# Patent

## Title
Expression vector with enhanced gene expression capacity and method for using the same

## Abstract

The present invention provides a novel expression vector which comprises a gene of interest, a nuclear anchoring element, and at least one inverted repeat element, preferably two inverted repeat elements. The expression vector is an episomal vector capable of transfecting a mammalian cell. The present invention further provides a method for enhancing gene expression by transfecting the expression vector to a mammalian cell, preferably a human cell.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel expression vector which is capable of enhancing gene expression through gene amplification in mammalian cells. The expression vector is organized as an inverted dimer (ID) and comprises, in addition to the gene to be expressed, a nuclear anchoring element and at least one inverted repeat element, preferably two inverted repeat elements. The present invention further relates to a method for enhancing gene expression by transfecting the expression vector to a mammalian cell, preferably a human cell.
2. Description of the Prior Art
A conventional gene expression method is used to express human recombinant proteins in a prokaryotic expression system. Most human recombinant proteins require post- and/or peri-translational modifications such as glycosylation, g-carboxylation, or g-hydroxylation. Therefore, a well-known problem with prokaryotic expression systems is that prokaryotic expression systems do not carry out post- and/or peri-translational modifications as mammalian expression systems do. Proteins requiring post- and/or peri-translational modifications for normal function cannot be expressed properly with prokaryotic expression systems.
Another conventional gene expression method widely used to express said human recombinant proteins is a mammalian expression system. A mammalian expression system uses mammalian cells because of their ability to undergo post-translational modifications and peri-translational modifications.
In particular, a mammalian expression system using Chinese hamster ovary (CHO) cells has become a routine and convenient expression system for expressing biopharmaceutical proteins for therapeutic and diagnostic uses.
A conventional method for enhancing gene expression in mammalian cells comprises a step of amplifying a gene by a step-by-step selection against a concentration-increasing selection agent. An embodiment of the conventional method for enhancing gene expression in mammalian cells uses Chinese hamster ovary (CHO) cells and amplifies a dihydrofolate recuctase (DHFR) gene with a step-by-step selection against methotrexate (MTX). See Omasa, 2002, J. Biosci. Bioeng. 94: 600-605.
However, the step-by-step selection is tedious and time-consuming. The process takes months to complete with cells expressing genes in high levels, and the post-translational modifications in non-human mammalian cells are not identical to that in human cells. Moreover, such conventional gene expression enhancing method with gene amplification has not been widely employed in human cells in the production of biopharmaceutical proteins for therapeutic and diagnostic uses. In general, human proteins generated in human cells should have beneficial properties in comparison with their counterparts produced in non-human systems.
It has been observed that a mismatch repair (MMR) system in human cells strongly suppresses gene amplification. See Lin et al., 2001, Nucleic Acids Res 29, 3304-3310; Chen et al., 2001, Proc Natl Acad Sci USA 98, 13802-13807. Thus gene expression enhanced by gene amplification is strongly suppressed in MMR+ (MMR-normal/MMR-proficient) cells. HCT116 cells are MMR− (MMR-deficient) due to a mutation in the hMLH1 gene. It is shown that HCT116 cells allow gene expression enhancement by gene amplification. In contrast, HCT116+Ch3 (MMR+ due to introduction of chromosome 3 which carries a wild-type hMLH1 gene) or HCT116+hMLH1 (MMR+ due to introduction of a cDNA which carries a wild-type hMLH1 gene) suppresses a drug resistant gene expression enhanced by gene amplification.
To overcome the shortcomings of the conventional gene expression methods and the conventional method for enhancing gene expression in mammalian cells (especially in human cells), the present invention provides a method to mitigate or obviate the aforementioned problems.
SUMMARY OF THE INVENTION
One aspect of the present invention is to provide an expression vector which comprises at least a gene; a nuclear anchoring element; and at least one inverted repeat (IR) element which comprises two lateral nucleic acid sequences, each being complementary to the other. The expression vector is an episomal vector and is further characterized as being capable of transfecting a mammalian cell. Preferably, the IR element contains a central sequence which is inserted between the two lateral nucleic acid sequences and is different from each of the two lateral nucleic acid sequences. Also preferably, the expression vector contains one IR element, such as pGT02-GFP, and most favorably, two IR elements, such as pGT03-GFP and pGT04-ID. In the case where the expression vector contains two IR elements, these two IR elements can be the same or different.
The gene in the expression vector either encodes a protein or can be transcribed into a non-coding RNA. The protein encoded by the gene includes, but is not limited to, signal peptide, growth factor, hormone, cytokine, chemokine, neuropeptide, antigen, antibody, enzyme, clotting factor, anti-angiogenic factor, pro-angiogenic factor, transport protein, receptor, ligand, regulatory protein, structural protein, reporter protein, transcription factor, ribozyme, fusion protein, and drug-resistance protein. The protein is of natural origin or artificially modified, such as EGFP (i.e., enhanced green fluorescent protein), which is a reporter protein.
The non-coding RNA includes, but is not limited to, tRNA, rRNA, antisense RNA, micro-RNA, and double stranded RNA.
The mammalian cell is preferably a human cell, such as a mismatch repair-deficient (MMR−) cell or a mismatch repair-proficient (MMR+) cell. Examples of the MMR− cell includes, but is not limited to, HCT116 (hMLH1−) cell or a DLD-1 (hMSH6−) cell. Examples of the MMR+ cell includes, but is not limited to HCT116+ch3 or HCT116+hMLH1 cells.
The nuclear-anchoring element is preferably an EBV (Epstein-Barr Virus) gene encoding an EBV nuclear protein EBNA-1 and an EBV replicon oriP.
The expression vector is capable of enhancing the expression of the gene in the mammalian cells.
Another aspect of the present invention provides an expression vector which is organized as an inverted dimer (ID). The expression vector is a circular nucleic acid molecule having two long nucleic acid sequences separated by two inverted repeat (IR) elements. Each of the two long nucleic acid sequences is complementary to the other, each containing at least a gene and a nuclear anchoring element; each of the two IR elements comprises two lateral nucleic acid sequences; each being complementary to the other. The two IR elements are identical to or different from each other. The ID is an episomal vector.
Finally, the present invention provides a method for expressing a gene in a mammalian cell by transfecting the expression vector to the mammalian cell to produce a transfected cell; culturing the transfected cells to allow the transfected cell to produce a protein which is encoded by the gene; and harvesting the protein from the mammalian cell.
The expression vector is transfected into the mammalian cell by electroporation.
Other objectives, advantages and novel features of the invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a map of an expression vector pGT01-GFP in accordance with the present invention;
FIG. 1B is a map of an expression vector pGT02-GFP in accordance with the present invention;
FIG. 1C is a map of an expression vector pGT03-GFP in accordance with the present invention;
FIG. 2 is a flow chart of generating an expression vector pGT04/ID from the expression vector pGT03-GFP in FIG. 1C;
FIG. 3A is a photograph of DLD-1 (hMSH6−) cells transfected with pGT03-GFP or pGT04/ID wherein the transfected cells were treated with 400 μg/ml or 1000 μg/ml of hygromycin B;
FIG. 3B is a photograph of HCT116 (hMLH1−) cells or HCT116+hMLH1 cells transfected with pGT02-GFP, pGT03-GFP or pGT04/ID wherein the transfected cells were treated with 1000 μg/ml of hygromycin B;
FIG. 3C is a bar chart showing the survival colonies of the transfected cells in FIG. 3A;
FIG. 3D is a bar chart showing the survival colonies of the cells transfected with pGT03-GFP or pGT04/ID in FIG. 3B;
FIG. 4A is a graph of cell counts versus green fluorescence intensity of DLD-1 cells transfected with pGT04/ID and treated with 50 μg/ml, 400 μg/ml or 1000 μg/ml of hygromycin B; and
FIG. 4B is a photograph of a Western-blotting analysis of EGFP expression of the cells in FIG. 4A. Non-transfected DLD-1 cells were used as negative control. Control of protein loading level was performed with anti-actin monoclonal antibody in the gel of cell extracts (data not shown).



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Definitions
The term “expression vector” as used herein, refers to a vector, and in particular, an episomal vector. A vector is generally a plasmid that is used to introduce and express a specific gene into a target cell. The expression vector allows production of large amounts of stable mRNA. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular transcription and translation machinery. The plasmid is engineered such that it contains a highly active promoter which causes the production of large amounts of mRNA. An episomal vector is capable of self-replicating autonomously within the host cells.
The term “gene,” as used herein, refers to a set of segments of nucleic acid that contains the information necessary to produce a functional RNA product in a controlled manner through a transcription process. This RNA can then be used directly (such as tRNA, rRNA, snRNAs and other non-coding RNAs (e.g., the SRP RNAs), anti-sense RNA, or micro-RNA) or to direct the synthesis of proteins. When the phrase “a gene encoding a protein” or “a protein is encoded by a gene” is used, it means that the gene is transcribed into an mRNA which then is translated into a protein, including post- and peri-translation which occur in the mammalian cells.
Examples of the protein which can be expressed in the mammalian cells via the enhanced replication system of the expression vector includes, but is not limited to, signal peptide, growth factor, hormone, cytokine, chemokine, neuropeptide, antigen, antibody, enzyme, clotting factor, anti-angiogenic factor, pro-angiogenic factor, transport protein, receptor, ligand, regulatory protein, structural protein, reporter gene, transcription factor, ribozyme, fusion protein, and drug-resistance protein. The protein is of natural origin or artificially modified.
The term “reporter gene,” as used herein, refers to a “gene” that is attached to another gene of interest in cell culture, animals or plants. Certain genes are chosen as reporters because they are easily identified and measured; or because they are selectable markers. Reporter genes are generally used to determine whether the gene of interest has been taken up by or expressed in the cell or organism population. Commonly used reporter genes include, but is not limited to, the gene that encodes jellyfish green fluorescent protein (GFP), which causes cells that express it to glow green under UV light; the enzyme luciferase, which catalyzes a reaction with a luciferin to produce light; the lacZ gene, which encodes the protein β-galactosidase which causes the host cells expressing the gene to appear blue; and chloramphenicol acetyltransferase (CAT) gene, which confers resistance to the antibiotic chloramphenicol. The protein that is encoded by the reporter gene is called “reporter protein” as used herein
The term “reporter gene,” as used herein, refers to a “gene” that is attached to another gene of interest in cell culture, animals or plants. Certain genes are chosen as reporters because they are easily identified and measured; or because they are selectable markers. Reporter genes are generally used to determine whether the gene of interest has been taken up by or expressed in the cell or organism population. Commonly used reporter genes include, but is not limited to, the gene that encodes jellyfish green fluorescent protein (GFP), which causes cells that express it to glow green under UV light; the enzyme luciferase, which catalyzes a reaction with a luciferin to produce light; the lacZ gene, which encodes the protein .beta.-galactosidase which causes the host cells expressing the gene to appear blue; and chloramphenicol acetyltransferase (CAT) gene, which confers resistance to the antibiotic chloramphenicol. The protein that is encoded by the reporter gene is called “reporter protein” as used herein The term “inverted repeat element” (hereinafter “IR”), as used herein, refers to a nucleic acid molecule having two lateral nucleic acid sequences and an optional central sequence. Each of the lateral nucleic acid sequences is complementary to the other. The two lateral nucleic acid sequences may form a palindrome. The central sequence is a short nucleic acid sequence different from each of the two lateral nucleic acid sequences, inserted between the two lateral nucleic acid sequences and disrupting the palindrome formed with the two lateral nucleic acid sequences. For example: a nucleic acid having a sequence of “5′-taatccgga tt tccggatta-3′” (SEQ ID NO: 1) wherein the two lateral nucleic acid sequences are “5′-taatccgga-3′” and “5′-tccggatta-3′” while the central sequence is “5′-tt-3′.”
Various IRs can be found in many different regions of the human genomes. The human genome IRs information is available to the public. For example, Boston University has an Inverted Repeats Database (IRDB) public repository, which provides information on IRs in genomic DNA and contains a variety of tools for their analysis. See tandem.bu.edu/cgi-bin/irdb/irdb.exe. These currently include, the Inverted Repeats Finder algorithm, query and filtering capabilities for finding particular repeats of interest, repeat clustering algorithms based on sequence similarity, PCR primer selection, and data download in a variety of formats. In addition, IRDB serves as a centralized research workbench. It provides storage space for results of analysis and permits collaborators to privately share their data and analysis. Other IRs include those described by Lyu, Lin and Liu (Lyu et al., 1999, J. Mol. Biol., 285:1485-1501). IR can also be artificially designed.
The term “inverted dimer”(hereinafter “ID”) as used herein, refers to a circular nucleic acid molecule which contains two long nucleic acid sequences separated by two IR elements. Each of the two long nucleic acid sequences is complementary to each other, each containing at least a gene and a nuclear anchoring element. The ID is produced from a linear DNA substrate containing two terminal IRs. Theoretically, the linear DNA substrate containing the two terminal IRs is converted to the dumbbell-like DNA intermediate via proceeding by exonuclease or helicase/nuclease within a cell. Two types of circular IDs can be generated depending on specificity of the exonuclease/helicase. In the first type, a double-stranded 5′→3′ or 3′→5′ exonuclease can expose the two terminal IRs as protruding single-stranded DNA on opposite DNA strands. Formation of terminal hairpin loops at the inverted repeat sites then converts the DNA into a dumbbell-like intermediate with terminal loops. Subsequent DNA replication converts the dumbbell-like DNA into the circular inverted dimer. In the second type, the same or a different exonuclease/helicase can convert the linear DNA substrate into a single-stranded DNA. This can be achieved by either a single exonuclease or helicase working processively from one end. Annealing the IRs on the single-stranded DNA results in the formation of dumbbell-like DNA with terminal loops. Subsequent DNA replication converts the dumbbell-like DNA into the circular inverted dimer. See Lin et al., 2001, Nucleic Acids Res. 29: 3529-3538, which is herein incorporated by reference.
The term “nuclear anchoring element” refers to a nuclei acid sequence of a nucleic acid molecule that is retained in a nucleus of a cell, especially in a nucleus of a human cell. This nucleic acid molecule, being a vector and having the nuclear anchoring element, anchors to a nuclear matrix of the nucleus. Examples of a nuclear anchoring element includes, but not limited to, an EBV gene encoding an EBV nuclear protein (e.g., nuclear antigen-1 [EBNA-1]) and an EBV origin (i.e., an EBV replicon, oriP); a papovavirus origin of replication and a papovavirus large T antigen; or a polyomavirus (Py) origin (PyOri) and large T (LT) antigen gene (PyLT). See Heffernan and Dennis, 1991, Nucleic Acids Res. 19:85-92).
The preferred nuclear anchoring element is the EBV gene and the EBV origin. It has been shown that the anchoring effect of the EBV gene and the EBV origin is due to a high-affinity matrix attachment of the oriP sequence (Jankelevich et al., 1992, EMBO J., 11, 1165-1176; Mattia et al., 1999, Virology 262, 9-17) and an interaction of oriP with the origin binding protein, EBNA-1 (Lupton and Levine, 1985, Mol. Cell Biol. 5, 2533-2542; Polvino-Bodnar and Schaffer, 1992, Virology 187, 591-603; Yates et al., 1984, Proc. Natl. Acad. Sci. USA 81, 3806-03810).
In accordance with of the present invention, the expression vectors comprising at least one inverted repeat element or organized as an inverted dimer are constructed. These vectors are demonstrated to be selected for increasing gene expression dramatically. When expressing a resistance gene against a selection agent, the cells may be selected with a high concentration selection agent in one step. Proteins expressed by the expression vectors can be obtained rapidly and effectively even in MMR+ human cells.
The method in accordance with the present invention is for producing at least one protein in cells and comprises the following steps of:
(a) preparing an expression vector comprising at least one nuclear anchoring element, at least one gene; and at least one inverted repeat element;
(b) transfecting the expression vector into a mammalian cell to generate a transfected cell;
(c) culturing the transfected cell and allowing the transfected cell to produce a protein which is encoded by the gene; and
(d) harvesting the protein.
The preferred mammalian cells to be used in expressing the genes carried by the expression vector include, but are not limited to, Chinese hamster ovarian (CHO) cells, human embryonic kidney (HEK293) cells, human colorectal carcinoma with hMLH1−/− cells (HCT116, HCT116+ch3, and HCT116+hMLH1), human colon cancer cells (DLD-1(hMSH6−)).
The following experimental designs are illustrative, but not limiting the scope of the present invention. Reasonable variations, such as those occur to reasonable artisan, can be made herein without departing from the scope of the present invention.
EXAMPLE 1
Construction and Transfection of Expression Vectors
With reference to FIG. 1A, a commercially available pEGFP-N1 plasmid (Clontech, Calif.), an EBV-based p220.2 plasmid derived from a commercially available pDR2 plasmid (Clontech, Calif.) and a commercially available pMAMneo plasmid (Clontech, Calif.) were used to construct a pGT01-GFP plasmid.
The pEGFP-N1 plasmid has multiple cloning sites comprising a BglII and a BamHI and an XhoI restriction sites.
The pEGFP-N1 plasmid was digested with BglII and BamHI restriction enzymes to eliminate the XhoI restriction site from the multiple cloning sites and to obtain a linear pEGFP-N1 DNA. The linear pEGFP-N1 DNA underwent self-ligation after agarose gel purification. The elimination of Xhol site was checked with the digestion by EcoRI. An XhoI-eliminated pEGFP-N1 plasmid was obtained. An EGFP gene having an EGFP coding sequence (CDS) encoding an EGFP protein and a CMV promoter driving the EGFP CDS were obtained by the digesting the XhoI-eliminated pEGFP-N1 plasmid with AflII and ApaLI restriction enzymes.
With reference to FIGS. 1A, 1B and 1C, a plasmid pGT01-GFP has a nuclear-anchoring element, a gene for hygromycin B resistance, a gene for ampicillin resistance, an HindIII restriction site, an AflII restriction site, a PvuII restriction site, an XhoI restriction site and a BamHI restriction site. The EGFP gene was inserted into the BamHI restriction site as a gene. The nuclear-anchoring element comprises an EBV gene encoding an EBV nuclear protein EBNA-1 and an EBV replicon oriP.
The plasmid, pGT01-GFP, was obtained primarily from the p220.2 plasmid with a BamHI-HindIII fragment of the pMAMneo plasmid.
An HPH fragment, being an inverted repeat element and comprising two H segments as lateral nucleic acid sequences and a P segment as a central sequence of an inverted repeat element, was obtained from a pHPH plasmid. The pHPH plasmid was generated from pBR322 (Bi and Liu, 1996, Proc. Natl. Acad. Sci. USA 93, 819-823). The HPH fragment was formed as an HPH/tet cassette in the pHPH plasmid and was a genetic switch controlling transcription of the functional tetracycline gene, depending on the orientation of the P segment.
The shortened HPH fragment was obtained from a pHPH plasmid (Lin et al., 1997, Nucleic Acids Res. 25, 3009-3016) by NruI digestion.
A plasmid, pGT02-GFP, was generated by cloning the HPH fragment to the blunted BamHI site as a first inverted repeat element and cloning an EGFP gene to the AflII restriction site.
A plasmid, pGT03-GFP, was generated by further cloning the HPH fragment as a second inverted repeat element to the PvuII restriction site of pGT02-GFP in an opposite orientation of P to the first inverted repeat element. The relative orientations of the first inverted repeat element and the second inverted repeat element were determined by restriction enzyme digestion.
With further reference to FIG. 2, pGT03-GFP was digested with XhoI enzyme and undergone gel-purification to obtain a linear pGT03-GFP DNA.
The linear pGT03-GFP DNA has two ends and the first inverted repeat element and the second repeat were respectively located near the ends.
The linear pGT03-GFP DNA was denatured to obtain a neutralized DNA solution containing a single stranded linear pGT03-GFP DNA. The denaturation of the linear pGT03-GFP DNA was achieved by alkali denaturation with a volume of 0.1 N NaOH.
An equal volume of 0.1 N HCl plus one tenth volume of 1 M Tris (pH 7.8) was added to the neutralized and thereby quench the DNA solution. The neutralized DNA solution was briefly incubated at 37° C. for 10 min and then kept on ice.
Each of the lateral nucleic acid sequences (H segments) of the first inverted repeat element was allowed to be annealed to the other; and so was each of the lateral nucleic acid sequences of the second repeat.
A hairpin-like structure was formed near each of the two ends of the single stranded linear pGT03-GFP DNA to obtain a self-annealing single stranded linear pGT03-GFP DNA. With reference to FIG. 2, a gap was formed between the ends of the self-annealing single stranded linear pGT03-GFP DNA. A DNA polymerase and a ligase may be employed to treat the a self-annealing single stranded linear pGT03-GFP DNA to seal the gap and to obtain a dumbbell-like DNA (Lin et al., 1997, Nucleic Acids Res. 25, 3009-3016; Lin et al., 2001, Nucleic Acids Res. 29: 3529-3538).
The self-annealing single stranded linear pGT03-GFP DNA was introduced into an E. coli DH5α (RecA−) to allow DNA replication and to generate a double stranded DNA plasmid pGT04/ID (SEQ ID NO: 2) from the dumbbell-like DNA as shown in FIG. 2.
The constructed pGT01-GFP, pGT02-GFP, pGT03-GFP and pGT04/ID, being used as expression vectors, were used to transfect into cells by electroporation using a commercially available Nuleofector system (Amaxa, Germany) according to the manufacturer's instructions. Transfected cells were cultured for 24-48 hours before addition of 50 μg/ml of hygromycin B for selecting stable clones.
The main objective in accordance with the present invention was to enhance the gene expression in transfected cells. To this end, the expression vectors capable of replicating in human cells was designed to allow for the persistence of molecules over prolonged periods of culture by containing the nuclear-anchoring element. In the example, the nuclear-anchoring element comprises the EBNA-1 and the oriP. A vector or an expression vector containing EBNA-1 and oriP is also known as an EBV vector. It has been shown that EBV vectors anchor to the nuclear matrix through a high-affinity matrix attachment region containing the oriP sequence (Jankelevich et al., 1992, EMBO J., 11, 1165-1176; Mattia et al., 1999, Virology, 262, 9-17). Furthermore, interaction of oriP with the origin binding protein, EBNA-1 is required for EBV vector replication, segregation, and retention in primate cells (Lupton and Levine, 1985, Mol. Cell. Biol. 5, 2533-2542; Polyino-Bodnar and Schaffer, 1992, Virology 187, 591-603; Yates et al., 1984, Proc. Natl. Acad. Sci. USA 81, 3806-3810). With further reference to FIGS. 1A, 1B and 1C, to demonstrate whether the DNA rearrangement mediated by at least one inverted repeat element can further enhance the gene expression through the increased drug selection and the gene copies, the expression vectors containing EBV elements and at least one inverted repeat (IR) element were constructed as described above. The EGFP gene encoding an EGFP protein driven by a CMV promoter was cloned into each of the expression vectors as a gene. With further reference to FIG. 2, the inverted dimer, pGT04/ID, was generated from pGT03-GFP through an IR-mediated rearrangement.
EXAMPLE 2
Preparation of Cells
HCT116 (human colorectal carcinoma with hMLH1−/−) cells were obtained from Dr. C. R. Boland (UCSD, Calif.) (Koi et al., 1994, Cancer Res. 54, 4308-4312; ATCC#CCL-247).
Plasmid pC9MLHWT harboring the wild-type hMLH1 cDNA (under the control of CMV promoter) were obtained from Dr. B. Vogelstein.
Plasmid pC9MLHWT was used to transfect HCT116 cells by electroporation to restore the mismatch repair function (Lin et al., Nucleic Acids Res. 29, 3304-3310). The resulting G418-resistant clone expressing the hMLH1 protein was termed HCT116+hMLH1. HCT116+hMLH1 cells were cultured in the presence of 100 μg/ml of G418.
Another colon cancer cell line DLD-1 (hMSH6−) (ATCC#CCL-221) was obtained from Dr. Thomas A. Kunkel (NIEHS, NC). DLD-1 cells were cultured in complete RPMI medium supplemented with 10% fetal bovine serum.
EXAMPLE 3
Enrichment Assay
About 1×104 of subconfluent cells were seeded in 100×20 mm culture dishes with complete RPMI-1640 or DMEM medium supplemented with 10% commercially available fetal bovine serum (Gibco) 24 hrs prior to drug selection. As nutrition of the medium consumed and cell wastes accumulated, the aged medium was renewed in a period of time with a fresh medium. Following medium renewal, hygromycin B was added in indicated concentrations to each plate. The culture dishes were incubated in a 37° C. incubator supplied with 5% CO2. Depending on the cytotoxity induced by the hygromycin B, cells were replenished with fresh medium after treatment for 2 to 4 days. Survival colonies were observed after 10-14 days of growth. The plating efficiency of cell lines was determined by seeding 500 cells in a 100×20 mm culture dish and counting the colonies after 10 days of growth.
EXAMPLE 4
(1) Measurement of Enhanced Green Fluorescence Protein (EGFP) Production
Cells were washed once with PBS, trypsinized, and resuspended in PBS. The green fluorescence of EGFP was measured at an excitation wavelength of 480 nm and an emission wavelength of 525 nm using a FACS Vantage cell sorter (Becton Dickinson, Mountain View, Calif.).
(2) Enhanced Recombinant Gene Expression
The human colon cancer cells, DLD-1, HCT116, and HCT116+hMLH1, were transfected with each of the expression vectors and selected for stable clones with 50 μg/ml of hygromycin B. Multiple stable clonal cells of each stable clone were cultured. 1×104 stable clonal cells of each human colon cancer cells were seeded and further cultured in medium containing either 400 μg/ml or 1000 μg/ml of hygromycin B.
With further reference to FIGS. 3A, 3B, 3C and 3D, colon cancer cell lines, DLD-1 (MMR−) and HCT116 (MMR−) cells, transfected with pGT04/ID gave rise to over 50 times more colonies than cells with at least one inverted repeat element upon selection with 1000 μg/ml of hygromycin B. With further reference to FIG. 4A, the increased yields of EGFP were observed in cells upon culture in 400 μg/ml or 1000 μg/ml of hygromycin B by flow cytometry.
EXAMPLE 5
Western Blotting
A cocktail of protease inhibitors is a mixture of protease inhibitors with broad specificity for the inhibition of proteases such as serine, cysteine, aspartic proteases and aminopeptidases. A cocktail of protease inhibitors may contain 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), pepstatinA, E-64, bestatin, leupeptin, and aprotinin. A cocktail of protease inhibitors may contain no metal chelators. A cocktail of protease inhibitors may be commercially available.
Cell extracts were prepared in lysis buffer (50 mM HEPES pH 7.6, 0.5% SDS, 1% sodium deoxycholate and 5 mM EDTA) containing a cocktail of protease inhibitors (Sigma). Same amounts of protein samples were separated on 12% SDS-PAGE gel and electroblotted to polyvinylidene fluoride membranes (Amersham). An antibody against GFP (Chemicon International) and an antibody against actin (1-19; Santa Cruz Biotechnology) were used to carry out Western-blotting. Secondary antibodies conjugated with horseradish peroxidase (Sigma) and enhanced chemiluminescence (ECL; Amersham) were used for detection.
With further reference to FIG. 4B, the increased yields of EGFP was observed in cells upon culture in 50 μg/ml, 400 μg/ml or 1000 μg/ml of hygromycin B by Western blotting.
RESULTS
The examples shown above clearly demonstrated that the expression vector containing both the nuclear anchoring element and the IR element(s) enhanced the transgene expression of genes in both MMR− and MMR+ cells.
The experiments of the present invention were carried out by transfecting an expression vector containing a nuclear anchoring element, one or two IR elements, two drug-resistance genes (i.e., hygromycin B-resistance gene and an ampicillin gene) and a reporter gene (i.e., an EGFP gene) to the human cells. As shown in FIG. 3, the transfected cells demonstrated a drastic improvement in drug-resistance (i.e., hygromycin B-resistance), as demonstrated by the finding that more clones survived in 1000 μg of hygromycin B. Also, as shown in FIG. 4, the concentration of EGFP in the clones that survived in higher concentration of hygromycin B was significantly higher than those survived in lower concentration of hygromycin B and in the control (which was not transfected by the expression vector), as demonstrated by the elevated intensity of fluorescence and the higher staining of EGFP in SDS-polyacrylamide gel electrophoresis. This result confirmed that the gene amplification and expression was enhanced in the transfected cells. Most significantly, the improvement was not only found in MMR-deficient cells (which did not suppress gene amplification and expression), but also in MMR-proficient cells (which should have the effect of suppressing gene amplification and expression).
Among the various vectors constructed in the present invention, the pGT04/ID expression vector structured as the circular inverted dimer (“ID”) provided the highest drug-resistance effect and EGFP concentration in the host cells.
A skilled artisan would understand that the drug-resistance genes and EGFP gene as used herein can be replaced with a gene encoding other proteins, such as growth factors, hormones, cytokines, chemokines, neuropeptides, antigens, antibodies, enzymes, clotting factors, anti-angiogenic factors, pro-angiogenic factors, transport proteins, receptors, ligands, regulatory proteins, structural proteins, transcription factors, and ribozymes. Therefore, the present invention has demonstrated that the expression vector can be used for large production of therapeutic proteins and for screening the best expression systems or cell lines for protein production.
Even though numerous characteristics and advantages of the present invention have been set forth in the foregoing description, together with details of the structure and features of the invention, the disclosure is illustrative only. Changes may be made in the details, especially in matters of shape, size, and arrangement of parts within the principles of the invention to the full extent indicated by the broad general meaning of the terms in which the appended claims are expressed.

## Claims

1. An isolated, circular, double-stranded, inverted dimer (ID) expression vector, consisting of:
a first inverted repeat (IR) element having a first end and a second end; a second IR element having a first end and a second end;
a first sequence located between said first end of said first IR element and said first end of said second IR element; and
a second sequence located between said second end of said first IR element and said second end of said second IR element,
wherein said first sequence and said second sequence are fully complementary to each other, each of said first and second sequences comprising a nuclear anchoring element and at least a gene,
wherein each of said first and second IR elements comprises two lateral nucleic acid sequences, each being complementary to the other, and a central sequence that is different from each of said two lateral nucleic acid sequences,
wherein said expression vector is organized as an inverted dimer (ID) for enhancing gene expression in mammalian cells,
wherein said expression vector is capable of transfecting mammalian cells,
wherein said expression vector is an episomal vector in transfected mammalian cells,
wherein said expression vector comprises pGT04-ID, and
wherein said gene encodes a protein which is selected from the group consisting of signal peptide, growth factor, hormone, cytokine, chemokine, neuropeptide, antigen, antibody, enzyme, clotting factor, anti-angiogenic factor, pro-angiogenic factor, transport protein, receptor, ligand, regulatory protein, structural protein, reporter protein, transcription factor, ribozyme, fusion protein, and drug-resistance protein.

2. The expression vector according to claim 1, wherein said protein is of natural origin or artificially modified.

3. The expression vector according to claim 2, wherein said reporter protein is an enhanced green fluorescent protein (EGFP).

4. The expression vector according to claim 1, wherein said gene is transcribed into an RNA which is one selected from the group consisting of tRNA, rRNA, antisense RNA, micro-RNA, and double stranded RNA.

5. The expression vector according to claim 1, wherein said mammalian cells are Chinese hamster ovary (CHO) cells.

6. The expression vector according to claim 1, wherein said mammalian cells are human cells.

7. The expression vector according to claim 6, wherein said human cells are mismatch repair-deficient (MMR−) cells.

8. The expression vector according to claim 7, wherein said MMR− cells are HCT116 (hMLH1−) cells or DLD-1 (hMSH6−) cells.

9. The expression vector according to claim 6, wherein said human cells are mismatch repair-proficient (MMR+) cells.

10. The expression vector according to claim 1, wherein the nuclear-anchoring element comprises an EBV (Epstein-Barr Virus) gene encoding an EBV nuclear protein EBNA-1 and an EBV replicon oriP.

11. A pGT04-ID expression vector comprising a gene selected from the group consisting of signal peptide, growth factor, hormone, cytokine, chemokine, neuropeptide, antigen, antibody, enzyme, clotting factor, anti-angiogenic factor, pro-angiogenic factor, transport protein, receptor, ligand, regulatory protein, structural protein, reporter protein, transcription factor, ribozyme, fusion protein, and drug-resistance protein.

12. The expression vector according to claim 1, wherein said expression vector is capable of enhancing expression of said gene in said mammalian cells.

13. An in vitro human cell comprising the expression vector according to claim 1.

14. The human cell according to claim 13, wherein said human cell is a mismatch repair-deficient (MMR−) cell.

15. The human cell according to claim 13, wherein said human cell is a mismatch repair-proficient (MMR+) cell.

16. A CHO cell comprising the expression vector according to claim 1.

17. The CHO cell according to claim 16, wherein said CHO cell is a mismatch repair-deficient (MMR−) cell.

18. The CHO cell according to claim 16, wherein said CHO cell is a mismatch repair-proficient (MMR+) cell.

19. A method for expressing a gene in a mammalian cell comprising:
transfecting the expression vector according to claim 1 into a mammalian cell in vitro to produce a transfected cell;
culturing said transfected cell to allow said transfected cell to produce a protein which is encoded by said gene; and
harvesting said protein from said cultured transfected cell.

20. The method according to claim 19, wherein said gene is transcribed into an RNA which is one selected from the group consisting of tRNA, rRNA, antisense RNA, micro-RNA, and double stranded RNA.

21. The method according to claim 19, wherein said mammalian cell is a human cell.

22. The method according to claim 21, wherein said human cell is a mismatch repair-deficient (MMR−) cell.

23. The method according to claim 22, wherein said MMR− cell is a HCT116 (hMLH1−) cell or a DLD-1 (hMSH6−) cell.

24. The method according to claim 21, wherein said human cell is a mismatch repair-proficient (MMR+) cell.

25. The method according to claim 19, wherein said mammalian cell is a CHO cell.

26. The method according to claim 25, wherein said CHO cell is a mismatch repair-deficient (MMR−) cell.

27. The method according to claim 25, wherein said CHO cell is a mismatch repair-proficient (MMR+) cell.