspond with thymine (T) (or uracil for RNA) in the second (and vice versa), and cytosine (C) in thefirst nucleic acid must correspond with guanine (G) in the second (and vice versa). The two nucleic acids must also be anƟparallel. If two nucleic acids are complementary, one may be described as the “reverse complement” of the other to indicate that their bases are complementary when one is in the 5’ to 3’ direcƟon and the other is in the 3’ to 5’ direcƟon. When one nucleic acid sequence is described as complementary to another, it is intended to indicate that the sequences are anƟparallel and able to base-pair with one another.

[0025] The “configuraƟon” of a nucleic acid refers to the presence, order and direcƟon of funcƟonal segments with the nucleic acid.

[0026] A ‘transposase’ is a polypepƟde that catalyzes the excision of a corresponding transposon from a donor nucleic acid, for example a vector, and (providing the transposase is not integraƟon-deficient) the subsequent integraƟon of the transposon into a target nucleic acid.

[0027] “TransposiƟon” refers to acƟon of a transposase in excising a transposon from one nucleic acid and then integraƟng it, either into a different site in the same nucleic acid, or into a second nucleic acid.

[0028] A “transposon” means a nucleic acid that can be excised from afirst nucleic acid, for instance, a vector, and be integrated into a second posiƟon in the same nucleic acid, or into a second nucleic acid, for instance, the genomic or extrachromosomal DNA of a cell, by the acƟon of a corresponding trans-acƟng transposase. A transposon comprises afirst transposon end and a second transposon end, which are nucleic acid sequences recognized by and transposed by a transposase. Thefirst and second transposon ends include inverted terminal repeats. Two copies of a transposon target site are usually present on the outside of the transposon ends (one on each side). A transposon usually further comprises a nucleic acid between the two transposon ends, which along with the two transposon ends is transposed bythe acƟon of the transposase. In natural transposons, the nucleic acid between the transposon ends is typically a corresponding transposase. Transposons of the present invenƟon are “syntheƟc transposons” comprising a heterologous nucleic acid, which is transposable by virtue of its juxtaposiƟon between two transposon ends. SyntheƟc transposons may or may not further compriseflanking nucleic acid sequence(s) outside the transposon ends, such as a sequence encoding a transposase, a vector sequence or sequence encoding a selectable marker.

[0029] A “transposon end” means the cis-acƟng nucleoƟde sequences that are sufficient for recogniƟon by and transposiƟon by a corresponding transposase. Transposon ends of piggyBac-like transposons comprise perfect or imperfect repeats such that the respecƟve repeats in the two transposon ends are reverse complements of each other. These are referred to as inverted terminal repeats (ITR) or terminal inverted repeats (TIR). A transposon end may or may not include addiƟonal sequence proximal to the ITR that promotes or augments transposiƟon.

[0030] The terms “corresponding transposon” and “corresponding transposase” are used to indicate an acƟvity relaƟonship between a transposase and a transposon. A transposase transposes its corresponding transposon. Many transposases correspond with a single transposon, and many transposons correspond with a single transposase. The term “orthogonal” refers to a lack of interacƟon between two systems. Afirst transposon and its correspondingfirst transposase and a second transposon and its corresponding second transposase are orthogonal if thefirst transposase does not excise or transpose the second transposon and the second transposase does not excise or transpose thefirst transposon.

[0031] A “target site” for a transposase is a site or sequence in a molecule into which a transposon can be inserted by a transposase. The piggyBac transposase from Trichoplusia ni inserts its transposon predominantly into the target sequence 5’-TTAA-3’. Other useable target sequences for piggyBac transposons are 5’-CTAA-3’, 5’-TTAG-3’, 5’-ATAA-3’, 5’-TCAA-3’, 5’-AGTT- 3’, 5’-ATTA-3’, 5’-GTTA-3’, 5’-TTGA-3’, 5’-TTTA-3’, 5’-TTAC-3’, 5’-ACTA-3’, 5’-AGGG-3’, 5’-CTAG-3’, 5’-GTAA-3’, 5’-AGGT-3’, 5’-ATCA -3’, , 5’- CTCC-3’, 5’- TAAA-3’, 5’-TCTC -3’, 5’-TGAA -3’, 5’- AAAT-3’, 5’- AATC-3’, 5’-ACAA -3’, 5’- ACAT-3’, 5’-ACTC -3’, 5’-AGTG -3’, 5’-ATAG -3’, 5’- CAAA-3’, 5’-CACA -3’, 5’-CATA -3’, 5’-CCAG -3’, 5’-CCCA -3’, 5’-CGTA -3’, 5’-CTGA -3’, 5’- GTCC-3’, 5’- TAAG-3’, 5’-TCTA -3’,5’-TGAG -3’, 5’-TGTT -3’, 5’-TTCA -3’, 5’- TTCT-3’ and 5’-TTTT -3’ (Li et al., 2013. Proc. Natl. Acad. Sci vol.110, no.6, E478-487) and 5’-TTAT. PiggyBac-like transposases transpose their transposons using a cut-and-paste mechanism, which results in duplicaƟon of their 4 base pair target sequence on inserƟon into a DNA molecule. The target sequence is thus found on each side of an integrated piggyBac-like transposon.

[0032] A “coupling element” or “translaƟonal coupling element” means a DNA sequence that allows the expression of afirst polypepƟde to be linked to the expression of a second polypepƟde. Internal ribosome entry site elements (IRES elements) and cis-acƟng hydrolase elements (CHYSEL elements) are examples of coupling elements.

[0033] A DNA sequence, segment of DNA, RNA sequence or RNA segment means a conƟguous nucleic acid sequence, which can be an oligonucleoƟde of 2 to 20 nucleoƟdes in length to a full-length genomic sequence of thousands or hundreds of thousands of base pairs.

[0034] A vector is a nucleic acid that facilitates any of transfecƟon, integraƟon, replicaƟon or expression of an open reading frame incorporated into the vector. An expression vector is a vector comprising a promoter which has been or can be operably linked to an open reading frame to be expressed. TransfecƟon of the expression vector into a cell allows the cell to express the open reading frame. An expression vector can be a geneƟcally engineered plasmid, virus, recombinant virus, or an arƟficial chromosome derived from, for example, a bacteriophage, adenovirus, adeno-associated virus, retrovirus, lenƟvirus, poxvirus, or herpesvirus. Such expression vectors can include sequences from bacteria, viruses or phages. Such vectors include chromosomal, episomal and virus-derived vectors, for example, vectors derived from bacterial plasmids, bacteriophages, yeast episomes, yeast chromosomal elements, and viruses, vectors derived from combinaƟons thereof, such as those derived from plasmid and bacteriophage geneƟc elements, cosmids and phagemids.

[0035] A “gene” refers to a transcripƟonal unit including a promoter and sequence to be expressed from it as an RNA or polypepƟde. The sequence to be expressed can be genomic or cDNA or one or more non-coding RNAs including siRNAs or microRNAs among other possibiliƟes. Other elements, such as introns, and other regulatory sequences may or may not be present.

[0036] A gene transfer system refers to an expression vector and opƟonally one or more other features to facilitate gene transfer. For example, a gene transfer system may comprise an expression vector and a lipid or viral packaging mix for enabling afirst nucleic acid to enter a cell, or it may comprise a nucleic acid that includes a transposon and a second nucleic acid encoding a corresponding transposase for genomic integraƟon of the transposon. A transposase and transposon of a gene transfer system may be on the same nucleic acid molecule or on different nucleic acid molecules.

[0037] Two elements are “heterologous” to one another if not naturally associated. For example, an open reading frame linked to a heterologous promoter means a promoter other than that which naturally drives expression of the open reading frame. A heterologous nucleic acidflanked by transposon ends or ITRs means a heterologous nucleic acid not naturallyflanked by those transposon ends or ITRs, such as a nucleic acid encoding a polypepƟde other than a transposase, including an anƟbody heavy or light chain. A nucleic acid is heterologous to a cell if not naturally found in the cell or if naturally found in the cell but in a different locaƟon (e.g., episomal or different genomic locaƟon) than the locaƟon described.

[0038] A “hyperacƟve” transposase is a transposase that is more acƟve than the naturally occurring transposase from which it is derived. “HyperacƟve” transposases are thus not naturally occurring sequences.

[0039] A transposase derived from a naturally occurring transposase of a specified SEQ ID NO. means a transposase having at least 90% idenƟty to the specified SEQ ID NO. over the full-length of the shorter of the sequences being compared. Such derived transposase includes transposases with any of the sequence variaƟons disclosed herein from the relevant naturally occurring transposase and any permutaƟons thereof.

[0040] An “IRES” or “internal ribosome entry site” means a specialized sequence that directly promotes ribosome binding, independent of a cap structure.

[0041] An ‘isolated’ object, such as a polypepƟde or nucleic acid, means the object has been either removed from its natural environment, produced using recombinant techniques, or chemically or enzymaƟcally synthesized (i.e., result from a non-natural syntheƟc process). Objects can also be purified, that is, provided at least 90%, 95% or 99% free w / w of othermaterials with which they are naturally associated or are used in their producƟon or purificaƟon. The terms isolated and purified do not exclude presence of other components not naturally associated with the object that facilitate its use, such as a heterologous promoter for an open reading frame, or pharmaceuƟcal excipient.

[0042] Unless otherwise apparent from the context, the terms “nucleoside” and “nucleoƟde” include those moieƟes which contain not only the standard purine and pyrimidine bases, but also other heterocyclic bases which have been modified. Such modificaƟons include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleosides or nucleoƟdes can also include modificaƟons on the sugar moiety, for example, where one or more of the hydroxyl groups are replaced with halogen, aliphaƟc groups, or is funcƟonalized as ethers, amines, or the like. The term “nucleoƟdic unit” encompasses nucleosides and nucleoƟdes.

[0043] An “Open Reading Frame” or “ORF” means a porƟon of a nucleic acid that, when translated into amino acids, contains no stop codons. An open reading frame presumpƟvely encodes a polypepƟde. The geneƟc code reads DNA sequences in groups of three base pairs, which means that a double-stranded DNA molecule can read in any of six possible reading frames-three in the forward direcƟon and three in the reverse. An ORF typically also includes an iniƟaƟon codon at which translaƟon may start.

[0044] The term “operably linked” refers to funcƟonal linkage between two sequences such that one sequence operaƟonally modifies the behavior of the other. For example, a promoter is operably linked to an open reading frame when the promoter can iniƟate transcripƟon of the open reading frame, opƟonally with subsequent translaƟon of the transcript. A promoter is operably linked to one or more tet-operators, when iniƟaƟon of transcripƟon by the promoter can be regulated by binding of a tet-repressor or modified tet- repressor to the tet-operon. A mature polypepƟde and signal pepƟde are operably linked when the signal pepƟde regulates secreƟon or subcellular locaƟon of the mature polypepƟde.

[0045] The term “overhang” or “DNA overhang” means the single-stranded porƟon at the end of a double-stranded DNA molecule. Complementary overhangs are those which will base-pair with each other.

[0046] A “piggyBac-like transposase” means a transposase with at least 20% amino acid sequence idenƟty as idenƟfied using the TBLASTP algorithm to the piggyBac transposase from Trichoplusia ni (SEQ ID NO:116), and as more fully described in Sakar, A. et. al., 2003. Mol. Gen. Genomics 270: 173-180. “Molecular evoluƟonary analysis of the widespread piggyBac transposon family and related ‘domesƟcated’ species”, and further characterized by a DDE-like DDD moƟf, with aspartate residues at posiƟons corresponding to D268, D346, and D447 of Trichoplusia ni piggyBac transposase on maximal alignment. PiggyBac-like transposases are also characterized by their ability to excise their transposons precisely with a high frequency. A “piggyBac-like transposon” means a transposon having transposon ends which are the same or at least 80% and preferably at least 90, 95, 96, 97, 98, 99% or 100% idenƟcal to the nucleoƟde sequences of the transposon ends of a naturally occurring transposon that encodes a piggyBac- like transposase. A piggyBac-like transposon includes an inverted terminal repeat (ITR) sequence of approximately 12-16 bases at each end. These repeats may be idenƟcal at the two ends, or the repeats at the two ends may differ at 1 or 2 or 3 or 4 posiƟons in the two ITRs. The transposon isflanked on each side by a 4 base sequence corresponding to the integraƟon target sequence which is duplicated on transposon integraƟon (the Target Site DuplicaƟon or Target Sequence DuplicaƟon or TSD). PiggyBac-like transposons and transposases occur naturally in a wide range of organisms including Argyrogramma agnate (GU477713), Anopheles gambiae (XP_312615; XP_320414; XP_310729), Aphis gossypii (GU329918), Acyrthosiphon pisum (XP_001948139), AgroƟs ypsilon (GU477714), Bombyx mori (BAD11135), Ciona intesƟnalis (XP_002123602), Chilo suppressalis (JX294476), Drosophila melanogaster (AAL39784), Daphnia pulicaria (AAM76342), Helicoverpa armigera (ABS18391), Homo sapiens (NP_689808), Heliothis virescens (ABD76335), Macdunnoughia crassisigna (EU287451), Macaca fascicularis (AB179012), Mus musculus (NP_741958), PecƟnophora gossypiella (GU270322), RaƩus norvegicus (XP_220453), Tribolium castaneum (XP_001814566) and Trichoplusia ni (AAA87375) and Xenopus tropicalis (BAF82026), although transposiƟon acƟvity has been described for almost none of these.

[0047] A regulatory element such as promoter is acƟve in a specified target cell, such as a mammalian cell, means a regulatory element configurable to result in a level of expression ofat least 1 transcript and opƟonally at least ten, 100 or 1000 transcripts per cell in a mammalian cell into which the regulatory element has been introduced.

[0048] Sequence idenƟty can be determined by aligning sequences using algorithms, such as BESTFIT, FASTA, and TFASTA in the Wisconsin GeneƟcs SoŌware Package Release 7.0, GeneƟcs Computer Group, 575 Science Dr., Madison, Wis.), using default gap parameters, or by inspecƟon, and the best alignment (i.e., resulƟng in the highest percentage of sequence similarity over a comparison window). Percentage of sequence idenƟty is calculated by comparing two opƟmally aligned sequences over a window of comparison, determining the number of posiƟons at which the idenƟcal residues occurs in both sequences to yield the number of matched posiƟons, dividing the number of matched posiƟons by the total number of matched and mismatched posiƟons not counƟng gaps in the window of comparison (i.e., the window size), and mulƟplying the result by 100 to yield the percentage of sequence idenƟty. Unless otherwise indicated the window of comparison between two sequences is defined by the longer of (a) enƟre length of the shorter of the two sequences being compared, or (b) at least 25 conƟguous nucleoƟdes or amino acids. Matched posiƟons in maximally aligned sequences can be referred to as corresponding to one another.

[0049] Specific binding between two enƟƟes refers to binding detectably higher in magnitude and disƟnguishable from non-specific binding of each of the enƟƟes to at least one unrelated target. Specific binding can be the result of formaƟon of bonds between parƟcular funcƟonal groups or parƟcular spaƟalfit (e.g., lock and key type) whereas nonspecific binding is usually the result of van der Waals forces. Exemplary specific binding affinity can be at least 107, 108, 109, or 1010M-1.

[0050] PreferenƟal binding between two enƟƟes refers to a substanƟal difference in binding affinity between two different condiƟons, such as presence or absence of tetracycline. For example, the affiniƟes can differ by a factor of a least 5, 10, 25, 50 or 100. PreferenƟal expression in one condiƟon compared with another likewise refers to variaƟon in expression by a factor of at least 5, 10, 25, 50 or 100 between the condiƟons.

[0051] A polypepƟde refers to any polymer of amino acids natural or syntheƟc regardless of length and thus includes full length proteins, fragments thereof, and pepƟdes.

[0052] A promoter can be represented by a single-stranded sequence of nucleoƟdes present on a transcribed strand or a double-stranded sequence of nucleoƟde formed of the single-stranded sequence of nucleoƟdes just described duplexed with its complement. Depending on the context, reference to a promoter may refer to either single- or double- stranded forms or both.

[0053] In eukaryoƟc cells, gene expression is regulated by several different classes of elements, including enhancers, promoters, introns, RNA export elements, polyadenylaƟon sequences and transcripƟonal terminators.

[0054] TransfecƟon is used generically for any process for introducing a nucleic into cells.

[0055] The term “comprising” indicates that other features besides those recited may or may not be present. The term “consisƟng essenƟally of” is used in accordance with convenƟon to refer to the basic and novel features of an invenƟon.

[0056] The term “about” indicates variaƟon without significant funcƟonal consequences, e.g., within + / -10% of a stated value. DETAILED DESCRIPTION I. GENERAL

[0057] To prepare a stable cell line using transposiƟon, a transposase is typically provided encoded on mRNA. This is because of concern that conƟnued presence of transposase protein would result in instability in the cell line, by causing transposons to be conƟnually excised from one chromosomal locaƟon and either lost or integrated into a new locaƟon with different expression characterisƟcs. Indeed, the FDA requires absence of both detectable transposase acƟvity and transposase-encoding nucleic acid sequences in stable cell lines to be used for expression of protein therapeuƟcs. The expectaƟon is therefore that conƟnuous expression of a transposase protein within a host cell whose genome comprises a corresponding transposon will be detrimental to the expression of a protein encoded on the transposon.

[0058] On the other hand, when a transposase is introduced into a cell encoded on a nucleic acid at the same Ɵme as a corresponding transposon, the transposase must be translated (and transcribed if the nucleic acid is a DNA molecule) before it can begin excising transposons from their original plasmids and integraƟng them into the host cell genome. Prior to synthesis of transposase protein, any introduced transposons will be subject to degradaƟon by cellular nucleases, reducing the average number of transposons surviving to be integrated into the host cell genome, thus reducing the average number of stably integrated transposon copies per genome which in turn reduces the average expression levels of any protein encoded on the transposon. Such degradaƟon can be reduced by introducing a transposon into a cell that already contains acƟve corresponding transposase protein. One way to achieve this is to introduce nucleic acids encoding the transposase before introducing the transposon, so that acƟve transposase protein has been expressed prior to introducƟon of the corresponding transposon, but this increases the number of manipulaƟons required for the cells which may be neither convenient for the operators nor good for the health of the cells for which exogenously introduced nucleic acids can be toxic.

[0059] We therefore decided to test a method for transient protein producƟon in which afirst nucleic acid comprising an open reading frame encoding a transposase is stably introduced into the genome of a host cell. Thefirst polynucleoƟde further comprises regulatory DNA sequences (including afirst promoter and afirst polyadenylaƟon signal) operably linked to the transposase-encoding open reading frame, such that the transposase is expressible within the host cell. Expression of the transposase from the genome before introducƟon of the transposon provides a mechanism to reduce degradaƟon of the transposon before integraƟon can occur, but also results in potenƟal instability due to further transposiƟon of integrated transposons within the genome. Surprisingly, despite the potenƟal instability due to conƟnued expression of the transposase, we found that cells transformed in this manner expressed substanƟally higher yields of a desired protein and / or expressed over longer periods over convenƟonal transient expression methods without integraƟon.II. OVERVIEW OF THE METHODS

[0060] The disclosed methods involve forming a cell line or other mammalian cell populaƟon that has integrated a nucleic acid encoding a transposase into the genomes of the cells in the populaƟon and is capable of stably maintaining the integrated nucleic acid through mulƟple rounds of cell propagaƟon and expressing the transposase. Such cells are then transfected with at least one transposon encoding a polypepƟde or polypepƟdes to be expressed. The transposase can then integrate the transposon(s) into the genome of cells of the mammalian cell populaƟon. IntegraƟon can occur at different Ɵmes aŌer introducƟon of the transposon(s) and at different genomic locaƟons in different cells in the transfected populaƟon. In some cells, a transposon can be integrated at a locaƟon in a cell and then undergo one or more further transposiƟon to different locaƟons. In some cells, mulƟple copies of a transposon can be integrated at different sites in the same genome. Some cells of the transfected populaƟon can receive a transposon but not integrate it. Some cells of the transfected populaƟon may fail to receive a transposon. Expression of polypepƟde(s) from the transposon(s) can occur both before transposiƟon and aŌer transposiƟon. Thus, the aggregate yield of polypepƟde(s) from a populaƟon of cells can reflect contribuƟons from both integrated and non-integrated transposons.

[0061] AŌer transfecƟon of a transposon into a populaƟon of cells with an integrated nucleic acid encoding a corresponding transposase, the cells resulƟng from the transfecƟon are cultured to permit polypepƟde(s) encoded by the transposon to be expressed, and usually propagaƟon of cells generaƟng progeny cells. The media can be any suitable culture media to support propagaƟon and expression of the cells. OpƟonally, the culturing includes selecƟon for cells that have received a transposon over cells which cells have not received a transposon, such that the proporƟon of the laƩer relaƟve to the former are reduced and thus increasing the nutrients in the culture medium available for cells with the transposon. Such selecƟon can be achieved by incorporaƟng a DNA segment encoding a selecƟve marker into the transposon, who expression allows cells to overcome a selecƟve agent, such as an anƟbioƟc supplied to the culture medium. AlternaƟvely, selecƟon can be achieved by incorporaƟng a second open reading frame encoding an enzyme, such as glutamine synthetase or dihydrofolate reductasethat allows cells to make an essenƟal nutrient (e.g., glutamine or tetrahydrofolate) omiƩed from the culture medium. If selecƟon is performed, polypepƟde can be expressed contemporaneously with the selecƟon process and subsequently collected, in contrast to the procedure for stable cell lines where selecƟon is a separate step preceding producƟon. Culturing can also be performed without selecƟon for cells receiving a transposon over cells that have not received a transposon, in which case the transposon(s) need not encode selecƟve markers.

[0062] Cell cultures can be fed addiƟonal nutrients as iniƟal nutrients in the culture media are depleted or can be passaged into fresh media. Temperature of the culture medium can be kept constant (e.g., ~37° C) or varied. A higher temperature e.g., (37° C) can be used iniƟally for cell propagaƟon with a shiŌ to a lower temperature (e.g., 32° C) to bias cells to polypepƟde producƟon and away from propagaƟon. The temperature shiŌ can occur for example, between one and four days post-transfecƟon. Feeding of cells at about between one and four days post-transfecƟon can also sƟmulate polypepƟde synthesis. If the nucleic acid encoding a transposase integrated into the cell genomes is operably linked to an inducible promoter, expression of the transposase can be induced or turned off by supplying or withholding the inducer. Typically, the inducer is supplied before introducing a transposon into the cells so that expressed transposase is available for transposiƟon as soon as a transposon enters cells, thereby reducing the window for loss of transposon molecules due to nuclease acƟon. However, aŌer transposiƟon has occurred further transposiƟon is unnecessary. Thus, further inducer need not be supplied in culturing of the cells. The polypepƟde(s) encoded by the transposon introduced into cells are typically expressed from a consƟtuƟve promoter because the goal is to obtain high level expression of such polypepƟdes over a short period. However, if such polypepƟde(s) are expressed from an inducible promoter, the expression of polypepƟde(s) can be controlled by supplying or withholding the relevant inducer.

[0063] AŌer introducƟon of transposon(s) into cells, isolaƟon of a clonal cell line is typically not performed. Thus, the cells being cultured vary in the genomic locaƟon(s), if any, of integrated transposon(s), copy number of integrated transposons and the Ɵme(s) at which integraƟon occurs aŌer introducƟon of the transposon into cells.

[0064] The yield of a polypepƟde or complex of polypepƟdes in a mulƟmeric enƟty expressed from transposon(s) in transposase-expressing cells can be higher than that obtained from convenƟonal transient polypepƟde expression, or a control system in which either a transposon is introduced into cells lacking the integrated nucleic acid encoding a corresponding transposase, or in which a nucleic acid that is not a transposon is introduced into cells comprising the integrated nucleic acid encoding a transposase. The yield can be higher due to a higher daily yield and / or more days of producƟon resulƟng in a higher total yield for a producƟon run. Typically in a producƟon run, levels of viable cells and expressed polypepƟde(s) iniƟally increase to a maximum value and then decline. HarvesƟng of polypepƟde(s) is preferably performed at or about (e.g., within + / - 5, 10 or 20%) the maximum level of polypepƟde producƟon, which can be assessed by contemporaneous monitoring or historical measurement under the same condiƟons. Whereas convenƟonal transient cell cultures are maintained for a period of about 7-14 days post-transfecƟon depending on cell type, the present cell cultures can be maintained for periods of days, weeks or even months post- transfecƟon, depending on cell type and how much polypepƟde is needed. For example, such a culture can be maintained for at least 1 week, 2 weeks, 3 weeks and up to a month or more. For example, culturing can be performed for 7-30 days post-transfecƟon. Longer culture Ɵmes may require addiƟonal steps to maintain cell viability by providing adequate nutrients and prevenƟng accumulaƟon of toxic waste products, for example by feeding and / or exchange of spent media with fresh media (for example perfusion or intensified fed batch methods). PolypepƟde(s) can be obtained in a higher yield on an accumulated basis (daily x days of producƟon) than for convenƟonal transient expression. For example, the yield from a producƟon run can be at least 1.2, 2, 3, 4, 5 or 10 fold higher than the yield from control system as discussed above expressed in mg / L cell culture media. An exemplary yield can be 1.2 -10 or 1.5-5 Ɵmes than the yield from control system as discussed above expressed in mg / L cell culture media. An exemplary yield of a polypepƟde or mulƟmeric complex of polypepƟdes from a producƟon run is in the range of 50-1500 mg / L.

[0065] PolypepƟde(s) can also be obtained more quickly than from an established stable cell line. For example, polypepƟde(s) producƟon can be started within 24 hours of transfecƟonand polypepƟde(s can be harvested within 10, 15, 20, 25, 30 days or month from transfecƟng. By contrast, it takes at least about two months to produce and scale-up a stable cell line before beginning protein producƟon.

[0066] GlycosylaƟon paƩerns of polypepƟde(s) produced by the present methods also can be more similar to those from stable cell lines with integrated DNA encoding polypepƟde(s) than is typically the case with convenƟonal transient expression systems. For example, convenƟonal transient expression is oŌen done in expiCHO cells, which is a CHO-S based system chosen for its high yield but with a very different glycan profile than CHO-K1 cells, which are oŌen used for making permanent cell lines. However, CHO-K1 cells can also be used in the present methods resulƟng in similar glycosylaƟon paƩerns to permanent cell lines. PolypepƟdes produced by such cultures, which are usually secreted into the culture medium, can be isolated from the media and further purified by convenƟonal methods. PurificaƟon can be performed, e.g., 7-30 days post -transfecƟon. PolypepƟdes can then be used for various purposes including non-clinical research with cells or non-human animals, clinical research or as industrial enzymes. III. TRANSPOSON SYSTEMS

[0067] Heterologous polynucleoƟdes may be more efficiently integrated into a target genome if they are part of a transposon, for example so that they may be integrated by a transposase. A parƟcular benefit of a transposon is that the enƟre polynucleoƟde between the transposon ITRs is integrated. This is in contrast with random integraƟon, where a polynucleoƟde introduced into a eukaryoƟc cell is oŌen fragmented at random in the cell, and only parts of the polynucleoƟde become incorporated into the target genome, usually at a low frequency. There are several different classes of transposon. piggyBac-like transposons include the piggyBac transposon from the looper moth Trichoplusia ni, Xenopus piggyBac-like transposons, Bombyx piggyBac-like transposons, Heliothis piggyBac-like transposons,Helicoverpa piggyBac-like transposons, Agro s piggyBac-like transposons, Amyelois piggyBac-like transposons, Myo s piggyBac-like transposons and Oryzias piggyBac-like transposons. hATtransposons include TcBuster and Tol2. Mariner transposons include Sleeping Beauty. Helitron transposons include Helraiser. Each of these transposons can be integrated into the genome ofa mammalian cell by a corresponding transposase. Heterologous polynucleoƟdes incorporated into transposons may be integrated into mammalian cells, as well as hepatocytes, neural cells, muscle cells, blood cells, embryonic stem cells, somaƟc stem cells, hematopoieƟc cells, embryos, zygotes and sperm cells (some of which are open to be manipulated in an in vitro seƫng). Preferred cells can also be pluripotent cells (cells whose descendants can differenƟate into several restricted cell types, such as hematopoieƟc stem cells or other stem cells) or toƟpotent cells (i.e., a cell whose descendants can become any cell type in an organism, e.g., embryonic stem cells).

[0068] Preferred gene transfer systems comprise a transposon in combinaƟon with a corresponding transposase protein that transposases the transposon, or a nucleic acid that encodes the corresponding transposase protein and is expressible in a cell transfected with a transposon. In some cell lines of the invenƟon, a DNA molecule encoding a transposase is integrated into the genome of a cell. In other applicaƟon, such as introducƟon of a transposon containing a transposase into a cellular genome, the transposase effecƟng this transposiƟon, which is typically orthogonal to the transposase encoded by the transposon, can be supplied as a protein or nucleic acid, which can be DNA or mRNA.

[0069] An exemplary cell, cell line or populaƟon of cells has a genome comprising a polynucleoƟde comprising an open reading frame encoding a polypepƟde with at least 95% and opƟonally 100% sequence idenƟty to an amino acid sequence selected from SEQ ID NOS:9-41, 46-69, 74, 158, 84, 89, 96, 101, 106, 157, 116, 130 or 141, operably linked to a promoter acƟve in the cell(s). The polynucleoƟde may further include transposon ends such that the open reading frame and operably linked promoter are transposable by a second transposase, but not by the transposase encoded by the open reading frame. If the open reading frame encodes a polypepƟde with at least 95% sequence idenƟty to an amino acid sequence selected from SEQ ID NOS:9-41, the polynucleoƟde may comprise a pair of ITRsflanking the open reading frame and operably linked promoter selected from SEQ ID NOS:42 and 43, or 70 and 71, or 75 and 76, or 80 and 81, or 85 and 86, or 90 and 91, or 97 and 98, or 102 and 103, or 107 and 108, or 112 and 113, or 128 and 129, or 137 and 138. Exemplary polynucleoƟde sequences comprising an open reading frame encoding a polypepƟde with at least 95% sequence idenƟty to an aminoacid sequence selected from SEQ ID NOS:9-41 transposable by an orthogonal transposase are nucleoƟde sequences SEQ ID NOS:150-156. If the open reading frame encodes a polypepƟde with at least 95% sequence idenƟty to an amino acid sequence selected from SEQ ID NOS:46- 69, the polynucleoƟde may comprise a pair of ITRsflanking the open reading frame and operably linked promoter selected from SEQ ID NOS:1 and 2, or 70 and 71, or 75 and 76, or 80 and 81, or 85 and 86, or 90 and 91, or 97 and 98, or 102 and 103, or 107 and 108, or 112 and 113, or 128 and 129, or 137 and 138. If the open reading frame encodes a polypepƟde with at least 95% sequence idenƟty to SEQ ID NO:74, the polynucleoƟde may comprise a pair of ITRs flanking the open reading frame and operably linked promoter selected from SEQ ID NOS:1 and 2, or 42 and 43, or 75 and 76, or 80 and 81, or 85 and 86, or 90 and 91, or 97 and 98, or 102 and 103, or 107 and 108, or 112 and 113, or 128 and 129, or 137 and 138. If the open reading frame encodes a polypepƟde with at least 95% sequence idenƟty to SEQ ID NO:158, the polynucleoƟde may comprise a pair of ITRsflanking the open reading frame and operably linked promoter selected from SEQ ID NOS:1 and 2, or 42 and 43, or 70 and 71, or 80 and 81, or 85 and 86, or 90 and 91, or 97 and 98, or 102 and 103, or 107 and 108, or 112 and 113, or 128 and 129, or 137 and 138. An exemplary polynucleoƟde sequence comprising an open reading frame encoding a polypepƟde with at least 95% sequence idenƟty to SEQ ID NO:158 transposable by an orthogonal transposase is nucleoƟde sequence SEQ ID NO:156. If the open reading frame encodes a polypepƟde with at least 95% sequence idenƟty to SEQ ID NO:84, the polynucleoƟde may comprise a pair of ITRsflanking the open reading frame and operably linked promoter selected from SEQ ID NOS:1 and 2, or 42 and 43, or 70 and 71, or 75 and 76, or 85 and 86, or 90 and 91, or 97 and 98, or 102 and 103, or 107 and 108, or 112 and 113, or 128 and 129, or 137 and 138. If the open reading frame encodes a polypepƟde with at least 95% sequence idenƟty to SEQ ID NO:89, the polynucleoƟde may comprise a pair of ITRsflanking the open reading frame and operably linked promoter selected from SEQ ID NOS:1 and 2, or 42 and 43, or 70 and 71, or 75 and 76, or 80 and 81, or 90 and 91, or 97 and 98, or 102 and 103, or 107 and 108, or 112 and 113, or 128 and 129, or 137 and 138. If the open reading frame encodes a polypepƟde with at least 95% sequence idenƟty to SEQ ID NO:96, the polynucleoƟde may comprise a pair of ITRsflanking the open reading frame and operably linked promoter selected from SEQ ID NOS:1and 2, or 42 and 43, or 70 and 71, or 75 and 76, or 85 and 86, or 80 and 81, or 97 and 98, or 102 and 103, or 107 and 108, or 112 and 113, or 128 and 129, or 137 and 138. An exemplary polynucleoƟde sequence comprising an open reading frame encoding a polypepƟde with at least 95% sequence idenƟty to SEQ ID NO:96 transposable by an orthogonal transposase is nucleoƟde sequence SEQ ID NO:153. If the open reading frame encodes a polypepƟde with at least 95% sequence idenƟty to an amino acid sequence selected from SEQ ID NOS:101 and 106, the polynucleoƟde may comprise a pair of ITRsflanking the open reading frame and operably linked promoter selected from SEQ ID NOS:1 and 2, or 42 and 43, or 70 and 71, or 75 and 76, or 80 and 81, or 85 and 86, or 90 and 91, or 107 and 108, or 112 and 113, or 128 and 129, or 137 and 138. If the open reading frame encodes a polypepƟde with at least 95% sequence idenƟty to SEQ ID NO:157, the polynucleoƟde may comprise a pair of ITRsflanking the open reading frame and operably linked promoter selected from SEQ ID NOS:1 and 2, or 42 and 43, or 70 and 71, or 75 and 76, or 80 and 81, or 85 and 86, or 97 and 98, or 90 and 91, or 102 and 103, or 112 and 113, or 128 and 129, or 137 and 138. An exemplary polynucleoƟde sequence comprising an open reading frame encoding a polypepƟde with at least 95% sequence idenƟty to SEQ ID NO:157 transposable by an orthogonal transposase is nucleoƟde sequence SEQ ID NO:154. If the open reading frame encodes a polypepƟde with at least 95% sequence idenƟty to SEQ ID NO:116, the polynucleoƟde may comprise a pair of ITRsflanking the open reading frame and operably linked promoter selected from SEQ ID NOS:1 and 2, or 42 and 43, or 70 and 71, or 75 and 76, or 80 and 81, or 85 and 86, or 97 and 98, or 90 and 91, or 102 and 103, or 107 and 108, or 128 and 129, or 137 and 138. An exemplary polynucleoƟde sequence comprising an open reading frame encoding a polypepƟde with at least 95% sequence idenƟty to SEQ ID NO:116 transposable by an orthogonal transposase is nucleoƟde sequence SEQ ID NO:155. If the open reading frame encodes a polypepƟde with at least 95% sequence idenƟty to SEQ ID NO:130, the polynucleoƟde may comprise a pair of ITRsflanking the open reading frame and operably linked promoter selected from SEQ ID NOS:1 and 2, or 42 and 43, or 70 and 71, or 75 and 76, or 80 and 81, or 85 and 86, or 97 and 98, or 90 and 91, or 102 and 103, or 107 and 108, or 112 and 113, or 137 and 138. If the open reading frame encodes a polypepƟde with at least 95% sequence idenƟty to SEQ ID NO:141, the polynucleoƟde may comprise a pair of ITRsflanking the openreading frame and operably linked promoter selected from SEQ ID NOS:1 and 2, or 42 and 43, or 70 and 71, or 75 and 76, or 80 and 81, or 85 and 86, or 97 and 98, or 90 and 91, or 102 and 103, or 107 and 108, or 112 and 113, or 128 and 129.When there are mulƟple components of a gene transfer system, for example one or more polynucleoƟdes comprising transposon endsflanking genes for expression in the target cell, and a transposase (which may be provided either as a protein or encoded by a nucleic acid), these components can be transfected into a cell at the same Ɵme, or sequenƟally. For example, a transposase protein or its encoding nucleic acid may be transfected into a cell prior to, simultaneously with or subsequently to transfecƟon of a corresponding transposon. AddiƟonally, administraƟon of either component of the gene transfer system may occur repeatedly, for example, by administering at least two doses of this component.

[0070] Transposase proteins may be encoded by polynucleoƟdes including RNA or DNA. Preferable RNA molecules include those with appropriate subsƟtuƟons to reduce toxicity effects on the cell, for example subsƟtuƟon of uridine with pseudouridine, and subsƟtuƟon of cytosine with 5-methyl cytosine. mRNA encoding the transposase may be prepared such that it has a 5’- cap structure to improve expression in a target cell. Exemplary cap structures are a cap analog (G(5’)ppp(5’)G), an anƟ-reverse cap analog (3’-O-Me-m7G(5’)ppp(5’)G, a clean cap (m7G(5’)ppp(5’)(2’OMeA)pG), an mCap (m7G(5’)ppp(5’)G). mRNA encoding the transposase may be prepared such that some bases are parƟally or fully subsƟtuted, for example uridine may be subsƟtuted with pseudo-uridine, cytosine may be subsƟtuted with 5-methyl-cytosine. Any combinaƟons of these caps and subsƟtuƟons may be made. Similarly, the nucleic acid encoding the transposase protein or the transposon of this invenƟon can be transfected into the cell as a linear fragment or as a circularized fragment, either as a plasmid or as recombinant viral DNA. If the transposase is introduced as a DNA sequence encoding the transposase, then the open reading frame encoding the transposase is preferably operably linked to a promoter that is acƟve in the target mammalian cell. 1. Xenopus

[0071] An advantageous piggyBac-like transposon for modifying the genome of a mammalian cell is a Xenopus transposon which comprises, from 5’ to 3’, afirst ITR with the withnucleoƟde sequence SEQ ID NO:1, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence SEQ ID NO:2. The transposon may further beflanked by a copy of the tetranucleoƟde 5’-TTAA-3’ on each side, or a copy of the tetranucleoƟde 5’-TTAT-3’ on each side, immediately adjacent to the ITRs and distal to the heterologous polynucleoƟde. The transposon may further comprise afirst addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably thefirst ITR, and proximal to the heterologous polynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:5 or 6. The transposon may further comprise a second addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably the second ITR, and proximal to the heterologous polynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:7 or 8. This transposon may be transposed by a corresponding Xenopus transposase comprising a polypepƟde sequence at least 90% idenƟcal to the polypepƟde sequence of SEQ ID NO:9 or 10, for example any of SEQ ID NOS:9-41. The Xenopus transposase may opƟonally be fused to a heterologous nuclear localizaƟon signal. Preferably the transposase is a hyperacƟve variant of a naturally occurring transposase. Preferably the hyperacƟve variant transposase comprises one or more of the following amino acid changes, relaƟve to the polypepƟde sequence of SEQ ID NO:9: Y6L, Y6H, Y6V, Y6I, Y6C, Y6G, Y6A, Y6S, Y6F, Y6R, Y6P, Y6D, Y6N, S7G, S7V, S7D, E9W, E9D, E9E, M16E, M16N, M16D, M16S, M16Q, M16T, M16A, M16L, M16H, M16F, M16I, S18C, S18Y, S18M, S18L, S18Q, S18G, S18P, S18A, S18W, S18H, S18K, S18I, S18V, S19C, S19V, S19L, S19F, S19K, S19E, S19D, S19G, S19N, S19A, S19M, S19P, S19Y, S19R, S19T, S19Q, S20G, S20M, S20L, S20V, S20H, S20W, S20A, S20C, S20Q, S20D, S20F, S20N, S20R, E21N, E21W, E21G, E21Q, E21L, E21D, E21A, E21P, E21T, E21S, E21Y, E21V, E21F, E21M, E22C, E22H, E22R, E22L, E22K, E22S, E22G, E22M, E22V, E22Q, E22A, E22Y, E22W, E22D, E22T, F23Q, F23A, F23D, F23W, F23K, F23T, F23V, F23M, F23N, F23P, F23H, F23E, F23C, F23R, F23Y, S24L, S24W, S24H, S24V, S24P, S24I, S24F, S24K, S24Y, S24D, S24C, S24N, S24G, S24A, S26F, S26H, S26V, S26Q, S26Y, S26W, S28K, S28Y, S28C, S28M, S28L, S28H, S28T, S28Q, V31L, V31T, V31I, V31Q, V31K, A34L, A34E, L67A, L67T, L67M, L67V, L67C, L67H, L67E, L67Y, G73H, G73N, G73K, G73F, G73V, G73D, G73S, G73W, G73L, A76L, A76R, A76E, A76I, A76V, D77N, D77Q, D77Y, D77L, D77T, P88A, P88E, P88N, P88H, P88D, P88L, N91D, N91R, N91A, N91L, N91H, N91V, Y141I, Y141M, Y141Q, Y141S, Y141E, Y141W, Y141V, Y141F, Y141A, Y141C, Y141K,Y141L, Y141H, Y141R, N145C, N145M, N145A, N145Q, N145I, N145F, N145G, N145D, N145E, N145V, N145H, N145W, N145Y, N145L, N145R, N145S, P146V, P146T, P146W, P146C, P146Q, P146L, P146Y, P146K, P146N, P146F, P146E, P148M, P148R, P148V, P148F, P148T, P148C, P148Q, P148H, Y150W, Y150A, Y150F, Y150H, Y150S, Y150V, Y150C, Y150M, Y150N, Y150D, Y150E, Y150Q, Y150K, H157Y, H157F, H157T, H157S, H157W, A162L, A162V, A162C, A162K, A162T, A162G, A162M, A162S, A162I, A162Y, A162Q, A179T, A179K, A179S, A179V, A179R, L182V, L182I, L182Q, L182T, L182W, L182R, L182S, T189C, T189N, T189L, T189K, T189Q, T189V, T189A, T189W, T189Y, T189G, T189F, T189S, T189H, L192V, L192C, L192H, L192M, L192I, S193P, S193T, S193R, S193K, S193G, S193D, S193N, S193F, S193H, S193Q, S193Y, V196L, V196S, V196W, V196A, V196F, V196M, V196I, S198G, S198R, S198A, S198K, T200C, T200I, T200M, T200L, T200N, T200W, T200V, T200Q, T200Y, T200H, T200R, S202A, S202P, L210H, L210A, F212Y, F212N, F212M, F212C, F212A, N218V, N218R, N218T, N218C, N218G, N218I, N218P, N218D, N218E, A248S, A248L, A248H, A248C, A248N, A248I, A248Q, A248Y, A248M, A248D, L263V, L263A, L263M, L263R, L263D, Q270V, Q270K, Q270A, Q270C, Q270P, Q270L, Q270I, Q270E, Q270G, Q270Y, Q270N, Q270T, Q270W, Q270H, S294R, S294N, S294G, S294T, S294C, T297C, T297P, T297V, T297M, T297L, T297D, E304D, E304H, E304S, E304Q, E304C, S308R, S308G, L310R, L310I, L310V, L333M, L333W, L333F, Q336Y, Q336N, Q336M, Q336A, Q336T, Q336L, Q336I, Q336G, Q336F, Q336E, Q336V, Q336C, Q336H, A354V, A354W, A354D, A354C, A354R, A354E, A354K, A354H, A354G, C357Q, C357H, C357W, C357N, C357I, C357V, C357M, C357R, C357F, C357D, L358A, L358F, L358E, L358R, L358Q, L358V, L358H, L358C, L358M, L358Y, L358K, L358N, L358I, D359N, D359A, D359L, D359H, D359R, D359S, D359Q, D359E, D359M, L377V, L377I, V423N, V423P, V423T, V423F, V423H, V423C, V423S, V423G, V423A, V423R, V423L, P426L, P426K, P426Y, P426F, P426T, P426W, P426V, P426C, P426S, P426Q, P426H, P426N, K428R, K428Q, K428N, K428T, K428F, S434A, S434T, S438Q, S438A, S438M, T447S, T447A, T447C, T447Q, T447N, T447G, L450M, L450V, L450A, L450I, L450E, A462M, A462T, A462Y, A462F, A462K, A462R, A462Q, A462H, A462E, A462N, A462C, V467T, V467C, V467A, V467K, I469V, I469N, I472V, I472L, I472W, I472M, I472F, L476I, L476V, L476N, L476F, L476M, L476C, L476Q, P488E, P488H, P488K, P488Q, P488F, P488M, P488L, P488N, P488D, Q498V, Q498L, Q498G, Q498H, Q498T, Q498C, Q498E, Q498M, L502I, L502M, L502V, L502G, L502F, E517M,E517V, E517A, E517K, E517L, E517G, E517S, E517I, P520W, P520R, P520M, P520F, P520Q, P520V, P520G, P520D, P520K, P520Y, P520E, P520L, P520T, S521A, S521H, S521C, S521V, S521W, S521T, S521K, S521F, S521G, N523W, N523A, N523G, N523S, N523P, N523M, N523Q, N523L, N523K, N523D, N523H, N523F, N523C, I533M, I533V, I533T, I533S, I533F, I533G, I533E, D534E, D534Q, D534L, D534R, D534V, D534C, D534M, D534N, D534A, D534G, D534F, D534T, D534H, D534K, D534S, F576L, F576K, F576V, F576D, F576W, F576M, F576C, F576R, F576Q, F576A, F576Y, F576N, F576G, F576I, F576E, K577L, K577G, K577D, K577R, K577H, K577Y, K577I, K577E, K577V, K577N, I582V, I582K, I582R, I582M, I582G, I582N, I582E, I582A, I582Q, Y583L, Y583C, Y583F, Y583D, Y583Q, L587F, L587D, L587R, L587I, L587P, L587N, L587E, L587S, L587Y, L587M, L587Q, L587G, L587W, L587K or L587T. 2. Bombyx

[0072] An advantageous piggyBac-like transposon for modifying the genome of a mammalian cell is a Bombyx transposon which comprises, from 5’ to 3’, afirst ITR with the with nucleoƟde sequence of SEQ ID NO:42, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence of SEQ ID NO:43. The transposon may further beflanked by a copy of the tetranucleoƟde 5’-TTAA-3’ on each side, or by a copy of the tetranucleoƟde 5’- TTAT-3’ on each side, immediately adjacent to the ITRs and distal to the heterologous polynucleoƟde. The transposon may further comprise afirst addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably thefirst ITR, and proximal to the heterologous polynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:44. The transposon may further comprise a second addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably the second ITR, and proximal to the heterologous polynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:45. This transposon may be transposed by a corresponding Bombyx transposase comprising a polypepƟde sequence at least 90% idenƟcal to the polypepƟde sequence of SEQ ID NO:46 or 47, for example any of SEQ ID NOS:46-69. The Bombyx transposase may opƟonally be fused to a heterologous nuclear localizaƟon signal. Preferably the transposase is a hyperacƟve variant of a naturally occurring transposase. Preferably the hyperacƟve variant transposase comprises one or more of the following amino acid changes, relaƟve to the polypepƟde sequence of SEQ ID NO:46: Q85E,Q85M, Q85K, Q85H, Q85N, Q85T, Q85F, Q85L, Q92E, Q92A, Q92P, Q92N, Q92I, Q92Y, Q92H, Q92F, Q92R, Q92D, Q92M, Q92W, Q92C, Q92G, Q92L, Q92V, Q92T, V93P, V93K, V93M, V93F, V93W, V93L, V93A, V93I, V93Q, P96A, P96T, P96M, P96R, P96G, P96V, P96E, P96Q, P96C, F97Q, F97K, F97H, F97T, F97C, F97W, F97V, F97E, F97P, F97D, F97A, F97R, F97G, F97N, F97Y, H165E, H165G, H165Q, H165T, H165M, H165V, H165L, H165C, H165N, H165D, H165K, H165W, H165A, E178S, E178H, E178Y, E178F, E178C, E178A, E178Q, E178G, E178V, E178D, E178L, E178P, E178W, C189D, C189Y, C189I, C189W, C189T, C189K, C189M, C189F, C189P, C189Q, C189V, A196G, L200I, L200F, L200C, L200M, L200Y, A201Q, A201L, A201M, L203V, L203D, L203G, L203E, L203C, L203T, L203M, L203A, L203Y, N207G, N207A, L211G, L211M, L211C, L211T, L211V, L211A, W215Y, T217V, T217A, T217I, T217P, T217C, T217Q, T217M, T217F, T217D, T217K, G219S, G219A, G219C, G219H, G219Q, Q235C, Q235N, Q235H, Q235G, Q235W, Q235Y, Q235A, Q235T, Q235E, Q235M, Q235F, Q238C, Q238M, Q238H, Q238V, Q238L, Q238T, Q238I, R242Q, K246I, K253V, M258V, F261L, S263K, C271S, N303C, N303R, N303G, N303A, N303D, N303S, N303H, N303E, N303R, N303K, N303L, N303Q, I312F, I312C, I312A, I312L, I312T, I312V, I312G, I312M, F321H, F321R, F321N, F321Y, F321W, F321D, F321G, F321E, F321M, F321K, F321A, F321Q, V323I, V323L, V323T, V323M, V323A, V324N, V324A, V324C, V324I, V324L, V324T, V324K, V324Y, V324H, V324F, V324S, V324Q, V324M, V324G, A330K, A330V, A330P, A330S, A330C, A330T, A330L, Q333P, Q333T, Q333M, Q333H, Q333S, P337W, P337E, P337H, P337I, P337A, P337M, P337N, P337D, P337K, P337Q, P337G, P337S, P337C, P337L, P337V, F368Y, L373C, L373V, L373I, L373S, L373T, V389I, V389M, V389T, V389L, V389A, R394H, R394K, R394T, R394P, R394M, R394A, Q395P, Q395F, Q395E, Q395C, Q395V, Q395A, Q395H, Q395S, Q395Y, S399N, S399E, S399K, S399H, S399D, S399Y, S399G, S399Q, S399R, S399T, S399A, S399V, S399M, R402Y, R402K, R402D, R402F, R402G, R402N, R402E, R402M, R402S, R402Q, R402T, R402C, R402L, R402V, T403W, T403A, T403V, T403F, T403L, T403Y, T403N, T403G, T403C, T403I, T403S, T403M, T403Q, T403K, T403E, D404I, D404S, D404E, D404N, D404H, D404C, D404M, D404G, D404A, D404Q, D404L, D404P, D404V, D404W, D404F, N408F, N408I, N408A, N408E, N408M, N408S, N408D, N408Y, N408H, N408C, N408Q, N408V, N408W, N408L, N408P, N408K, S409H, S409Y, S409N, S409I, S409D, S409F, S409T, S409C, S409Q, N441F, N441R, N441M, N441G, N441C, N441D, N441L, N441A, N441V, N441W, G448W, G448Y, G448H, G448C, G448T,G448V, G448N, G448Q, E449A, E449P, E449T, E449L, E449H, E449G, E449C, E449I, V469T, V469A, V469H, V469C, V469L, L472K, L472Q, L472M, C473G, C473Q, C473T, C473I, C473M, R484H, R484K, T507R, T507D, T507S, T507G, T507K, T507I, T507M, T507E, T507C, T507L, T507V, G523Q, G523T, G523A, G523M, G523S, G523C, G523I, G523L, I527M, I527V, Y528N, Y528W, Y528M, Y528Q, Y528K, Y528V, Y528I, Y528G, Y528D, Y528A, Y528E, Y528R, Y543C, Y543W, Y543I, Y543M, Y543Q, Y543A, Y543R, Y543H, E549K, E549C, E549I, E549Q, E549A, E549H, E549C, E549M, E549S, E549F, E549L, K550R, K550M, K550Q, S556G, S556V, S556I, P557W, P557T, P557S, P557A, P557Q, P557K, P557D, P557G, P557N, P557L, P557V, H559K, H559S, H559C, H559I, H559W, V560F, V560P, V560I, V560H, V560Y, V560K, N561P, N561Q, N561G, N561A, V562Y, V562I, V562S, V562M, V567I, V567H, V567N, S583M, E601V, E601F, E601Q, E601W, E605R, E605W, E605K, E605M, E605P, E605Y, E605C, E605H, E605A, E605Q, E605S, E605V, E605I, E605G, D607V, D607Y, D607C, D607N, D607W, D607T, D607A, D607H, D607Q, D607E, D607L, D607K, D607G, S609R, S609W, S609H, S609V, S609Q, S609G, S609T, S609K, S609N, S609Y, L610T, L610I, L610K, L610G, L610A, L610W, L610D, L610Q, L610S, L610F or L610N. 3. MyoƟs

[0073] An advantageous piggyBac-like transposon for modifying the genome of amammalian cell is a Myo s transposon which comprises, from 5’ to 3’, afirst ITR with the withnucleoƟde sequence of SEQ ID NO:70, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence of SEQ ID NO:71. The transposon may further beflanked by a copy of the tetranucleoƟde 5’-TTAA-3’ on each side, or a copy of the tetranucleoƟde 5’- TTAT-3’ on each side, immediately adjacent to the ITRs and distal to the heterologous polynucleoƟde. The transposon may further comprise afirst addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably thefirst ITR, and proximal to the heterologous polynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:72. The transposon may further comprise a second addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably the second ITR, and proximal to the heterologous polynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:73. This transposon may betransposed by a corresponding Myo s transposase comprising a polypepƟde sequence at least90% idenƟcal to the polypepƟde sequence of SEQ ID NO:74. The Myo s transposase mayopƟonally be fused to a heterologous nuclear localizaƟon signal. Preferably the transposase is a hyperacƟve variant of a naturally occurring transposase. Preferably the hyperacƟve variant transposase comprises one or more of the following amino acid changes, relaƟve to the sequence of SEQ ID NO:74: A14V, D475G, P491Q, A561T, T546T, T300A, T294A, A520T, G239S, S5P, S8F, S54N, D9N, D9G, 1345 V, M481V, E11G, K130T, G9G, R427H, S8P, S36G, D10G, S36G. 4. piggyBac

[0074] An advantageous piggyBac-like transposon for modifying the genome of a mammalian cell is a Trichoplusia transposon which comprises, from 5’ to 3’, afirst ITR with the with nucleoƟde sequence of SEQ ID NO:75, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence of SEQ ID NO:76. The transposon may further be flanked by a copy of the tetranucleoƟde 5’-TTAA-3’ on each side, or a copy of the tetranucleoƟde 5’-TTAT-3’ on each side, immediately adjacent to the ITRs and distal to the heterologous polynucleoƟde. The transposon may further comprise afirst addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably thefirst ITR, and proximal to the heterologous polynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:77. The transposon may further comprise a second addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably the second ITR, and proximal to the heterologous polynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:78. This transposon may be transposed by a corresponding Trichoplusia transposase comprising a polypepƟde sequence at least 90% idenƟcal to the polypepƟde sequence of SEQ ID NO:158. The Trichoplusia transposase may opƟonally be fused to a heterologous nuclear localizaƟon signal. Preferably the transposase is a hyperacƟve variant of a naturally occurring transposase. Preferably the hyperacƟve variant transposase comprises one or more of the following amino acid changes, relaƟve to the sequence of SEQ ID NO:158: G2C, S3N, S5P, S8F, S8P, D9N, D9G, G9G, D10G, E11G, T13C, A14V, M14V, T22C, C23T, G25A, A26G, A29G, I30V, A32G, S36G, Q40R, C41T, T43A, A46S, A46T, A51T, S54N, S61R, R65A, R65E, C74T, I82W, R95A, R95E, R97A, R97E, S103P, S103T, A103T, A104C, A106G, T118A, Q118P, R119P, C125A, C125L, K130T, I30A, R135A, R135E, C137T, G138T, T140C, T150C, C153A, R161A, R161E, G161A, G165S, S171E, K176A,K176E, Y177K, Y177H, A179C, F180L, F180I, F180V, M185L, M185V, A187G, R192A, R192E, M194V, K195A, K195E, S103P, G165S, F200W, G201A, G202A, V207P, R208A, R208E, V209F, M226F, L235R, T236A, G239S, V240K, F241L, P243K, N258S, D270N, C277T, A279G, R281G, M282V, M282Q, M282L, M282I, M282A, T294A, L296W, L296Y, L296F, M298V, M298A, M298L, T300A, P311V, P311I, R315K, G316E, T319G, Y327R, Y328V, C340G, C340L, I345V, A351G, A389C, D421H, I426V, R427H, V436I, M456Y, G456A, L470F, D475G, M481V, S486K, P491Q, Q497L, M503I, M503L, N505D, S509G, A520T, N538K, N538R, T546T, V552K, A561T, A570T, N570S, N571S, Q573L, Q591P, Q591R, F594L, A687G, G715A, A880G, A898G, A1033G, T1050C, Tl155C, G1280A, A1356G, A1424G, A1441G, C1472A, G1558A, T1572A, T1638C, T1641C or G1681A. 5. Amyelois

[0075] An advantageous piggyBac-like transposon for modifying the genome of a mammalian cell is an Amyelois transposon which comprises, from 5’ to 3’, afirst ITR with the with nucleoƟde sequence of SEQ ID NO:80, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence of SEQ ID NO:81. The transposon may further be flanked by a copy of the tetranucleoƟde 5’-TTAA-3’ on each side, or a copy of the tetranucleoƟde 5’-TTAT-3’ on each side, immediately adjacent to the ITRs and distal to the heterologous polynucleoƟde. The transposon may further comprise afirst addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably thefirst ITR, and proximal to the heterologous polynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:82. The transposon may further comprise a second addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably the second ITR, and proximal to the heterologous polynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:83. This transposon may be transposed by a corresponding Amyelois transposase comprising a polypepƟde sequence at least 90% idenƟcal to the polypepƟde sequence of SEQ ID NO:84. The Amyelois transposase may opƟonally be fused to a heterologous nuclear localizaƟon signal. Preferably the transposase is a hyperacƟve variant of a naturally occurring transposase. Preferably the hyperacƟve variant transposase comprises one or more of the following amino acid changes, relaƟve to the sequence of SEQ ID NO:84: P65E, P65D, R95S, R95T, V100I, V100L,V100M, L115D, L115E, E116P, H121Q, H121N, K139E, K139D, T159N, T159Q, V166F, V166Y, V166W, G179N, G179Q, W187F, W187Y, P198R, P198K, L203R, L203K, I209L, I209V, I209M, N211R, N211K, E238D, L273I, L273V, L273M, D304K, D304R, I323L, I323M, I323V, Q329G, Q329R, Q329K, T345L, T345I, T345V, T345M, K362R, T366R, T366K, T380S, L408M, L408I, L408V, E413S, E413T, S416E, S416D, I426M, I426L, I426V, S435G, L458M, L458I, L458V, A472S, A472T, V475I, V475L, V475M, N483K, N483R, I491M, I491V, I491L, A529P, K540R, S560K, S560R, T562K, T562R, S563K, S563R. 6. Heliothis

[0076] An advantageous piggyBac-like transposon for modifying the genome of a mammalian cell is a Heliothis transposon which comprises, from 5’ to 3’, afirst ITR with the with nucleoƟde sequence of SEQ ID NO:85, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence of SEQ ID NO:86. The transposon may further beflanked by a copy of the tetranucleoƟde 5’-TTAA-3’ on each side, or a copy of the tetranucleoƟde 5’- TTAT-3’ on each side, immediately adjacent to the ITRs and distal to the heterologous polynucleoƟde. The transposon may further comprise afirst addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably thefirst ITR, and proximal to the heterologous polynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:87. The transposon may further comprise a second addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably the second ITR, and proximal to the heterologous polynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:88. This transposon may be transposed by a corresponding Heliothis transposase comprising a polypepƟde sequence at least 90% idenƟcal to the polypepƟde sequence of SEQ ID NO:89. The Heliothis transposase may opƟonally be fused to a heterologous nuclear localizaƟon signal. Preferably the transposase is a hyperacƟve variant of a naturally occurring transposase. Preferably the hyperacƟve variant transposase comprises one or more of the following amino acid changes, relaƟve to the sequence of SEQ ID NO:89: S41V, S41I, S41L, L43S, L43T, V81E, V81D, D83S, D83T, V85L, V85I, V85M, P125S, P125T, Q126S, Q126T, Q131R, Q131K, Q131T, Q131S, S136V, S136I, S136L, S136M, E140C, E140A, N151Q, K169E, K169D, N212S, I239L, I239V, I239M, H241N, H241Q, T268D, T268E, T297C, M300R, M300K, M305N, M305Q, L312I, C316A, C316M, L321V,L321M, N322T, N322S, P351G, H357R, H357K, H357D, H357E, K360Q, K360N, E379P, K397S, K397T, Y421F, Y421W, V450I, V450L, V450M, Y495F, Y495W, A447N, A447D, A449S, A449V, K476L, V492A, I500M, L585K and T595K. 7. Oryzias

[0077] An advantageous piggyBac-like transposon for modifying the genome of a mammalian cell is an Oryzias transposon which comprises, from 5’ to 3’, afirst ITR with the with nucleoƟde sequence of SEQ ID NO:90, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence of SEQ ID NO:91. The transposon may further beflanked by a copy of the tetranucleoƟde 5’-TTAA-3’ on each side, or a copy of the tetranucleoƟde 5’- TTAT-3’ on each side, immediately adjacent to the ITRs and distal to the heterologous polynucleoƟde. The transposon may further comprise afirst addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably thefirst ITR, and proximal to the heterologous polynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:94. The transposon may further comprise a second addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably the second ITR, and proximal to the heterologous polynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:95. This transposon may be transposed by a corresponding Oryzias transposase comprising a polypepƟde sequence at least 90% idenƟcal to the polypepƟde sequence of SEQ ID NO:96. The Oryzias transposase may opƟonally be fused to a heterologous nuclear localizaƟon signal. Preferably the transposase is a hyperacƟve variant of a naturally occurring transposase. Preferably the hyperacƟve variant transposase comprises one or more of the following amino acid changes, relaƟve to the sequence of SEQ ID NO:96: E22D, A124C, Q131D, Q131E, L138V, L138I, L138M, F149R, D160E, Y164F, Y164W, I167L, I167V, I167M, T202R, T202K, I206L, I206V, I206M, I210L, I210V, I210M, N214D, N214E, V253I, V253L, V253M, V258L, V258I, V258M, A284L, A284I, A284M, A284V, V386I, V386M, V386L, M400L, M400I, M400V, S408E, S408D, L409I, L409V, L409M, V458L, V458M, V458I, V467I, V467M, V467L, L468I, L468V, L468M, A514R, A514K, V515I, V515M, V515L, R548K, D549K, D549R, D550R, D550K, S551K and S551R.8. AgroƟs

[0078] An advantageous piggyBac-like transposon for modifying the genome of amammalian cell is an Agro s transposon which comprises, from 5’ to 3’, afirst ITR with the withnucleoƟde sequence of SEQ ID NO:97, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence of SEQ ID NO:98. The transposon may further beflanked by a copy of the tetranucleoƟde 5’-TTAA-3’ on each side, or a copy of the tetranucleoƟde 5’- TTAT-3’ on each side, immediately adjacent to the ITRs and distal to the heterologous polynucleoƟde. The transposon may further comprise afirst addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably thefirst ITR, and proximal to the heterologous polynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:99. The transposon may further comprise a second addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably the second ITR, and proximal to the heterologous polynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:100. This transposon may betransposed by a corresponding Agro s transposase comprising a polypepƟde sequence at least90% idenƟcal to the polypepƟde sequence of SEQ ID NO:101. The Agro s transposase mayopƟonally be fused to a heterologous nuclear localizaƟon signal. Preferably the transposase is a hyperacƟve variant of a naturally occurring transposase. 9. Helicoverpa

[0079] An advantageous piggyBac-like transposon for modifying the genome of a mammalian cell is a Helicoverpa transposon which comprises, from 5’ to 3’, afirst ITR with the with nucleoƟde sequence of SEQ ID NO:102, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence of SEQ ID NO:103. The transposon may further be flanked by a copy of the tetranucleoƟde 5’-TTAA-3’ on each side, or a copy of the tetranucleoƟde 5’-TTAT-3’ on each side, immediately adjacent to the ITRs and distal to the heterologous polynucleoƟde. The transposon may further comprise afirst addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably thefirst ITR, and proximal to the heterologous polynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:104. The transposon may further comprise a second addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably the second ITR, and proximal to the heterologouspolynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:105. This transposon may be transposed by a corresponding Helicoverpa transposase comprising a polypepƟde sequence at least 90% idenƟcal to the polypepƟde sequence of SEQ ID NO:106. The Helicoverpa transposase may opƟonally be fused to a heterologous nuclear localizaƟon signal. Preferably the transposase is a hyperacƟve variant of a naturally occurring transposase. 10. Sleeping Beauty

[0080] An advantageous Mariner transposon for modifying the genome of a mammalian cell is a Sleeping Beauty transposon, which comprises, from 5’ to 3’, afirst ITR with the with nucleoƟde sequence of SEQ ID NO:107, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence of SEQ ID NO:108. The transposon may further comprise afirst addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably thefirst ITR, and proximal to the heterologous polynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:109. The transposon may further comprise a second addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably the second ITR, and proximal to the heterologous polynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:157. This transposon may be transposed by a corresponding Sleeping Beauty transposase comprising a polypepƟde sequence at least 90% idenƟcal to the polypepƟde sequence of SEQ ID NO:157, including hyperacƟve variants thereof. Preferably the hyperacƟve variant transposase comprises one or more of the following amino acid changes, relaƟve to the sequence of SEQ ID NO:157, K13A, K14R, K30R, K33A, V34A, L64A, E69A, L72A, T83A, M90A, L91A and G95A, II00L, R115H, R143L, R147E, A205K, H207V, K208R, D210E, R214D, K215A, E216V, N217Q, M243A, M243Q, M243H, K252A, V254A, D260A, E267D, S270A, P277A, T314N, G317E. 11. Tc Buster

[0081] An advantageous hAT transposon for modifying the genome of a mammalian cell is a TcBuster transposon, which comprises, from 5’ to 3’, afirst ITR with the with nucleoƟde sequence of SEQ ID NO:112, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence of SEQ ID NO:113. The transposon may further comprise afirst addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably thefirst ITR, and proximalto the heterologous polynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:114. The transposon may further comprise a second addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably the second ITR, and proximal to the heterologous polynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:115. This transposon may be transposed by a corresponding Tc Buster transposase comprising a polypepƟde sequence at least 90% idenƟcal to the polypepƟde sequence of SEQ ID NO:116, including hyperacƟve variants thereof. Preferably the hyperacƟve variant transposase comprises one or more of the following amino acid changes, relaƟve to the sequence of SEQ ID NO:116: D189A, V377T, E469K, K573E, E578L. 12. Tol2

[0082] An advantageous hAT transposon for modifying the genome of a mammalian cell is a Tol2 transposon, which comprises, from 5’ to 3’, afirst ITR with the with nucleoƟde sequence of SEQ ID NO:137, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence of SEQ ID NO:138. The transposon may further comprise afirst addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably thefirst ITR, and proximal to the heterologous polynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:139. The transposon may further comprise a second addiƟonal polynucleoƟde immediately adjacent to one ITR, preferably the second ITR, and proximal to the heterologous polynucleoƟde, whose nucleoƟde sequence is at least 95% idenƟcal to SEQ ID NO:140. This transposon may be transposed by a corresponding Tol2 transposase comprising a polypepƟde sequence at least 90% idenƟcal to the polypepƟde sequence of SEQ ID NO:141, including hyperacƟve variants thereof. 13. Helitron

[0083] An advantageous Helitron transposon for modifying the genome of a mammalian cell is a Helraiser transposon, which comprises, from 5’ to 3’, afirst transposon end with the with nucleoƟde sequence of SEQ ID NO:128, a heterologous polynucleoƟde to be transposed and a second transposon end with nucleoƟde sequence of SEQ ID NO:129. This transposon may be transposed by a corresponding Helitron transposase comprising a polypepƟde sequence atleast 90% idenƟcal to the polypepƟde sequence of SEQ ID NO:130, including hyperacƟve variants thereof.

[0084] A transposase protein can be introduced into a cell as a protein or as a nucleic acid encoding the transposase, for example as a ribonucleic acid, including mRNA or any polynucleoƟde recognized by the translaƟonal machinery of a cell; as DNA, e.g. as extrachromosomal DNA including episomal DNA; as plasmid DNA, or as viral nucleic acid. Furthermore, the nucleic acid encoding the transposase protein can be transfected into a cell as a nucleic acid vector such as a plasmid, or as a gene expression vector, including a viral vector. The nucleic acid can be circular or linear. DNA encoding the transposase protein can be stably inserted into the genome of the cell or into a vector for consƟtuƟve or inducible expression. Where the transposase protein is transfected into the cell or inserted into the vector as DNA, the transposase encoding sequence is preferably operably linked to a heterologous promoter. There are a variety of promoters that could be used including consƟtuƟve promoters, Ɵssue- specific promoters, inducible promoters, species-specific promoters, cell-type specific promoters and the like. All DNA or RNA sequences encoding transposase proteins are expressly contemplated. AlternaƟvely, the transposase may be introduced into the cell directly as protein, for example using cell-penetraƟng pepƟdes (e.g., as described in Ramsey and Flynn, 2015. Pharmacol. Ther.154: 78-86 “Cell-penetraƟng pepƟdes transport therapeuƟcs into cells”); using small molecules including salt plus propanebetaine (e.g., as described in Astolfo et. al., 2015. Cell 161: 674-690); or electroporaƟon (e.g., as described in Morgan and Day, 1995. Methods in Molecular Biology 48: 63-71 “The introducƟon of proteins into mammalian cells by electroporaƟon”). IV. OPEN READING FRAMES

[0085] Open reading frames are segments of nucleic acids encoding a polypepƟde. Examples of polypepƟdes includes therapeuƟc proteins, proteins associated with a disease phenotype, enzymes, and proteins used as selecƟon markers. Open reading frame can also encode a signal pepƟde to permit secreƟon of polypepƟde encoded by the open reading frame. Some examples of therapeuƟc proteins include anƟbodies or their component heavy and light chains, or heavy and light chain fused to one another as a single-chain anƟbody, anƟbodiesengineered to produce heteromeric mulƟ-chain molecules capable of binding more than one target protein (e.g. bispecific or mulƟ-specific anƟbodies having e.g., three or four chains), T cell engagers, chimeric anƟgen receptors combining an anƟgen binding region with transmembrane and T cell signaling domains, Fc fusion proteins, anƟgens from pathogens (e.g. for use in vaccines), anƟcoagulants, blood factors, bone morphogeneƟc proteins, enzymes, growth factor hormones, interferons, interleukins and thrombolyƟcs. Proteins associated with disease are oŌen mutated forms of human proteins. Some examples of such proteins are hunƟngƟn, cysƟc fibrosis trans-membrane regulator, hemoglobin, alpha-1 anƟtrypsin, phenylalanine hydroxylase, beta-hexosaminidase, amyloid precursor protein, alpha-synuclein prion protein, transthyreƟn, crystallin and p53.

[0086] For expression of mulƟmeric polypepƟdes, the component polypepƟdes can be encoded by the same reading frame separated by intervening pepƟdes, such as CHYSEL elements, part of separate opening reading frames in the same transcripƟonal unit separated by IRES elements, or part of separate transcripƟonal units. Separate transcripƟonal units may be provided as part of the same polynucleoƟde (transposon), or they may be provided on separate polynucleoƟdes, opƟonally as part of separate transposons. If provided as separate transposons, both transposons can be transposable by corresponding transposase(s) encoded by an integrated DNA segment(s) within a cell genome.

[0087] A selecƟon marker is a nucleic acid or expression product that allows for selecƟon of a molecule or cell containing the marker oŌen under parƟcular condiƟons. These markers can encode an acƟvity, such as, producƟon of RNA, pepƟde, or protein, or can provide a binding site for RNA, pepƟdes, proteins, inorganic and organic compounds or composiƟons. Examples of selectable markers include: (1) DNA segments that encode products which provide resistance against otherwise toxic compounds (e.g., anƟbioƟcs, such as, puromycin, hygromycin blasƟcidin, zeocin and neomycin); (2) DNA segments that encode products which are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); (3) DNA segments that encode products which suppress the acƟvity of a gene product; (4) DNA segments that encode products which can be readily idenƟfied (e.g., phenotypic markers such as beta-galactosidase, greenfluorescent protein (GFP), and cell surface proteins); (5) DNA segments that bind productswhich are otherwise detrimental to cell survival and / or funcƟon; (6) DNA segments that otherwise inhibit the acƟvity of any of the DNA segments described in Nos.1-5 above (e.g., anƟsense oligonucleoƟdes); (7) DNA segments that bind products that modify a substrate (e.g. restricƟon endonucleases); (8) DNA segments that can be used to isolate a desired molecule (e.g. specific protein binding sites); (9) DNA segments that encode a specific nucleoƟde sequence which can be otherwise non-funcƟonal (e.g., for PCR amplificaƟon of subpopulaƟons of molecules); and / or (10) DNA segments, which when absent, directly or indirectly confer sensiƟvity to parƟcular compounds. Some examples of selecƟon markers include glutamine synthetase, dihydrofolate reductase, blasƟcidin-resistance, neo- resistance, hygromycin- resistance, puromycin-resistance and zeocin-resistance. A polypepƟde to be expressed and a selecƟon marker can be part of the same reading frame separated by CHYSEL elements, part of first and second open reading frames in the same transcripƟonal unit separated by an IRES element orfirst and second open reading frames in separate transcripƟonal units. V. REGULATORY SEQUENCES

[0088] The transcripƟonal units encoding transposases, and those encoding polypepƟdes to be expressed from transposons all include regulatory sequences as well as an open reading frame. Regulatory sequences include any or all of promoters, enhancers, 5’ and 3’ UTRs, introns, RNA export elements, polyadenylaƟon sequences and transcripƟonal terminators. The open reading frame for the transposase element typically does not include a signal pepƟde sequence but may include a heterologous nuclear localizaƟon signal because the transposase acts intracellularly (and within the nucleus). Open reading frames encoding a polypepƟde to be expressed and secreted comprise an open reading frame encoding a signal pepƟde upstream of the polypepƟde to permit its secreƟon. A variety of promoters can be used including consƟtuƟve promoters, Ɵssue-specific promoters, inducible promoters, species- specific promoters, cell-type specific promoters and the like. Examples of consƟtuƟve promoters include promoters from immediate early genes 1, 2 or 3 of cytomegalovirus (CMV) from either human, primate or rodent cells, EF1a (elongaƟon factor 1-alpha from a mammalian gene), SV40, PGK1 (phosphoglycerate kinase), human ubiquiƟn C, and beta acƟn from a mammalian or avian gene. Examples of inducible promoters include tetracycline and cumateinducible systems (see, e.g., Minshull, US20220307056, Kallunki et la., Cells.2019 Jul 30;8(8):796. doi: 10.3390 / cells8080796. PMID: 31366153; PMCID: PMC6721553) and small molecule inducible promoters (see Doshi et al., Crit Rev Biotechnol.2020 Dec; 40(8): 1131– 1150). Enhancers are cis acƟng sequences oŌen 5’ to a promoter but can also be in other locaƟons such as within an intron or 3’ to a coding sequence. Examples of enhancers include an enhancer from immediate early genes 1, 2 or 3 of cytomegalovirus (CMV) from either human, primate or rodent cells, an enhancer from the adenoviral major late protein enhancer or an enhancer from SV40 VI. INTRODUCTION OF NUCLEIC ACIDS INTO CELLS

[0089] Nucleic acids encoding transposase and transposon elements of the invenƟon require introducƟon into cells. Nucleic acids can be transfected into one or more cells by techniques such as parƟcle bombardment, electroporaƟon, microinjecƟon, combining the components with lipid-containing vesicles, such as caƟonic lipid vesicles, DNA condensing reagents (example, calcium phosphate, polyline or polyethyleneimine).

[0090] The nucleic acid encoding a transposase can be introduced into cells as a component of a vector, e.g., a retroviral vector, such as a lenƟviral vector, or a transposon, or as a nucleic acid fragment without vector sequences, or with otherflanking sequence(s). The nucleic acid is typically in the form of a transcripƟonal unit including regulatory sequences operably linked to an open reading frame encoding the transposase to permit expression of the transposase within cells. The regulatory sequences include at least a promoter. The nucleic acid can be configured to integrate by random or site-specific integraƟon, the laƩer of which can be achieved by adding to the nucleic acid homologousflanking sequences to a desired target site within the host cell genome or site specific integraƟon sequences, such as the CRE- LOX system, wherein the host cell comprises a complementary target lox site for integraƟon in its genomic DNA. The nucleic acid may also beflanked with transposon inverted repeats and integrated using a corresponding (second) transposase. Preferably, the nucleic acid encoding thefirst transposase is a component of a second transposon orthogonal to thefirst transposase and is introduced by transposiƟon by a second transposase, which can transpose the orthogonal second transposon. The second transposase can be supplied in the form of a protein, or mRNAor DNA to be expressed. Use of mRNA encoding the second transposase is preferred such that the second transposase is expressed and integrates transposon encoding thefirst transposase into the cell genome, but the second transposase does not persist long in cells aŌer the introducƟon has occurred. AŌer the transposon encoding afirst transposase has been introduced into cells and integrated into their genomes, by transposiƟon, or otherwise, a clonal cell line can be established into which one or more copies of the nucleic acid encoding thefirst transposase has integrated. Such a cell line should be capable of stably maintaining the integrated nucleic acid encoding thefirst transposase through propagaƟon of the cell line and expressing the integrated nucleic acid to produce the encode transposase either consƟtuƟvely or inducibly depending on the expression system used in the integrated nucleic acid. As an alternaƟve to establishing a cell line encoding a transposase, a populaƟon or pool of transfected cells can be used, which includes some or all cells having integrated DNA encoding a transposase albeit in varying locaƟons and copy number.

[0091] Some cell lines include at a plurality of copies of a nucleic acid encoding a transposase (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10). Once a cell line has been created and posiƟon of integrated nucleic acids encoding a transposase mapped, a similar cell can be reproduced by site directed targeƟng of the nucleic acid to those locaƟons.

[0092] AŌer establishing a cell populaƟon with an integrated nucleic acid capable of expressing afirst transposase, the cell populaƟon is transfected with a corresponding transposon. The corresponding transposon includes at least one open reading frame encoding a polypepƟde to be expressed operably linked to a promoter and at least one other regulatory sequence. For a secreted protein, the open reading frame also includes a signal pepƟde to permit secreƟon of the polypepƟde encoded by the open reading frame. The transposon can be introduced as a component of a vector or as nucleic acid lacking vector sequences. The nucleic acid encoding the transposon can be capable of episomal replicaƟon in recipient cells or not. The transposon can be circular or linear.

[0093] AŌer transfecƟon, transposons can remain in the cytoplasm, in the nucleus (e.g., as an episome) or can be integrated into the genome. IntegraƟon of a nucleic acid into the genome of a host cell generally makes it heritable, by subjecƟng it to the same mechanisms thatensure the replicaƟon and division of genomic DNA. Such heritability contributes to achieving high level consistent expression over a longer period that for convenƟonal transient expression. VII. CELL TYPES AND CULTURE MEDIA

[0094] Mammalian cells used in the invenƟon can be e.g., human, primate, or rodent, such as mouse or rat. Examples of such cells include HEK, e.g., HEK293 and HEK293T, CHO, e.g., CHO K1, HeLa, COS, U20S, 3T3, SP2, NS0. The cells iniƟally used for introducƟon of a nucleic acid encoding a transposase can be from a cell line or a heterogenous populaƟon, such an isolate of primary cells. Cells are cultured in a media to support cell propagaƟon and protein expression from a transposon within the cells. Culture media can contain one or more of a carbon source (e.g., glucose), a buffering system, such as HEPES, a pH indicator, serum, metabolites, vitamins and minerals, and one or more selecƟve agents, such as anƟbioƟcs. Examples of media include Eagle’s minimal essenƟal medium, Dulbecco’s modified Eagle’s medium, RPMI-1640, IMDM (Iscoveʹs Modified Dulbeccoʹs Medium) McCoy’s 5A and Ham’s F12. Preferred media are chemically defined and lack serum, such as BalanCD (Irvine ScienƟfic) and Advanced CHO Fed-batch (Sigma).

[0095] All publicaƟons, patents and patent applicaƟons, accession numbers, websites and the like menƟoned in this specificaƟon are incorporated by reference to the same extent as if each individual publicaƟon, patent or patent applicaƟon was so individually denoted. To the extent different content is associated with an accession number or other reference at different Ɵmes, the content in effect as of the effecƟvefiling date of this applicaƟon is meant. The effecƟvefiling date is the date of the earliest priority applicaƟon disclosing the accession number in quesƟon. Unless otherwise apparent from the context any element, embodiment, step, feature or aspect of the invenƟon can be performed in combinaƟon with any other. EXAMPLES Example 1 Enhanced transient protein expression from transposons in cell pools stably expressing a corresponding transposase

[0096] Afirst open reading frame encoding a Xenopus transposase with amino acid sequence SEQ ID NO:16 fused to an N-terminal heterologous nuclear localizaƟon signal wascloned onto afirst Amyelois transposon. Thefirst Amyelois transposon (“523802” with nucleoƟde sequence SEQ ID NO:152) comprised, from 5’ to 3’, afirst ITR with the with nucleoƟde sequence of SEQ ID NO:117, which is an embodiment of SEQ ID NO:80, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence of SEQ ID NO:118 which is an embodiment of SEQ ID NO:81. Thefirst Amyelois transposon was flanked by a copy of the tetranucleoƟde 5’-TTAA-3’ on each side, immediately adjacent to the ITRs and distal to the heterologous polynucleoƟde. Thefirst Amyelois transposon further comprised afirst addiƟonal polynucleoƟde immediately adjacent to thefirst ITR, and proximal to the heterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:82. Thefirst Amyelois transposon further comprised a second addiƟonal polynucleoƟde immediately adjacent to the second ITR, and proximal to the heterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:83. Thefirst Amyelois transposon further comprised a second open reading frame encoding blasƟcidin deaminase, operably linked to regulatory elements such that the blasƟcidin deaminase was consƟtuƟvely expressible in a mammalian cell. Thefirst open reading frame encoding the Xenopus transposase was operably linked to a PGK promoter with nucleoƟde sequence SEQ ID NO:119 and a polyadenylaƟon signal with nucleoƟde sequence SEQ ID NO:120 such that it was consƟtuƟvely expressible in a mammalian cell.

[0097] Afirst open reading frame encoding a Xenopus transposase with amino acid sequence SEQ ID NO:16 fused to an N-terminal heterologous nuclear localizaƟon signal was cloned onto a second Amyelois transposon. The second Amyelois transposon (“523809” with nucleoƟde sequence SEQ ID NO:151) comprised, from 5’ to 3’, afirst ITR with the with nucleoƟde sequence of SEQ ID NO:117, which is an embodiment of SEQ ID NO:80, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence of SEQ ID NO:118 which is an embodiment of SEQ ID NO:81. The second Amyelois transposon was flanked by a copy of the tetranucleoƟde 5’-TTAA-3’ on each side, immediately adjacent to the ITRs and distal to the heterologous polynucleoƟde. The second Amyelois transposon further comprised afirst addiƟonal polynucleoƟde immediately adjacent to thefirst ITR, and proximal to the heterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:82. The second Amyelois transposon further comprised a second addiƟonal polynucleoƟde immediatelyadjacent to the second ITR, and proximal to the heterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:83. The second Amyelois transposon further comprised a second open reading frame encoding blasƟcidin deaminase, operably linked to regulatory elements such that the blasƟcidin deaminase was consƟtuƟvely expressible in a mammalian cell. Thefirst open reading frame encoding the Xenopus transposase was operably linked to an EF1 promoter with nucleoƟde sequence SEQ ID NO:121 and an EF1 intron with nucleoƟde sequence SEQ ID NO:122 and a polyadenylaƟon signal with nucleoƟde sequence SEQ ID NO:120 such that it was consƟtuƟvely expressible in a mammalian cell.

[0098] Afirst open reading frame encoding a Xenopus transposase with amino acid sequence SEQ ID NO:16 fused to an N-terminal heterologous nuclear localizaƟon signal was cloned onto a third Amyelois transposon. The third Amyelois transposon (“523810” with nucleoƟde sequence SEQ ID NO:150) comprised, from 5’ to 3’, afirst ITR with the with nucleoƟde sequence of SEQ ID NO:117, which is an embodiment of SEQ ID NO:80, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence of SEQ ID NO:118 which is an embodiment of SEQ ID NO:81. The third Amyelois transposon was flanked by a copy of the tetranucleoƟde 5’-TTAA-3’ on each side, immediately adjacent to the ITRs and distal to the heterologous polynucleoƟde. The third Amyelois transposon further comprised afirst addiƟonal polynucleoƟde immediately adjacent to thefirst ITR, and proximal to the heterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:82. The third Amyelois transposon further comprised a second addiƟonal polynucleoƟde immediately adjacent to the second ITR, and proximal to the heterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:83. The third Amyelois transposon further comprised a second open reading frame encoding blasƟcidin deaminase, operably linked to regulatory elements such that the blasƟcidin deaminase was consƟtuƟvely expressible in a mammalian cell. Thefirst open reading frame encoding the Xenopus transposase was operably linked to a doxycycline-inducible promoter with nucleoƟde sequence SEQ ID NO:123 and a polyadenylaƟon signal with nucleoƟde sequence SEQ ID NO:120. The third Amyelois transposon further comprised a third open reading frame encoding a modified tet-repressor fused to a VP16 transcripƟonal acƟvator with amino acid sequence SEQ ID NO:124; the third open reading frame was operably linked toan EF1 promoter with nucleoƟde sequence SEQ ID NO:125, and EF1 intron with nucleoƟde sequence SEQ ID NO:126 and a BGH polyadenylaƟon signal with nucleoƟde sequence SEQ ID NO:120 such that the third open reading frame was consƟtuƟvely expressible in a mammalian cell. The Xenopus transposase was inducibly expressible in mammalian cells in response to doxycycline from the third Amyelois transposon.

[0099] Thefirst, second and third Amyelois transposons were each separately introduced into afirst, second and third pool of CHO-K1 cells respecƟvely, together with mRNA encoding a corresponding Amyelois transposase fused to a heterologous nuclear localizaƟon signal with amino acid sequence of SEQ ID NO:127. Intofive million cells, 25 µg of transposon and 3 µg of transposase mRNA were electroporated; the cells were then cultured in Advanced CHO Fed-batch media (Sigma) in the presence of 2 µg / ml blasƟcidin unƟl the cells recovered to >95% viability at which Ɵme banks of cells were cryopreserved over liquid nitrogen. Prior to subsequent lipid-based transfecƟon, cells were adapted to BalanCD media (Irvine ScienƟfic).

[0100] Afirst Xenopus transposon was constructed. The transposon was designed to be transposable by the Xenopus transposase expressible from thefirst, second and third Amyelois transposons respecƟvely integrated into thefirst, second and third CHO-K1 pools. Thefirst Xenopus transposon (“532006”) comprised, from 5’ to 3’, afirst ITR with the with nucleoƟde sequence of SEQ ID NO:3, which is an embodiment of SEQ ID NO:1, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence of SEQ ID NO:4 which is an embodiment of SEQ ID NO:2. Thefirst Xenopus transposon wasflanked by a copy of the tetranucleoƟde 5’-TTAA-3’ on each side, immediately adjacent to the ITRs and distal to the heterologous polynucleoƟde. Thefirst Xenopus transposon further comprised afirst addiƟonal polynucleoƟde immediately adjacent to thefirst ITR, and proximal to the heterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:131, which is 95% idenƟcal to SEQ ID NO:6. Thefirst Xenopus transposon further comprised a second addiƟonal polynucleoƟde immediately adjacent to the second ITR, and proximal to the heterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:8. Thefirst Xenopus transposon further comprised afirst transcripƟonal unit comprising afirst open reading frame encoding puromycin-N- acetyltransferase, operably linked to regulatory elements (including an HSV-TK promoter withnucleoƟde sequence SEQ ID NO:145, and an SV40 polyadenylaƟon signal with nucleoƟde sequence SEQ ID NO:148) such that the puromycin-N-acetyltransferase was consƟtuƟvely expressible in a mammalian cell. Thefirst Xenopus transposon further comprised a second transcripƟonal unit comprising an open reading frame encoding a mature anƟbody light chain with amino acid sequence SEQ ID NO:132 fused to an N-terminal secreƟon signal sequence and an open reading frame encoding a mature anƟbody heavy chain with amino acid sequence SEQ ID NO:133 fused to an N-terminal secreƟon signal sequence. The two open reading frames were operably linked by an internal ribosome entry site with nucleic acid sequence SEQ ID NO:134, with the light chain open reading frame to the 5’ of the IRES and the heavy chain open reading frame to the 3’ of the IRES. The second transcripƟonal unit was operably linked to a hybrid mouse-human CMV enhancer-promoter with nucleoƟde sequence SEQ ID NO:135 and a globin polyadenylaƟon sequence. Thefirst Xenopus transposon had nucleoƟde sequence SEQ ID NO:136.

[0101] Thefirst Xenopus transposon was transfected into thefirst, second and third CHO-K1 pools whose genomes comprised respecƟvely thefirst, second and third Amyelois transposons. Thefirst Xenopus transposon was also transfected into the parental CHO-K1 host cell line, either alone or as a co-transfecƟon with afirst transient transposase expression plasmid comprising an open reading frame encoding a Xenopus transposase with amino acid sequence SEQ ID NO:16 fused to an N-terminal heterologous nuclear localizaƟon signal, wherein the open reading frame was operably linked to a human CMV promoter and a polyadenylaƟon signal such that the transposase was expressible in a CHO-K1 cell. Each transfecƟon comprised 31 µg of thefirst Xenopus transposon. One transfecƟon into the parental CHO-K1 host cell line further comprised 8 µg of thefirst transient transposase expression plasmid. All transfecƟons were performed using 25 ml of cells at 3 million cells per milliliter using FectoPro (Polyplus / Sartorius) according to the manufacturer’s recommended procedures. Cultures were grown at 37ºC in BalanCD media (Irvine ScienƟfic) in 125 mlflasks at 120 shaker speed and 5% CO2. AŌer 24 hours, cells were fed by addiƟon of transfectory supplement (Irvine ScienƟfic) Fectoboost (Polyplus / Sartorius) anƟ-clump (Thermo Fisher), all according to the manufacturers’ specificaƟons, and 145 µl of 50% glucose, and the temperaturewas reduced to 32ºC. ConcentraƟons of secreted anƟbody were measured at 11 and 14 days post-transfecƟon in the culture supernatant using an Octet (Sartorius), these values are shown in Table 1 columns C and D respecƟvely. AŌer 14 days, secreted anƟbody was purified the culture supernatant, the amount of anƟbody quanƟfied and this value was used to calculate the Ɵter of purifiable anƟbody present in the supernatant, this value is shown in Table 1 column E.

[0102] Table 1 shows that transfecƟon of thefirst Xenopus transposon into the parental CHO-K1 cell line resulted in anƟbody Ɵters (as measured by Octet) of around 134 mg / L 11 days post-transfecƟon and 153 mg / L 14 days post-transfecƟon. When thefirst transient transposase expression plasmid was co-transfected with thefirst Xenopus transposon into the parental CHO- K1 cell line, Ɵters were substanƟally increased to around 280 mg / L 11 days post-transfecƟon and 267 mg / L 14 days post-transfecƟon. This shows that transient protein expression from a transposon can be increased by transiently expressing a corresponding transposase in the cell. By integraƟng the transposon into the host cell genome, transposon loss is reduced and expression is boosted. TransfecƟon of thefirst Xenopus transposon into thefirst, second and third CHO-K1 pools resulted in anƟbody Ɵters (as measured by Octet) from thefirst CHO-K1 pool of around 594 mg / L 11 days post-transfecƟon and 511 mg / L 14 days post-transfecƟon, from the second CHO-K1 pool of around 589 mg / L 11 days post-transfecƟon and 501 mg / L 14 days post-transfecƟon and from the third CHO-K1 pool of around 593 mg / L 11 days post- transfecƟon and 498 mg / L 14 days post-transfecƟon. It is not surprising that the anƟbody Ɵter did not increase between days 11 and 14, because cells had not been fed since 24 hours post- transfecƟon, and by day 11 viability for all pools was around 50%. The Ɵters of purifiable anƟbody (Table 1 column E) follow the same paƩern: more than twice as much anƟbody could be purified from the culture supernatants of cells where thefirst Xenopus transposon was introduced into cells whose genomes comprised thefirst, second or third Amyelois transposon (Table 1 rows 3-5, column E), than could be purified from the supernatant of cells where the first transient transposase expression plasmid was co-transfected with thefirst Xenopus transposon (Table 1 row 2, column E). The 14 day Ɵters from each of the pools whose genomes comprised transcripƟonal units expressing a Xenopus transposase, the transposase being capable of transposing thefirst Xenopus transposon into the CHO genome, were all aboutdouble the Ɵters obtained from a CHO-K1 cell line whose genomes did not comprise transcripƟonal units expressing a Xenopus transposase. Even though the expression of the Xenopus transposase was not induced from the doxycycline-inducible third Amyelois transposon, this inducible system is leaky and expresses some transposase in the absence of doxycycline. We conclude that consƟtuƟve expression of a transposase in a CHO-K1 cell enhances protein expression from a subsequently introduced corresponding transposon, and that this effect is more beneficial than the enhancement of expression obtained when a transcripƟonal unit encoding the corresponding transposase is introduced into the CHO-K1 cell at the same Ɵme as the corresponding transposon. Example 2 Enhanced transient protein expression from drug-selected transposons in cell pools stably expressing a corresponding transposase

[0103] Expression of an anƟbody from pools of CHO-K1 cells whose genomes comprised thefirst or second Amyelois transposon as described for Example 1, were compared for three different polynucleoƟdes encoding the anƟbody. Expression was compared both with and without puromycin selecƟon.

[0104] Thefirst anƟbody-encoding polynucleoƟde (“526014”) comprised afirst transcripƟonal unit comprising afirst open reading frame encoding puromycin-N- acetyltransferase, operably linked to an HSV promoter with nucleoƟde sequence SEQ ID NO:145 and an SV40 polyadenylaƟon signal with nucleoƟde sequence SEQ ID NO:148 such that the puromycin-N-acetyltransferase was consƟtuƟvely expressible in a mammalian cell. Thefirst anƟbody-encoding polynucleoƟde further comprised a second transcripƟonal unit comprising an open reading frame encoding a mature anƟbody light chain with amino acid sequence SEQ ID NO:132 fused to an N-terminal secreƟon signal sequence and an open reading frame encoding a mature anƟbody heavy chain with amino acid sequence SEQ ID NO:133 fused to an N-terminal secreƟon signal sequence. The two open reading frames were operably linked by an internal ribosome entry site with nucleic acid sequence SEQ ID NO:134, with the light chain open reading frame to the 5’ of the IRES and the heavy chain open reading frame to the 3’ of the IRES. The second transcripƟonal unit was operably linked to a hybrid mouse-human CMV enhancer-promoter with nucleoƟde sequence SEQ ID NO:135 and a globin polyadenylaƟonsequence. Thefirst anƟbody-encoding polynucleoƟde further comprised an open reading frame encoding the Epstein Barr virus (EBV) nuclear anƟgen (EBNA) with amino acid sequence SEQ ID NO:144 operably linked to a promoter and polyadenylaƟon sequence such that the EBNA was expressible in the CHO-K1 host. Thefirst anƟbody-encoding polynucleoƟde further comprised an EBV origin of replicaƟon (OriP) with nucleoƟde sequence SEQ ID NO:143. The EBV OriP supports episomal replicaƟon of DNA molecules in the presence of EBNA. The presence of the EBV OriP and expressible open reading frame encoding EBNA is expected to improve the persistence of thefirst anƟbody-expressing polynucleoƟde in the CHO-K1 host. Thefirst anƟbody-encoding polynucleoƟde had nucleoƟde sequence SEQ ID NO:142.

[0105] The second anƟbody-encoding polynucleoƟde was thefirst Xenopus transposon (532006) described in Example 1. The third anƟbody-encoding polynucleoƟde was a second Xenopus transposon (“535624”) which was idenƟcal to thefirst Xenopus transposon (532006) except for the regulatory elements operably linked to the open reading frame encoding puromycin-N-acetyltransferase, where the HSV-TK promoter for 532006 was replaced with a PGK promoter with nucleoƟde sequence SEQ ID NO:146 and the SV40 polyadenylaƟon signal was replaced with a human globin polyadenylaƟon signal with nucleoƟde sequence SEQ ID NO:149. The second Xenopus transposon (535624) had nucleoƟde sequence SEQ ID NO:142.

[0106] The three anƟbody-encoding polynucleoƟdes were each independently transfected into thefirst and second CHO-K1 pools whose genomes comprised respecƟvely the first and second Amyelois transposons, prepared as described in Example 1. Each transfecƟon comprised 31 µg of the respecƟve anƟbody-encoding polynucleoƟde. All transfecƟons were performed using 25 ml of cells at 3 million cells per milliliter using FectoPro (Polyplus / Sartorius) according to the manufacturer’s recommended procedures. Cultures were grown at 37ºC in BalanCD media (Irvine ScienƟfic) in 125 mlflasks at a shaker speed of 120 rpm in the presence of 5% CO2. AŌer 24 hours, cells were diluted to 1,250,000 cells / ml and puromycin was added to afinal concentraƟon of 0, 5, 10 or 15 µg / ml. Four days post-transfecƟon, cells were fed by addiƟon of transfectory supplement (Irvine ScienƟfic), Fectoboost (Polyplus / Sartorius) and anƟ- clump (Thermo Fisher), all according to the manufacturers’ specificaƟons, and 145 µl of 50% glucose, and the temperature was reduced to 32ºC. ConcentraƟons of secreted anƟbody weremeasured for each culture at various Ɵmes post-transfecƟon in the culture supernatant using an Octet (Sartorius); cell viability and viable cell density were measured using a Vi-CELL; these values are shown in Table 2.

[0107] Table 2 rows 1-4 and 13-16 show the measurements for cells transfected with the first anƟbody expressing polynucleoƟde (526014). This polynucleoƟde comprises an open reading frame encoding puromycin acetyl transferase and EBV oriP but does not comprise transposon ends. The polynucleoƟde can therefore confer resistance to puromycin and can be amplified episomally by the EBNA that it encodes, but it is not a substrate for the Xenopus transposase expressed by either of the host cell pools. As seen in Table 2 row 1, transfecƟon of 526014 into the CHO-K1 pool whose genomes comprise thefirst Amyelois transposon (523802) resulted in a transfected cell pool with 6.35 million cells per ml at day 4 in the absence of puromycin selecƟon. Viable cell numbers climbed to 9.66 million on day 8, fell slightly to 8.86 million on day 11 before falling to around 4 million cells per ml on days 15 and 20. Cell viability remained at around 95% unƟl day 11, but by day 15 viability had fallen to around 38%. These culture dynamics reflect a cell populaƟon that has exhausted its food supply between day 11 and day 15. The Ɵter of anƟbody in the culture was around 100 mg / L at day 8 and rose to 168 mg / L on day 11 and a liƩle over 200 mg / L on days 15 and 20. The paƩern of viability, cell density and anƟbody Ɵter was very similar when polynucleoƟde 526014 was transfected into the CHO-K1 pool whose genomes comprise the second Amyelois transposon (523809), and incubated in the absence of puromycin, as shown in Table 2 row 13. Puromycin inhibits protein synthesis and thus prevents cell growth in cells that are not expressing puromycin acetyl transferase. Thefirst anƟbody expressing polynucleoƟde (526014) comprises an open reading frame encoding puromycin acetyl transferase expressible in the CHO-K1 host. When puromycin was added to the culture to 5 µg / ml at 24 hours post-transfecƟon, the viable cell density was significantly lower than in the corresponding no puromycin culture when measured on days 4, 8 and 11 because protein synthesis and growth were inhibited in the cells that had not received thefirst anƟbody-expressing polynucleoƟde (compare Table 2 row 2 with row 1, and row 14 with row 13). Cell viabiliƟes at these days post-transfecƟon were also lower than the corresponding no puromycin cultures, reflecƟng the killing of cells not expressing puromycinacetyl transferase (compare Table 2 row 2 with row 1, and row 14 with row 13). At days 15 and 20 post-transfecƟon, cell densiƟes and viabiliƟes without puromycin fell substanƟally when the cultures ran out of nutrients. However, in the cultures containing 5 µg / ml puromycin, cell densiƟes did not fall, and cell viability remained high throughout the 20 days of the culture. This is because these puromycin-containing cultures had not exhausted their nutrients; in parƟcular they had not used the nutrients to fuel the growth of non-producer cells that had not received thefirst anƟbody expressing polynucleoƟde. Because puromycin selected for cells containing thefirst anƟbody expressing polynucleoƟde, anƟbody Ɵters at each Ɵme point in the presence of 5 µg / ml puromycin were approximately double the Ɵters seen in the absence of puromycin for both CHO-K1 host pools (compare Table 2 row 2 with row 1, and row 14 with row 13). The addiƟon of higher concentraƟons of puromycin (10 or 15 µg / ml) to the culture did not result in significantly higher anƟbody Ɵters, but it did reduce both the viable cell density and the cell viability at all Ɵme points (compare Table 2 row 2 with rows 3-4 and Table 2 row 14 with rows 15-16). This can be explained as follows. Higher expression of puromycin acetyl transferase is required to overcome higher concentraƟons of puromycin. The viable cell density is a reflecƟon of the number of cells that contain enough copies of thefirst anƟbody-encoding polynucleoƟde to confer resistance to puromycin. Although more copies of thefirst anƟbody-expressing polynucleoƟde in a cell lead to higher expression levels for the encoded anƟbody, the fracƟon of cells possessing this number of copies is smaller than the number of cells carrying enough copies to confer resistance to 5 µg / ml puromycin. Higher concentraƟons of puromycin thus lead to smaller numbers of higher producing cells.

[0108] Table 2 rows 5-8 and 17-20 show the measurements for cells transfected with the second anƟbody expressing polynucleoƟde (532006). This polynucleoƟde comprises an open reading frame encoding puromycin acetyl transferase and Xenopus transposon ends, making it a substrate for the Xenopus transposase expressed by either of the host cell pools. TransfecƟon of 532006 into the CHO-K1 pool whose genomes comprise thefirst Amyelois transposon (523802) resulted in a transfected cell pool with 4.68 million cells per ml at day 4 in the absence of puromycin selecƟon (Table 2 row 5). Viable cell numbers climbed to 6.08 million on day 8, fell slightly to 5.56 million on day 11, 5.91 million on days 15 before falling to 4.23 million cells perml on day 20. Cell viability remained above 90% unƟl day 15, by day 20 viability had fallen to around 50%. These culture dynamics reflect a cell populaƟon that has begun to exhaust its food supply between day 15 and day 20. The Ɵter of anƟbody in the culture was around 321 mg / L at day 8 and rose to 431 mg / L on day 11, a liƩle over 700 mg / L on day 15 and 647 mg / L on day 20. The paƩern of viability, cell density and anƟbody Ɵter was similar when polynucleoƟde 532006 was transfected into the CHO-K1 pool whose genomes comprise the second Amyelois transposon (523809), although cell densiƟes were a liƩle lower and anƟbody Ɵters were a liƩle higher, as shown in Table 2 row 17.

[0109] The anƟbody Ɵters from either of the CHO-K1 cell pools transfected with the second anƟbody-expressing polynucleoƟde, which comprises transposon ends transposable by the transposase expressed within the CHO-K1 pools, were approximately three Ɵmes higher than the Ɵters from the same CHO-K1 cell pools transfected with thefirst anƟbody-expressing polynucleoƟde which does not comprise such transposon ends, when no puromycin was present in the cultures. This increase in anƟbody Ɵters was seen both for the CHO-K1 pool comprising thefirst Amyelois transposon (523802), in which the open reading frame encoding the Xenopus transposase is operably linked to a PGK promoter (compare Table 2 rows 1 and 5), and for the CHO-K1 pool comprising the second Amyelois transposon (523809), in which the open reading frame encoding the Xenopus transposase is operably linked to an EF1 promoter (compare Table 2 rows 13 and 17), in each case in the absence of puromycin. An advantageous method for expressing a polypepƟde in a CHO-K1 host cell comprises (i) incorporaƟng an open reading frame encoding the polypepƟde and its operably linked regulatory elements that make the polypepƟde expressible in the CHO-K1 host into a transposon such that the transcripƟonal unit for expression of the polypepƟde is transposable by a corresponding transposase, and (ii) transfecƟng the transposon into a CHO-K1 host stably expressing the corresponding transposase.

[0110] The anƟbody Ɵters from either of the CHO-K1 cell pools transfected with the second anƟbody-expressing polynucleoƟde, which comprises transposon ends transposable by the transposase expressed within the CHO-K1 pools, were also higher than the Ɵters from the same CHO-K1 cell pools transfected with thefirst anƟbody-expressing polynucleoƟde whichdoes not comprise such transposon ends, when 5 µg / ml puromycin was added to the cultures aŌer 24 hours. AnƟbody Ɵters from the CHO-K1 cell pool comprising thefirst Amyelois transposon (523802) transfected with the second anƟbody-expressing polynucleoƟde, which comprises transposon ends transposable by the transposase expressed within the CHO-K1 pool, were between 1.6 and 1.9 Ɵmes higher than the same CHO-K1 pool transfected with thefirst anƟbody-expressing polynucleoƟde which does not comprise such transposon ends when 5 µg / ml puromycin was added to the cultures aŌer 24 hours (compare Table 2 rows 2 and 6). For the CHO-K1 pool comprising the second Amyelois transposon (523809), anƟbody Ɵters were between 1.9 and 2.5 Ɵmes greater when the host was transfected with the second anƟbody- expressing polynucleoƟde, which comprises transposon ends transposable by the transposase expressed within the CHO-K1 pool, compared with the same CHO-K1 pool transfected with the first anƟbody-expressing polynucleoƟde which does not comprise such transposon ends when 5 µg / ml puromycin was added to the cultures aŌer 24 hours (compare Table 2 rows 14 and 18). An advantageous method for expressing a polypepƟde in a CHO-K1 host cell comprises (i) incorporaƟng an open reading frame encoding the polypepƟde and its operably linked regulatory elements that make the polypepƟde expressible in the CHO-K1 host into a transposon such that the transcripƟonal unit for expression of the polypepƟde is transposable by a corresponding transposase, wherein the transposon further comprises a selectable marker, (ii) transfecƟng the transposon into a CHO-K1 host stably expressing the corresponding transposase, and (iii) culturing the cells under condiƟons that favor cells expressing the selectable marker. The addiƟon of higher concentraƟons of puromycin (10 or 15 µg / ml) to the cultures did not result in significantly higher anƟbody Ɵters, but it did reduce both the viable cell density and the cell viability at all Ɵme points (compare Table 2 row 6 with rows 7-8 and Table 2 row 18 with rows 19-20). As described above, higher concentraƟons of puromycin lead to smaller numbers of higher producing cells.

[0111] Similar results were obtained with the third anƟbody-expressing polynucleoƟde. In the absence of puromycin from the culture, anƟbody Ɵters from CHO-K1 cell pools transfected with the third anƟbody-expressing polynucleoƟde (535624), which comprises transposon ends transposable by the transposase expressed within the CHO-K1 pools, werebetween 2.2 and 2.7 Ɵmes higher than from CHO-K1 cell pools transfected with thefirst anƟbody-expressing polynucleoƟde which does not comprise such transposon ends for the CHO-K1 pool comprising thefirst Amyelois transposon (523802), (compare Table 2 rows 1 and 9), and between 2.9 and 4.6 Ɵmes higher for the CHO-K1 pool comprising the second Amyelois transposon (523809), (compare Table 2 rows 13 and 21). In the presence of 5 µg / ml puromycin in the culture, anƟbody Ɵters from the CHO-K1 cell pool comprising thefirst Amyelois transposon (523802) transfected with the third anƟbody-expressing polynucleoƟde, which comprises transposon ends transposable by the transposase expressed within the CHO-K1 pool, were between 1.3 and 1.7 Ɵmes higher than the same CHO-K1 pool transfected with thefirst anƟbody-expressing polynucleoƟde which does not comprise such transposon ends (compare Table 2 rows 2 and 10). For the CHO-K1 pool comprising the second Amyelois transposon (523809), anƟbody Ɵters were between 1.5 and 2.6 Ɵmes greater when the host was transfected with the third anƟbody-expressing polynucleoƟde, which comprises transposon ends transposable by the transposase expressed within the CHO-K1 pool, compared with the same CHO-K1 pool transfected with thefirst anƟbody-expressing polynucleoƟde which does not comprise such transposon ends (compare Table 2 rows 14 and 22).

[0112] Although thefirst anƟbody-expressing polynucleoƟde, which lacked transposon ends, yielded 2-3-fold higher Ɵters at all Ɵme points measured up to 20 days post-transfecƟon when 5 µg / ml puromycin was added to the culture than when no puromycin was added, the same paƩern was not seen for the second and third anƟbody-expressing polynucleoƟdes, which comprised transposon ends. For these transposon polynucleoƟdes, puromycin showed liƩle benefit unƟl 20 days post-transfecƟon. This is because stable expression of the transposase within the cells results in efficient integraƟon of anƟbody-encoding transposons into the cell genome. Once in the cell genome, the transposons are stably replicated. In contrast, non- integrated polynucleoƟdes, even when they comprise viral replicaƟon elements, have a high tendency to be lost from cells, a tendency that is counter-acted by the presence of puromycin.

[0113] The Ɵter of anƟbody expressed when polynucleoƟdes comprising anƟbody- encoding open reading frames were introduced into a pool of cells cell stably expressing a transposase was between 1.5- and 5-fold higher when the polynucleoƟde comprisedcorresponding transposon ends such that the anƟbody-encoding open reading frames and their operably linked regulatory elements were transposable by the transposase than when the polynucleoƟde lacked such transposon ends. Example 3: Enhanced transient protein expression from transposons in stable cell pools expressing 4 different transposases

[0114] We wished to determine how general the advantage for expression observed in Examples 1 and 2 would be, when different transposases were expressed in a mammalian cell populaƟon, and a corresponding transposon transposable by the transposase was introduced into the populaƟon, wherein the corresponding transposon comprised an open reading frame encoding an expressible polypepƟde operably linked to regulatory sequences such that the expressible protein was expressible in cells of the populaƟon into which the transposon was introduced. To do this we compared expression of the expressible polypepƟde when the open reading frame encoding an expressible polypepƟde was introduced on a corresponding transposon or a non-corresponding transposon, relaƟve to the transposase expressed within the populaƟon of cells.

[0115] Four transposase-expressing transposons were constructed by independently cloning open reading frames encoding 4 different transposases (a Xenopus piggyBac-like transposase with amino acid sequence SEQ ID NO:16, an Oryzias piggyBac-like transposase with amino acid sequence SEQ ID NO:96, a Trichoplusia piggyBac transposase with amino acid sequence SEQ ID NO:158 and an hAT transposase Tc Buster with amino acid sequence SEQ ID NO: 116) onto Amyelois transposons. Each transposon comprised, from 5’ to 3’, afirst ITR with the with nucleoƟde sequence of SEQ ID NO:117, which is an embodiment of SEQ ID NO:80, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence of SEQ ID NO:118 which is an embodiment of SEQ ID NO:81. Each transposon wasflanked by a copy of the tetranucleoƟde 5’-TTAA-3’ on each side, immediately adjacent to the ITRs and distal to the heterologous polynucleoƟde. Each transposon further comprised afirst addiƟonal polynucleoƟde immediately adjacent to thefirst ITR, and proximal to the heterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:82. Each transposon further comprised a second addiƟonal polynucleoƟde immediately adjacent to the second ITR, and proximal to theheterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:83. Each transposon further comprised a second open reading frame encoding blasƟcidin deaminase, operably linked to regulatory elements such that the blasƟcidin deaminase was consƟtuƟvely expressible in a mammalian cell. The open reading frame encoding each transposase was operably linked to a PGK promoter with nucleoƟde sequence SEQ ID NO:119 and a polyadenylaƟon signal with nucleoƟde sequence SEQ ID NO:120 such that it was consƟtuƟvely expressible in a mammalian cell. The transposon nucleoƟde sequences were SEQ ID NOs:152, 153, 156 and 155 respecƟvely.

[0116] Each transposase-expressing Amyelois transposon was separately introduced into a pool of CHO-K1 cells, together with mRNA encoding a corresponding Amyelois transposase fused to a heterologous nuclear localizaƟon signal with amino acid sequence of SEQ ID NO:127. Intofive million cells, 25 µg of transposon and 3 µg of transposase mRNA were electroporated; the cells were then cultured in Advanced CHO Fed-batch media (Sigma) in the presence of 2 µg / ml blasƟcidin unƟl the cells recovered to >95% viability at which Ɵme banks of cells were cryopreserved over liquid nitrogen. Prior to subsequent lipid-based transfecƟon, cells were adapted to BalanCD media (Irvine ScienƟfic).

[0117] Four different expression transposons, for expression of an anƟbody (the expressible polypepƟde), were constructed, with one transposon corresponding to the transposase in each CHO-K1 cell pool comprising a different transposase-expressing Amyelois transposon. Each transposon comprised afirst transcripƟonal unit comprising afirst open reading frame encoding puromycin-N-acetyltransferase, operably linked to regulatory elements (including an HSV-TK promoter with nucleoƟde sequence SEQ ID NO:145, and an SV40 polyadenylaƟon signal with nucleoƟde sequence SEQ ID NO:148) such that the puromycin-N- acetyltransferase was consƟtuƟvely expressible in a mammalian cell. Each transposon further comprised a second transcripƟonal unit comprising an open reading frame encoding a mature anƟbody light chain with amino acid sequence SEQ ID NO:132 fused to an N-terminal secreƟon signal sequence and an open reading frame encoding a mature anƟbody heavy chain with amino acid sequence SEQ ID NO:133 fused to an N-terminal secreƟon signal sequence. The two open reading frames were operably linked by an internal ribosome entry site with nucleic acid sequence SEQ ID NO:134, with the light chain open reading frame to the 5’ of the IRES and theheavy chain open reading frame to the 3’ of the IRES. The second transcripƟonal unit was operably linked to a hybrid mouse-human CMV enhancer-promoter with nucleoƟde sequence SEQ ID NO:135 and a globin polyadenylaƟon sequence.

[0118] The Xenopus expression transposon (“532006”) was as described in Example 1.

[0119] The Oryzias expression transposon comprised, from 5’ to 3’, afirst ITR with the with nucleoƟde sequence of SEQ ID NO:92, which is an embodiment of SEQ ID NO:90, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence of SEQ ID NO:93 which is an embodiment of SEQ ID NO:91. The Oryzias expression transposon wasflanked by a copy of the tetranucleoƟde 5’-TTAA-3’ on each side, immediately adjacent to the ITRs and distal to the heterologous polynucleoƟde. The Oryzias expression transposon further comprised afirst addiƟonal polynucleoƟde immediately adjacent to thefirst ITR, and proximal to the heterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:94. The Oryzias transposon further comprised a second addiƟonal polynucleoƟde immediately adjacent to the second ITR, and proximal to the heterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:95.

[0120] The Trichoplusia piggyBac expression transposon comprised, from 5’ to 3’, afirst ITR with the with nucleoƟde sequence of SEQ ID NO:75, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence of SEQ ID NO:76. The Trichoplusia piggyBac expression transposon wasflanked by a copy of the tetranucleoƟde 5’-TTAA-3’ on each side, immediately adjacent to the ITRs and distal to the heterologous polynucleoƟde. The Trichoplusia piggyBac expression transposon further comprised afirst addiƟonal polynucleoƟde immediately adjacent to thefirst ITR, and proximal to the heterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:77. The Trichoplusia piggyBac transposon further comprised a second addiƟonal polynucleoƟde immediately adjacent to the second ITR, and proximal to the heterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:78. The Tc Buster expression transposon comprised, from 5’ to 3’, afirst ITR with the with nucleoƟde sequence of SEQ ID NO:112, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence of SEQ ID NO:113. The Tc Buster expression transposon wasflanked by a copy of the octanucleoƟde 5’-CTCTAGAG-3’ on eachside, immediately adjacent to the ITRs and distal to the heterologous polynucleoƟde. The Tc Buster expression transposon further comprised afirst addiƟonal polynucleoƟde immediately adjacent to thefirst ITR, and proximal to the heterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:114. The Tc Buster transposon further comprised a second addiƟonal polynucleoƟde immediately adjacent to the second ITR, and proximal to the heterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:115.

[0121] Each of the four expression transposons was independently transfected into each of the four CHO-K1 pools whose genomes comprised one of the transposase-expressing Amyelois transposons. All transfecƟons were performed using 25 ml of cells at 3 million cells per milliliter using FectoPro (Polyplus / Sartorius) according to the manufacturer’s recommended procedures. Cultures were grown at 37ºC in BalanCD media (Irvine ScienƟfic) in 125 mlflasks at 120 shaker speed and 5% CO2. AŌer 24 hours, cells were fed by addiƟon of transfectory supplement (Irvine ScienƟfic) Fectoboost (Polyplus / Sartorius) anƟ-clump (Thermo Fisher), all according to the manufacturers’ specificaƟons, and 145 µl of 50% glucose, and the temperature was reduced to 32ºC. ConcentraƟons of secreted anƟbody were measured at 10 and 14 days post-transfecƟon in the culture supernatant using an Octet (Sartorius), these values are shown in Table 3 columns C and D respecƟvely. AŌer 14 days, secreted anƟbody was purified the culture supernatant, the amount of anƟbody quanƟfied and this value was used to calculate the Ɵter of purifiable anƟbody present in the supernatant, this value is shown in Table 3 column E.

[0122] Table 3 row 1 shows that when a cell pool expressing a Xenopus piggyBac-like transposase is transfected with a corresponding transposon with Xenopus ITRs, it produces around 480 mg / L anƟbody that can be purified from the culture supernatant on day 14 (Table 3 row 1 column E). This is very comparable to the results seen in Example 1 and Table 1. In contrast, when non-corresponding transposons with Oryzias or Trichoplusia piggyBac transposon ITRs or hAT transposon Tc Buster ITRs were transfected into the Xenopus piggyBac- like transposase expressing cell pool, purifiable anƟbody Ɵters were between about 10 and 15% of this level (Table 3 column E, rows 2-4).

[0123] Table 3 row 6 shows that when a cell pool expressing an Oryzias piggyBac-like transposase is transfected with a corresponding transposon with Oryzias ITRs, it producesaround 40 mg / L anƟbody that can be purified from the culture supernatant on day 14 (Table 3 row 6 column E). In contrast, when non-corresponding transposons with Xenopus or Trichoplusia piggyBac transposon ITRs or hAT transposon Tc Buster ITRs were transfected into the Oryzias piggyBac-like transposase expressing cell pool, purifiable anƟbody Ɵters were between about 15 and 25% of this level (Table 3 column E, rows 5, 7 and 8).

[0124] Table 3 row 11 shows that when a cell pool expressing a Trichoplusia piggyBac transposase is transfected with a corresponding transposon with Trichoplusia piggyBac ITRs, it produces around 74 mg / L anƟbody that can be purified from the culture supernatant on day 14 (Table 3 row 11 column E). In contrast, when non-corresponding transposons with Xenopus or Oryzias piggyBac-like transposon ITRs or hAT transposon Tc Buster ITRs were transfected into the Trichoplusia piggyBac transposase expressing cell pool, purifiable anƟbody Ɵters were between about 15 and 25% of this level (Table 3 column E, rows 9, 10 and 12).

[0125] Table 3 row 16 shows that when a cell pool expressing a Tc Buster hAT transposase is transfected with a corresponding transposon with hAT transposon Tc Buster ITRs, it produces around 21 mg / L anƟbody that can be purified from the culture supernatant on day 14 (Table 3 row 11 column E). In contrast, when non-corresponding transposons with Xenopus or Oryzias piggyBac-like transposon ITRs or hAT transposon Tc Buster ITRs were transfected into the Trichoplusia piggyBac transposase expressing cell pool, purifiable anƟbody Ɵters were between about 15 and 25% of this level (Table 3 column E, rows 13-15).

[0126] In all cases tested, consƟtuƟve expression of a transposase in a mammalian cell pool resulted in ~ 4-fold or greater increases in producƟon levels of a polypepƟde encoded on a transposon with ITRs corresponding to the transposase, compared to the levels obtained when the same polypepƟde was encoded on a transposon with ITRs that do not correspond to the transposase expressed in the cell pool. This result was not limited to transposases in the piggyBac superfamily, but also extends to other transposase superfamilies including the hAT transposase superfamily.

[0127] We observed that the maximum Octet Ɵters and the purifiable Ɵters of anƟbody from the different transposase-expressing cell pools differed significantly. This is not parƟcularly surprising, since each pool was adapted from Advanced CHO Fed-batch media (Sigma) toBalanCD media (Irvine ScienƟfic) to enable lipid-based transfecƟon. The adaptaƟon process took about 2 months, and during that Ɵme cell characterisƟcs can change, especially since the culture was a pool rather than clonally derived. One obvious factor that can affect overall Ɵters from a transient transfecƟon process is transfectability. The more DNA that can be introduced into a cell, and the greater fracƟon of a cell populaƟon that will take up DNA will both increase producƟvity. To measure transfectability of the four transposase-expressing pools, we introduced a GFP-expressing plasmid using the same transfecƟon condiƟons described above. Table 4 shows the fracƟon of GFP-expressing cells observed in duplicate transfecƟons, 2 and 3 days post-transfecƟon. Table 4 (rows 1 and 2) show that around 40% of cells expressing Xenopus piggyBac-like transposase were transfectable. In contrast, only around 10% of the cells expressing Oryzias piggyBac-like transposase (Table 4 rows 3 and 4) or Trichoplusia piggyBac transposase (Table 4 rows 5 and 6) were transfectable., and only around 4% of the cells expressing Tc Buster hAT transposase (Table 4 rows 7 and 8). We conclude that differences in absolute protein expression levels from cells expressing transposases can result from mulƟple other cell properƟes including transfectability. However, cells expressing a transposase will express higher levels of an expressible polypepƟde encoded on a transposon with corresponding ITRs than on a plasmid or other polynucleoƟde that lacks corresponding ITRs. Example 4 IsolaƟon of clonal cell lines stably expressing a transposase

[0128] A pool of cells into which a transposon and its corresponding transposase has been transfected typically comprises a populaƟon of cells descended from many different parental cells, with each parental cell comprising one or more transposons integrated into the cell genome, wherein the number and genomic locaƟon of said integrated transposons are different for each parental cell. In addiƟon, integraƟon of transposons into the genomes of each individual parental cell generally also leads to differences in other cell properƟes such as growth rate, transfectability and propensity to produce aggregates in secreted proteins. The properƟes of a cell pool can thus change over Ɵme, depending on the populaƟon dynamics of the cells of which it is composed. Cell lines with more predictable long-term properƟes can be obtained by isolaƟng individual cells and expanding them into clonal cell lines.

[0129] Individual cells were isolated from a parental stable pool of CHO-K1 cells prepared by stable transfecƟon with thefirst Amyelois transposon (“523802” with nucleoƟde sequence SEQ ID NO:152) comprising afirst open reading frame encoding a Xenopus transposase with amino acid sequence SEQ ID NO:16 fused to an N-terminal heterologous nuclear localizaƟon signal, as described in Example 1. Eleven clonal cell lines were derived from this stable pool, and tested for transfectability. TransfecƟons were performed using 25 ml of cells at 1.25 million cells per milliliter using FectoPro (Polyplus / Sartorius) according to the manufacturer’s recommended procedures. Two different condiƟons were tested for each clonal cell line: in thefirst, thefinal concentraƟon of DNA in the transfecƟon was 0.8 µg / ml and the raƟo of FectoPro to DNA was 1:1 (w / v); in the second thefinal concentraƟon of DNA in the transfecƟon was 1 µg / ml and the raƟo of FectoPro to DNA was 1.2:1 (w / v). Cultures were grown at 37ºC in BalanCD media (Irvine ScienƟfic) in 125 mlflasks at 120 shaker speed and 5% CO2. AŌer 48 hours, cells were analyzed byflow cytometry and data is shown in Table 5.

[0130] We observed a significant range of transfectability in the eleven clones. Of these the three most transfectable under both tested condiƟons were selected for further study: these were C71 (85% and 69% GFP posiƟve cells and MFI 1,716 and 547 under thefirst and second transfecƟon condiƟons respecƟvely), C35 (84% and 75% GFP posiƟve cells and MFI 2,007 and 968 under thefirst and second transfecƟon condiƟons respecƟvely) and C554 (77% and 88% GFP posiƟve cells and MFI 738 and 2,448 under thefirst and second transfecƟon condiƟons respecƟvely).

[0131] The three selected clones were each tested for anƟbody producƟon by transfecƟon with thefirst Xenopus transposon (532006) described in Example 1. Transposon 532006 was transfected into each of the selected CHO-K1 clonal lines. All transfecƟons were performed using 25 ml of cells at 2 million cells per milliliter using FectoPro (Polyplus / Sartorius) according to the manufacturer’s recommended procedures. Three different condiƟons were tested for each clonal cell line: in thefirst, thefinal concentraƟon of DNA in the transfecƟon was 0.8 µg / ml and the raƟo of FectoPro to DNA was 1:1 (w / v); in the second thefinal concentraƟon of DNA in the transfecƟon was 1 µg / ml and the raƟo of FectoPro to DNA was 1:1 (w / v) ; in the third thefinal concentraƟon of DNA in the transfecƟon was 1 µg / ml and the raƟo of FectoPro toDNA was 1.2:1 (w / v). Cultures were grown at 37ºC in BalanCD media (Irvine ScienƟfic) in 125 ml flasks at 120 shaker speed and 5% CO2. AŌer 72 hours, cells were fed by addiƟon of transfectory supplement (Irvine ScienƟfic) Fectoboost (Polyplus / Sartorius) anƟ-clump (Thermo Fisher), all according to the manufacturers’ specificaƟons, and 145 µl of 50% glucose, and the temperature was reduced to 32ºC. ConcentraƟons of secreted anƟbody were measured at 7, 10, 12 and 14 days post-transfecƟon in the culture supernatant using an Octet (Sartorius), these values are shown in Table 6 columns L, M, N and O respecƟvely. AŌer 14 days, secreted anƟbody was purified the culture supernatant, the amount of anƟbody quanƟfied and this value was used to calculate the Ɵter of purifiable anƟbody present in the supernatant, this value is shown in Table 6 column P. A control transfecƟon with the parental stable pool is shown in row 10. Of the 3 clones tested, C71 and C554 both resulted in Ɵters over 800 mg / L at day 14.

[0132] Clonal cell lines C71 and C554 each comprised at least one copy of thefirst Amyelois transposon (“523802” with nucleoƟde sequence SEQ ID NO:152) comprising afirst open reading frame encoding a Xenopus transposase with amino acid sequence SEQ ID NO:16 fused to an N-terminal heterologous nuclear localizaƟon signal, as described in Example 1. Each of these clonal cell lines was sequenced using Oxford Nanopore Technologies systems to idenƟfy the places in the CHO-K1 genome where the transposons were inserted. We determined that the genome of C71 comprised eight inserƟons of transposon with SEQ ID NO:152, and the genome of C554 comprised six inserƟons of transposon with SEQ ID NO:152. Table 7 shows the locaƟons of these inserƟons. Example 5 Enhanced transient protein expression from transposons in HEK 293 cell pools stably expressing a corresponding transposase

[0133] Thefirst Amyelois transposon (“523802” with nucleoƟde sequence SEQ ID NO:152) comprising afirst open reading frame encoding a Xenopus transposase with amino acid sequence SEQ ID NO:16 fused to an N-terminal heterologous nuclear localizaƟon signal was as described in Example 1.

[0134] Thefirst Amyelois transposon was introduced into a pool of suspension-adapted HEK293 cells, together with mRNA encoding a corresponding Amyelois transposase fused to a heterologous nuclear localizaƟon signal with amino acid sequence of SEQ ID NO:127. Into twomillion cells, 7.5 µg of transposon and 1 µg of transposase mRNA were transfected using FectoPro (Polyplus / Sartorius) according to the manufacturer’s recommended procedures; the cells were then cultured in Expi293 media (ThermoFisher) in the presence of 2 µg / ml blasƟcidin unƟl the cells recovered to >95% viability at which Ɵme banks of cells were cryopreserved over liquid nitrogen.

[0135] Afirst Xenopus transposon was constructed. The transposon was designed to be transposable by the Xenopus transposase expressible from thefirst Amyelois transposon integrated into the HEK 293 pool. Thefirst Xenopus transposon (“523811”) comprised, from 5’ to 3’, afirst ITR with the with nucleoƟde sequence of SEQ ID NO:3, which is an embodiment of SEQ ID NO:1, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence of SEQ ID NO:4 which is an embodiment of SEQ ID NO:2. Thefirst Xenopus transposon wasflanked by a copy of the tetranucleoƟde 5’-TTAA-3’ on each side, immediately adjacent to the ITRs and distal to the heterologous polynucleoƟde. Thefirst Xenopus transposon further comprised afirst addiƟonal polynucleoƟde immediately adjacent to thefirst ITR, and proximal to the heterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:131, which is 95% idenƟcal to SEQ ID NO:6. Thefirst Xenopus transposon further comprised a second addiƟonal polynucleoƟde immediately adjacent to the second ITR, and proximal to the heterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:8. Thefirst Xenopus transposon further comprised afirst transcripƟonal unit comprising an open reading frame encoding a mature anƟbody light chain with amino acid sequence SEQ ID NO:132 fused to an N- terminal secreƟon signal sequence and an open reading frame encoding a mature anƟbody heavy chain with amino acid sequence SEQ ID NO:133 fused to an N-terminal secreƟon signal sequence. The two open reading frames were operably linked by an internal ribosome entry site with nucleic acid sequence SEQ ID NO:134, with the light chain open reading frame to the 5’ of the IRES and the heavy chain open reading frame to the 3’ of the IRES. Thefirst transcripƟonal unit was operably linked to a human CMV enhancer-promoter with nucleoƟde sequence SEQ ID NO:159, and a globin polyadenylaƟon sequence. Thefirst Xenopus transposon had nucleoƟde sequence SEQ ID NO:160.

[0136] A control plasmid (258743) was also constructed. This plasmid also comprised a first transcripƟonal unit comprising an open reading frame encoding a mature anƟbody light chain with amino acid sequence SEQ ID NO:132 fused to an N-terminal secreƟon signal sequence and an open reading frame encoding a mature anƟbody heavy chain with amino acid sequence SEQ ID NO:133 fused to an N-terminal secreƟon signal sequence. The two open reading frames were operably linked by an internal ribosome entry site with nucleic acid sequence SEQ ID NO:134, with the light chain open reading frame to the 5’ of the IRES and the heavy chain open reading frame to the 3’ of the IRES. Thefirst transcripƟonal unit was operably linked to a human CMV enhancer-promoter with nucleoƟde sequence SEQ ID NO:159, and an SV40 polyadenylaƟon sequence. Control plasmid 258473 further comprised an Epstein Barr virus OriC replicaƟon origin with nucleoƟde sequence SEQ ID NO:162 and an open reading frame encoding EBNA (Epstein Barr virus nuclear anƟgen) with amino acid sequence SEQ ID NO:163, operably linked to promoter and polyadenylaƟon sequences such that the EBNA protein was consƟtuƟvely expressible in a mammalian cell. The effect of the EBNA is to induce replicaƟon of episomal plasmids comprising the OriC replicaƟon origin in mammalian cells. This results in increased copy number for such plasmids (including control plasmid 258473), leading in turn to increased expression of genes encoded thereon. Control plasmid 258473 had nucleoƟde sequence SEQ ID NO:161.

[0137] Xenopus transposon 523811 and control plasmid 258473 were separately transfected into 100 ml cultures of HEK293. In each transfecƟon 80 µg of plasmid DNA was introduced into 100 ml of cells at 3 million cells per milliliter using FectoPro (Polyplus / Sartorius) according to the manufacturer’s recommended procedures. AŌer 24 hours 0.5 µl FectoPro Boost and 8 µl glucose soluƟon (50% w / v) were added per ml of culture. Cells were cultured for a total of 7 days and anƟbody Ɵters were measured using an Octet. AnƟbody Ɵters in culture supernatant are shown in Table 8. The benefit of the EBNA viral amplificaƟon sequences in HEK 293 cells is clear. The HEK cells transfected with the control plasmid containing these sequences yielded anƟbody Ɵters over 350 mg / L (Table 8 row 1), compared with just over 100 mg / L yielded from HEK 293 cells transfected with the Xenopus transposon.

[0138] The results were very different when the same plasmids were transfected into a pool of HEK293 cells whose genomes comprised thefirst Amyelois transposon (“523802”) comprising an open reading frame encoding a Xenopus transposase operably linked to regulatory sequences such that the transposase was consƟtuƟvely expressed in the HEK293 cells. Xenopus transposon 523811 and control plasmid 258473 were separately transfected into 50 ml cultures of this transposase-expressing HEK293 pool. In each transfecƟon plasmid DNA was introduced into 100 ml of cells at 3 million cells per milliliter using FectoPro (Polyplus / Sartorius) according to the manufacturer’s recommended procedures. Three different condiƟons were tested for each plasmid: in thefirst, thefinal concentraƟon of DNA in the transfecƟon was 0.8 µg / ml and the raƟo of FectoPro to DNA was 0.8:1 (w / v); in the second the final concentraƟon of DNA in the transfecƟon was 0.8 µg / ml and the raƟo of FectoPro to DNA was 1:1 (w / v) ; in the third thefinal concentraƟon of DNA in the transfecƟon was 1 µg / ml and the raƟo of FectoPro to DNA was 0.8:1 (w / v). AŌer 24 hours 0.5 µl FectoPro Boost and 8 µl glucose soluƟon (50% w / v) were added per ml of culture. Cells were cultured for a total of 7 days and anƟbody Ɵters were measured using an Octet. AnƟbody Ɵters in culture supernatant are shown in Table 9. For each of the three condiƟons used, anƟbody Ɵters were higher in the cell pools transfected with Xenopus transposon 523811 than control plasmid 258473: compare Table 9 rows 1 and 2 (Ɵters from Xenopus transposon 523811 were about 170% those of control plasmid 258473), Table 9 rows 3 and 4 (Ɵters from Xenopus transposon 523811 were about 170% those of control plasmid 258473) and Table 9 rows 5 and 6 (Ɵters from Xenopus transposon 523811 were about 135% those of control plasmid 258473), despite the benefit of the EBNA viral amplifier sequences present in control plasmid 258473.

[0139] The Ɵter of anƟbody expressed when polynucleoƟdes comprising anƟbody- encoding open reading frames were introduced into a pool of HEK293 cells stably expressing a transposase was between 1.35- and 1.7-fold higher when the polynucleoƟde comprised corresponding transposon ends such that the anƟbody-encoding open reading frames and their operably linked regulatory elements were transposable by the transposase than when the polynucleoƟde lacked such transposon ends.Example 6 Enhanced transient protein expression from PCR-amplified transposons in HEK 293 cell pools stably expressing a corresponding transposase

[0140] The pool of HEK293 cells stably transfected with thefirst Amyelois transposon (“523802” with nucleoƟde sequence SEQ ID NO:152) comprising afirst open reading frame encoding a Xenopus transposase with amino acid sequence SEQ ID NO:16 fused to an N- terminal heterologous nuclear localizaƟon signal was prepared as described in Example 5.

[0141] A plasmid comprising a Xenopus transposon (“518540”) was constructed wherein the transposon comprised, from 5’ to 3’, afirst ITR with the with nucleoƟde sequence of SEQ ID NO:3, which is an embodiment of SEQ ID NO:1, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence of SEQ ID NO:4 which is an embodiment of SEQ ID NO:2. Xenopus transposon 518540 wasflanked by a copy of the tetranucleoƟde 5’-TTAA-3’ on each side, immediately adjacent to the ITRs and distal to the heterologous polynucleoƟde. The first Xenopus transposon further comprised afirst addiƟonal polynucleoƟde immediately adjacent to thefirst ITR, and proximal to the heterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:131, which is 95% idenƟcal to SEQ ID NO:6. Xenopus transposon 518540 further comprised a second addiƟonal polynucleoƟde immediately adjacent to the second ITR, and proximal to the heterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:8. Xenopus transposon 518540 further comprised afirst transcripƟonal unit comprising an open reading frame encoding a polypepƟde comprising a secreƟon signal fused to a human tumor necrosis factor receptor 1B fused to an IgG1 Fc domain, the mature polypepƟde had amino acid sequence SEQ ID NO:164. The open reading frame was operably linked to a human CMV enhancer-promoter with nucleoƟde sequence SEQ ID NO:159, and an SV40 polyadenylaƟon sequence. The plasmid comprising Xenopus transposon 518540 had nucleoƟde sequence SEQ ID NO:165.

[0142] The transposon was amplified from plasmid 518540 by PCR with primers with nucleoƟde sequences SEQ ID NOs: 166 and 167, and PCR product was purified using Macherey- Nagel kits according to the manufacturer’s direcƟons. PCR product was transfected into either naïve HEK293 cells, or into a pool of cells which expressed a Xenopus transposase from genomically integrated copies of thefirst Amyelois transposon (“523802” with nucleoƟdesequence SEQ ID NO:152). Nine different transfecƟon condiƟons were tested: thefirst three used 0.5 µg of DNA per ml offinal culture, with either 1.4 µl, 2.8 µl or 4.2 µl of FectoPro transfecƟon reagent per ml offinal culture used to create the transfecƟon complex; the second three used 1 µg of DNA per ml offinal culture, with either 1.4 µl, 2.8 µl or 4.2 µl of FectoPro transfecƟon reagent per ml offinal culture used to create the transfecƟon complex; the last three used 1.5 µg of DNA per ml offinal culture, with either 1.4 µl, 2.8 µl or 4.2 µl of FectoPro transfecƟon reagent per ml offinal culture used to create the transfecƟon complex. TransfecƟons were performed with 1 ml cells at 3 million cells per ml in a 96-deep well culture plate. AŌer 24 hours 0.5 µl FectoPro Boost and 8 µl glucose soluƟon (50% w / v) were added per ml of culture. Cells were cultured for a total of 7 days and protein Ɵters were measured using an Octet. Protein Ɵters in culture supernatant are shown in Table 10. For every condiƟon used, protein Ɵters were higher in the cell pools expressing Xenopus transposase from thefirst Amyelois transposon (“523802” with nucleoƟde sequence SEQ ID NO:152), than from control naïve HEK293 cells plasmid 258473. The highest increases observed were for 1 µg of DNA with 4.2 µl of transfecƟon reagent: comparing Table 10 rows 11 and 12 the Xenopus transposase- expressing HEK293 pools produced 153% of the Ɵters produced by the HEK293 cells not expressing a transposase; and for 1.5 µg of DNA with 4.2 µl of transfecƟon reagent: comparing Table 10 rows 17 and 18 the Xenopus transposase-expressing HEK293 pools produced 156% of the Ɵters produced by the HEK293 cells not expressing a transposase. The lowest increases observed were for 0.5 µg of DNA with 1.4 µl of transfecƟon reagent: comparing Table 10 rows 1 and 2 the Xenopus transposase-expressing HEK293 pools produced 107% of the Ɵters produced by the HEK293 cells not expressing a transposase.

[0143] The Ɵter of protein expressed when PCR-amplified linear polynucleoƟdes comprising protein-encoding open reading frames and transposon ends were introduced into a pool of cells stably expressing a corresponding transposase such that the protein -encoding open reading frames and their operably linked regulatory elements were transposable by the transposase was between 1.07- and 1.5-fold higher than when the same polynucleoƟdes were introduced into cells that did not express the corresponding transposase.Example 7: Enhanced transient protein expression from transposons in stable cell pools expressing Sleeping Beauty transposase

[0144] We tested whether Sleeping Beauty would confer the same kind of expression advantage in mammalian cells as shown for other corresponding transposon-transposase pairs in Example 3. To do this we compared expression of an expressible polypepƟde by a pool of mammalian cells expressing Sleeping Beauty transposase when the open reading frame encoding the expressible polypepƟde was introduced on a Sleeping Beauty transposon with expression of the same expressible polypepƟde introduced on a transposon that was not transposable by the Sleeping Beauty transposase.

[0145] A Sleeping Beauty transposase-expressing transposon was constructed by cloning an open reading frames encoding hyperacƟve Sleeping Beauty transposase with amino acid sequence SEQ ID NO: 157 onto an Amyelois transposon. The transposon comprised, from 5’ to 3’, afirst ITR with the with nucleoƟde sequence of SEQ ID NO:117, which is an embodiment of SEQ ID NO:80, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence of SEQ ID NO:118 which is an embodiment of SEQ ID NO:81. The transposon wasflanked by a copy of the tetranucleoƟde 5’-TTAA-3’ on each side, immediately adjacent to the ITRs and distal to the heterologous polynucleoƟde. The transposon further comprised afirst addiƟonal polynucleoƟde immediately adjacent to thefirst ITR, and proximal to the heterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:82. The transposon further comprised a second addiƟonal polynucleoƟde immediately adjacent to the second ITR, and proximal to the heterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:83. The transposon further comprised a second open reading frame encoding blasƟcidin deaminase, operably linked to regulatory elements such that the blasƟcidin deaminase was consƟtuƟvely expressible in a mammalian cell. The open reading frame encoding the Sleeping Beauty transposase was operably linked to a PGK promoter with nucleoƟde sequence SEQ ID NO:119 and a polyadenylaƟon signal with nucleoƟde sequence SEQ ID NO:120 such that it was consƟtuƟvely expressible in a mammalian cell. The transposon nucleoƟde sequence was SEQ ID NO:168.

[0146] The Sleeping Beauty transposase-expressing Amyelois transposon was introduced into a pool of CHO-K1 cells, together with mRNA encoding a corresponding Amyelois transposase fused to a heterologous nuclear localizaƟon signal with amino acid sequence of SEQ ID NO:127. Intofive million cells, 25 µg of transposon and 3 µg of transposase mRNA were electroporated; the cells were then cultured in Advanced CHO Fed-batch media (Sigma) in the presence of 2 µg / ml blasƟcidin unƟl the cells recovered to >95% viability at which Ɵme banks of cells were cryopreserved over liquid nitrogen. Prior to subsequent lipid-based transfecƟon, cells were adapted to BalanCD media (Irvine ScienƟfic).

[0147] In addiƟon to the four different expression transposons for expression of an anƟbody (the expressible polypepƟde) described in Example 3 (the Xenopus expression transposon (“532006”), Oryzias expression transposon, the Trichoplusia piggyBac expression transposon and the Tc Buster expression transposon) afiŌh expression transposon was constructed to be transposable by the Sleeping Beauty transposase. The Sleeping Beauty expression transposon comprised afirst transcripƟonal unit comprising afirst open reading frame encoding puromycin-N-acetyltransferase, operably linked to regulatory elements (including an HSV-TK promoter with nucleoƟde sequence SEQ ID NO:145, and an SV40 polyadenylaƟon signal with nucleoƟde sequence SEQ ID NO:148) such that the puromycin-N- acetyltransferase was consƟtuƟvely expressible in a mammalian cell. The Sleeping Beauty expression transposon further comprised a second transcripƟonal unit comprising an open reading frame encoding a mature anƟbody light chain with amino acid sequence SEQ ID NO:132 fused to an N-terminal secreƟon signal sequence and an open reading frame encoding a mature anƟbody heavy chain with amino acid sequence SEQ ID NO:133 fused to an N-terminal secreƟon signal sequence. The two open reading frames were operably linked by an internal ribosome entry site with nucleic acid sequence SEQ ID NO:134, with the light chain open reading frame to the 5’ of the IRES and the heavy chain open reading frame to the 3’ of the IRES. The second transcripƟonal unit was operably linked to a hybrid mouse-human CMV enhancer-promoter with nucleoƟde sequence SEQ ID NO:135 and a globin polyadenylaƟon sequence. The Sleeping Beauty expression transposon comprised, from 5’ to 3’, afirst ITR with the with nucleoƟde sequence of SEQ ID NO:109, a heterologous polynucleoƟde to betransposed and a second ITR with nucleoƟde sequence of SEQ ID NO:110. The Sleeping Beauty expression transposon wasflanked by a copy of the octanucleoƟde 5’-ATATATAT-3’ on each side, immediately adjacent to the ITRs and distal to the heterologous polynucleoƟde.

[0148] Each of thefive expression transposons were independently transfected into the Sleeping Beauty transposase-expressing CHO-K1 pool whose genome comprised the Sleeping Beauty transposase-expressing Amyelois transposon. All transfecƟons were performed using 25 ml of cells at 3 million cells per milliliter using FectoPro (Polyplus / Sartorius) according to the manufacturer’s recommended procedures. Cultures were grown at 37ºC in BalanCD media (Irvine ScienƟfic) in 125 mlflasks at 120 shaker speed and 5% CO2. AŌer 24 hours, cells were fed by addiƟon of transfectory supplement (Irvine ScienƟfic) Fectoboost (Polyplus / Sartorius) anƟ-clump (Thermo Fisher), all according to the manufacturers’ specificaƟons, and 145 µl of 50% glucose, and the temperature was reduced to 32ºC. ConcentraƟons of secreted anƟbody were measured at 10 and 14 days post-transfecƟon in the culture supernatant using an Octet (Sartorius), these values are shown in Table 11 columns C and D respecƟvely. AŌer 14 days, secreted anƟbody was purified the culture supernatant, the amount of anƟbody quanƟfied and this value was used to calculate the Ɵter of purifiable anƟbody present in the supernatant, this value is shown in Table 11 column E.

[0149] Table 11 row 4 shows that when a cell pool expressing a Sleeping Beauty Mariner transposase is transfected with a corresponding transposon with Mariner transposon Sleeping Beauty ITRs, it produces around 29 mg / L anƟbody that can be purified from the culture supernatant on day 14 (Table 11 row 4 column E). In contrast, when non-corresponding transposons with Xenopus, Trichoplusia or Oryzias piggyBac-like transposon ITRs or hAT transposon Tc Buster ITRs were transfected into the Sleeping Beauty Mariner expressing cell pool, purifiable anƟbody Ɵters were between about 37 and 62% of this level (Table 11 column E, rows 1-3 and 5).

[0150] Thus for the Mariner transposase Sleeping Beauty, consƟtuƟve expression of a transposase in a mammalian cell pool resulted in significantly greater producƟon levels of a polypepƟde encoded on a transposon with ITRs corresponding to the transposase, compared to the levels obtained when the same polypepƟde was encoded on a transposon with ITRs that donot correspond to the transposase expressed in the cell pool. This result is thus not limited to transposases in the piggyBac and hAT superfamilies, but also extends to other transposase superfamilies including the Mariner transposase superfamily. Example 8 Enhanced transient protein expression from PCR-amplified transposons in CHO-K1 cell pools stably expressing a corresponding transposase

[0151] A plasmid comprising a Xenopus transposon (“547187”) was constructed wherein the transposon comprised, from 5’ to 3’, afirst ITR with the with nucleoƟde sequence of SEQ ID NO:3, which is an embodiment of SEQ ID NO:1, a heterologous polynucleoƟde to be transposed and a second ITR with nucleoƟde sequence of SEQ ID NO:4 which is an embodiment of SEQ ID NO:2. Xenopus transposon 547187 wasflanked by a copy of the tetranucleoƟde 5’-TTAA-3’ on each side, immediately adjacent to the ITRs and distal to the heterologous polynucleoƟde. Xenopus transposon 547187 further comprised afirst addiƟonal polynucleoƟde immediately adjacent to thefirst ITR, and proximal to the heterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:131, which is 95% idenƟcal to SEQ ID NO:6. Xenopus transposon 547187 further comprised a second addiƟonal polynucleoƟde immediately adjacent to the second ITR, and proximal to the heterologous polynucleoƟde, with nucleoƟde sequence SEQ ID NO:8. Xenopus transposon 547187 further comprised afirst transcripƟonal unit comprising an open reading frame encoding a mature anƟbody light chain with amino acid sequence SEQ ID NO:132 fused to an N-terminal secreƟon signal sequence and an open reading frame encoding a mature anƟbody heavy chain with amino acid sequence SEQ ID NO:133 fused to an N-terminal secreƟon signal sequence. The two open reading frames were operably linked by an internal ribosome entry site with nucleic acid sequence SEQ ID NO:134, with the light chain open reading frame to the 5’ of the IRES and the heavy chain open reading frame to the 3’ of the IRES. Thefirst transcripƟonal unit was operably linked to a hybrid mouse-human CMV enhancer-promoter with nucleoƟde sequence SEQ ID NO:135 and a globin polyadenylaƟon sequence.

[0152] A second plasmid (“563552”) was constructed, lacking the transposon-related sequences of transposon 547187 but otherwise idenƟcal. Thus plasmid 563552 comprised an open reading frame encoding a mature anƟbody light chain with amino acid sequence SEQ IDNO:132 fused to an N-terminal secreƟon signal sequence and an open reading frame encoding a mature anƟbody heavy chain with amino acid sequence SEQ ID NO:133 fused to an N-terminal secreƟon signal sequence. The two open reading frames were operably linked by an internal ribosome entry site with nucleic acid sequence SEQ ID NO:134, with the light chain open reading frame to the 5’ of the IRES and the heavy chain open reading frame to the 3’ of the IRES. Thefirst transcripƟonal unit was operably linked to a hybrid mouse-human CMV enhancer-promoter with nucleoƟde sequence SEQ ID NO:135 and a globin polyadenylaƟon sequence.

[0153] Thefirst transcripƟon unit was independently amplified from plasmid 563552 or Xenopus transposon 547187 by PCR with primers with nucleoƟde sequences SEQ ID NOs: 166 and 167, and PCR product was purified using Macherey-Nagel kits according to the manufacturer’s direcƟons. PCR product from plasmid 563552 had nucleoƟde sequence SEQ ID NO:170, PCR product from Xenopus transposon 547187 had nucleoƟde sequence SEQ ID NO:169.

[0154] Each PCR product was independently introduced into CHO-K1 clone C554, which consƟtuƟvely expressed afirst open reading frame encoding a Xenopus transposase with amino acid sequence SEQ ID NO:16 fused to an N-terminal heterologous nuclear localizaƟon signal, as described in Example 4. PCR product was introduced into the CHO cells in four different ways. One sample was transfected with 1 µg of DNA per ml offinal culture, with 1.2 µl of FectoPro transfecƟon reagent per ml offinal culture -Lipid(2)- using PCR product as eluted from the Macherey-Nagel kit. For the other samples, the PCR product was concentrated to 1.5 mg / ml to be suitable for electroporaƟon. A lipid control transfecƟon was performed using this concentrated PCR product -Lipid(1)- with thefinal raƟos of DNA, transfecƟon reagent and cells being the same as for Lipid(2): 1 µg of DNA per ml offinal culture, with 1.2 µl of FectoPro transfecƟon reagent per ml offinal culture. One electroporaƟon was performed using a Lonza nucleofector according to the manufacturer’s direcƟons. One electroporaƟon was performed using a Maxcyte electroporator according to the manufacturer’s direcƟons. In each case a total of 15 million cells were transfected and the culture volume adjusted to 10 ml. Cultures were grown at 37ºC in BalanCD media (Irvine ScienƟfic) in TPPs at 120 shaker speed and 5% CO2.AŌer 24 hours, cells were fed by addiƟon of transfectory supplement (Irvine ScienƟfic) Fectoboost (Polyplus / Sartorius) anƟ-clump (Thermo Fisher), all according to the manufacturers’ specificaƟons, and 145 µl of 50% glucose, and the temperature was reduced to 32ºC. ConcentraƟons of secreted anƟbody were measured at 7 and 14 days post-transfecƟon in the culture supernatant using an Octet (Sartorius), these values are shown in Table 12 columns C and D respecƟvely.

[0155] In every case, the Ɵter obtained using a PCR product comprising a transposon transposable by the transposase expressed in the CHO-K1 cells was more than 2-fold higher than when the PCR product did not comprise a transposon transposable by the transposase expressed in the CHO-K1 cells. Compare Table 12 row 1 with row 2, row 3 with row 4, row 5 with row 6 and row 7 with row 8.

[0156] We conclude that in both HEK and CHO cells transfected with either plasmid or linear DNA (for example PCR product), expression of protein encoded on the transfected DNA is enhanced if the expressible sequences are part of a transposon that is transposable by a corresponding transposase expressed within the cell.

[0157] TABLES

[0158] Table 1. AnƟbody Titers From CHO-K1 Cell Cultures. Stable CHO-K1 cell pools comprising Amyelois transposons were prepared as described in Example 1. Cell pools were transfected with afirst Xenopus transposon comprising a transcripƟonal unit comprising open reading frames encoding an anƟbody as described in Example 1. The idenƟƟes of the transfected pools are shown in column A. Column B shows the pool that was co-transfected with thefirst Xenopus transposon and afirst transient transposase expression plasmid comprising an open reading frame encoding a Xenopus transposase. Octet Ɵters of anƟbody in culture supernatants 11 and 14 days post-transfecƟon are shown in columns C and D respecƟvely. The concentraƟon of anƟbody in the supernatant at day 14 that was purifiable is shown in column E.

[0159] Table 2. AnƟbody Titers and Cell DensiƟes and ViabiliƟes From CHO-K1 Cell Cultures. Stable CHO-K1 cell pools comprising Amyelois transposons encoding Xenopus transposase were prepared as described in Example 1. Cell pools were transfected with one ofthree different polynucleoƟdes, each comprising a transcripƟonal unit comprising open reading frames encoding an anƟbody as described in Example 2. The idenƟty of the Amyelois transposon integrated into the genome of the CHO-K1 host is shown in column A. Column B shows the idenƟty of the anƟbody-encoding polynucleoƟde transfected into the host. Column C shows the concentraƟon of puromycin added 4 days post-transfecƟon. Columns D-G show the anƟbody Ɵter measured in the culture supernatant in mg / L at various days post-transfecƟon: column D 8 days, column E 11 days, column F 15 days, column G 20 days. Columns H-L show the viable cell density measured in the culture in millions of cells per ml at various days post- transfecƟon: column H 4 days, column I 8 days, column J 11 days, column K 15 days, column L 20 days. Columns M-Q show the % cell viability in the culture at various days post-transfecƟon: column M 4 days, column N 8 days, column O 11 days, column P 15 days, column Q 20 days.

[0160] Table 3. AnƟbody Ɵters from transposase-expressing CHO-K1 cell cultures. Stable CHO-K1 cell pools comprising transposase-expressing Amyelois transposons were prepared as described in Example 3. The transposase expressed in each cell pool is shown in column A. Cell pools were transfected with 1 of 4 different expression transposons, each comprising a transcripƟonal unit comprising open reading frames encoding an anƟbody and flanked by ITRs corresponding to one of the transposases as described in Example 3. The ITRs on the expression transposon are shown in column B. Octet Ɵters of anƟbody in culture supernatants 10 and 14 days post-transfecƟon are shown in columns C and D respecƟvely. The concentraƟon of anƟbody in the supernatant at day 14 that was purifiable is shown in column E.

[0161] Table 4. Transfectability transposase-expressing CHO-K1 cell pools. Stable CHO-K1 cell pools comprising Xenopus-transposase-expressing Amyelois transposons were prepared as described in Example 3. The transposase expressed in each cell pool is shown in column A. Cell pools were transfected with a GFP-encoding plasmid, and the percentage of GFP- expressing cells was measured 2 (column B) and 3 (column C) days post-transfecƟon.

[0162] Table 5. Transfectability of transposase-expressing CHO-K1 clonal cell lines. Stable clonal CHO-K1 cell lines comprising Xenopus-transposase-expressing Amyelois transposons were prepared as described in Example 4. The cell line idenƟfier is shown in column A. Cell pools were transfected with a GFP-encoding plasmid, the concentraƟon of DNAin the transfecƟon is shown in column B and the w / v raƟo of FectoPro to DNA is shown in column C. Two days aŌer transfecƟon, the percentage of GFP-expressing cells was measured by FACS (column D) and the meanfluorescent intensity was also measured (column E). Cell density in the transfected culture (in million cells per ml) is shown in column F, and the percentage of cells that were viable is shown in column G. The stable pool from which these clonal lines were derived is shown in row 1.

[0163] Table 6. AnƟbody producƟon from transposase-expressing CHO-K1 clonal cell lines. Stable clonal CHO-K1 cell lines comprising Xenopus-transposase-expressing Amyelois transposons were prepared as described in Example 4. The cell line idenƟfier is shown in column A. Cell pools were transfected with an anƟbody-encoding transposon (532006), the concentraƟon of DNA in the transfecƟon is shown in column B and the w / v raƟo of FectoPro to DNA is shown in column C. Three days aŌer transfecƟon, cells were fed and temperature shiŌed, as described in Example 4. The cell density was measured at 7, 10, 12 and 14 days post- transfecƟon, these values (in million cells per ml) are shown in columns D, E, F and G respecƟvely. The cell viability was measured at 7, 10, 12 and 14 days post-transfecƟon, these values (in % viable cells) are shown in columns H, I, J and K respecƟvely. AnƟbody Ɵters in the culture supernatant were measured using an Octet at 7, 10, 12 and 14 days post-transfecƟon, these values (in mg / L) are shown in columns L, M, N and O respecƟvely. AnƟbody was purified from cultures that survived to day 14 using protein A affinity capture, and quanƟfied using A280 absorbance; the anƟbody yield was used to calculate a purifiable Ɵter of anƟbody shown in column P (in mg / L). The stable pool from which these clonal lines were derived is shown in row 10. n / d = not done: cultures were typically not conƟnued if the viability fell below 50%.

[0164] Table 7. Genomic loci of transposase-expressing transposons in CHO-K1 clonal cell lines. Chromosomal inserƟon points were determined by targeted genome sequencing using Oxford Nanopore Technologies systems. The clonal line idenƟty is shown in column A, inserƟon # in column B and the chromosomal locaƟon in column C. The CHO-K1 genome has not yet been fully assembled, so chromosomal locaƟons are oŌen defined as parƟally assembled scaffolds rather than fully assembled chromosomes. Genomic informaƟon can be obtained by copying the locus informaƟon in column C into a genome web browser such as theNCBI version that can be found at world wide web ncbi.nlm.nih.gov / gdv / ?org=cricetulus- griseus. If the inserƟon is within a gene, the gene name is given in column D, the approximate locaƟon within the gene is given in column E. If the inserƟon is not within a gene, the name of a nearby gene is given in column F.

[0165] Table 8. AnƟbody producƟon from HEK293 cells. HEK293 cells were transfected either with an anƟbody-encoding plasmid (258743) or with an anƟbody-encoding transposon (523811), as described in Example 5. The presence of transposon ends is indicated in column B, the presence of EBNA viral amplifier sequences is indicated in column C. AnƟbody Ɵters in the culture supernatant were measured using an Octet at 7days post-transfecƟon, these values (in mg / L) are shown in column D.

[0166] Table 9. AnƟbody producƟon from transposase-expressing HEK293 stable pools. Stable HEK293 cell pools comprising Xenopus-transposase-expressing Amyelois transposons were prepared as described in Example 5. Cell pools were transfected either with an anƟbody-encoding transposon (523811), or an anƟbody-encoding control plasmid (258743) lacking transposon ends but instead comprising components of an EBNA viral amplificaƟon system. The transfected construct idenƟfier is indicated in column A, the presence of transposon ends is indicated in column B, the presence of EBNA viral amplifier sequences is indicated in column C. The concentraƟon of DNA in the transfecƟon is shown in column D and the w / v raƟo of FectoPro to DNA is shown in column E. AnƟbody Ɵters in the culture supernatant were measured using an Octet at 7 days post-transfecƟon, these values (in mg / L) are shown in column F.

[0167] Table 10. Protein producƟon from transposase-expressing HEK293 stable pools. HEK293 cells, or stable HEK293 cell pools comprising Xenopus-transposase-expressing Amyelois transposons (prepared as described in Example 5) were transfected with a PCR- amplified linear DNA molecule comprising a protein-encoding transposon (518540). Whether or not the cells expressed a Xenopus transposase is indicated in column A, thefinal DNA concentraƟon of the transposon in the transfecƟon is shown in column B, the volume of transfecƟon reagent used per ml of transfected cells is shown in column C. Four independent transfecƟons were performed for each DNA / transfecƟon reagent raƟo in each cell type. ProteinƟters in the culture supernatant were measured using an Octet at 7 days post-transfecƟon, these values (in mg / L) are shown in columns D, E, F and G. The average (mean) of the Ɵters for the 4 independent transfecƟons is shown in column H. The protein Ɵter in the HEK293 cells expressing transposase is shown as a percentage of the protein Ɵter in the HEK293 cells that do not express a transposase in column I.

[0168] Table 11. AnƟbody Ɵters from Sleeping Beauty transposase-expressing CHO-K1 cell cultures. Stable CHO-K1 cell pools comprising transposase-expressing Amyelois transposons were prepared as described in Example 7. The transposase expressed in each cell pool is shown in column A. Cell pools were transfected with 1 of 5 different expression transposons, each comprising a transcripƟonal unit comprising open reading frames encoding an anƟbody andflanked by ITRs corresponding to one of the transposases as described in Example 7. The ITRs on the expression transposon are shown in column B. Octet Ɵters of anƟbody in culture supernatants 10 and 14 days post-transfecƟon are shown in columns C and D respecƟvely. The concentraƟon of anƟbody in the supernatant at day 14 that was purifiable is shown in column E.

[0169] Table 12. Protein producƟon from PCR product transfected into a transposase- expressing CHO-K1 clone. CHO-K1 clone C554 consƟtuƟvely expressing a Xenopus transposase (prepared as described in Example 4) was transfected with PCR-amplified linear DNA molecules encoding heavy and light chains of an anƟbody, as described in Example 8. Whether or not the PCR product comprised transposon ends transposable by the Xenopus transposase expressed by the CHO-K1 cells is indicated in column A, the transfecƟon method is shown in column B. Protein Ɵters in the culture supernatant were measured using an Octet at 7 and 14 days post- transfecƟon, these values (in mg / L) are shown in columns C and D respecƟvely. The protein Ɵter in the CHO-K1 cells transfected by transposable PCR product is shown as a percentage of the protein Ɵter in the CHO-K1 cells transfected by non-transposable PCR product in column E.

[0170] TABLE 1TABLE 4TABLE 5TABLE 7TABLE 8TABLE 9TABLE 11TABLE 12

Claims

What is claimed is:

1. A method for expressing a protein, the method comprising (i) transfecƟng a mammalian cell populaƟon, the genomes of which comprise a nucleic acid encoding a transposase operably linked to regulatory sequences including a promoter and a polyadenylaƟon signal such that the transposase is expressible in the mammalian cell populaƟon, with a corresponding transposon transposable by the transposase, wherein the corresponding transposon comprises an open reading frame encoding an expressible polypepƟde operably linked to regulatory sequences including a promoter and a polyadenylaƟon signal such that the expressible protein is expressible in cells of the populaƟon into which the transposon has bene introduced; and (ii) culturing the transfected mammalian cell populaƟon under condiƟons such that the expressible polypepƟde is expressed.

2. The method of claim 1, wherein the transposase is a piggyBac-like transposase, an hAT transposase, a Mariner transposase or a Helitron transposase.

3. The method of claim 1 or 2, wherein the transposase is a piggyBac-like transposase selected from a transposase derived from a naturally occurring transposase from the looper moth Trichoplusia ni, a transposase derived from a naturally occurring transposase from a Xenopus species, a transposase derived from a naturally occurring transposase from a Bombyx species, a transposase derived from a naturally occurring transposase from a Heliothis species, a transposase derived from a naturally occurring transposase from a Helicoverpaspecies, a transposase derived from a naturally occurring transposase from an Agro s species, atransposase derived from a naturally occurring transposase from an Amyelois species, atransposase derived from a naturally occurring transposase from a Myo s species and atransposase from an Oryzias species.

4. The method of claim 1, wherein the transposase is a Sleeping Beauty transposase, a Tol2 transposase, a Tc Buster transposase or a Helraiser transposase.

5. The method of any preceding claim, wherein the promoter operably linked to the open reading frame encoding the transposase is a consƟtuƟve promoter.

6. The method of any one of claims 1-5, wherein the promoter operably linked to the open reading frame encoding the transposase is an inducible promoter, and the method further comprises inducing the promoter before transfecƟng the mammalian cell populaƟon with the corresponding transposon.

7. The method of any preceding claim, wherein the culturing step is performed with selecƟon for cells comprising the transposon over cells lacking the transposon.

8. The method of claim 7, wherein the corresponding transposon further comprises a second open reading frame encoding a selecƟve marker that allows cells comprising the transposon to overcome a selecƟve agent (e.g., puromycin, hygromycin, blasƟcidin, zeocin, neomycin) and the culturing step further comprises supplying to culture medium the selecƟve agent to inhibit growth of cells that do not comprise the corresponding transposon.

9. The method of claim 7, wherein the corresponding transposon comprises a second open reading frame encoding an enzyme required to provide the nutrient (glutamine synthetase, dihydrofolate reductase) and the culturing step is performed in media lacking the nutrient (e.g., glutamine, tetrahydrofolate) thereby inhibiƟng growth of cells that do not comprise the corresponding transposon.

10. The method of any preceding claim, wherein the open reading frame encoding the transposase in the genomes of the mammalian cell populaƟon is within a second transposon not transposable by the transposase.

11. The method of any of claims 1-10, wherein the open reading frame encoding the transposase in the genomes of the mammalian cell populaƟon is within a retroviral vector, such as a lenƟviral vector.

12. The method of claim 11, wherein the open reading frame encoding the transposase in the genomes of the mammalian cell populaƟon was introduced into the genomes by random integraƟon.

13. The method of any preceding claim, wherein the mammalian cell populaƟon is a CHO cell or a HEK cell populaƟon.

14. The method of any preceding claim, wherein the expressible polypepƟde is an anƟbody chain, or an Fc fusion protein or a chain of a 3-chain or 4-chain mulƟ-specific anƟbody.

15. The method of claim 14, wherein the expressible polypepƟde comprises a plurality of anƟbody chains separated by CHYSEL elements.

16. The method of any one of claims 1-14, wherein the corresponding transposon comprises a plurality of open reading frames encoding a plurality of expressible polypepƟdes, each operably linked to regulatory sequences.

17. The method of claim 16, wherein the plurality of expressible polypepƟdes are chains of a mulƟ-chain anƟbody.

18. The method of claim 17, wherein the plurality of open reading frames are in the same transcripƟonal unit separated by IRES elements.

19. The method of any one of claims 1-14, further comprising transfecƟng the mammalian cell populaƟon with a second corresponding transposon transposable by the transposase, wherein the second corresponding transposon comprises a second open reading frame encoding a second expressible polypepƟde operably linked to regulatory sequences including a promoter and a polyadenylaƟon signal such that the second expressible polypepƟde is expressible in cells of the populaƟon into which the second corresponding transposon has been introduced; wherein in the culturing step the second expressible polypepƟde is expressed.

20. The method of claim 19, wherein the expressible polypepƟde and second expressible polypepƟde assemble into a mulƟmeric polypepƟde, e.g., an anƟbody.

21. The method of any preceding claim, wherein the culturing comprises feeding the cells.

22. The method of any preceding claim further comprises changing the incubaƟon temperature of the cells during the culturing step to bias the cells to producƟon of the polypepƟde over propagaƟon.

23. The method of any preceding claim, wherein the culturing of the cells results in propagaƟon of the cells.

24. The method of any preceding claim, performed without clonal selecƟon of a cell line from the cultured cells.

25. The method of any preceding claim, where the cultured cells and their progeny integrate the transposon at different locaƟons in their genomes.

26. The method of claim 25, wherein the locaƟons of the transposon in the genomes of the cultured cells and their progeny change with Ɵme.

27. The method of any preceding claim, further comprising purifying the expressible polypepƟde from the cultured transfected cells.

28. The method of claim 27, wherein the purifying step is performed within 7-30 days of the transfecƟng step.

29. The method of claim 27, wherein the purifying step is performed when the concentraƟon of the expressible polypepƟde is within ± 20% of its maximum concentraƟon.

30. The method of any preceding claim, wherein the purified expressible polypepƟde includes polypepƟde expressed during selecƟon of the cultured cells for presence of a selecƟon marker encoded by the transposon.

31. The method of any preceding claim, further comprising freezing the cultured transfected cells.

32. The method of any preceding claim, wherein the maximum yield of the expressible protein is 50-1500 mg / L.

33. The method of any preceding claim, wherein the culturing is performed from 7-30 days.

34. The method of any preceding claim, wherein the expressible protein is used for non-clinical research.

35. The method of any preceding claim, wherein the corresponding transposon is a component of an episome.

36. The method of any one of claims 1-34, wherein the corresponding transposon is not a component of an episome.

37. A cell line having a genome comprising an open reading frame encoding a polypepƟde with at least 95% and opƟonally 100% sequence idenƟty to an amino acid sequence selected from SEQ ID NOS:9-41, 46-69, 74, 79, 158, 84, 89, 96, 101, 106, 111, 157, 116, 130 or 141, operably linked to a heterologous promoter acƟve in the cell line.

38. The cell line of claim 37, wherein the promoter has a sequence selected from SEQ ID NOS:1 and 2, or 42 and 43, or 75 and 76, or 80 and 81, or 85 and 86, or 90 and 91, or 97 and 98, or 102 and 103, or 107 and 108, or 112 and 113, or 128 and 129, or 137 and 138.

39. The cell line of claim 37, which is a CHO K1 cell line having a genome comprising a nucleic acid encoding a transposase of SEQ ID NO:16 fused at its N-terminus to a nuclear localizaƟon signal, operably linked to the heterologous promoter.

40. The CHO K1 cell line of claim 39, comprising six copies of the transposase at six locaƟons of the genome.

41. The CHO K1 cell line of claim 39, wherein the six locaƟons are as shown in Table 7.

42. The cell line of any one of claims 39-41, wherein the nucleic acid encoding the transposase comprises SEQ ID NO:152.

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