Vector composition and use thereof

Abstract

A vector composition containing i) a first vector that comprises a double-stranded cargo nucleic acid, wherein the double-stranded cargo nucleic acid comprises a first expression cassette of a gene of interest, a second expression cassette of a selectable marker, a ubiquitous chromatin opening element (UCOE) arranged upstream of and right before the first expression cassette or the second expression cassette, and a pair of transposon derived inverted terminal repeats (ITRs), wherein the first expression cassette, the second expression cassette and the UCOE are operably connected and flanked by the pair of transposon derived ITRs, and ii) a second vector that encodes and expresses a transposase, wherein the transposase is able to recognize the pair of transposon derived ITRs in the first vector and transpose the double-stranded cargo nucleic acid.

Description

VECTOR COMPOSITION AND USE THEREOFCROSS-REFERENCES

[0001] This application claims priority to US provisional patent application No. 63 / 652,263 filed May 28, 2024.

[0002] The foregoing application, and all documents cited therein or during its prosecution (”appln cited documents’7) and all documents cited or referenced herein (including without limitation all literature documents, patents, published patent applications cited herein) (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. Any Genbank sequences mentioned in this disclosure are incorporated by reference with the Genbank sequence to be that of the earliest effective filing date of this disclosure.REFERENCE TO A “SEQUENCE LISTING”

[0003] The instant application contains a Sequence Listing XML labeled “55375- 00006SequenceListingXML” which was created on May 14, 2025 and is 24 kb in size. The entire content of the sequence listing is incorporated herein by reference in its entirety.TECHNICAL FIELD OF THE INVENTION

[0004] The present application generally relates to a vector composition that may comprise i) a first vector that may comprise a double-stranded cargo nucleic acid, and ii) a second vector that may encode and express a transposase, wherein transposase is able to recognize and transpose the double-stranded cargo nucleic acid. The cargo nucleic acid may contain a ubiquitous chromatin opening element (UCOE) for stable expression of a gene of interest (GOI) in the cargo nucleic acid, and a pair of transposon derived inverted terminal repeats (ITRs) for interaction with the transposase and transposition of the cargo nucleic acid.BACKGROUND OF THE INVENTION

[0005] There is a widespread demand for stable and efficient expression of recombinant proteins in the biopharmaceutical industry.

[0006] Creation of stable cell lines is one of the key steps in successful recombinant protein expression. During the traditional process of generating stable cell populations, DNA fragments containing the target protein coding sequence(s) are randomly inserted into the host cell genome with a low integration probability. Despite a long selection time, the obtained cells may produce proteins at a low level.

[0007] The engineered transposon may be used as a vector to introduce foreign sequences into host cells, and ease the stable cell line generation process by increasing the foreign sequence integration efficiency. DNA transposons are repetitive sequences naturally present in the genomes of animal cells. The DNA transposon usually consists of a transposase gene flanked by two inverted terminal repeats (ITRs). The transposase may recognize the two ITRs and cleave the transposon from the genome, and later integrate the cleaved transposon into a new genomic location. The transposase gene may be replaced with a gene of interest (GOI) in an engineered transposon, which may be introduced to cells with a transposase-expressing vector, enabling efficient integration of the GOI into the cell genome. Currently, the transposon systems derived from Sleeping Beauty and piggyBac are the most promising ones for e.g., gene transfer, persistent heterologous protein expression and even gene therapy, and the piggyBac derived transposon system may have higher transposition activity and usually leave no 3 bp footprints after excision, thus causing less genomic damage, as compared to the Sleeping Beauty derived transposon system (Wu SC etal., (2006) piggyBac is a flexible and highly active transposon as compared to sleeping beauty, Tol2, and Mosl in mammalian cells. Proc Natl Acad Sci USA 103:15008-13; Wang W et al., (2008) Chromosomal transposition of Piggy Bac in mouse embryonic stem cells. Proc Natl Acad Sci USA 105:9290-5). In particular, the piggyBac transposition system can transfer GOI-containing sequences between genomes and plasmids via a "cut and paste" mechanism, precisely inserting the GOI into TTAA chromosomal sites.

[0008] Another challenge for heterologous protein expression is the unstable production levels, often due to e.g., the cytosine methylation or histone posttranslational modification-mediated epigenetic silencing of the GOI integrated into the host cell genome. The production instability is observed in up to 63% of therecombinant Chinese hamster ovary (CHO) cell lines. Multiple gene regulatory elements that may reduce or eliminate repressive chromatin, including ubiquitous chromatin opening elements (UCOE), stabilizing anti-repressor elements (STAR), Scaffold or matrix attachment regions (S / MAR) and the DNase I hypersensitive site 4 insulator of the chicken beta-globin locus control region (cHS4), were tested in parallel for their ability to confer sustained transgene expression, and a UCOE was found to improve the transgene expression to a greater extent (Saunders F et al., (2015) Chromatin function modifying elements in an industrial antibody production platform comparison of UCOE, MAR, STAR and cHS4 elements. PLoS One 10(4):e0120096). The UCOE was initially discovered at the HNRPA2B1-CBX3 locus in human cells, the promoter regions of which are encompassed by extensive unmethylated CpG islands. The UCOE can overcome gene silencing by preventing methylation of surrounding sequences and maintaining the chromatin in an open, transcriptionally active state. Currently, the UCOEs are increasingly used in cell and gene therapy as well as recombinant protein expression, and the commonly used UCOEs include the human A2UCOE and mouse Rps3UCOE.

[0009] Some commercial expression vectors have been reported to use the piggyBac transposon system or UCOE elements individually. However, more than 15 days are required for selection of stable cell pools transfected with these expression vectors, and the protein yields are not very high.

[0010] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.SUMMARY OF THE INVENTION

[0011] The inventors of the present application have designed a vector composition that contains i) a vector containing a double-stranded cargo nucleic acid which contains i-1) one or more UCOEs each located upstream of an expression cassette and i-2) a pair of transposon derived inverted terminal repeats (ITRs) located at the ends, and ii) a vector for expressing a transposase that is able to recognize and transpose the doublestranded cargo nucleic acid. A UCOE upstream of the expression cassette of a gene of interest (GOI) surprisingly accelerated the stable cell selection timeline as compared to the cargo nucleic acid containing the transposon derived ITRs but lacking UCOEs. Further, the presence of one UCOE upstream of the GOI expression cassette and one UCOE upstream of the expression cassette of a selectable marker in the cargo nucleicacid unexpectedly shortened the time required for selection of stable cell lines and / or increased the GOI expression as compared to the cargo nucleic acid containing the transposon derived ITRs but no UCOEs, and also the cargo nucleic acid containing two UCOEs but no transposon derived ITRs.

[0012] Without wishing to be bound by theory, the inventors of the present application believe that the UCOE located right before or after the GOI expression cassette may prevent methylation-induced silencing of the elements in the GOI expression cassette, including the promoter, the GOI sequence and the poly(A) tail-encoding sequence, if absent, thereby rendering sustained and stable expression of the GOI. As far as the inventors know, no one has ever used a UCOE upstream of and right after the poly(A) tail-encoding sequence to enable stable expression of the poly(A) tail before the effective filing date of the present application. The inventors of the application also believe that the UCOE right before a selectable marker expression cassette may prevent methylation-induced silencing of the promoter and the selectable marker gene in this cassette, thereby significantly reducing the time required for stable cell pool selection. And it is believed that no one has ever used a UCOE to prevent silencing of a selectable marker before the effective filing date of the present application.

[0013] Therefore, in a first aspect, the present application provides a vector composition, which may comprise: i) a first vector that may comprise a double-stranded cargo nucleic acid, wherein the double-stranded cargo nucleic acid may comprise a first expression cassette of a gene of interest, a second expression cassette of a selectable marker, a ubiquitous chromatin opening element (UCOE) that may be situated upstream of and right before the first expression cassette or the second expression cassette, and a pair of transposon derived inverted terminal repeats (ITRs), wherein the first expression cassette, the second expression cassette and the UCOE may be operably connected to each other and flanked by the pair of transposon derived ITRs, and ii) a second vector that may encode and express a transposase, wherein the transposase may recognize the pair of transposon derived ITRs in the first vector and transpose the double-stranded cargo nucleic acid.

[0014] The first vector may be a DNA vector, e.g., a double-stranded DNA vector. The first vector may be a linear or circular vector. The first vector may be a plasmid vector, a bacterial vector, or a viral vector (e.g., an adenoviral vector, an adenovirus- related virus). In certain embodiments, the first vector may be a plasmid vector.

[0015] The double-stranded cargo nucleic acid may be 5000 to 11000 bp in size.

[0016] The first expression cassette may comprise i) a promoter, ii) a gene of interest coding for a biomolecule, e.g., a protein or an RNA (e.g., a non-coding RNA), or alternatively a restriction site for inserting a target sequence, and optionally iii) apoly(A) tail-encoding sequence, in said order. The first expression cassette may comprise a first strand that may comprise, from 5’ end to 3’ end. i) a promoter, ii) a gene of interest coding for a biomolecule, e.g., a protein or an RNA, or alternatively a restriction site, and optionally iii) a poly(A) tail-encoding sequence, and a second strand complementary to the first strand. The gene of interest coding for a protein may be an open-reading frame (ORF). The promoter may be a CMV promoter, a Ubc promoter or a SV40 promoter. In certain embodiments, the promoter may be a CMV promoter comprising the nucleic acid sequence of e.g., SEQ ID NO: 10. The restriction site may be a multiple restriction site. The restriction site may be used to insert a gene of interest. The biomolecule may be a pharmaceutical agent, e.g., a fusion protein, an antibody or an antigen-binding portion thereof, or a non-coding RNA.

[0017] The second expression cassette may comprise i) a promoter, ii) a selectable marker, or alternatively a restriction site for inserting a selectable marker gene, and optionally iii) a poly(A) tail-encoding sequence, in said order. The second expression cassette may comprise a first strand that may comprise, from 5’ end to 3' end, i) a promoter, ii) a selectable marker, or alternatively a restriction site, and optionally iii) a poly(A) tail-encoding sequence, and a second strand complementary to the first strand. The promoter may be a SV40 promoter, a CMV promoter or a Ubc promoter. In certain embodiments, the promoter may be a SV40 promoter comprising the nucleic acid sequence of e.g.. SEQ ID NO: 12. In certain embodiments, the promoter may be a CMV promoter comprising the nucleic acid sequence of e.g., SEQ ID NO: 10. The selectable marker may be a gene that may provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients lacked in or absent from the culture media. The selectable marker may be a gene that confers an ability to utilize a certain carbohydrate or amino acid supply, or a drug resistance gene (e.g., an antibiotics resistance gene). In certain embodiments, the selectable marker may be a DNA fragment coding for glutamine synthetase, such as an open reading frame for encoding glutamine synthetase. The restriction site may be a multiple restriction site. The restriction site may be used to insert a selectable marker.

[0018] The double-stranded cargo nucleic acid may, in certain embodiments, comprisea UCOE upstream of and right before the first expression cassette and a UCOE upstream of and right before the second expression cassette. In certain embodiments, the double-stranded cargo nucleic acid may comprise a UCOE upstream of and right before the first expression cassette, and a UCOE downstream of and right after the first expression.

[0019] The double-stranded cargo nucleic acid may further comprise an additional expression cassette of a gene of interest. In certain embodiments, the double-stranded cargo nucleic acid may further comprise one or more (e.g., 1, 2, or 3) additional expression cassettes of a gene of interest. The additional expression cassette(s) may each comprise i) a promoter, ii) a gene of interest coding for a biomolecule, e.g., a protein or an RNA (e.g., a non-coding RNA). or alternatively a restriction site for inserting a target sequence, and optionally iii) a poly(A) tail-encoding sequence, in said order. The additional expression cassette(s) may each comprise a first strand that may comprise, from 5’ end to 3’ end, i) a promoter, ii) a gene of interest coding for a biomolecule, e.g., a protein or an RNA, or alternatively a restriction site, and optionally iii) a poly(A) tail-encoding sequence, and a second strand complementary to the first strand. The gene of interest coding for a protein may be an open-reading frame (OFR). The promoter may be a CMV promoter, a SV40 promoter, or a Ubc promoter. In certain embodiments, the promoter may be a CMV promoter comprising e.g., the nucleic acid sequence of SEQ ID NO: 10. The first vector may further comprise a UCOE upstream of and right before an additional expression cassette of a gene of interest. In certain embodiments, the first vector may further comprise a UCOE upstream of and right before each additional expression cassette of a gene of interest.

[0020] The double-stranded cargo nucleic acid may further comprise an additional expression cassette of a selectable marker. In certain embodiments, the double-stranded cargo nucleic acid may further comprise one or more (e.g., 1, 2, or 3) additional expression cassettes of a selectable marker. The additional expression cassette(s) of a selectable marker may each comprise i) a promoter, ii) a selectable marker, or alternatively a restriction site for inserting a selectable marker gene, and optionally iii) a poly(A) tail-encoding sequence, in said order. The additional expression cassette(s) of a selectable marker may each comprise a first strand that may comprise, from 5’ end to 3’ end, i) a promoter, ii) a selectable marker, or alternatively a restriction site, and optionally iii) a poly(A) tail-encoding sequence, and a second strand complementary to the first strand. The selectable marker may be an open-reading frame (OFR). Theselectable marker may be a gene that may provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. The selectable marker may be a resistance gene that confers an ability to utilize a certain carbohydrate or amino acid supply, or a drug resistance gene (e.g., an antibiotics resistance gene). In certain embodiments, the selectable marker may be different from the selectable marker mentioned above in the second expression cassette. The promoter may be a SV40 promoter, a CMV promoter or a Ubc promoter. In certain embodiments, the promoter may be a SV40 promoter comprising the nucleic acid sequence of e.g., SEQ ID NO: 12. In certain embodiments, the promoter may be a CMV promoter comprising e.g., the nucleic acid sequence of SEQ ID NO: 10. The first vector may further comprise a UCOE upstream of and right before an additional expression cassette of a selectable marker. In certain embodiments, the first vector may further comprise a UCOE upstream of and right before each additional expression cassette of a selectable marker.

[0021] The UCOE situated upstream of and right before the expression cassette, including those situated upstream of and right before the first expression cassette, the second expression cassette, the additional expression cassette of a gene of interest, and the additional expression cassette of a selectable marker, in the first vector may be derived from A2UCOE. TBP / PSMB1, SURF1 / SURF2 or Rps3 UCOE. In certain embodiments, the UCOE may be derived from A2UCOE. In certain embodiments, the UCOE may be derived from Rps3 UCOE. In certain embodiments, the UCOE may comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99% or 100% sequence identity to SEQ ID NO: 1. In certain embodiments, the UCOE may comprise the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the UCOE may comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 2. In certain embodiments, the UCOE may comprise the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the UCOE may comprise an amino acid sequence having at least 90%, 91%, 92%, 93%. 94%. 95%. 96%. 97%. 98%. 99% or 100% sequence identity to SEQ ID NO: 3. In certain embodiments, the UCOE may comprise the amino acid sequence of SEQ ID NO: 3. When the double-stranded cargo nucleic acid comprises more than one UCOE. these UCOEs may be the same or different between or among each other.

[0022] The transposase expressed by the second vector may recognize the pair oftransposon derived ITRs in the first vector and transpose the double-stranded cargo nucleic acid to e.g., a target nucleic acid locus. The pair of transposon derived ITRs in the first vector and the transposase expressed by the second vector may be derived from a same transposon, such as a same DNA transposon, e.g., piggyBac transposon or Sleeping Beauty transposon. In certain embodiments, the pair of transposon derived ITRs in the first vector and the transposase expressed by the second vector may be derived from piggyBac transposon. In certain embodiments, the pair of transposon derived ITRs comprise amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 4 and 5, respectively. In certain embodiments, the pair of transposon derived ITRs comprise the amino acid sequences of SEQ ID NOs: 4 and 5, respectively.

[0023] The second vector may be a DNA vector or an RNA vector. The second vector may be a linear or a circular vector. The second vector may be an mRNA, a circular RNA, a plasmid vector, a bacterial vector, or a viral vector (e.g., an adenoviral vector, an adenovirus-related virus). In certain embodiments, the second vector may be an mRNA which may comprise, from 5’ end to 3’ end, a 5’ cap, a 5’UTR, an open reading frame encoding a transposase, a 3’UTR, and a poly(A) tail. The transposase may be derived from a DNA transposon, including, but not limited to, piggyBac transposon, and Sleeping Beauty transposon. In certain embodiments, the transposase may be derived from piggyBac transposon. In certain embodiments, the transposase may comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 6. In certain embodiments, the transposase may comprise the amino acid sequence of SEQ ID NO: 6. The 5'UTR and the 3’UTR may comprise the amino acid sequences of SEQ ID NOs: 14 and 15, respectively.

[0024] The first expression cassette, the second expression cassette, and optionally the additional expression cassette(s) of a gene of interest or a selectable marker, may be arranged in any order. In certain embodiments, the first expression cassette and the optional additional expression cassette(s) of a gene of interest may the located upstream of the second expression cassette and the optional additional expression cassette(s) of a selectable marker.

[0025] The first vector, in certain embodiments, may comprise 5’ transposon derived ITR, a UCOE, a first expression cassette of a gene of interest, a second expressioncassette of a selectable marker, and 3‘ transposon derived ITR, in said order, wherein these elements are operably connected to each other. The first vector may comprise a first strand that may comprise, from 5’ end to 3’ end, 5’ transposon derived ITR, a UCOE, a first expression cassette of a transgene, a second expression cassette of a selectable marker, and 3’ transposon derived ITR, and a second strand that may be complementary to the first strand.

[0026] The first vector, in certain embodiments, may comprise 5’ transposon derived ITR, a UCOE, a first expression cassette of a gene of interest, an additional expression cassette of a gene of interest, a second expression cassette of a selectable marker, and 3’ transposon derived ITR, in said order, wherein these elements are operably connected to each other. The first vector may comprise a first strand that may comprise, from 5’ end to 3’ end, 5’ transposon derived ITR, a UCOE, a first expression cassette of a transgene, an additional expression cassette of a gene of interest, a second expression cassette of a selectable marker, and 3’ transposon derived ITR, and a second strand that may be complementary to the first strand.

[0027] The first vector, in certain embodiments, may comprise 5’ transposon derived ITR, a UCOE, a first expression cassette of a gene of interest, a UCOE, a second expression cassette of a selectable marker, and 3’ transposon derived ITR, in said order, wherein these elements are operably connected to each other. The first vector may comprise a first strand that may comprise, from 5’ end to 3’ end, 5’ transposon derived ITR, a UCOE, a first expression cassette of a gene of interest, a UCOE, a second expression cassette of a selectable marker, and 3’ transposon derived ITR, and a second strand that may be complementary to the first strand.

[0028] The first vector, in certain embodiments, may comprise 5’ transposon derived ITR, a UCOE, a first expression cassette of a gene of interest, a UCOE, an additional expression cassette of a gene of interest, a UCOE, a second expression cassette of a selectable marker, and 3’ transposon derived ITR, in said order, wherein these elements are operably connected to each other. The first vector may comprise a first strand that may comprise, from 5’ end to 3’ end. 5‘ transposon derived ITR. a UCOE, a first expression cassette of a gene of interest, a UCOE, an additional expression cassette of a gene of interest, a UCOE, a second expression cassette of a selectable marker, and 3’ transposon derived ITR, and a second strand that may be complementary to the first strand.

[0029] In a second aspect, the disclosure provides the use of the vector compositionof the disclosure in generating a cell line.

[0030] Specifically the disclosure may provide a method for generating a cell line, comprising the steps of i) providing a first vector of the disclosure, and optionally inserting a gene of interest (GOI) or a selectable marker at a restriction site in the first vector as required, ii) providing a second vector of the disclosure, iii) introducing or delivering the first vector and the second vector into a cell or cell population, and iv) culturing the cell or cell population under a condition for selection, to obtain cells or cell populations having the double-stranded cargo nucleic acid of the first vector integrated into the cell genome(s).

[0031] The method may further comprise step v) of testing the cells or cell populations obtained in step iv) for GOI expression level.

[0032] The method may further comprise step vi) of selecting the cells or cell populations with high GOI expression level, based on the results of step v).

[0033] The step of inserting a gene of interest or a selectable marker at a restriction site may be done with a restriction enzyme.

[0034] The step of introducing or delivering the first vector and the second vector into a cell or cell population may be done with e.g., calcium phosphate, liposome, polymers, nanoparticles, electroporation, or microinjection.

[0035] The step of introducing or delivering the first vector and the second vector into a cell or cell population may comprise introducing or delivering the first vector and the second vector in a 10: 1 to 1: 1 weight ratio. In certain embodiments, the step of introducing or delivering the first vector and the second vector into a cell or cell population may comprise introducing or delivering a plasmid vector as the first vector and an mRNA as the second vector in a 10: 1 weight ratio.

[0036] The step of culturing the cell or cell population under a condition for selection may comprise culturing the cell or cell population in a medium containing a selection agent, e.g.. an antibiotic, a drug, or a toxin. In certain embodiments, the first vector may comprise a DNA fragment coding for glutamine synthetase, such as an open reading frame for encoding glutamine synthetase, as the selectable marker, and the step of culturing the cell or cell population under a condition for selection may comprise culturing the cell or cell population in a medium containing a glutamine synthetase inhibitor, e.g., methionine sulfoximine (MSX).

[0037] The cell or cell population used in step iii) may be a mammalian cell or a mammalian cell population. In certain embodiments, the cell or cell population may be e.g., a Chinese hamster ovary (CHO) cell or a CHO cell population. In certain embodiments, the cell or cell population may be a suspension-adapted CHO cell or a suspension-adapted CHO cell population.

[0038] The method may reduce or shorten the time step iv) requires to obtain the table cell lines. In certain embodiments, the method may reduce the time step iv) requires to obtain the stable cell lines by up to 66.7% compared to a method using a vector containing neither transposon derived ITRs nor UCOEs.

[0039] In a third aspect, the disclosure provides the use of the vector composition of the disclosure in producing biomolecules.

[0040] Specifically, the disclosure may provide a method for producing a biomolecule, comprising the steps of: i) obtaining cells or cell populations having a double-stranded cargo nucleic acid of a first vector of the disclosure integrated into the cell genome(s), according to the method of the disclosure for generating a cell line, wherein the double-stranded cargo nucleic acid in the first vector may comprise a gene coding for a biomolecule as the gene of interest in the first expression cassette, ii) optionally testing the cells or cell populations obtained in step i) for the gene of interest expression level, to obtain cells or cell populations with high gene of interest expression level, and iii) culturing the cells or cell populations under a condition for production of the biomolecule.

[0041] Step iii) may comprise culturing the cell or cell population via fed-batch culture.

[0042] The method may comprise: i) providing a first vector of the disclosure, and optionally inserting a gene of interest or a selectable marker at a restriction site in the first vector as required, ii) providing a second vector of the disclosure, iii) introducing or delivering the first vector and the second vector into a cell or cell population, iv) culturing the cell or cell population under a condition for selection, to obtain cells or cell populations having the double-stranded cargo nucleic acid of the first vector integrated into the cell genome(s), v) optionally testing the cells or cell populations obtained in step iv) for the geneof interest expression level, to obtain cells or cell populations with high gene of interest expression level, and vi) culturing the cells or cell populations under a condition for production of the biomolecule.

[0043] The method may comprise producing one or more biomolecules. The method may comprise producing one biomolecule by using a first vector comprising a first expression cassette comprising a gene of interest coding for a biomolecule. The method may comprise producing more than one biomolecules by using a first vector comprising a first expression cassette comprising a gene of interest coding for a biomolecule and one or more additionally expression cassettes of a gene of interest each comprising a gene of interest coding for the additional biomolecule(s).

[0044] Step vi) may comprise culturing the cell or cell population via fed-batch culture.

[0045] The method may increase the production level of the biomolecule. In certain embodiments, the method may use fed-batch culture and increase the production level of the biomolecule by up to 250% compared to a method using a vector containing neither transposon derived ITRs nor UCOEs.

[0046] The biomolecules as produced may be used for e.g., investigational new drug (IND) application and clinical trials. The method of the disclosure may be used for e.g., clinical batch production and commercial-scale production.

[0047] This invention combines the UCOE with the PiggyBac transposon system, leveraging the advantages of both to achieve better production yields compared to using UCOE or PiggyBac transposon system alone, and shortening the screening timeline of the stable cell pools.

[0048] Other features and advantages of the instant disclosure will be apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all references, Genbank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0049] Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meetthe written description and enablement requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53(c) EPC and Rule 28(b) and (c) EPC. All rights to explicitly disclaim any embodiments that are the subject of any granted patent(s) of applicant in the lineage of this application or in any other lineage or in any prior filed application of any third party is explicitly reserved. Nothing herein is to be construed as a promise.

[0050] It is noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of' and "consists essentially of' have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.DESCRIPTION OF THE DRAWINGS

[0051] The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

[0052] FIG. 1 is a schematic drawing of cargo nucleic acids containing a pair of transposon derived ITRs and two UCOEs (A), two UCOEs but no transposon derived ITRs (B), a pair of transposon derived ITRs but no UCOEs (C) or with no transposon derived ITRs or UCOEs (D), to be ligated into a plasmid.

[0053] FIG. 2 shows the time for selection of stable cell lines transfected with the plasmids containing UCOEs and / or a pair of transposon derived ITRs.

[0054] FIG. 3 shows the concentrations of heterologous proteins as expressed by fed- batch cultured CHO cells transfected with the plasmids containing UCOEs and / or a pair of transposon derived ITRs.

[0055] FIG. 4 show s the time for selection of stable cell lines transfected with the plasmids containing human or murine derived UCOEs and / or a pair of transposon derived ITRs.

[0056] FIG. 5 shows the concentrations of heterologous proteins as expressed by fed-batch cultured CHO cells transfected with the plasmids containing human or murine derived UCOEs and / or a pair of transposon derived ITRs.

[0057] FIG. 6 is a schematic drawing of cargo nucleic acids containing no transposon derived ITRs or UCOEs (A), a pair of transposon derived ITRs but no UCOEs (B), a pair of transposon derived ITRs and one UCOE (C), or a pair of transposon derived ITRs and two UCOEs (D), to be ligated into a plasmid.

[0058] FIG. 7 shows the time for selection of stable cell lines transfected with the plasmids containing one or two UCOEs and / or a pair of transposon derived ITRs.

[0059] FIG. 8 shows the concentrations of heterologous proteins as expressed by fed- batch cultured CHO cells transfected with the plasmids containing one or two UCOEs and / or a pair of transposon derived ITRs.

[0060] FIG. 9 shows the time for selection of stable cell lines transfected with the plasmids containing UCOEs and / or a pair of transposon derived ITRs.

[0061] FIG. 10 shows the concentrations of heterologous antibodies as expressed by fed-batch cultured CHO cells transfected with the plasmids containing UCOEs and / or a pair of transposon derived ITRs.DETAILED DESCRIPTION OF THE INVENTION

[0062] The present disclosure is now further described with the non-limiting examples below.

[0063] As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including", "includes", "having", "has", "with", or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising".

[0064] The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one or more than one standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0065] The term “vector” refers to a substance that is used to carry or include a nucleic acid sequences, including for example, in order to introduce a nucleic acid sequence into a host cell and optionally express a nucleic acid sequence in the host cell. Vectorsapplicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, to provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more nucleic acid molecules are to be co-expressed, both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art. Particularly, an “expression vector” is associated with actual expression of a coding DNA fragment into an mRNA and a protein in the target cell or organism, and usually contains all the regulatory sequences, such as promoter, ribosomal binding site, transcription initiation site, translation initiation site, which are essential for getting maximum expression. The first vector of the disclosure may be a DNA vector that is used to deliver the cargo nucleic acid into a host cell, and / or replicate the cargo nucleic acid. The second vector of the disclosure may be a DNA vector that is used to deliver a transposase-encoding fragment into a host cell and express the transposase in a host cell. The second vector of the disclosure may be an RNA vector, e.g., an mRNA. that is used to carry a transposase-encoding fragment into a host cell and express the transposase in a host cell. The mRNA may be a safer vector than DNA as mRNA is a transient molecule and does not interact w ith the host genome. The vector may be linear or circular, singlestranded or double stranded.

[0066] The term “cargo nucleic acid” or “double-stranded cargo nucleic acid” refers to a nucleic acid fragment that is located in the vector, specifically the first vector, andto be delivered into a host cell with the vector and then integrated into the host cell genome.

[0067] The term “expression cassette” refers to a combination of nucleic acid sequences or elements that are expressed together or are operably linked for expression. In some embodiments, an expression cassette refers to the combination of regulatory- elements (e.g., a promoter), a gene or genes to which they are operably linked for expression, and optionally a poly(A) tail-encoding sequence.

[0068] The term “gene of interest”, “GOI” or “transgene” refers to a piece of genetic material that is to be transposed to and expressed in a certain organism. More specifically, the gene of interest is a DNA fragment that is to be delivered into a host cell and integrated into the host cell genome, to render its expression in the host cell. The gene of interest may code for an RNA (e.g., a non-coding RNA) or a protein.

[0069] The term “promoter” as used herein, generally refers to the regulatory DNA region which controls transcription or expression of a gene and which may be located adjacent to or overlapping a nucleotide or region of nucleotides at which RNA transcription is initiated. A promoter may contain specific DNA sequences which bind protein factors, often referred to as transcription factors, which facilitate binding of RNA polymerase to the DNA leading to gene transcription.

[0070] The term "expression", as used herein, generally refers to the process by which a nucleic acid sequence or a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0071] The term “open reading frame” or “ORF” refers to a DNA or RNA fragment that consists of a series of codons that specify the amino acid sequence of a protein the DNA or RNA fragment codes for, usually starting with a start codon and ending with a downstream in-frame stop codon.

[0072] The term “ubiquitous chromatin opening element” or “UCOE” refers to a genomic DNA fragment that is able to consistently confer stable, site of integrationindependent GOI expression that is proportional to copy number, including from within regions of heterochromatin. The term “heterochromatin” refers to a form of chromatin that is densely packed and found in e.g.. the nucleus of eukaryotic cells. It is in such a condensed structure that it does not enable DNA and RNA polymerases to access theDNA, therefore preventing DNA replication and transcription.

[0073] The term "upstream" or “upstream of’ refers to a DNA fragment 5’ to a given reference point on the DNA, i.e., the DNA fragment is close to the 5’ end than the reference point. The term “right before” refers to a DNA fragment located upstream of and adjacent to a given reference point on the DNA. The UCOE arranged upstream of and right before an expression cassette means the UCOE is located 5’ to and adjacent to the promoter of the expression cassette. The term “downstream” or “downstream of’ refers to a DNA fragment 3’ to a given reference point on the DNA, i.e., the DNA fragment is close to the 3’ end than the reference point. The term “right after” refers to a DNA fragment located downstream of and adjacent to a given reference point on the DNA.

[0074] The term “transposon” is a kind of transposable element that is able to move within the genome, or move between the genome and vectors, with the transposase. There are two types of transposon, the RNA transposon which functions via reverse transcription of an RNA intermediate, and the DNA transposon which generally move by a cut-and-paste mechanism in which the transposon is excised from one location and reintegrated elsewhere. A DNA transposon usually consists of a transposase gene flanked by two inverted terminal repeats (ITRs), referred to as 5’ ITR and 3’ ITR respectively. The transposase may recognize the t o ITRs, cleave the transposon and integrate the cleaved transposon into a nucleic acid locus. The “3' end” of a nucleic acid refers the end having a free hydroxy group which is on the right-hand side of a sequence, w hile the “5' end” of a nucleic acid refers to the end having a free phosphate group which is on the left-hand side of a sequence.

[0075] The term “selectable marker”, “selective gene” or “selectable marker gene” may refer to the gene that can for example provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Examples of the selectable marker include the antibiotic resistance genes, and a gene coding for glutamine synthetase.

[0076] As used herein, “operably connected” or “operably linked” generally refer to juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a UCOE, which may prevent methylation of surrounding sequences and maintain the chromatin in an open, transcriptionally active state, can be operatively linked to an expression cassette to promote expression of the gene in the expression cassette. There may be interveningresidues between these elements so long as this functional relationship is maintained.

[0077] The term ”poly(A) tail” refers to a long chain of adenine nucleotides that is added to a RNA, especially a messenger RNA (mRNA) molecule to increase the stability of the RNA molecule, while “a poly(A) tail-encoding sequence” is a nucleotide sequence that codes for the poly(A) tail, including a sequence containing continuous adenine nucleotides, and a sequence containing several continuous adenine nucleotide- containing fragments with a short intervening nucleotide segment between each two continuous adenine nucleotide-containing fragments.

[0078] The term “introducing. . . into a cell” or “introduction.. . into a cell” refers to the entry of a substance, e.g., a vector or a vector composition of the disclosure, into a cell through the cell membrane by any means such as transfection.

[0079] The terms percent “identity” or “sequence identity” as used herein in the context of two or more nucleic acids or proteins / peptides, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to. BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof.

[0080] One nucleic acid strand “complementary to” to another nucleic acid strand means the two strands, when aligned anti-parallel to each other, the nucleotide bases pair according to the A-T. C-G and A-U pairing rule. The term “substantially complementary to” means more than 85%, 90% or 95% of the bases pair between two strands, while “completely complementary to” means almost 100%, e.g., 98% or 99%, or 100% of the bases pair between two strands.

[0081] Amongst the most important outputs of the biopharmaceutical industry are recombinant proteins, the stable and efficient expression of which are needed.

[0082] The product on of recombinant proteins begins with transfection of host cells with vectors carrying a recombinant protein-encoding fragment. The transfection can be classified into two types, namely stable and transient transfection. Stable transfection refers to sustaining long-term expression of a gene of interest by integrating the gene of interest into the host cell genome, while transient transfection does notrequire integrating nucleic acids into the host cell genome. The generation of stable cell lines offers several advantages, including improved batch-to-batch product consistency and favorable growth profiles, but also face some challenges such as a long time it takes for cell selection.

[0083] The engineered transposon system may be used as a vector to introduce foreign sequences into host cells, and ease the stable cell line generation process by increasing the foreign sequence integration efficiency. There are two classes of transposons, the RNA transposon (or retrotransposon) and the DNA transposon. Retrotransposons employ an RNA intermediate that is first reverse transcribed into a complementary single-stranded DNA strand by a reverse transcriptase encoded by the retrotransposon, and then the single-stranded DNA is converted into a double-stranded DNA that later integrates into the host cell genome. This results several new copies of retrotransposons expanding throughout the host genome, which may create genomic instability. The DNA transposons translocate via a “non-replicative mechanism.” whereby two Terminal Inverted Repeats (ITRs) are recognized and cleaved by a transposase enzyme, releasing the cognate DNA transposons with free DNA ends, and the excised DNA transposons then integrate into a new genomic region where target sites are recognized and cut by the same transposase (Tipanee J etal., (2017) Transposons: Moving Forward from Preclinical Studies to Clinical Trials. Hum Gene Ther. 28(11): 1087-1104).

[0084] Therefore, the DNA transposon, having lower risk of causing host cell genome instability, may be engineered to introduce a gene of interest into host cells. The DNA transposon usually consists of a transposase gene flanked by two inverted terminal repeats (ITRs). The transposase may recognize the two ITRs and cleave the transposon from the genome, and later integrate the cleaved transposon into a new genomic location. A two-component transposon system can be created by separating the transposase gene from the pair of ITRs. In particular, the transposase gene may be replaced with a gene of interest (GOI) in an engineered transposon system, which maybe introduced to cells with a transposase-expressing vector, enabling efficient integration of the GOI into the cell genome. Expressing the transposase from a plasmid vector may provide sustained transposase expression at a low level which may result in continuous transposon mobilization and integration, causing genome instability-. An mRNA may be a safer vector to deliver the transposase gene and express the transposase, which may be shortly degraded in the host cell after the transposition of the GOI. The short-term presence of the mRNA and thus the transposase enables thetransposition of the GOI into the host cell genome with low risk of causing genome instability.

[0085] Currently, the transposon systems derived from Sleeping Beauty and piggyBac are the most promising ones for e.g., gene transfer, persistent heterologous protein expression and even gene therapy. The piggyBac derived transposon system may have higher transposition activity and usually leave no 3 bp footprints after excision, thus causing less genomic damage, as compared to the Sleeping Beauty derived transposon system, the piggyBac transposition system can precisely insert the GOI into TTAA chromosomal sites.

[0086] Another challenge for heterologous protein expression is the unstable production levels, often due to e.g., the cytosine methylation or histone posttranslational modification-mediated epigenetic silencing of the GOI integrated into the host cell genome. CHO cells are the most widely used mammalian platform, providing 84% of monoclonal antibodies in 2018. However, up to 63 % of the recombinant Chinese hamster ovary (CHO) cell lines have production instability issue, the impact of this problem might be substantial. The production instability is highly unpredictable, requiring lengthy stability' studies, which are labor-intensive and costly.

[0087] The epigenetic silencing can be ameliorated by genetic elements, known as insulators, which can stop the spread of repressive chromatin. Multiple types of gene regulatory elements have been tested for their ability to sustain gene of interest expression, including ubiquitous chromatin opening elements (UCOE), stabilizing antirepressor elements (STAR), Scaffold or matrix attachment regions (S / MAR) and the DNase I hypersensitive site 4 insulator of the chicken beta-globin locus control region (cHS4), a UCOE was found to improve the gene of interest expression to a greater extent than any other genetic element as tested.

[0088] UCOE is a genomic DNA fragment that is encompassed by extensive unmethylated CpG islands and is able to consistently confer stable, site of integrationindependent gene of interest expression, including from within regions of heterochromatin. The UCOEs have become popular for biomanufacturing applications, as a way to reduce production instability.

[0089] Four fragments of DNA have been classified as UCOEs to date: human TBP / PSMB1 UCOE. human A2UCOE, human SURF1 / SURF2 UCOE, and the murine Rps3 UCOE. The four UCOEs all have CpG islands, and the modification state of histones at UCOEs may be important for the expression stability (Feichtinger J et al.,(2016) Comprehensive genome and epigenome characterization of CHO cells in response to evolutionary pressures and over time. Biotechnol Bioeng 113(10):2241-53; Hernandez I et al., (2019) Epigenetic regulation of gene expression in Chinese Hamster Ovary cells in response to the changing environment of a batch culture. Biotechnol Bioeng 116(3):677-92). However, the efficiency of the UCOEs may be dependent on the promoter in vicini ty. The 1.5 kb A2UCOE fragment outperformed the Rsp3 UCOE in protein expression when used in combination with the mCMV, rCMV or Ubc promoters, but the increased productivity was not observed when A2UCOE was combined w ith the gCMV or SRa promoters (Nair AR et al., (2011) Effect of different UCOE-promoter combinations in creation of engineered cell lines for the production of Factor VIII. BMC Res Notes 10(4): 178).

[0090] The inventors of the application used the UCOE in the binary transposon system, and found the combination of the UCOE and the transposon system surprisingly shortened the time as required for selection of stable cell lines and / or increased the gene of interest expression by the selected stable cell lines.

[0091] Specifically, the inventors constructed a transposon system which contains a cargo nucleic acid-containing vector and a transposase-encoding vector, wherein the cargo nucleic acid-containing vector may contain 5’ transposon derived ITR, a UCOE, an expression cassette of a gene of interest, an expression cassette of a selectable marker, and 3?transposon derived ITR, in said order. The UCOE upstream of the expression cassette of the gene of interest surprisingly accelerated the stable cell selection timeline as compared to the cargo nucleic acid containing the transposon derived ITRs but lacking UCOEs. The expression cassette of the selected marker, although located far away from the UCOE, may have been affected by the UCOE and thus promoted selectable marker expression. Alternatively, the cargo nucleic acid-containing vector may contain 5’ transposon derived ITR, a UCOE, an expression cassette of a gene of interest, a UCOE, an expression cassette of a selectable marker, and 3’ transposon derived ITR, in said order. The presence of one UCOE upstream of the gene of interest expression cassette and one UCOE upstream of the expression cassette of a selectable marker unexpectedly shortened the time required for selection of stable cell lines and / or increased the GOI expression as compared to the cargo nucleic acid containing the transposon derived ITRs but no UCOEs, and also the cargo nucleic acid containing tw o UCOEs but no transposon derived ITRs. The two UCOEs located before and after the gene of interest expression cassette may have prevented the methylation-inducedsilencing of the promoter at the 5' end of the expression cassette and methylation- induced silencing of the gene of interest sequence and the poly(A) tail at the 3' end of the expression cassette.

[0092] The inventors of the application further compared the use of A2UCOE and Rps3 UCOE, it turned out that these two UCOEs provided comparable effects.

[0093] Therefore, the present application provides a vector composition, which may comprise i) a first vector that may comprise a double-stranded cargo nucleic acid, wherein the double-stranded cargo nucleic acid may comprise a first expression cassette of a gene of interest, a second expression cassette of a selectable marker, a ubiquitous chromatin opening element (UCOE) that may be situated upstream of and right before the first expression cassette or the second expression cassette, and a pair of transposon derived inverted terminal repeats (ITRs), wherein the first expression cassette, the second expression cassette and the UCOE may be operably connected to each other and flanked by the pair of transposon derived ITRs, and ii) a second vector that may encode and express a transposase. wherein the transposase may recognize the pair of transposon derived ITRs and transpose the double-stranded cargo nucleic acid.

[0094] The UCOE arranged upstream of and right before the first expression cassette may promote the expression of the gene of interest, increasing biomolecule production yield. The UCOE arranged upstream of and right before the second expression cassette may promote the expression of the selectable marker, to shorten the time required for stable cell selection. The UCOE right after the poly(A) tail-encoding sequence may enable stable expression of the poly(A) tail, further improving GOI expression levels. The UCOE may be designed to be upstream of the second expression cassette only when a quick stable cell selection is required while the gene of interest expression level is not an issue of interest.

[0095] Particularly, the double-stranded cargo nucleic acid may comprise a UCOE upstream of and right before the first expression cassette and a UCOE upstream of and right before the second expression cassette, to shorten the selection time and / or increase GOI expression.

[0096] The first vector may be a DNA vector. The first vector may be a linear or circular vector. The first vector may be a plasmid vector, a bacterial vector, or a viral vector (e.g., an adenoviral vector, an adenovirus-related virus). In certain embodiments,the first vector may be a plasmid vector.

[0097] The second vector may be a DNA vector or an RNA vector. The second vector may be a linear or a circular vector. The second vector may be a non-integrative DNA vector, e.g., a non-integrative plasmid, bacterial or viral vector (e.g., an adenoviral vector, an adenovirus-related virus). The second vector may be an mRNA, or a circular RNA. In certain embodiments, the second vector may be an mRNA, which may comprise, from 5’ end to 3’ end. a 5?cap. a 5’UTR. an open reading frame encoding a transposase, a 3’UTR, and a poly(A) tail. Short-term expression of the transposase may be required, as sustained transposase expression may lead to continuous cargo nucleic acid transposition.

[0098] The expression cassette in the first vector may each comprise a promoter, a gene (a gene of interest or a selectable marker), and an optional poly(A) tail-encoding sequence. The expression cassette in the first vector may each comprise a promoter, a restriction site, and an optional poly(A) tail-encoding sequence. The restriction site may be used to insert any gene (e.g., a gene of interest or a selectable marker) as required, with a restriction enzyme. The promoter may be a CMV promoter, a SV40 promoter, or a Ubc promoter, especially a CMV promoter.

[0099] The transposase expressed by the second vector may recognize the pair of transposon derived ITRs in the first vector and transpose the double-stranded cargo nucleic acid to e.g., a target nucleic acid locus. The pair of transposon derived ITRs in the first vector and the transposase expressed by the second vector may be derived from a same transposon, such as a same DNA transposon, e.g., piggyBac transposon or Sleeping Beauty transposon. In certain embodiments, the pair of transposon derived ITRs in the first vector and the transposase expressed by the second vector may be derived from piggyBac transposon.

[0100] The selectable marker may be a gene that confers an ability to utilize a certain carbohydrate or amino acid supply, or a drug resistance gene (e.g., an antibiotics resistance gene), allowed for positive or negative selection of cells introduced with the vector composition of the disclosure.

[0101] The application also provides the use of the vector composition of the disclosure in generating a cell line.

[0102] Specifically the disclosure may provide a method for generating a cell line, comprising the steps of:i) providing a first vector of the disclosure, and optionally inserting a gene of interest or a selectable marker at a restriction site as required, ii) providing a second vector of the disclosure, iii) introducing or delivering the first vector and the second vector into a cell or cell population, and iv) culturing the cell population under a condition for selection, to obtain the cell or cell population having the double-stranded cargo nucleic acid of the first vector integrated into the cell genome.

[0103] The step of introducing or delivering the first vector and the second vector into a cell or cell population may be done with e.g., calcium phosphate, liposome, polymers, nanoparticles, electroporation, or mi croinj ection.

[0104] The step of culturing the cell or cell population under a condition for selection may comprise culturing the cell or cell population in a medium containing a selection agent, e.g., an antibiotic, a drug, or a toxin. In certain embodiments, the first vector may comprise a DNA fragment coding for glutamine synthetase, such as an open reading frame for encoding glutamine synthetase, as the selectable marker, and the step of culturing the cell or cell population under a condition for selection may comprise culturing the cell or cell population in a medium containing a glutamine synthetase inhibitor, e g., methionine sulfoximine (MSX).

[0105] In CHO cell expression systems, the dihydrofolate reductase (DHFR) and the glutamine synthetase (GS) selection systems are the most commonly used. In cell lines deficient in the DHFR gene, e.g., CHO-DG44 cells, selection is performed in the medium without hypoxanthine and thymidine. Amplification steps through rounds of increasing concentration of methotrexate (MTX) are often included to improve productivity. However, this step increases the timeline for cell line generation. The GS selection system offers a time advantage because it requires fewer gene copies for the survival of the stable cells, and thus allow s faster selection of high producers. The GS selection system uses the essential activity of GS in catalyzing the ATP-dependent condensation of glutamate and ammonia to generate glutamine. In cell lines with sufficient level of GS, such as CHO cells, addition of the GS inhibitor, MSX allows the selection of productive stable cells (Lin PC et al., (2019) Attenuated glutamine synthetase as a selection marker in CHO cells to efficiently isolate highly productive stable cells for the production of antibodies and other biologies. MAbs. 11(5):965-976).

[0106] The method may further comprise step v) of testing the cells or cell populationsobtained in step iv) for the GOI expression level.

[0107] The cell or cell population used in step iii) may be a mammalian cell or a mammalian cell population. In certain embodiments, the cell or cell population may be e.g., a Chinese hamster ovary (CHO) cell or a CHO cell population. The cell or cell population used in step iii) may be also human embry onic kidney (HEK293), babyhamster kidney (BHK), Escherichia coli, or Sf9 cell or cell population.

[0108] The application may further provide the use of the vector composition of the disclosure in producing biomolecules.

[0109] Specifically, the disclosure may provide a method for producing a biomolecule, comprising the steps of: i) obtaining cells or cell populations having a double-stranded cargo nucleic acid of a first vector of the disclosure integrated into the cell genome, according to the method of the disclosure for generating a cell line, wherein the double-stranded cargo nucleic acid in the first vector may comprise a gene coding for a biomolecule as the gene of interest in the first expression cassette, ii) optionally testing the cells or cell populations obtained in step i) for the GOI level, to obtain cells or cell populations with high gene of interest expression level, and iii) culturing the cells or cell populations under a condition for production of the biomolecule.

[0110] Step iii) may comprise culturing the cell or cell population via fed-batch culture.

[0111] The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.EXAMPLESExample 1. Construction and characterization of vectors containing PiggyBac ITRs and two UCOEs

[0112] A vector containing an expression cassette of the gene of interest (GOI) and an expression cassette of the selectable marker flanked by two transposon-derived inverted terminal repeats (ITRs). with a UCOE upstream of each expression cassette, was constructed, and introduced into cells. The time as required to screen out the stable cell lines, and the GOI expression in these cell lines were measured, as compared to vectors that i) contained the ITRs but no UCOE, ii) contained two UCOEs but no ITRs, or iii) contained no ITRs or UCOE.

[0113] Specifically, a DNA fragment was synthesized to contain in the coding strand,from 5’ end to 3 ’end, a 5’ PiggyBac ITR (SEQ ID NO: 4). a 2.2 kb A2UCOE fragment (SEQ ID NO: 1). a CMV promoter (SEQ ID NO: 10), a sequence encoding a Fc-fusion protein (about 1300 nt in length), a polyA sequence (SEQ ID NO: 11), a 2.2 kb A2UCOE fragment (SEQ ID NO: 1), a SV40 promoter (SEQ ID NO: 12), a sequence encoding glutamine synthetase (the amino acid sequence set forth in SEQ ID NO: 13), a polyA sequence (SEQ ID NO: 11), and a 3’ PiggyBac ITR (SEQ ID NO: 5) (see FIG. 1 (A)). The DNA fragment was inserted into an in house made plasmid via CloneEZ™ Next-Generation Molecular Cloning sendee provided by Genscript. The resultant plasmid was referred to as Plasmid A.

[0114] Similarly, a DNA fragment was synthesized to contain in the coding strand, from 5’ end to 3’end, a 2.2 kb A2UCOE fragment (SEQ ID NO: 1), a CMV promoter (SEQ ID NO: 10), a sequence encoding the Fc-fusion protein (about 1300 nt in length), a polyA sequence (SEQ ID NO: 11), a 2.2 kb A2UCOE fragment (SEQ ID NO: 1), a SV40 promoter (SEQ ID NO: 12), a sequence encoding glutamine synthetase (the amino acid sequence set forth in SEQ ID NO: 13), and a polyA sequence (SEQ ID NO: 11) (see FIG. 1 (B)). The DNA fragment was inserted into the same plasmid as above. The resulting plasmid was referred to as Plasmid B.

[0115] Another DNA fragment was synthesized to contain in the coding strand, from 5’ end to 3’end, a 5’ PiggyBac ITR (SEQ ID NO: 4), a CMV promoter (SEQ ID NO: 10), a sequence encoding a Fc-fusion protein (about 1300 nt in length), a polyA sequence (SEQ ID NO: 1 1), a SV40 promoter (SEQ ID NO: 12), a sequence encoding glutamine synthetase (the amino acid sequence set forth in SEQ ID NO: 13), a polyA sequence (SEQ ID NO: 11), and a 3’ PiggyBac ITR (SEQ ID NO: 5) (see FIG. 1 (C)). The DNA fragment was inserted into the same plasmid as above . The obtained plasmid was referred to as Plasmid C.

[0116] A DNA fragment was synthesized to contain in the coding strand, from 5’ end to 3’end, a CMV promoter (SEQ ID NO: 10), a sequence encoding a Fc-fusion protein (about 1300 nt in length), a polyA sequence (SEQ ID NO: 11), a SV40 promoter (SEQ ID NO: 12), a sequence encoding glutamine synthetase (the amino acid sequence set forth in SEQ ID NO: 13), and a polyA sequence (SEQ ID NO: 11) (see FIG. 1 (D)). The DNA fragment was inserted into the same plasmid as above. The obtained plasmid was referred to as Plasmid D.

[0117] An mRNA for expression of the Piggy Bac transposase was constructed, whichcontained from the 5’ end to the 3‘ end, a 5’-cap (Cap 0), a 5‘UTR (SEQ ID NO: 14), a sequence for encoding the PiggyBac transposase (amino acid sequence being SEQ ID NO: 6), a 3‘UTR (SEQ ID NO: 15), and a poly(A) tail (containing 120 nt As).

[0118] The plasmids were used to transfect suspension-adapted CHO cells (GenScript) with the transposase-encoding mRNA. Briefly, on the day of transfection, 1E7 of the CHO cells, at the density of between 1E6-4E6 cells / mL with viability7greater than 90%, were centrifuged at 1500 rpm for 10 minutes, and resuspended, after supernatants discarded, in 200 pL CD CHO medium (ThermoFisher, Cat. No: 10743029). The plasmids A, B, C, and D obtained above, each at the amount of 30 pg, were respectively mixed with 3 pg of the mRNA coding for the PiggyBac transposase, and then added separately to the CHO cell suspensions prepared above. Electroporation was performed for the transfection, using a Bio-Rad Gene Pulser Xcell™ electroporator, with parameters set to 250 V, 30 ms, and 4 mm square wave pulse.

[0119] Forty-eight (48) hours post transfection, the CHO cells were added with CD CHO medium containing 20 p M MSX (Sigma-Aldrich, Cat. No: m5379). referred to as the selection medium, wherein the cell density of the CHO cells was 1-2E5 cells / mL. Then, the cell suspensions containing the cells transfected with one of the 4 plasmids were plated onto a 24-well plate, 0.5 mL cell suspension per well, for mini cell pool selection. The selection medium was refreshed every 4-6 days, and the cells were observed under a microscope before medium replacement.

[0120] Stable cell pools were generated when more than 1 / 3 of the mini cell pools transfected with one of the 4 plasmids adapted to the selection pressure, i.e., the cells resumed normal growth with increased total cell count and improved viability as compared to the earlier stage. The cell supernatants were collected, and the concentrations of the Fc-fusion proteins in the supernatants were determined by ELISA, using the coating / capture antibody (Goat anti Human IgG-UNLB, Southern Biotech, Cat. No: 2040-01) and ELISA detection antibody (Goat anti-Human IgG Fc Cross- Adsorbed Secondary Antibody, HRP, ThermoFisher, Cat. No: 31413).

[0121] The top 4 mini cell pools with the highest protein expression yields were identified from each group of cells that were transfected with one of the 4 plasmids and expanded from the 24-well plate to a 6-well plate.

[0122] Once the viability of all cell pools transfected with one of the 4 plasmids reached around 90%. the cells in each well were diluted to 0.4E6 cells / mL inproduction-use Dynamis medium (ThermoFisher, Cat. No: A2661501) and placed on a shaker for fed-batch cultures using EfficientFeed™ B+ 3X Supplement (ThermoFisher, Cat. No: A3937501), with the feeding volume being 4% of the basal medium volume even- 2 days. After 14 days, the supernatants from the cell cultures were collected, and measured for the protein expression yields by ELISA.

[0123] As shown in FIG. 2. the time as required to obtain stable cells in the selection step was much shorter in the cells transfected with plasmid A than in the cells transfected with the other three plasmids, suggested that plasmid A greatly accelerated the screening timeline. Further, it can be seen from FIG. 3 that the protein titers were significantly higher in the cell cultures with plasmid A transfection than those in the cell cultures with plasmid B. C or D transfection.Example 2. Construction and characterization of vectors containing PiggyBac ITRs and murine-derived Rsp3UCOE

[0124] Vectors with the similar structure to the plasmid vectors of Example 1 were constructed using the murine-derived Rsp3UCOE or the 1.5 kb human-derived A2UCOE, and observed for their ability to generate stable cell lines and confer stable high-level GOI expression.

[0125] Specifically, a DNA fragment was synthesized to contain in the coding strand, from 5‘ end to 3'end. a 5’ PiggyBac ITR (SEQ ID NO: 4). a 1.5 kb A2UCOE fragment (SEQ ID NO: 2). a CMV promoter (SEQ ID NO: 10), a sequence encoding Aflibercept (a Fc fusion protein, with amino acid sequence being SEQ ID NO: 7), a poly A sequence (SEQ ID NO: 11), a 1.5 kb A2UCOE fragment (SEQ ID NO: 2), a SV40 promoter (SEQ ID NO: 12), a sequence encoding glutamine synthetase ((the amino acid sequence set forth in SEQ ID NO: 13), a polyA sequence (SEQ ID NO: 11), and a 3’ PiggyBac ITR (SEQ ID NO: 5). The DNA fragment was inserted into the same in house made plasmid as used in Example 1 via CloneEZ™ Next-Generation Molecular Cloning service provided by Genscript. The resultant plasmid was referred to as Plasmid A.

[0126] Plasmids B to D were constructed following the protocol above, except that the plasmid B contained two 1.5 kb A2UCOE fragments but no PiggyBac derived ITRs. the plasmid C contained the PiggyBac derived ITRs but no UCOEs, and the plasmid D did not contain A2UCOE elements or ITR sequences.

[0127] A DNA fragment that contained in the coding strand, from 5’ end to 3 ’end, a 5’ PiggyBac ITR (SEQ ID NO: 4), a Rsp3UCOE fragment (SEQ ID NO: 3), a CMV promoter (SEQ ID NO: 10), a sequence encoding Aflibercept (amino acid sequencebeing SEQ ID NO: 7), a polyA sequence (SEQ ID NO: 11), a Rsp3UCOE fragment (SEQ ID NO: 3). a SV40 promoter (SEQ ID NO: 12), a sequence encoding glutamine synthetase (the amino acid sequence set forth in SEQ ID NO: 13), a polyA sequence (SEQ ID NO: 11), and a 3’ PiggyBac ITR (SEQ ID NO: 5), was prepared and used to construct Plasmid E.

[0128] An mRNA encoding the PiggyBac transposase was prepared as described in Example I.

[0129] The 5 plasmids were used to transfect suspension-adapted CHO cells with the Piggy Bac transposase-encoding mRNA, and the stable cell pools were identified and fed-batch cultured for GOI expression, following the protocol of Example 1.

[0130] The results were shown in FIG. 4 and FIG. 5. It took about 7 days to generate stable cell pools in the plasmid A or plasmid E transfection group, which was shorter than the time it took to generate stable cell pools in groups with transfection of the other three plasmids. Further, the cells transfected with plasmid A or plasmid E produced much more proteins than the cells transfected with the other three plasmids.

[0131] All the data indicated that the mouse-derived Rps3UCOE and the human- derived A2UCOE might function similarly in terms of stable cell generation and recombinant protein production. When combined with the piggybac transposon system, both significantly accelerated the stable cell selection timeline and enhanced heterologous protein expression.Example 3. Construction and characterization of vectors containing PiggyBac ITRs and one or two UCOEs

[0132] A vector was constructed with the GOI of Example 1 but with only one 2.2kb A2UCOE, and observed for its ability to generate stable cell lines and confer stable high-level GOI expression.

[0133] Specifically, a DNA fragment was synthesized to contain in the coding strand, from 5' end to 3 ’end, a 5’ PiggyBac ITR (SEQ ID NO: 4), a 2.2 kb A2UCOE fragment (SEQ ID NO: 1), a CMV promoter (SEQ ID NO: 10), a sequence encoding a Fc-fusion protein (about 1300 nt in length), a polyA sequence (SEQ ID NO: I I), a SV40 promoter (SEQ ID NO: 12), a sequence encoding glutamine synthetase (the amino acid sequence set forth in SEQ ID NO: 13), a polyA sequence (SEQ ID NO: 11), and a 3’ PiggyBac ITR (SEQ ID NO: 5). The DNA fragment was inserted into the same in house made plasmid as used in Example 1 via CloneEZ™ Next-Generation Molecular Cloning service provided by Genscript. The resultant plasmid was referred to as PlasmidI xUCOE.

[0134] The plasmid 2xUCOE, plasmid PiggyBac and the control plasmid were constructed following the protocol above, except that the 2xUCOE further contained a 2.2 kb A2UCOE fragment (SEQ ID NO: 1) between the first polyA sequence and the SV40 promoter, the plasmid PiggyBac contained the Piggy Bac derived ITRs but no UCOEs, and the control plasmid did not contain A2UCOE elements or ITR sequences. See FIG. 6 for the detailed structures.

[0135] An mRNA encoding the PiggyBac transposase was prepared as described in Example 1.

[0136] The 4 plasmids were used to transfect suspension-adapted CHO cells with the PiggyBac transposase-encoding mRNA, and the stable cell pools were identified and fed-batch cultured for GOI expression, following the protocol of Example 1.

[0137] The results were shown in FIG. 7 and FIG. 8. The presence and the number / location of the UCOE were quite important in accelerating stable cell screening timeline. It took about 16 days to generate stable cell pools in the group with plasmid 1 xUCOE transfection, shortened by 23.8% compared to the control group of about 21 days. When the constructed plasmid contained two UCOEs, stable cell pools were obtained after about 7 days of pressure selection, the time it took was shortened by 66.7% compared to the control group. It can be seen the presence of the UCOEs upstream of the GOI promoter and the selectable marker promoter did have a significant effect on accelerating the cell pool screening timeline. The presence of the piggybac transposon system did not improve the cell pool screening procedure but greatly increased the production of the GOI protein.Example 4. Antibody expression bv cells transfected with transposon derived ITRs and UCOE-containing vectors

[0138] Four vectors were constructed as described in Example 1 using the antibodyencoding sequences as the GOI.

[0139] Specifically, a DNA fragment was synthesized to contain in the coding strand, from 5’ end to 3’end. a 5’ PiggyBac ITR (SEQ ID NO: 4). a 2.2 kb A2UCOE fragment (SEQ ID NO: 1), a CMV promoter (SEQ ID NO: 10), a sequence encoding an antibody light chain (amino acid sequence being SEQ ID NO: 8), a polyA sequence (SEQ ID NO: 11), a CMV promoter (SEQ ID NO: 10), a sequence encoding an antibody heavy chain (amino acid sequence being SEQ ID NO: 9), a polyA sequence (SEQ ID NO: 11 ),a 2.2 kb A2UC0E fragment (SEQ ID NO: 1), a SV40 promoter (SEQ ID NO: 12), a sequence encoding glutamine synthetase (the amino acid sequence set forth in SEQ ID NO: 13), a poly A sequence (SEQ ID NO: 11), and a 3’ PiggyBac ITR (SEQ ID NO: 5). The DNA fragment was inserted into the same in house made plasmid as used in Example 1 via CloneEZ™ Next-Generation Molecular Cloning service provided by Genscript. The resultant plasmid was referred to as Plasmid A.

[0140] Plasmids B to D were constructed following the protocol above, except that the plasmid B contained two 2.2 kb A2UCOE fragments but no PiggyBac derived ITRs, the plasmid C contained the PiggyBac derived ITRs but no UCOEs, and the plasmid D did not contain A2UCOE elements or ITR sequences.

[0141] An mRNA encoding the PiggyBac transposase was prepared as described in Example 1.

[0142] The 4 plasmids were used to transfect suspension-adapted CHO cells with the Piggy Bac transposase-encoding mRNA, and the stable cell pools were identified and fed-batch cultured for GOI expression, following the protocol of Example 1. The highest-expressing mini pools from each group of cells that were transfected with one of the 4 plasmids were identified by ELISA using the capture antibody (Goat anti Human IgG-UNLB, Southern Biotech, Cat. No.: 2040-01) and the detection antibody (Goat Anti Human Kappa-HRP, Southern Biotech, Cat. No.: 2060-05), expanded from the 24-well plate to the 6-well plate, and fed-batch cultured for GOI expression, following the protocol of Example 1.

[0143] The results were shown in FIG. 9 and FIG. 10. In consistent with the data of Example 1. the transfection of plasmid A significantly shortened the time to generate stable cell pools and enhanced heterologous protein expression by these stable cell pools, suggesting the presence of the UCOEs and the transposon derived ITRs in the plasmid vectors were important for stable cell generation and heterologous protein expression, no matter what the GOI it was that was inserted in the plasmids.SEQ ID NO 1: 2.2kb A2UCOE sequenceACGCGTCCCCCACCCGGGAGCGCGGGCCTCGTGGTCAGCGCATCCGCGGG GAGAAACAAAGGCCGCGGCACGGGGGCTCAAGGGCACTGCGCCACACCG CACGCGCCTACCCCCGCGCGGCCACGTTAACTGGCGGTCGCCGCAGCCTC GGGACAGCCGGCCGCGCGCCGCCAGGCTCGCGGACGCGGGACCACGCGCAGGGGGCGCCTCCGCCGGAACGCGGGTGGGGGAGGGGAGGGGGAAATGCGCTTTGTCTCGAAATGGGGCAACCGTCGCCACAGCTCCCTACCCCGTCGAGGGCAGAGCAGTCCCCCCACTAACTACCGGGCTGGCCGCGCGCCAGGCCAGCCGCGAGGCCACCGCCCGACCCTCCACTCCTTCCCGCAGCTCCCGGCGCGGGGTCCGGCGAGAAGGGGAGGGGAGGGGAGCGGAGAACCGGGCCCCCGGGACGCGTGTGGCATCTGAAGCACCACCAGCGAGCGAGAGCTAGAGAGAAGGAAAGCCACCGACTTCACCGCCTCCGAGCTGCTCCGGGTCGCGGGTCTGCAGCGTCTCCGGCCCTCCGCGCCTACAGCTCAAGCCACATCCGAAGGGGGAGGGAGCCGGGAGCTGCGCGCGGGGCCGCCGGGGGGAGGGGTGGCACCGCCCACGCCGGGCGGCCACGAAGGGCGGGGCAGCGGGCGCGCGCGCGGCGGGGGGAGGGGCCGGCGCCGCGCCCGCTGGGAATTGGGGCCCTAGGGGGAGGGCGGAGGCGCCGACGACCGCGGCACTTACCGTTCGCGGCGTGGCGCCCGGTGGTCCCCAAGGGGAGGGAAGGGGGAGGCGGGGCGAGGACAGTGACCGGAGTCTCCTCAGCGGTGGCTTTTCTGCTTGGCAGCCTCAGCGGCTGGCGCCAAAACCGGACTCCGCCCACTTCCTCGCCCGCCGGTGCGAGGGTGTGGAATCCTCCAGACGCTGGGGGAGGGGGAGTTGGGAGCTTAAAAACTAGTACCCCTTTGGGACCACTTTCAGCAGCGAACTCTCCTGTACACCAGGGGTCAGTTCCACAGACGCGGGCCAGGGGTGGGTCATTGCGGCGTGAACAATAATTTGACTAGAAGTTGATTCGGGTGTTTCCGGAAGGGGCCGAGTCAATCCGCCGAGTTGGGGCACGGAAAACAAAAAGGGAAGGCTACTAAGATTTTTCTGGCGGGGGTTATCATTGGCGTAACTGCAGGGACCACCTCCCGGGTTGAGGGGGCTGGATCTCCAGGCTGCGGATTAAGCCCCTCCCGTCGGCGTTAATTTCAAACTGCGCGACGTTTCTCACCTGCCTTCGCCAAGGCAGGGGCCGGGACCCTATTCCAAGAGGTAGTAACTAGCAGGACTCTAGCCTTCCGCAATTCATTGAGCGCATTTACGGAAGTAACGTCGGGTACTGTCTCTGGCCGCAAGGGTGGGAGGAGTACGCATTTGGCGTAAGGTGGGGCGTAGAGCCTTCCCGCCATTGGCGGCGGATAGGGCGTTTACGCGACGGCCTGACGTAGCGGAAGACGCCTTAGTGGGGGGGAAGGTTCTAGAAAAGCGGCGGCAGCGGCTCTAGCGGCAGTAGCAGCAGCGCCGGGTCCCGTGCGGAGGTGCTCCTCGCAGAGTTGTTTCTCCAGCAGCGGCAGTTCTCACTACAGCGCCAGGACGAGTCCGGTTCGTGTTCGTCCGCGGAGATCTCTCTCATCTCGCTCGGCTGCGGGAAATCGGGCTGAAGCGACTGAGTCCGCGATGGAGGTAACGGGTTTGAAATCAATGAGTTATTGAAAAGGGCATGGCGAGGCCGTTGGCGCCTCAGTGGAAGTCGGCCAGCGGCTTAAGGTTTATGAACGGGGTCTTGAGCGGAGGCCTGAGCGTACAAACAGCTTCCCCACCCTCAGCCTCCCGGCGCCATTTCCCTTCACTGGGGGTGGGGGATGGGGAGCTTTCACATGGCGGACGCTGCCCCGCTGGGGTGAAAGTGGGGCGCGGAGGCGGGACTTCTTATTCCCTTTCTAAAGCACGCTGCTTCGGGGGCCACGGCGTCTCCTCGGACGGCCGSEQ ID NO 2: 1.5kb A2UCOE sequenceACGCGTGGCCCTCCGCGCCTACAGCTCAAGCCACATCCGAAGGGGGAGGGAGCCGGGAGCTGCGCGCGGGGCCGCCGGGGGGAGGGGTGGCACCGCCCACGCCGGGCGGCCACGAAGGGCGGGGCAGCGGGCGCGCGCGCGGCGGGGGGAGGGGCCGGCGCCGCGCCCGCTGGGAATTGGGGCCCTAGGGGGAGGGCGGAGGCGCCGACGACCGCGGCACTTACCGTTCGCGGCGTGGCGCCCGGTGGTCCCCAAGGGGAGGGAAGGGGGAGGCGGGGCGAGGACAGTGACCGGAGTCTCCTCAGCGGTGGCTTTTCTGCTTGGCAGCCTCAGCGGCTGGCGCCAAAACCGGACTCCGCCCACTTCCTCGCCCGCCGGTGCGAGGGTGTGGAATCCTCCAGACGCTGGGGGAGGGGGAGTTGGGAGCTTAAAAACTAGTACCCCTTTGGGACCACTTTCAGCAGCGAACTCTCCTGTACACCAGGGGTCAGTTCCACAGACGCGGGCCAGGGGTGGGTCATTGCGGCGTGAACAATAATTTGACTAGAAGTTGATTCGGGTGTTTCCGGAAGGGGCCGAGTCAATCCGCCGAGTTGGGGCACGGAAAACAAAAAGGGAAGGCTACTAAGATTTTTCTGGCGGGGGTTATCATTGGCGTAACTGCAGGGACCACCTCCCGGGTTGAGGGGGCTGGATCTCCAGGCTGCGGATTAAGCCCCTCCCGTCGGCGTTAATTTCAAACTGCGCGACGTTTCTCACCTGCCTTCGCCAAGGCAGGGGCCGGGACCCTATTCCAAGAGGTAGTAACTAGCAGGACTCTAGCCTTCCGCAATTCATTGAGCGCATTTACGGAAGTAACGTCGGGTACTGTCTCTGGCCGCAAGGGTGGGAGGAGTACGCATTTGGCGTAAGGTGGGGCGTAGAGCCTTCCCGCCATTGGCGGCGGATAGGGCGTTTACGCGACGGCCTGACGTAGCGGAAGACGCCTTAGTGGGGGGGAAGGTTCTAGAAAAGCGGCGGCAGCGGCTCTAGCGGCAGTAGCAGCAGCGCCGGGTCCCGTGCGGAGGTGCTCCTCGCAGAGTTGTTTCTCCAGCAGCGGCAGTTCTCACTACAGCGCCAGGACGAGTCCGGTTCGTGTTCGTCCGCGGAGATCTCTCTCATCTCGCTCGGCTGCGGGAAATCGGGCTGAAGCGACTGAGTCCGCGATGGAGGTAACGGGTTTGAAATCAATGAGTTATTGAAAAGGGCATGGCGAGGCCGTTGGCGCCTCAGTGGAAGTCGGCCAGCCGCCTCCGGTTTATGAACGGGGTCTTGAGCGGAGGCCTGAGCGTACAAACAGCTTCCCCACCCTCAGCCTCCCGGCGCCATTTCCCTTCACTGGGGGTGGGGGATGGGGAGCTTTCACATGGCGGACGCTGCCCCGCTGGGGTGAAAGTGGGGCGCGGAGGCGGGACTTCTTATTCCCTTTCTAAAGCACGCTGCTTCGGGGGCCACGGCGTCTCCTCGGACGGCCGSEQ ID NO 3: 3 kb Rsp3UCOE sequenceACCACTAAGCCATCTCTCCAGCCCTGAGTCATGGTTTTAGTGTGAGAGGCATCATTGAATTTTCTGAGCACGGCCATCAGGGTAGCTGGCACAGGTCTTCAGATACAAGGAGATAGTTATAAGAAGGCAGCCATGGCTGTGGTGCACTAGAAATGGAGAAACAGCTTCATCAGGTGACAGACCAGTCTGACTCTGTCCCATGATTAGAAGCCATCTTGTTACAAGGTCAAAATAAGTTCATTCCTGTTTTCTGTAACACTTGGGTTTGATCCTGTCGTCAACCCATTTTCTGGAATTTGACATGTTCCATACTCCATTATACCCTGACTTCCACCCTGATAAGATGTTCTGCCAAGTTCCTGTGTAGCCAACATTCCCCTGGAAATCTCTCTTCCCTTGGAAACCACCTAGTCTTAGAAATTTTGAGTTATATAAATTCCACTTCTATGTTTGATGCTATTCTTTAAAACTCCACTTTAGGGAGATAGCCCTGTCTGATAGAAAATAAAACTTGCTTAATTTGTCTAAAAGATTTTAAGTAATAGTTTTTACTTTTGTTCCGTGGGATTAGTACAGGGTGAAACAGACTCCCGTGTTTCCAGTGTGAAGTGAGCCACACACTGCAGTACAAGTTATATCAGCAGGTTCTGCCTCTGCGCAATGAACTTTTGCTTGTGTGGACATCAGGGTCTGTGTGAAGGGAAGGTCCTATGGCCTAGTTTTATACTATTCAACAGTCTGTCCCCGAAGCCCTGGTGCTTTATTATTTTGACAAGCCCCTGCTGCTGGTATTCCACCCTGCTGCGAGTCAAAAAAGTTCCTGTCTCGGAAAAACAAAACAAAACAAAACAACCAAAAAATAAATTTTTTTTTCCCACAGGTTCTAGTGGAGGTGCTCACTACCAGAAATCCTACAAATAAGCCCATCTCATGGATCAGGGTTTACCTTTGTAATAATATTAAATCTGTGTGCATGTGCGCACGCATGTGTTTTATGCTTGCATATATGTATACGCAGCCATGGTTTTCTACTGTCCCACTCACTCTGTAACTTACTGAGCCATCCAGCTGGTCCTCTAAATACATTTCAATGAAAGTTTTCATTAGCGTGAACGTGAAGGTGGTAAAATCTGTTAGTGTGTGCTTATGCCTGTGGTTTGCACCTCTAGTCTGAAGGTTGCTCTTTTCAAATTTTTTATTTATTTACGTTTTTACTTCTGAGTCAGAAACTCATAAAGGCCATGGCCTCGAATTCGCTATGTAGTCAACGATGACCTTAAACTTGTGACCCTCTACTTCGTTAGTGCTGGAACCCCAAGTCCGCCTACATTACAGGTTGCTAGGTTACACCCCCCCTACGCCGTTTTAGACGCAAAACTTCATTTCCCATGCAAAACTTCATTTCCCATGAACACTTGCAAGGGTCGCCGCGCTGCGCGGCGTCATTGCTCCCGCCCTATATACCTACTTCCGCCCGCGAGCCACTTCCTTTCCTTTCAGCGGCGCGCGGCTGCAAGATGGCGGTGCAGATTTCCAAGAAGAGGAAGGTAAGCGTCTGGGCCCGGTTCGGGAGTCCGCCGCGGGTTCTACAAGTGCCAGGGAGGCCTGTGGCTCCGTAATCAGTCCTGTGGAGCGTCTGGGGCCGCCTGCCGTCTCTTCGAGCCTCGGATGGCCGTAGATTGTGTATTGGGCCGGAGCCGGGCGAGTGCTGTGTGCCTGGGCAAGGGAGGGACAAACTCCTCGAGTTCTGGACCGACTCGAACACCGGGCGCCTCCAGTTCCGGACTAGACACCTTTGAGCGTTTCTTGGTCTCCATAATAGTAATCCTGTGGCACAGTTAGAGGGCGTGTGCCATCAGATCTAGTCCAGTCTCTTTAGTAAGTGAAGTTTAGCAGTCCCTTCTCTTAGTCGCGTGATCCTGCAAGTGGCCATAGTTGAAAGCCTACTTACTGACTGCTGCCGTGTTCACTCGGGACCCGGAGCTGCAGCGTCCCTGTGGTTATCATTTCATGGGGGAAAAGTGTGCAGGTTGCCAGGTTTAGAAATAGATGGTCTGTCGTTTGTGCTTATGCACACAGATGATAAACCTGTTTTGAGTCAGGATTCCTCTCCTATCCGAGGTACAACTTACAGTCCCAGCTGTACATGTGCTACTTGGAGACAGATTTTTCTTTGTCTCTTGGGTGTAGATTATGCCGTAGAGCCCTTCGATGAAGAGGTGATGACGAGTCTGAGTAGGAAGTGTTGTCTTTGTCCAAGATGCCTCACTATGCTGCGTTCTGTGGCACAGCTGAAAGCACTGTGGTCAAAAGAAACTTCCTAAAGATGACCAAGAGGCATTTGTCTGAGAAGGGTTGCTGCTTTTCTGTAGGGCCATTGGGCTTGCTCTGACTAACCCTGTCTTCACCTCAGAGGTAACTTGTTTCCTTTGGTTCAGTTTGTAGCTGATGGCATCTTCAAAGCTGAGCTGAATGAATTTCTCACTCGGGAGCTGGCTGAAGATGGCTACTCTGGAGTTGAAGTCCGAGTTACACCAACCAGGACAGAAATCATTATTTTAGCCACCAGGTAGAAATACCATTGATTGTCACCTGTAAATATTGTGTGTACTGAGATGCTGTGTAAACTTGGGCCAACCAAGCAGTAAATCTGGCCTCAGTGGGTGTAACTGCTTTGTTAGAACTGCATTTGGGAAGAACTTACCTTCCATTTAACCTGTGTGCTGGCGTTGTGGTGGGCGGCAGGTGGGATCTTGAGTAAATGGTTGCGCTTCCCCTCTACAGGACACAGAATGTTCTTGGGGAGAAGGGTCGTCGGATCAGAGAGTTGACCGCAGTTGTCCAGAAGCGCTTTGGCTTCCCTGAAGGCAGCGTAGAGGTGAGTTCCTCTGCTTTATCTCCCGGGGGTTTTAGACTGAGTTGGGATGTGGCTTCTGCTATAGAATTGTACTTCTGAAAACCTGACATGGCCAGTGACAGTCACAGGTACTTGATGCTSEQ ID NO 4: Piggybac 5?ITR sequenceCCCTAGAAAGATAGTCTGCGTAAAATTGACGCATGSEQ ID NO 5: Piggybac 3’ ITR sequenceCCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTA AAATTGACGCATGSEQ ID NO 6: Piggybac transposaseMGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQSDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNKHCWSTSKSTRRSR VSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESM TSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRD RFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMINGMPYLGRGTQTNG VPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEPYKLTIVGTVRSN KREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVYLLSSCDEDASIN ESTGKPQMVMYYNQTKGGVDTLDQMC S VMTC SRKTNRWPMALLYGMINI ACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLEAPTLKRYLRD NISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASCKKCKKVICR EHNIDMCQSCFSEQ ID NO 7: AfliberceptSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSE MKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGSEQ ID NO 8: PD-1 antibody light chainEIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIY LASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTK VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGECSEQ ID NO 9: PD-1 antibody heavy chainQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMG GINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDY RFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVD HKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS RWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKSEQ ID NO 10: CMV promoterGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCC CGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACG TATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTG GAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATAT GCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC ATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCT ACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCA ATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCC ATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTG TACGGTGGGAGGTCTATATAAGCAGAGCTSEQ ID NO 11: BGH poly ACctcgactgtgcctctagtgccagccatctgtgtttgcccctcccccgtgcctcctgaccctggaaggtgccactccca ctgtccttcctaataaaatgaggaaatgcatcgcatgtctgagtaggtgtcatctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggSEQ ID NO 12: SV40 promoterGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAASEQ ID NO 13: glutamine synthetaseMATSASSHLNKNIKQMYLCLPQGEKVQAMYIWVDGTGEGLRCKTRTLDCEPKCVEELPEWNFDGSSTFQSEGSNSDMYLSPVAMFRDPFRRDPNKLVFCEVFKYNRKPAETNLRHSCKRIMDMVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADKAYGRDIVEAHYRACLYAGVKITGTNAEVMPAQWEFQIGPCEGIRMGDHLWVARFILHRVCEDFGVIATFDPKPIPGNWNGAGCHTNFSTKAMREENGLKHIEEAIEKLSKRHRYHIRAYDPKGGLDNARRLTGFHETSNINDFSAGVANRSASIRIPRTVGQEKKGYFEDRRPSANCDPFAVTEAIVRTCLLNETGDEPFQYKNSEQ ID NO 14: 5’ UTRGAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCSEQ ID NO 15: 3’ UTRCTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCAC***

[0144] Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

Claims

We claim:

1. A vector composition, comprising: i) a first vector that comprises a double-stranded cargo nucleic acid, wherein the double-stranded cargo nucleic acid comprises a first expression cassette of a gene of interest (GOI), a second expression cassette of a selectable marker, a ubiquitous chromatin opening element (UCOE) arranged upstream of and right before the first expression cassette or the second expression cassette, and a pair of transposon derived inverted terminal repeats (ITRs), wherein the first expression cassette, the second expression cassette and the UCOE are operably connected and flanked by the pair of transposon derived ITRs, and ii) a second vector that encodes and expresses a transposase. wherein the transposase is able to recognize the pair of transposon derived ITRs in the first vector and transpose the double-stranded cargo nucleic acid.

2. The vector composition of claim 1, comprising a UCOE upstream of and right before the first expression cassette, and a UCOE upstream of and right before the second expression cassette.

3. The vector composition of claim 1, wherein the UCOE is derived from A2UC0E, TBP / PSMB1. SURF1 / SURF2 or Rps3 UCOE.

4. The vector composition of claim 3, wherein the UCOE comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 1, 2 or 3.

5. The vector composition of claim 1, wherein the transposase and the transposon derived ITRs are derived from piggyBac transposon or Sleeping Beauty’ transposon.

6. The vector composition of claim 5. wherein the transposase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

6. and the pair of transposon derived ITRs comprise amino acid sequences having at least 90% sequence identity to SEQ ID NOs: 4 and 5, respectively.

7. The vector composition of claim 1, wherein first expression cassette comprises i) a promoter, ii) a gene of interest coding for a biomolecule, or a restriction site, andoptionally iii) a poly(A) tail-encoding sequence, in said order.

8. The vector composition of claim 7, wherein the promoter is a CMV promoter, a SV40 promoter, or a Ubc promoter.

9. The vector composition of claim 1, wherein the second expression cassette comprises, i) a promoter, ii) a selectable marker, or a restriction site, and optionally hi) a poly(A) tail-encoding sequence, in said order.

10. The vector composition of claim 9, wherein the promoter is a SV40 promoter, a CMV promoter or a Ubc promoter.

11. The vector composition of claim 1, wherein the selectable marker is a gene that confers an abi 1 i ty to utilize a certain carbohydrate or amino acid supply, or a drug resistance gene.

12. The vector composition of claim 1, wherein the double-stranded cargo nucleic acid further comprises one or more additional expression cassettes of a gene of interest, wherein each additional expression cassette of a gene of interest comprises i) a promoter, ii) a gene of interest coding for a biomolecule, or a restriction site, and optionally hi) a poly(A) tail-encoding sequence, in said order.

13. The vector composition of claim 12, wherein the double-stranded cargo nucleic acid further comprises a UCOE arranged upstream of and right before each of the additional expression cassettes of a gene of interest.

14. The vector composition of claim 1, wherein the first vector is a linear or circular DNA vector.

15. The vector composition of claim 1, wherein the first vector comprises i) 5’ transposon derived ITR, a UCOE, a first expression cassette of a gene of interest, a second expression cassette of a selectable marker, and 3’ transposon derived ITR, in said order, ii) 5?transposon derived ITR, a UCOE, a first expression cassette of a gene of interest, a UCOE, a second expression cassette of a selectable marker, and 3’transposon derived ITR, in said order, iii) 5’ transposon derived ITR, a UCOE, a first expression cassette of a gene of interest, an additional expression cassette of a gene of interest, a UCOE, a second expression cassette of a selectable marker and 3’ transposon derived ITR, in said order, or iv) 5’ transposon derived ITR, a UCOE, a first expression cassette of a gene of interest, a UCOE, an additional expression cassette of a gene of interest, a UCOE, a second expression cassette of a selectable marker, and 3‘ transposon derived ITR, in said order.

16. The vector composition of claim 1, wherein the second vector is an mRNA.

17. A method for generating a cell line using the vector composition of any one of claims 1 to 16, comprising the steps of: i) providing a first vector, and optionally inserting a gene of interest or a selectable marker at a restriction site in the first vector as required, ii) providing a second vector, iii) introducing or delivering the first vector and the second vector into a cell or cell population, and iv) culturing the cell or cell population under a condition for selection, to obtain a cell or cell population having the double-stranded cargo nucleic acid of the first vector integrated into cell genome.

18. The method of claim 17, wherein the cell or cell population used in step iii) is a mammalian cell or cell population, e.g., a Chinese hamster ovary (CHO) cell or cell population.

19. The method of claim 17, wherein step iii) is done with calcium phosphate, liposome, polymers, nanoparticles, electroporation, or microinjection.

20. The method of claim 19, wherein the second expression cassette in the first vector comprises a gene coding for glutamine synthetase as the selectable marker, and step iv) comprises culturing the cell or cell population in a medium containing methionine sulfoximine (MSX).

21. A method for producing a biomolecule, comprising the steps of:i) obtaining cells or cell populations having a double-stranded cargo nucleic acid of a first vector integrated into cell genome(s), according to the method of any one of claims 17 to 20, wherein the double-stranded cargo nucleic acid comprises a gene coding for a biomolecule as the gene of interest in the first expression cassette, ii) optionally testing the cells or cell populations obtained in step i) for the gene of interest expression level, to obtain cells or cell populations with high gene of interest expression level, and iii) culturing the cells or cell populations under a condition for production of the biomolecule.

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