Novel promoters for high level expression

Abstract

The present invention provides a novel promoter for high level expression. The present invention more specifically relates to functional chimeric gene regulatory units capable of driving strong and sustained heterologous gene expression.

Description

[0001] This application is a divisional application of an invention patent application with an application date of June 17, 2015, application number 2015800327853, and invention name “Novel promoter for high-level expression”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application makes reference to and claims the benefit of priority from Singapore patent application duly assigned application number 10201403371T filed on 18 June 2014. The contents of said application filed on 18 June 2014 are incorporated herein by reference for all purposes including the incorporation of any element or part thereof not contained herein but referred to in PCT Rule 20.5(a) pursuant to PCT Rule 4.18 in its description, claims or drawings. Technical Field

[0004] The present invention relates to the field of recombinant promoter variants capable of driving strong and sustained heterologous gene expression in in vitro and in vivo applications such as gene therapy and recombinant protein expression. Background Art

[0005] Strong promoters are required for high-level recombinant protein production in order to provide large quantities of the desired recombinant protein, thereby allowing for a wide variety of possible uses, including industrial processes, diagnostics, and disease treatment.

[0006] A typical promoter for recombinant protein production comprises a promoter element, an immediate upstream enhancer and other cis-acting regulatory elements if necessary. Optionally, transcription factors that act synergistically to enhance transcriptional activity are recruited to the corresponding site by specific sequence motifs.

[0007] Improving recombinant protein expression by improving transcription and by preventing promoter silencing is desirable for optimizing yield. In order to be able to provide functional proteins, mammalian proteins are usually expressed in mammalian cell lines because these proteins ensure the required post-translational modifications, such as "natural" mammalian glycosylation patterns and molecular folding steps. Therefore, mammalian cells are important hosts for the preparation of clinically relevant recombinant proteins. The most widely used method for this purpose is to establish a cell line with actively expressed recombinant genes stably integrated in its genome. Alternatively, proteins can be produced briefly in cells without requiring recombinant gene integration within a limited period of time.

[0008] Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for large-scale commercial preparation of therapeutic proteins. The first CHO cell line was obtained by single cell cloning in 1957. Subsequently, the cell line CHO-K1 was obtained through this progenitor cell line, which contains a significantly lower amount of DNA than the original CHO. Later, another derivative of the original CHO cell line was mutagenized to obtain CHO-DG44, which is a cell line in which both dhfr alleles are deleted. Although CHO cells are used for genetics, toxicity screening, nutrition and gene expression research, the most prominent use is recombinant protein expression.

[0009] Process development using CHO cell lines focuses on achieving maximum yield of active product. Optimization of the amount of active product can be achieved via increasing specific productivity (i.e., product per cell) and / or through cell line development. Cell line development can include subcloning of cell lines to select high-yield clones, as well as using gene amplification.

[0010] Another way to achieve higher recombinant protein production is to increase the cell yield of the process (i.e., the number of cells per unit volume). This can be achieved through process development (e.g., batch, fed-batch, perfusion, etc.) and culture medium development. By increasing the number of cells per unit volume per day, higher levels of product can be produced.

[0011] However, even though the efficiency and yield of recombinant protein production have increased substantially in recent years, there remains a need in the art for alternative methods that allow even higher expression levels to be achieved. Summary of the invention

[0012] The inventors of the present application have found that said need can be met by novel chimeric gene regulatory units.

[0013] Thus, in a first aspect, the present invention relates to an isolated nucleic acid molecule comprising a functional chimeric gene regulatory unit comprising (a) a functional enhancer nucleotide sequence, (b) a functional core promoter nucleotide sequence, and (c) at least one nucleotide sequence encoding an intron, wherein the enhancer nucleotide sequence is 5' to the promoter nucleotide sequence and the intron nucleotide sequence is 3' to the promoter sequence, and wherein at least one nucleotide sequence of the enhancer nucleotide sequence, the promoter nucleotide sequence or the at least one nucleotide sequence encoding an intron is derived from a species different from the other nucleotide sequence.

[0014] In various embodiments, the isolated nucleic acid molecule also comprises at least one nucleotide sequence encoding a polypeptide, peptide or RNA molecule, wherein the sequence is operably connected to the chimeric gene regulatory unit. The at least one nucleotide sequence encoding an interested polypeptide, peptide or RNA molecule can be located at the 3' of an intron nucleotide sequence, preferably directly adjacent to the intron sequence. In various embodiments, if the nucleotide sequence encodes an interested polypeptide, the interested polypeptide is a polypeptide chain of a naturally occurring immunoglobulin or an artificial immunoglobulin. In various embodiments, the interested polypeptide can be an antibody or a fragment thereof. The antibody can be a human antibody or a humanized antibody or a fragment thereof.

[0015] In various embodiments, the chimeric gene regulatory unit has increased resistance to transcriptional silencing.

[0016] In various embodiments, the isolated nucleic acid molecule further comprises at least one nucleotide sequence encoding a restriction endonuclease recognition site. The at least one nucleotide sequence encoding a restriction endonuclease recognition site can be (i) 3' of the enhancer nucleotide sequence and 5' of the promoter nucleotide sequence, or (ii) 3' of the promoter nucleotide sequence and 5' of the at least one nucleotide sequence encoding an intron.

[0017] In various embodiments, the enhancer sequence is derived from a virus, preferably a double-stranded DNA virus. The virus may be a member of the Herpesviridae and Polyomaviridae families, preferably a member of the group consisting of human cytomegalovirus, murine cytomegalovirus, and simian virus 40.

[0018] In various embodiments, any one or more of the enhancer sequence, the promoter sequence and the intron are derived from human cytomegalovirus, mouse cytomegalovirus, simian virus 40, human EF-1α gene or chicken β-actin gene. More preferably, the promoter may be derived from human cytomegalovirus, mouse cytomegalovirus, simian virus 40, human EF-1α gene or chicken β-actin gene, the enhancer may be derived from human cytomegalovirus, mouse cytomegalovirus or simian virus 40, and / or the intron sequence may be derived from human cytomegalovirus, human EF-1α gene or chicken β-actin gene.

[0019] In various embodiments, the enhancer sequence comprises, consists essentially of, or consists of: (i) a nucleotide sequence as shown in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or a complementary sequence thereof; or (ii) a nucleotide sequence that shares at least 75% sequence identity with the nucleotide sequence of (i), or a complementary sequence thereof.

[0020] In various embodiments, the promoter sequence comprises, consists essentially of, or consists of: (i) a nucleotide sequence as shown in any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or its complementary sequence; or (ii) a nucleotide sequence that shares at least 75% sequence identity with the nucleotide sequence of (i), or its complementary sequence.

[0021] In various embodiments, the intron nucleotide sequence comprises, consists essentially of, or consists of: (i) a nucleotide sequence as shown in any one of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or a complementary sequence thereof; or (ii) a nucleotide sequence that shares at least 75% sequence identity with the nucleotide sequence of (i), or a complementary sequence thereof.

[0022] In various embodiments, the chimeric gene regulatory unit comprises, consists essentially of, or consists of:

[0023] (1) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 11 or the complementary sequence thereof;

[0024] (2) the nucleotide sequences shown in SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 11 or their complementary sequences;

[0025] (3) the nucleotide sequences shown in SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 11 or their complementary sequences;

[0026] (4) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 9 or the complementary sequence thereof;

[0027] (5) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 11 or the complementary sequence thereof;

[0028] (6) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 9 or the complementary sequence thereof;

[0029] (7) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 11 or its complementary sequence;

[0030] (8) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 11 or the complementary sequence thereof;

[0031] (9) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 7 and SEQ ID NO: 9 or the complementary sequence thereof;

[0032] (10) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 10 or a complementary sequence thereof;

[0033] (11) the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 10 or a complementary sequence thereof;

[0034] (12) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 6 and SEQ ID NO: 11 or its complementary sequence;

[0035] (13) the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 10 or a complementary sequence thereof;

[0036] (14) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 10 or a complementary sequence thereof;

[0037] (15) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 11 or its complementary sequence;

[0038] (16) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 9 or the complementary sequence thereof;

[0039] (17) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 7 and SEQ ID NO: 11 or its complementary sequence;

[0040] (18) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 8 and SEQ ID NO: 10 or a complementary sequence thereof;

[0041] (19) the nucleotide sequence shown in SEQ ID NO: 2; SEQ ID NO: 5 and SEQ ID NO: 10 or its complementary sequence;

[0042] (20) the nucleotide sequence shown in SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:11 or its complementary sequence;

[0043] (21) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 9 or a complementary sequence thereof;

[0044] (22) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 10 or a complementary sequence thereof;

[0045] (23) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 11 or a complementary sequence thereof;

[0046] (24) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 7 and SEQ ID NO: 10 or a complementary sequence thereof;

[0047] (25) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 10 or a complementary sequence thereof; or

[0048] (26) A nucleotide sequence that shares at least 75% sequence identity with a nucleotide sequence of (1) to (25) or its complementary sequence.

[0049] In various embodiments, the isolated nucleic acid molecule of the present invention comprising a functional chimeric gene regulatory unit and the at least one nucleotide sequence encoding a polypeptide, peptide or RNA molecule of interest has increased expression activity in CHO (Chinese Hamster Ovary) K1 or CHO DG44 cells for expressing a polypeptide, peptide or RNA molecule of interest compared to an isolated nucleic acid molecule comprising a naturally occurring gene regulatory unit and a nucleotide sequence encoding the same polypeptide, peptide or RNA molecule of interest. The chimeric gene regulatory unit having increased expression activity in CHO K1 cells may comprise, consist essentially of, or consist of (i) a nucleotide sequence as shown in SEQ ID NO: 30, SEQ ID NO: 35, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 65, or a complementary sequence thereof; or (ii) a nucleotide sequence that shares at least 75% sequence identity with the nucleotide sequence of (i), or a complementary sequence thereof. The chimeric gene regulatory unit with increased expression activity in CHO DG44 cells comprises, consists essentially of or consists of: (i) a nucleotide sequence as shown in SEQ ID No. 34, 36, 37, 45, 47-52, 56, 57, 59-62, 64-67, 71 or a complementary sequence thereof; or (ii) a nucleotide sequence or a complementary sequence thereof that shares at least 75% sequence identity with the nucleotide sequence of (i).

[0050] In various embodiments, the promoter comprises at least one binding site for a transcription factor. The transcription factor may be a specialized protein 1 (Sp1) transcription factor. The Sp1 transcription factor may comprise, consist essentially of, or consist of a polypeptide sequence as set forth in any one of SEQ ID NO:74 and SEQ ID NO:75, or a fragment thereof. In various embodiments, at least one binding site for the transcription factor comprises, consists essentially of, or consists of a nucleotide sequence as set forth in SEQ ID NO:76 (5'-(G / T)GGGCGG(G / A)(G / A)(C / T)-3').

[0051] In another aspect, the present invention relates to a vector, preferably a plasmid, comprising the isolated nucleic acid molecule as described herein.

[0052] In yet another aspect, the present invention also relates to a host cell comprising the isolated nucleic acid molecule or vector as described herein.The host cell may be a eukaryotic cell, such as a CHO cell, preferably a CHO K1 cell or a CHO DG44 cell.

[0053] Another aspect of the present invention relates to the use of an isolated nucleic acid molecule of the present invention in promoting or enhancing the expression of a polypeptide, peptide or RNA of interest, wherein the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide, peptide or RNA of interest, wherein the nucleotide sequence encoding the polypeptide, peptide or RNA of interest is operably linked to the chimeric gene regulatory unit of the isolated nucleic acid molecule.

[0054] Yet another aspect relates to a method for preparing a polypeptide, peptide or RNA of interest, comprising:

[0055] (i) providing an isolated nucleic acid molecule as described herein, wherein the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide, peptide or RNA of interest, wherein the nucleotide sequence encoding the polypeptide, peptide or RNA of interest is operably linked to a chimeric gene regulatory unit of the isolated nucleic acid molecule; and

[0056] (ii) preparing the polypeptide, peptide or RNA of interest by in vitro transcription and translation or in a suitable host cell under conditions that allow the production of the polypeptide, peptide or RNA of interest. In such methods, the host cell can be a eukaryotic cell, such as a CHO cell, preferably a CHO K1 cell or a CHO DG44 cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and accompanying drawings.

[0058] Figure 1 An overview schematic diagram of the design of chimeric gene regulatory units is shown. Five natural gene regulatory units present upstream of the human cytomegalovirus immediate early gene (hCMV), the mouse cytomegalovirus immediate early gene (mCMV), the simian virus 40 (SV40), the human elongation factor-1α gene (hEF-1α), and the chicken β-actin gene (cA) were split into enhancers (E), core promoters (CP), and introns (I). These enhancers, core promoters, and introns from different sources were then combined to generate fifty-seven new enhancer-core promoter and enhancer-core promoter-intron combinations, referred to as chimeric gene regulatory units or hybrid promoters.

[0059] Figure 2Schematic diagrams of monoclonal antibody expression vectors used to compare different chimeric promoters are shown. (A) Antibody expression vectors used to compare chimeric promoters in CHO K1 cells. (B) Antibody expression vectors used to compare chimeric promoters in CHO DG44 cells. ChiP: chimeric gene regulatory unit; LC: antibody light chain coding sequence; HC: antibody heavy chain coding sequence; Zeo: bleomycin coding sequence; DHFR: dihydrofolate reductase coding sequence; IRESwt: wild-type encephalomyocarditis virus internal ribosome entry site; mIRES: mutant IRES with reduced translation efficiency; pA: polyadenylation signal.

[0060] Figure 3 Shows the Figure 2 Comparison of expression levels of the newly generated chimeric gene regulatory units and wild-type promoters in stably transfected CHO K1 (A) and CHO DG44 (B) cells of Table 2. Stably transfected libraries were generated by transfecting CHO K1 / CHO DG44 cells with monoclonal antibody (mAb) expression vectors containing different chimeric promoters and selecting stable transfectants using zeocin. Antibody titers of the stably transfected libraries were determined in shake flask batch cultures. The black bars represent titers from the strongest wild-type promoter. Seven hybrid promoters showed higher recombinant protein yields in CHO K1 cells than any wild-type promoter. 21 hybrid promoters showed higher expression in CHO DG44 cells than any wild-type promoter. DETAILED DESCRIPTION

[0061] The specific details and embodiments of the present invention are described in detail below by way of example. These embodiments are described in sufficient detail to enable those skilled in the art to practice the present invention. Without departing from the scope of the present invention, other embodiments may be utilized, and structural and logical modifications may be made. Various embodiments may not necessarily be mutually exclusive, because some embodiments may be combined with one or more other embodiments to form new embodiments.

[0062] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In the event of a conflict, the present document, including definitions, will control.

[0063] It is an object of the present invention to provide novel chimeric gene regulatory units for high-level expression of molecules of interest.

[0064] In a first aspect, the present invention relates to an isolated nucleic acid molecule comprising a functional chimeric gene regulatory unit, the unit comprising (a) a functional enhancer nucleotide sequence, (b) a functional promoter nucleotide sequence, and (c) at least one nucleotide sequence encoding an intron, wherein the enhancer nucleotide sequence is 5' to the promoter nucleotide sequence and the intron nucleotide sequence is 3' to the promoter sequence, and wherein at least one nucleotide sequence of the enhancer nucleotide sequence, the promoter nucleotide sequence or the at least one nucleotide sequence encoding an intron is derived from a species different from the other nucleotide sequence.

[0065] As used herein, the term "functional" refers to an entity that has the native biological activity of a naturally occurring entity of its type, or any specific desired activity, such as in the case of a promoter, as determined by its ability to initiate transcription of a gene.

[0066] In the present context, the term "isolated nucleic acid molecule" relates to a nucleic acid molecule that can occur independently of its natural genetic environment and / or background and is preferably separated from other nucleic acids or cellular components. Isolation can be performed by purification, and various techniques for purification are known in the art.

[0067] The term "nucleic acid molecule" refers to a phosphate polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecule") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine or deoxycytidine; "DNA molecule"), any phosphate analogs thereof, such as thiophosphates and thioesters, and artificial nucleic acid analogs, such as peptide nucleic acids, morpholino nucleic acids and locked nucleic acids, as well as glycol nucleic acids and threose nucleic acids. Each of these artificial nucleic acid analogs is distinguished from naturally occurring DNA or RNA by changes in the molecular backbone. In preferred embodiments, the isolated nucleic acid molecule is a DNA molecule.

[0068] The term "at least one" herein refers to one or more, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0069] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, "element" means at least one element and may include more than one element.

[0070] The term "sequence" herein with reference to nucleic acids relates to the primary sequence of the nucleic acid molecule.

[0071] As used herein, the term "functional chimeric gene regulatory unit" or "hybrid promoter" refers to a combination of an enhancer, a core promoter and at least one intron as described herein.

[0072] In the present context, the term "chimeric" or "heterozygous" refers to the fact that different elements of the regulatory unit are derived from different genes, i.e., the combination of elements of the regulatory unit herein does not exist in nature. This can be achieved by using at least one element from a different species and combining it with other elements of the regulatory unit. At least one of the enhancer, core promoter and intron is therefore heterologous with respect to at least one of the other two.

[0073] In the present context, the term "functional enhancer" refers to a short DNA region that can activate transcription (of a gene) (activator), for example by being able to bind a protein.

[0074] Generally, the term "promoter" refers to a region of DNA that initiates transcription of a specific gene.

[0075] As used herein, the term "functional promoter" or "core promoter" refers to the core region in a promoter, which is the minimal promoter portion required to correctly initiate gene transcription.

[0076] As used herein, the term "intron" is a nucleotide sequence within a gene that is removed by RNA shearing during maturation of the final RNA product. The term intron refers to both the DNA sequence within a gene and the corresponding sequence in the RNA transcript. The sequences that are linked together in the final mature RNA after RNA shearing are exons.

[0077] The term "5" herein refers to the directionality of a single strand of nucleic acid, i.e., the end-to-end chemical orientation. Chemical transformation of the named carbon atoms in the nucleotide sugar ring produces the 5-end and the 3-end numerically. The relative position of the structure along the nucleic acid chain (including genes and various protein binding sites) is usually referred to as upstream (towards the 5-end) or downstream (towards the 3-end). This naming convention is of great significance because nucleic acids can only be synthesized in the body in a 5 to 3' direction, because the polymerase that assembles the new chain only attaches the new nucleotide to the 3-hydroxyl (-OH) group through a phosphodiester bond.

[0078] In various embodiments, the isolated nucleic acid molecule also comprises at least one nucleotide sequence encoding an interested peptide, polypeptide or RNA molecule. If high expression levels are desired, the molecules of interest are those that will be expressed using novel chimeric gene regulatory units. Preferably, they are recombinant polypeptides or proteins.

[0079] As used herein, the term "peptide" refers to two or more nucleic acids linked by a peptide bond, and thus includes dipeptides, oligopeptides and polypeptides.

[0080] The term "polypeptide" herein refers to a long continuous peptide chain preferably of at least 50 amino acids in length.

[0081] The term "protein" herein refers to one or more polypeptides arranged in a biologically functional manner. A protein may consist of more than one polypeptide chain, such as an antibody, which consists of two light chains and two heavy chains, each of which is a polypeptide. A protein may bind to a ligand, such as a coenzyme and a cofactor, or to another protein or other macromolecule.

[0082] In various embodiments, the sequence encoding the molecule of interest is operably linked to the chimeric gene regulatory unit. In a preferred embodiment, the at least one nucleotide sequence encoding the molecule of interest is located 3' (downstream) relative to the intron nucleotide sequence. In a more preferred embodiment, the at least one nucleotide sequence encoding the molecule of interest is directly adjacent to the intron sequence.

[0083] "Immediately adjacent" means that the intron sequence and the coding sequence are directly linked by a phosphodiester bond and that there is no linker nucleotide sequence intervening between the two elements.

[0084] As used herein, the term "operably linked" means linked in such a way that the chimeric gene regulatory unit can control the expression of a molecule of interest.

[0085] Advantageously, the at least one nucleotide sequence encoding the molecule of interest is directly adjacent to an intronic sequence, as this increases the chances of correct and high-level expression.

[0086] In various embodiments, the at least one nucleotide sequence encoding a molecule of interest encodes a polypeptide of interest, the polypeptide being a naturally occurring immunoglobulin or a polypeptide chain of an artificial immunoglobulin. In a preferred embodiment, the polypeptide of interest is a protein, and the protein is an antibody. In a more preferred embodiment, the antibody is a human antibody or a humanized antibody or a fragment thereof. It should be understood that in such embodiments, where the expression of an immunoglobulin, specifically an antibody, is desired, the isolated nucleic acid molecule comprises a nucleotide sequence encoding more than one polypeptide. For example, in the case of desired antibody expression, the isolated nucleic acid molecule may comprise two nucleic acid sequences encoding a polypeptide of interest, i.e., one sequence encoding a heavy chain and one sequence encoding a light chain. In such embodiments, different coding sequences may be directly connected or may be separated by a linker nucleotide sequence. The linker nucleotide sequence may be functional because they allow ribosomes to bind and may therefore, for example, comprise an internal ribosome entry site (IRES).

[0087] As used herein and consistent with the above, the term "immunoglobulin" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes.

[0088] In various embodiments, the chimeric gene regulatory unit has increased resistance to transcriptional silencing.

[0089] The term "transcriptional silencing" refers to any mechanism by which gene expression is downregulated at the transcriptional level, for example, via DNA methylation, histone modification, and chromatin remodeling, which renders the DNA permanently inoperable for further transcription.

[0090] Advantageously, if the chimeric gene regulatory unit has increased resistance to transcriptional silencing, this will result in a higher overall yield of expressed product.

[0091] In various embodiments, the nucleic acid molecule of this separation also comprises the nucleotide sequence of at least one coding restriction endonuclease recognition site.In preferred embodiments, the nucleotide sequence of the at least one coding restriction endonuclease recognition site is at 3' of the enhancer nucleotide sequence and 5' of the promoter nucleotide sequence.Or, in various preferred embodiments, the nucleotide sequence of the coding restriction endonuclease recognition site is at 3' of the promoter nucleotide sequence and 5' of the nucleotide sequence of the at least one coding intron.This position makes it possible to separate and combine the different elements of the control unit in a specific manner, for example, connect enhancer, promoter and intron sequence.

[0092] As used herein, the term "restriction endonuclease" is intended to mean an enzyme that recognizes a specific nucleotide sequence in a nucleic acid and cuts the nucleic acid. Restriction endonucleases can recognize sequences of, for example, 4, 5, 6, 7 or more nucleotides in length. Restriction endonucleases can recognize more than one sequence, for example, two or more variants of a degenerate sequence that contains one of two or more different nucleotides at a specific position. Alternatively, a restriction endonuclease can be specific for a single recognition sequence.

[0093] In this article, the term "recognition site" is intended to mean a portion of a nucleic acid that specifically binds to a specific binding moiety such as a restriction endonuclease, more specifically a substrate recognition and binding site for a restriction endonuclease. Typically, a restriction endonuclease recognition site is cut by a restriction endonuclease.

[0094] In various embodiments, the enhancer sequence is derived from a virus. In a preferred embodiment, the enhancer sequence is derived from a double-stranded DNA virus. In a more preferred embodiment, the enhancer sequence is derived from a virus consisting of the group of Herpesviridae and Polyomaviridae. In a further preferred embodiment, the enhancer sequence is derived from the group consisting of human cytomegalovirus, murine cytomegalovirus, and simian virus 40.

[0095] Cytomegalovirus is a genus of viruses under the family Herpesviridae, abbreviated as CMV. The species that infect humans is commonly referred to as human CMV (hCMV). Other CMV viruses, such as murine cytomegalovirus in mice, exist in several mammalian species, but the species isolated from animals differ from hCMV in genome structure.

[0096] Simian virus 40 (SV40) is a polyomavirus found in both monkeys and humans.

[0097] In various embodiments, the enhancer sequence comprises, consists essentially of, or consists of: (i) a nucleotide sequence as set forth in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or a complementary sequence thereof; or (ii) a nucleotide sequence that shares at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98.0%, 98.5%, 99.0%, or 99.5% sequence identity with the nucleotide sequence of (i), or a complementary sequence thereof.

[0098] "Complementary sequence" herein refers to a nucleic acid molecule that is complementary to another nucleic acid molecule when the two nucleic acid molecules are aligned antiparallel to each other, because substantially all of the nucleotides of either nucleic acid molecule form Watson-Crick base pairs with corresponding nucleotides on the other molecule. In various embodiments, the complementary sequence is a fully complementary sequence, because each nucleotide of the corresponding molecule or sequence forms a Watson-Crick base pair with a corresponding nucleotide on the other strand.

[0099] The term "sequence identity" herein is usually expressed as a percentage and refers to the percentage of identical amino acid residues or nucleotides (as the case may be) between two sequences when optimally aligned. For purposes of the present invention, sequence identity means sequence identity determined using the well-known Basic Local Alignment Search Tool (BLAST), which is publicly available through the National Cancer Institute / National Institutes of Health (Bethesda, Maryland) and described in printed publications (see, e.g., Altschul et al., J. Mol. Biol., 215 (3): 403-10 (1990)).

[0100] In various embodiments, the sequence of the core promoter is derived from the group consisting of human cytomegalovirus, murine cytomegalovirus, simian virus 40, human EF-1α gene promoter, and chicken β-actin gene promoter.

[0101] Elongation factor 1-alpha 1 (EF-1α) is a protein that in humans is encoded by the EEF1A1 gene. This gene encodes an isoform of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome.

[0102] In various embodiments, the core promoter sequence comprises, consists essentially of, or consists of: (i) a nucleotide sequence as shown in any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or a complementary sequence thereof; or (ii) a nucleotide sequence that shares at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98.0%, 98.5%, 99.0% or 99.5% sequence identity with the nucleotide sequence of (i), or a complementary sequence thereof.

[0103] In various embodiments, the intronic nucleotide sequence is selected from the group consisting of sequences derived from human cytomegalovirus, human EF-1α gene, and chicken β-actin gene.

[0104] In various embodiments, the intron nucleotide sequence comprises, consists essentially of, or consists of: (i) a nucleotide sequence as shown in any one of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or a complementary sequence thereof; (ii) a nucleotide sequence that shares at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98.0%, 98.5%, 99.0% or 99.5% sequence identity with the nucleotide sequence of (i), or a complementary sequence thereof.

[0105] The above elements may be combined such that the chimeric gene regulatory unit comprises, consists essentially of, or consists of:

[0106] (1) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 11 or the complementary sequence thereof;

[0107] (2) the nucleotide sequences shown in SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 11 or their complementary sequences;

[0108] (3) the nucleotide sequences shown in SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 11 or their complementary sequences;

[0109] (4) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 9 or the complementary sequence thereof;

[0110] (5) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 11 or its complementary sequence;

[0111] (6) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 9 or the complementary sequence thereof;

[0112] (7) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 11 or the complementary sequence thereof; or

[0113] (8) A nucleotide sequence or its complementary sequence that has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98.0%, 98.5%, 99.0% or 99.5% sequence identity with a nucleotide sequence of (1)-(7).

[0114] Such chimeric gene regulatory units are advantageous because, surprisingly, they were found to show higher levels of recombinant protein production in CHO KG1 cells compared to a wild-type gene regulatory unit or a different chimeric gene regulatory unit.

[0115] The term "wild type" (WT) herein refers to the typical, most common or most traditional form as it occurs in nature.

[0116] In various embodiments, the isolated nucleic acid molecules of the present invention comprising a functional chimeric gene regulatory unit and the at least one nucleotide sequence encoding a molecule of interest have increased expression activity because they express higher levels of the molecule of interest in Chinese hamster ovary (CHO) K1 cells relative to an isolated nucleic acid molecule comprising a naturally occurring gene regulatory unit and a nucleotide sequence encoding the same molecule of interest. In preferred embodiments, the increased expression activity of the functional chimeric gene regulatory unit is at least 1.1 times, 1.15 times, 1.2 times, 1.4 times, 1.5 times, 1.6 times, or 1.75 times higher than that of the naturally occurring gene regulatory unit.

[0117] CHO cells are advantageous for the following reasons: their ability to produce glycoproteins with post-translational modifications compatible with humans, their resistance to infection by human viruses, the availability of well-defined gene amplification systems for CHO cells, and the ability of the cells to adapt and grow in serum-free suspension culture. These properties make CHO cells ideal for large-scale high-titer culture in industry.

[0118] In various embodiments, the chimeric gene regulatory unit comprises, consists essentially of, or consists of (i) a nucleotide sequence as shown in SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, or a complementary sequence thereof; or (ii) a nucleotide sequence that shares at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98.0%, 98.5%, 99.0% or 99.5% sequence identity with the nucleotide sequence of (i), or a complementary sequence thereof. It was found that the construct provides increased expression activity in CHO K1 cells. Particularly preferred are chimeric gene regulatory units comprising, consisting essentially of or consisting of the nucleotide sequence as shown in SEQ ID NO: 35, or nucleotide sequences sharing at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98.0%, 98.5%, 99.0% or 99.5% sequence identity with said nucleotide sequence of SEQ ID NO: 35, or the corresponding complementary sequences of these sequences. It has been found that said sequence is particularly advantageous even though it does not provide higher titers, because the time for generating a stably transfected cell bank with said chimeric gene regulatory unit is significantly shorter, e.g. three weeks instead of two weeks, compared to the chimeric promoters as shown in SEQ ID No. 30, 59, 60, 62, 64 and 65.

[0119] In various other embodiments, the chimeric gene regulatory unit comprises, consists essentially of, or consists of:

[0120] (1) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 11 or its complementary sequence;

[0121] (2) the nucleotide sequence shown in SEQ ID NO: 2; SEQ ID NO: 5 and SEQ ID NO: 11 or its complementary sequence;

[0122] (3) the nucleotide sequence shown in SEQ ID NO: 1; SEQ ID NO: 5 and SEQ ID NO: 11 or its complementary sequence;

[0123] (4) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 7 and SEQ ID NO: 9 or the complementary sequence thereof;

[0124] (5) the nucleotide sequences shown in SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 10 or their complementary sequences;

[0125] (6) the nucleotide sequences shown in SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:10 or their complementary sequences;

[0126] (7) the nucleotide sequence as shown in SEQ ID NO: 1, SEQ ID NO: 6 and SEQ ID NO: 11 or the complementary sequence thereof;

[0127] (8) the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 10 or the complementary sequence thereof;

[0128] (9) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 10 or the complementary sequence thereof;

[0129] (10) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 11 or its complementary sequence;

[0130] (11) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 9 or the complementary sequence thereof;

[0131] (12) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 7 and SEQ ID NO: 11 or its complementary sequence;

[0132] (13) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 8 and SEQ ID NO: 10 or a complementary sequence thereof;

[0133] (14) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 10 or a complementary sequence thereof;

[0134] (15) the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 11 or its complementary sequence;

[0135] (16) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 11 or its complementary sequence;

[0136] (17) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 9 or the complementary sequence thereof;

[0137] (18) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 10 or its complementary sequence;

[0138] (19) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 11 or its complementary sequence;

[0139] (20) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 7 and SEQ ID NO: 10 or a complementary sequence thereof;

[0140] (21) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 10 or a complementary sequence thereof; or

[0141] (22) A nucleotide sequence or its complementary sequence that shares at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98.0%, 98.5%, 99.0% or 99.5% sequence identity with a nucleotide sequence of (1)-(21).

[0142] Such chimeric gene regulatory units are advantageous because they were surprisingly found to show higher levels of recombinant protein production in CHO DG44 cells compared to a wild-type gene regulatory unit or a different chimeric gene regulatory unit.

[0143] Therefore, in various embodiments, the isolated nucleic acid molecules of the present invention comprising a functional chimeric gene regulatory unit and the at least one nucleotide sequence encoding a molecule of interest have increased expression activity in Chinese hamster ovary (CHO) DG44 cells expressing a molecule of interest compared to the isolated nucleic acid molecules comprising a naturally occurring gene regulatory unit and a nucleotide sequence encoding a molecule of interest. In preferred embodiments, the increased expression activity of the functional chimeric gene regulatory unit is at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times or 1.7 times higher than the naturally occurring gene regulatory unit.

[0144] In various embodiments, the chimeric gene regulatory unit with increased expression activity in CHO DG44 cells comprises, consists essentially of, or consists of: (i) a nucleotide sequence as shown in SEQ ID No. 34, 36, 37, 45, 47-52, 56, 57, 59-62, 64-67, 71, or a complementary sequence thereof; or (ii) a nucleotide sequence that shares at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98.0%, 98.5%, 99.0% or 99.5% sequence identity with the nucleotide sequence of (i), or a complementary sequence thereof.

[0145] Such chimeric gene regulatory units are advantageous because, surprisingly, they have been found to exhibit higher levels of recombinant protein production than achieved with a wild-type gene regulatory unit or a different chimeric gene regulatory unit.

[0146] In various embodiments, the chimeric gene regulatory unit comprises at least one binding site for a transcription factor.

[0147] In a preferred embodiment, at least one binding site for the transcription factor is contained in an enhancer, a core promoter or an intron.

[0148] As used herein, the term "transcription factor" refers to a protein that binds to a specific DNA sequence to control the rate of transcription of genetic information from DNA to messenger RNA (mRNA). Transcription factors function alone or with other proteins in a complex by promoting (as activators) or blocking (as repressors) the recruitment of RNA polymerase to a specific gene. The defining feature of transcription factors is that they contain one or more DNA binding domains (DBDs) that are attached to specific DNA sequences adjacent to the genes they regulate. Herein, it is preferred that the recruited transcription factors activate RNA polymerase binding and function.

[0149] In a preferred embodiment, the transcription factor is a specialized protein 1 (Sp1) transcription factor. This can be advantageous because it has been shown that the SP1 binding site can enhance expression during long-term culture. The Sp1 transcription factor may comprise, consist essentially of, or consist of a polypeptide sequence as shown in SEQ ID NO:74 or SEQ ID NO:75, or may be a fragment thereof, and the fragment is functional in that it retains at least 50% of the activity of the full-length sequence. Alternatively, in other preferred embodiments, at least one binding site of the transcription factor contained in the nucleic acid molecule described herein comprises, consists essentially of, or consists of a nucleotide sequence as shown in SEQ ID NO:76 (5'-(G / T)GGGCGG(G / A)(G / A)(C / T)-3').

[0150] The use of transcription factors is advantageous since they represent an additional tool to control and improve the rate of transcription of messages from DNA to messenger RNA and thus achieve higher expression levels.

[0151] In yet another aspect, the present invention relates to a vector comprising the isolated nucleic acid molecule of the present invention.

[0152] In this article, the term "vector" refers to a nucleic acid molecule used as a vehicle for artificially carrying foreign genetic material into another cell, and the genetic material can be replicated and / or expressed in the cell. Four main types of vectors are plasmids, viral vectors, cosmids and artificial chromosomes. The common point of all engineered vectors is the origin of replication, multiple cloning sites and selective markers. The vector itself is usually composed of an insertion sequence (transgene) and a larger sequence serving as the "backbone" of the vector. The purpose of the vector that transmits genetic information to another cell is usually to separate the insertion sequence, multiply the insertion sequence in the target cell or express the insertion sequence in the target cell. In a preferred embodiment, the vector is a plasmid.

[0153] In a further aspect, the present invention relates to a host cell comprising the isolated nucleic acid molecule of the present invention and / or the vector of the present invention.

[0154] The term "host cell" herein refers to an organism having a nucleic acid molecule comprising a chimeric gene regulatory unit as described herein. They may be integrated into the genome of the host cell or exist in the cell in a separate form.

[0155] In various embodiments, the host cell is a eukaryotic cell. In a preferred embodiment, the host cell is a mammalian cell. In a further preferred embodiment, the host cell is a Chinese hamster ovary (CHO) cell. In a more preferred embodiment, the host cell is a CHO K1 cell or a CHO DG44 cell.

[0156] The nucleic acid molecules described herein can be used to promote or enhance the expression of a given molecule of interest in a cell. It should be understood that in such embodiments, the isolated nucleic acid molecule also comprises a nucleotide sequence encoding a molecule of interest, which is operably linked to the chimeric gene regulatory unit of the isolated nucleic acid molecule.

[0157] In such methods for expressing a molecule of interest, especially a polypeptide, the actual expression may be performed by in vitro transcription and translation or, more preferably, in a recombinant manner in a suitable host cell under conditions allowing the production of the molecule of interest.

[0158] The host cell may be a cell as described above.

[0159] Conditions that allow production of the molecule of interest include various parameters of cell culture, including the choice of culture medium and culture conditions such as temperature, time, etc. All of these factors are well known to those skilled in the art and can be easily adjusted using routine techniques.

[0160] It is to be understood that all embodiments disclosed herein with respect to the nucleic acids of the invention apply analogously to the vectors, host cells, uses and methods described herein, and vice versa.

[0161] The present invention is further illustrated by the following examples. However, it should be understood that the present invention is not limited to these illustrative embodiments.

[0162] Example

[0163] Five natural promoters existing upstream of the human cytomegalovirus immediate early gene (hCMV), the mouse cytomegalovirus immediate early gene (mCMV), the simian virus 40 (SV40), the human elongation factor-1α gene (hEF-1α), and the chicken β-actin gene (cA) were segmented into enhancers (E), core promoters (CP), and introns (I) ( Figure 1 ). These enhancers, core promoters, and introns from different sources were then recombined to generate fifty-seven new enhancer-core promoter and enhancer-core promoter-intron combinations, called chimeric gene regulatory units or hybrid promoters.

[0164] Table 1 shows the different elements used to generate chimeric gene regulatory units, while Table 2 shows the combinations tested.

[0165] Table 1: Naturally occurring enhancers, promoters and introns

[0166]

[0167] Table 2: Gene regulatory units evaluated

[0168]

[0169]

[0170]

[0171] The different chimeric gene regulatory units were then inserted into antibody expression vectors to compare chimeric promoters in CHO K1 and CHO DG44 cells, where the different elements were arranged in the vectors. Figure 2 It is schematically shown in FIG.

[0172] By dividing l×10 7 Stably transfected CHO K1 libraries were generated by transfecting 10 CHO K1 cells with 5 μg of the appropriate linearized mAb expression plasmid containing a specific chimeric promoter and the zeocin selectable marker gene ( Figure 2 A). Transfection was performed using the Nucleofactor kit V (Lonza, VCA1003) and program U-24 on the Nucleofector II system (Lonza, Cologne, Germany) according to the manufacturer's instructions. The transfected cells were then resuspended in 2 mL of protein-free medium in a 6-well suspension culture plate. 24 hours after transfection, the transfected cell culture was centrifuged at ~100xg for 5 minutes. The cell pellet was then resuspended in 25 mL of protein-free medium containing 600 μg / mL zeocin in a 125 mL shake flask to select stable transfectants. When the viability of the stably transfected pool was restored to more than 95%, the stably transfected pool was considered to have been successfully generated. The yield of each stably transfected pool was determined in a 125 mL shake flask batch culture. The cells were cultured at 2×10 5 Cells were plated at 100 cells / mL. Cell density and viability were monitored daily using Vi-Cell until viability dropped below 50%. Supernatants were collected at the end of the culture and analyzed for mAb concentration using turbidity on an IMMAGE 800 Immunochemistry System (Beckman Coulter, Buckinghamshire, England). The IMMAGE 800 system used an anti-human Fc region antibody to detect IgG. The average titer of the replicate pools generated using each promoter was Figure 3 Shown in A.

[0173] Transfection of CHO DG44 cells was performed using the same protocol as for CHO K1 cells. The transfected cells were then resuspended in 2 mL of protein-free medium containing hypoxanthine and thymidine (HT) in a 6-well suspension culture plate. 24 hours after transfection, the transfected cell culture was centrifuged at ~100×g for 5 minutes. The cell pellet was then resuspended in 25 mL of protein-free medium (without HT) in a 125 mL shake flask to select stable transfectants. When the viability of the stably transfected library was restored to more than 95%, stepwise methotrexate (MTX, Sigma, M8407) amplification was performed at concentrations of 50 nM to 500 nM. The yield of the amplified library at 500 nM was determined using the same protocol as for stably transfected CHO K1 cells. The average titer of the replicate libraries generated using each promoter was Figure 3 Shown in b.

Claims

1. An isolated nucleic acid molecule comprising a functional chimeric gene regulatory unit, the unit comprising: (a) a functional enhancer nucleotide sequence, (b) a functional core promoter nucleotide sequence, and (c) at least one nucleotide sequence encoding an intron, wherein the enhancer nucleotide sequence is located 5' to the promoter nucleotide sequence and the intron nucleotide sequence is located 3' to the promoter sequence, And wherein at least one nucleotide sequence among the enhancer nucleotide sequence, the promoter nucleotide sequence or the at least one nucleotide sequence encoding an intron is derived from a species different from the other nucleotide sequences.

2. The isolated nucleic acid molecule according to claim 1, further comprising at least one nucleotide sequence encoding a polypeptide, peptide or RNA molecule, wherein said sequence is operably linked to said chimeric gene regulatory unit.

3. The isolated nucleic acid molecule according to claim 2, wherein the at least one nucleotide sequence encoding a polypeptide, peptide or RNA molecule of interest is located 3' to the intronic nucleotide sequence.

4. An isolated nucleic acid molecule according to claim 3, wherein the at least one nucleotide sequence encoding a polypeptide, peptide or RNA molecule of interest is directly adjacent to the sequence of the intron.

5. The isolated nucleic acid molecule according to any one of claims 2 to 4, wherein the polypeptide of interest is a polypeptide chain of a naturally occurring immunoglobulin or an artificial immunoglobulin.

6. The isolated nucleic acid molecule of claim 5, wherein the polypeptide of interest is an antibody or a fragment thereof.

7. The isolated nucleic acid molecule of claim 6, wherein the antibody is a human antibody or a humanized antibody or a fragment thereof.

8. The isolated nucleic acid molecule of any one of claims 1-7, wherein the chimeric gene regulatory unit has increased resistance to transcriptional silencing.

9. according to the isolated nucleic acid molecule described in any one of claims 1-8, wherein said isolated nucleic acid molecule also comprises at least one nucleotide sequence encoding a restriction endonuclease recognition site.

10. An isolated nucleic acid molecule according to claim 9, wherein the at least one nucleotide sequence encoding a restriction endonuclease recognition site is: (i) 3' to the enhancer nucleotide sequence and 5' to the promoter nucleotide sequence, or (ii) 3' to the promoter nucleotide sequence and 5' to the at least one nucleotide sequence encoding an intron.

11. The isolated nucleic acid molecule according to any one of claims 1 to 10, wherein the enhancer sequence is derived from a virus, preferably from a double-stranded DNA virus.

12. The isolated nucleic acid molecule of claim 11, wherein the enhancer sequence is derived from a virus consisting of the group consisting of Herpesviridae and Polyomaviridae.

13. The isolated nucleic acid molecule of claim 12, wherein the enhancer sequence is derived from the group consisting of human cytomegalovirus, murine cytomegalovirus, and simian virus 40.

14. The isolated nucleic acid molecule according to any one of claims 1-13, wherein any one or more of the enhancer sequence, the promoter sequence and the intron are independently derived from human cytomegalovirus, murine cytomegalovirus, simian virus 40, human EF-1α gene or chicken β-actin gene.

15. An isolated nucleic acid molecule according to claim 14, wherein (i) the promoter is derived from human cytomegalovirus, mouse cytomegalovirus, simian virus 40, human EF-1α gene or chicken β-actin gene, (ii) the enhancer is derived from human cytomegalovirus, mouse cytomegalovirus or simian virus 40, and / or (iii) the intron sequence is derived from human cytomegalovirus, human EF-1α gene or chicken β-actin gene.

16. The isolated nucleic acid molecule of any one of claims 1-15, wherein the enhancer sequence comprises, consists essentially of, or consists of: (i) a nucleotide sequence as shown in any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or a complementary sequence thereof; or (ii) a nucleotide sequence or a complementary sequence thereof that shares at least 75% sequence identity with the nucleotide sequence of (i).

17. The isolated nucleic acid molecule of any one of claims 1-15, wherein the promoter sequence comprises, consists essentially of, or consists of: (i) a nucleotide sequence as shown in any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or a complementary sequence thereof; or (ii) a nucleotide sequence or a complementary sequence thereof that shares at least 75% sequence identity with the nucleotide sequence of (i).

18. The isolated nucleic acid molecule of any one of claims 1 to 17, wherein the intron nucleotide sequence comprises, consists essentially of, or consists of: (i) a nucleotide sequence as shown in any one of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or a complementary sequence thereof; or (ii) a nucleotide sequence or a complementary sequence thereof that shares at least 75% sequence identity with the nucleotide sequence of (i).

19. The isolated nucleic acid molecule of any one of claims 1-18, wherein the chimeric gene regulatory unit comprises, consists essentially of, or consists of: (27) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 11 or a complementary sequence thereof; (28) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 11 or its complementary sequence; (29) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 11 or its complementary sequence; (30) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 9 or a complementary sequence thereof; (31) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 11 or a complementary sequence thereof; (32) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 9 or the complementary sequence thereof; (33) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 11 or a complementary sequence thereof; (34) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 11 or the complementary sequence thereof; (35) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 7 and SEQ ID NO: 9 or the complementary sequence thereof; (36) the nucleotide sequence shown in SEQ ID NO: 2; SEQ ID NO: 6 and SEQ ID NO: 10 or a complementary sequence thereof; (37) the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 10 or the complementary sequence thereof; (38) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 6 and SEQ ID NO: 11 or a complementary sequence thereof; (39) the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 10 or a complementary sequence thereof; (40) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 10 or a complementary sequence thereof; (41) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 11 or its complementary sequence; (42) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 9 or a complementary sequence thereof; (43) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 7 and SEQ ID NO: 11 or its complementary sequence; (44) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 8 and SEQ ID NO: 10 or a complementary sequence thereof; (45) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 10 or a complementary sequence thereof; (46) the nucleotide sequence shown in SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:11 or its complementary sequence; (47) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 9 or a complementary sequence thereof; (48) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 10 or a complementary sequence thereof; (49) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 11 or a complementary sequence thereof; (50) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 7 and SEQ ID NO: 10 or a complementary sequence thereof; (51) the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 10 or a complementary sequence thereof; or (52) A nucleotide sequence that shares at least 75% sequence identity with a nucleotide sequence of (1)-(25) or its complementary sequence.

20. The isolated nucleic acid molecule according to any one of claims 1 to 19, wherein the isolated nucleic acid molecule of the present invention comprising the functional chimeric gene regulatory unit and the at least one nucleotide sequence encoding a polypeptide, peptide or RNA molecule of interest has increased expression activity in CHO (Chinese Hamster Ovary) K1 or CHO D44 cells to express the polypeptide, peptide or RNA molecule of interest compared to an isolated nucleic acid molecule comprising a naturally occurring gene regulatory unit and a nucleotide sequence encoding the same polypeptide, peptide or RNA molecule of interest.

21. The isolated nucleic acid molecule according to any one of claims 1 to 20, wherein the chimeric gene regulatory unit having increased expression activity in CHO K1 cells comprises, consists essentially of, or consists of: (i) a nucleotide sequence as shown in SEQ ID NO:30, SEQ ID NO:35, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:65, or a complementary sequence thereof; or (ii) a nucleotide sequence or a complementary sequence thereof that shares at least 75% sequence identity with the nucleotide sequence of (i).

22. The isolated nucleic acid molecule according to any one of claims 1-20, wherein the chimeric gene regulatory unit having increased expression activity in CHO DG44 cells comprises, consists essentially of, or consists of: (i) a nucleotide sequence as shown in SEQ ID No. 34, 36, 37, 45, 47-52, 56, 57, 59-62, 64-67, 71, or a complementary sequence thereof; or (ii) a nucleotide sequence or a complementary sequence thereof that shares at least 75% sequence identity with the nucleotide sequence of (i).

23. The isolated nucleic acid molecule of any one of claims 1-22, wherein the promoter comprises at least one binding site for a transcription factor.

24. A vector comprising the isolated nucleic acid molecule according to any one of claims 1-23.

25. A host cell comprising the isolated nucleic acid molecule according to any one of claims 1-23 or the vector according to claim 24.

26. The host cell of claim 25, wherein the host cell is a eukaryotic cell.

27. The host cell according to claim 26, wherein the host cell is a CHO cell, preferably a CHO K1 cell or a CHO DG44 cell.

28. A use, which is the use of an isolated nucleic acid molecule according to any one of claims 1 to 23 to promote or enhance the expression of a polypeptide, peptide or RNA of interest, wherein the isolated nucleic acid molecule comprises a nucleotide sequence encoding the polypeptide, peptide or RNA of interest, wherein the nucleotide sequence encoding the polypeptide, peptide or RNA of interest is operably linked to the chimeric gene regulatory unit of the isolated nucleic acid molecule.

29. A method for preparing a polypeptide, peptide or RNA of interest, comprising: (i) providing an isolated nucleic acid molecule according to any one of claims 1 to 23, wherein the isolated nucleic acid molecule comprises a nucleotide sequence encoding the polypeptide, peptide or RNA of interest, wherein the nucleotide sequence encoding the polypeptide, peptide or RNA of interest is operably linked to the chimeric gene regulatory unit of the isolated nucleic acid molecule; as well as (ii) preparing the polypeptide, peptide or RNA of interest by in vitro transcription and translation or in a suitable host cell under conditions that allow the production of the polypeptide, peptide or RNA of interest.

30. The method of claim 29, wherein the host cell is a eukaryotic cell.

31. The method according to claim 30, wherein the eukaryotic cell is a CHO cell, more preferably a CHO K1 cell or a CHO DG44 cell.

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