WO2014133468A1

WO2014133468A1 - Chimeric promoters for high gene expression level and stability in mammalian cells

Info

Publication number: WO2014133468A1
Application number: PCT/SG2014/000100
Authority: WO
Inventors: Yuansheng Yang; Cheng Leong Steven HO
Original assignee: Agency for Science Technology and Research Singapore
Current assignee: Agency for Science Technology and Research Singapore
Priority date: 2013-02-28
Filing date: 2014-02-28
Publication date: 2014-09-04
Anticipated expiration: 2015-08-28

Abstract

This invention relates to an isolated nucleic acid molecule comprising a functional chimeric gene regulatory unit comprising a functional enhancer nucleotide sequence and a functional promoter nucleotide sequence, the use said isolated nucleic acid molecule for facilitating or enhancing expression, a vector or a host cell comprising said nucleic acid molecule and a method of producing a polypeptide, peptide or RNA molecule of interest.

Description

CHIMERIC PROMOTERS FOR HIGH GENE EXPRESSION LEVEL AND STABILITY

IN MAMMALIAN CELLS

FIELD OF THE INVENTION

[0001] The present invention lies in the field of biochemistry and relates to an isolated nucleic acid molecule comprising a functional chimeric gene regulatory unit comprising a functional enhancer nucleotide sequence and a functional promoter nucleotide sequence. The present invention also relates to the use of said isolated nucleic acid molecule and to a vector or a host cell comprising the isolated nucleic acid molecule. Further, the present invention relates to a method of producing a polypeptide, a peptide or a RNA molecule.

BACKGROUND OF THE INVENTION

[0002] Transfer of exogenous genes carried on plasmid vectors into various cell types for transgene expression or recombinant expression is a technique commonly used in fields like biologies production, cell engineering or gene therapy. During production of biologies like monoclonal antibodies (mAb), it is critical that the recombinant protein expression levels remain stable and high during extended culture to ensure consistent product and maximal yield. Stable expression refers to maintenance of transgene expression over extended periods of up to two to three months without any selection drugs. Use of selection drugs should be avoided as it adds to costs of production and can complicate the downstream purification processes. Studies conducted to determine the stability of monoclonal antibody (mAb) expression in transfected mammalian cells have reported loss of expression level in some clones upon extended culture (Chusainow et al. 2009 Osterlehner et al. 201 1). The unstable protein expression was attributed to either loss of gene copies or a decrease in mRNA levels (Chusainow et al. 2009; Jun et al. 2006;). Transgene copies are lost probably due to homologous recombination and nonhomologous DNA repair (Hastings et al. 2009). The observed decrease in mRNA level without any drop in gene copies in some unstable cell lines indicated the possibility of transcriptional silencing (Barnes et al. 2004).

[0003] Silenced gene expression due to transcriptional silencing is commonly associated with methylation of cytosines in CpG dinucleotides in promoters (Klose and Bird 2006). This happens by either direct inhibition of transcription factor binding due to the cytosine methylation or through recruitment of methyl-CpG-binding proteins which can silence gene expression with other transcriptional co-repressors that mediate subsequent modification of the chromatin structure (Klose and Bird 2006). An increase in CpG methylation on the promoter was also observed for mAb producing cell lines which lost mAb expression levels upon extended culture (Yang et al. 2010).

[0004] Gene expression and stability for gene therapy was shown to be improved by using different enhancer and promoter combinations (Magnusson et al. 201 1). This could be due to different cell lines having varying abundance of transcription factors resulting in cell line specific promoter activity level and stability. Transcription factor binding confers methylation resistance to the corresponding promoter regions. Analysis of a methylation free DNA region's binding factor footprints identified binding sites for the transcription factor specificity protein 1 (Spl) (Senigl et al. 2008). Spl elements were also observed to promote CpG island demethylation (Brandeis et al. 1994). Transfected vectors were also protected from transcriptional silencing by a CpG island element that had multiple Spl sites (Senigl et al. 2008). Very few combinations of hybrid enhancer-promoters from different sources have been tested. It is still unclear which combinations work best for recombinant protein expression in mammalian cells.

[0005] One way to increase the expression of recombinant proteins is to include introns downstream of the promoter. Introns can affect transcription by containing regions with enhancer- or repressor-like elements, containing splicing signals that enhances transcription initiation and RNA polymerase II activity or allowing formation of ordered nucleosome arrays around the promoter (Le Hir et al. 2003). Inclusion of introns into a basic enhancer-core promoter combination has been shown to improve recombinant protein expression levels in mammalian cells (Kang et al. 2005; Mariati et al. 2010). The increased effectiveness achieved by adding introns is promoter and cell line specific. Expression levels in CHO cells from the human cytomegalovirus major immediate early gene promoter (hCMV) could be increased by only 2 fold while expression from the murine cytomegalovirus major immediate early gene promoter (mCMV) was increased 8 fold by addition of the first intron from the human EF-la gene (Kim et al. 2002). The same study also observed marked differences in the effect of the human EF-la first intron in different mammalian cell lines. While in some cases the intron did not affect gene expression, the improvements observed ranged from 1.2 to 23.1 fold (Kim et al. 2002). In another study, addition of the human -globin second intron was almost 5-fold more effective in combination with the mCMV promoter compared to hCMV in U937 and CHO cells (Kang et al. 2005). The effect of the second intron of human β-globin downstream of a mCMV promoter also exhibited cell line specific effects with over 10 fold increase of expression in luciferase assays in HeLa and U937 cells, 6 fold increase in CHO cells and no significant improvements in four other mammalian cell lines (Kang et al. 2005).

[0006] Hence, there is need in the art for transcriptional promoter sequences that allow stable and high transgene expression in mammalian cell lines or mammalian tissue, in particular in biotechnologically important CHO cells.

SUMMARY OF THE INVENTION

[0007] It is an object of the present invention to meet the above need by providing nucleic acid sequences allowing stable and high transgene expression. Surprisingly, the inventors have found that these requirements are met by the isolated nucleic acid molecules of the present invention that comprise a functional chimeric gene regulatory unit comprising (I) a functional enhancer nucleotide sequence and (II) a functional promoter sequence. Optionally, said isolated nucleic acid molecules may further comprise an intron sequence. The isolated nucleic acid molecules of the invention mediate increased and stable gene expression. Based on this properties said isolated nucleic acid molecule can be used in methods for of producing a polypeptide, peptide or R A of interest.

[0008] Three commonly used wild-type (WT) promoters for biologies production are the promoter of the human cytomegalovirus major immediate-early gene (hCMV), the promoter of the murine cytomegalovirus major immediate-early gene (mCMV) and the promoter from the simian virus 40 (SV40). The hCMV promoter is a commonly used strong promoter but is unstable. In order to identify better performing enhancer and promoter combinations, the WT promoters were further separated into enhancer and core promoter segments to generate a series of chimeric promoters. These promoters were used to express a recombinant protein in Chinese hamster ovary (CHO) cells to identify one that can provide high transgene expression levels and stability. Three different introns were tested for use with the best performing chimeric promoter to improve expression. The above functional chimeric gene regulatory units allow stable and high transgene expression in mammalian cell lines or mammalian tissue. To obtain both high level and sustainable gene expression, the best chimeric gene regulatory unit is the combination of the mCMV enhancer and the SV40 core promoter. This chimeric gene regulatory unit becomes even more efficient by addition of an intron nucleotide sequence, preferably the first intron of hCMV. The isolated nucleic acid molecules of the present invention can be used in recombinant protein production and possibly for cell engineering and gene therapy.

[0009] In a first aspect, the present invention is thus directed to an isolated nucleic acid molecule comprising a functional chimeric gene regulatory unit comprising a functional enhancer nucleotide sequence and a functional promoter nucleotide sequence, wherein the enhancer nucleotide sequence is 5' to the promoter sequence and derived from a first species of organisms and the promoter nucleotide sequence is derived from a second species of organisms, wherein the first species and the second species are not the same species.

[00010] In various embodiments of the invention, the isolated nucleic acid molecule further comprises at least one nucleotide sequence encoding for a polypeptide, peptide or RNA molecule of interest, wherein said sequence is operably linked to the chimeric gene regulatory unit, preferably lies 3' to the promoter sequence, more preferably lies directly adjacent to the promoter sequence. In certain embodiments, the nucleotide sequence encodes for a polypeptide of interest, the polypeptide preferably being a polypeptide chain of a naturally occurring or artificial immunoglobulin, preferably an antibody, more preferably a human or humanized antibody, or a fragment thereof.

[00011] In various embodiments, the enhancer sequence, the promoter sequence or both are derived from viruses. Preferably, the enhancer sequence, the promoter sequence or both are derived from double-stranded DNA viruses. More preferably, the enhancer sequence, the promoter sequence or both are derived from viruses selected from the group consisting of Herpesviridae and Polyomaviridae.

[00012] In various embodiments, the chimeric gene regulation unit has an increased resistance to transcriptional silencing, for example inactivation by methylation. The resistance to transcriptional silencing is preferably increased in comparison with the resistance to transcriptional silencing of the naturally occurring gene regulation unit of which the promoter or enhancer is derived from.

[00013] In various embodiments, the virus from which the promoter sequence, the enhancer sequence or both are derived is selected from the group consisting of human cytomegalovirus; murine cytomegalovirus; and simian virus 40.

[00014] In various embodiments, the promoter sequence comprises, consists essentially of or consists of (I) a nucleotide sequence as set forth in any one of SEQ ID NO. l; SEQ ID NO:3; SEQ ID NO:5; or a complement thereof; (II) a nucleotide sequence that shares at least 75% sequence identity with a nucleotide sequence of (I) or a complement thereof.

[00015] 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:2; SEQ ID NO:4; SEQ ID NO:6; or a complement thereof; (II) a nucleotide sequence that shares at least 75% sequence identity with a nucleotide sequence of (I) or a complement thereof.

[00016] In various embodiments, the chimeric gene regulatory unit comprises, consists essentially of or consists of (I) a nucleotide sequence as set forth in any one of SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO: 10; SEQ ID NO.l l ; SEQ ID NO:12; or a complement thereof; (II) a nucleotide sequence that shares at least 75% sequence identity with a nucleotide sequence of (I) or a complement thereof, preferably the chimeric gene regulatory unit comprises, consists essentially of or consists of (I) a nucleotide sequence as set forth in SEQ ID NO:7; (II) a nucleotide sequence that shares at least 75% sequence identity with a nucleotide sequence of (I) or a complement thereof.

[00017] In various embodiments, the isolated nucleic acid molecule further comprises at least one nucleotide sequence encoding for an intron. Said intron nucleotide sequence preferably lies 3 ' to the promoter nucleotide sequence and 5' to the nucleotide sequence encoding for a polypeptide, peptide or RNA molecule of interest.

[00018] In various embodiments, the intron/ sequence comprises, consists essentially of or consists of (I) a nucleotide sequence as set forth in any one of SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; or a complement thereof; (II) a nucleotide sequence that shares at least 75% sequence identity with a nucleotide sequence of (I) or a complement thereof, preferably said nucleotide sequence comprises, consists essentially of or consists of (I) a nucleotide sequence as set forth in any one of SEQ ID NO: 17; or a complement thereof; (II) a nucleotide sequence that shares at least 75% sequence identity with a nucleotide sequence of (I) or a complement thereof.

[00019] In various embodiments, the promoter comprises at least one binding site for a transcription factor. In various embodiments, the transcription factor is specificity protein 1 (Spl) transcription factor. In some embodiments, the S l transcription factor may be of human origin as set forth in SEQ ID NO:20 (NCBI Reference Sequence: NP_001238754; Version: NP_001238754.1 GI:352962149) or mouse origin as set forth in SEQ ID NO:21 (NCBI Reference Sequence: NP_038700.2; Version: NP_038700.2 GI:119226255).

[00020] In various embodiments the isolated nucleic acid molecule of the invention further comprises at least one nucleotide sequence encoding for a recognition site of a restriction endonuclease. In various embodiments, said at least one nucleotide sequence encodes for a recognition site of a restriction endonuclease that is 3' to the enhancer nucleotide sequence and 5' to the promoter nucleotide sequence. In various embodiments, the recognition site of the restriction endonuclease is a Spel recognition site, for example as set forth in SEQ ID NO:22.

[00021] In various embodiments, the isolated nucleic acid molecule of the invention comprises a nucleotide sequence encoding for a recognition site of a restriction endonuclease, wherein said nucleotide sequence encoding for a second recognition site of a restriction endonuclease is 3' to the promoter nucleotide sequence and 5' to the at least one nucleotide sequence encoding for an intron. In various embodiments, this recognition site of the restriction endonuclease is a Notl recognition site, for example as set forth in SEQ ID NO:23.

[00022] In various embodiments, the isolated nucleic acid molecule of the invention comprises at least one nucleotide sequence encoding for a recognition site of a restriction endonucJease that is 3' to the enhancer nucleotide sequence and 5' to the promoter nucleotide sequence, preferably a Spel site, and a nucleotide sequence encoding for a recognition site of a restriction endonucleases that is 3' to the promoter nucleotide sequence and 5' to the at least one nucleotide sequence encoding for an intron, preferably a NotI site. In various embodiments, the isolated nucleotide of the invention is set forth as SEQ ID NO:24.

[00023] In various embodiments the at least one binding site for a transcription factor, preferably Spl transcription factor, comprises, consists essentially of or consists of the nucleotide sequence set forth in SEQ ID NO: 13 (5'-(G/T)GGGCGG(G/A)(G/A)(C/T)-3').

[00024] In a further aspect, the invention provides a vector comprising the isolated nucleic acid molecule of the invention.

[00025] In a further aspect, the invention provides a host cell comprising the isolated nucleic acid molecule or vector as defined herein. The host cell may be a prokaryotic or eukaryotic cell, preferably a eukaryotic cell. More preferably the host cell is a Chinese hamster ovary (CHO) cell.

J00026J In a still further aspect, the invention provides for the use of the isolated nucleic acid molecule of the invention for facilitating or enhancing the expression of a polypeptide, peptide or RNA of interest, wherein said isolated nucleic acid molecule comprises a nucleotide sequence encoding the polypeptide, peptide or RNA of interest, wherein said nucleotide sequence encoding the polypeptide, peptide or RNA of interest being operably linked to the chimeric gene regulatory unit of the isolated nucleic acid molecule.

[00027] In another aspect, the invention also provides for a method of producing a polypeptide, peptide or RNA of interest comprising: providing the isolated nucleic acid molecule according to claims 1 to 21, wherein said isolated nucleic acid molecule comprises a nucleotide sequence encoding the polypeptide, peptide or RNA of interest, wherein said nucleotide sequence encoding the polypeptide, peptide or RNA of interest being operably linked to the chimeric gene regulatory unit of the isolated nucleic acid molecule; and producing the polypeptide, peptide or RNA of interest by in vitro transcription and translation or in a suitable host cell under conditions that allow production of the polypeptide, peptide or RNA of interest. The host cell may be a prokaryotic or eukaryotic cell, preferably a eukaryotic cell. More preferably the host cell is a Chinese hamster ovary (CHO) cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[00028] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings.

[00029] Figure 1 shows the vectors used for recombinant protein expression, (a) Wild- type and chimeric promoters as listed in Figure 3 are compared, (b) Schematic drawing for promoter optimization by addition of introns to the chimeric gene regulatory unit to enhance mAb expression. Enh: Enhancer, cPro: Core promoter, GFP: Green fluorescent protein cDNA, IRESatt: Attenuated internal ribosome entry site element with lower translation efficiency (Rees et al. 1996), mNeo: Mutant neomycin phosphotransferase selection marker (D261G) (Sautter and Enenkel 2005), SpA: Simian virus 40 early polyadenylation signal, LC: Monoclonal antibody light chain cDNA, HC: Monoclonal antibody heavy chain gene, DHFR: dihydrofolate reductase selection marker.

[00030] Figure 2 shows the enhancer sequence and promoter sequence combinations investigated in expression and stability experiments.

[00031] Figure 3 shows promoters tested for expression level and expression stability.

[00032] Figure 4 shows stable expression levels of clones generated using different promoters. 18 clones were selected from pools transfected with vectors carrying each promoter. GFP expression was measured as the mean fluorescence intensity (MFI). hCMV, mCMV and SV40 are wild-type promoters while the rest are combinations of enhancer and promoter sequences from the corresponding wild-type sequences. Each point represents pool generated from a single clone. The rectangle and error bars represent the average MFI of the 18 clones.

[00033] Figure 5 shows the measurement of expression stability through retention of expression. Clones were passaged for 8 weeks without any selection pressure. MFI was measured at the end of the 8 weeks. The percentage of expression left as compared to the start of passaging was determined and expressed as retention of expression. Each point represents a single clone. The rectangle and error bar represents the average retained expression of the 18 clones for each promoter.

[00034] Figure 6 shows the absolute fluorescence intensity measured after 8 weeks of continuous expression. Values are calculated from the measurements shown in Figures 4 and 5.

[00035] Figure 7 shows monoclonal antibody expression using the mCS promoter with introns. mAb titer was collected at the end of culturing mAb expressing cell lines that have been generated by using four different mAb vectors driven by mCS alone or combined with either first intron from the human cytomegalovirus immediate early gene (SEQ ID NO: 17) (mCSvI), or the human EFl-α gene (SEQ ID NO: 18) (mCSfl), or the chicken /3-actin gene (SEQ ID NO: 19) (mCSal). Each bar and error bar represents the average of two individually transfected pools.

DETAILED DESCRIPTION OF THE INVENTION

[00036] The present inventors surprisingly found that that isolated nucleic acid molecules comprising a functional chimeric gene regulatory unit comprising a functional enhancer nucleotide sequence and a functional promoter nucleotide sequence mediate increased and stable gene expression.

[00037] Thus, in a first aspect, the invention relates to an isolated nucleic acid molecule comprising a functional chimeric gene regulatory unit comprising a functional enhancer nucleotide sequence and a functional promoter nucleotide sequence, wherein the enhancer nucleotide sequence is 5' to the promoter sequence and derived from a first species of organisms and the promoter nucleotide sequence is derived from a second species of organisms, wherein the first species and the second species are not the same species.

[00038] In various embodiments of the invention, the isolated nucleic acid molecule further comprises at least one nucleotide sequence encoding for a polypeptide, peptide or RNA molecule of interest, wherein said sequence is operably linked to the chimeric gene regulatory unit, preferably lies 3' to the promoter sequence, more preferably lies directly adjacent to the promoter sequence. In certain embodiments, the nucleotide sequence encodes for a polypeptide of interest, the polypeptide preferably being a polypeptide chain of a naturally occurring or artificial immunoglobulin, preferably an antibody, more preferably a human or humanized antibody, or a fragment thereof.

[00039] In various embodiments, the enhancer sequence, the promoter sequence or both are derived from viruses. Preferably, the enhancer sequence, the promoter sequence or both ard derived from double-stranded DNA viruses. More preferably, the enhancer sequence, the promoter sequence or both are derived from viruses selected from the group consisting of Herpesviridae and Polyomaviridae.

[00040] In various embodiments, the chimeric gene regulation unit has an increased resistance to transcriptional silencing, for example inactivation by methylation. The resistance to transcriptional silencing is preferably increased in comparison with the resistance to transcriptional silencing of the naturally occurring gene regulation unit the promoter or enhancer is derived from.

[00041] In various embodiments, the virus from which the promoter sequence, the enhancer sequence or both are derived is selected from the group consisting of human cytomegalovirus; murine cytomegalovirus; and simian virus 40.

[00042] In various embodiments, the promoter sequence comprises, consists essentially of or consists of a (I) nucleotide sequence as set forth in any one of SEQ ID NO. l; SEQ ID NO:3; SEQ ID NO:5; or a complement thereof; (II) a nucleotide sequence that shares at least 75% sequence identity with a nucleotide sequence of (I) or a complement thereof.

[00043] 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:2; SEQ ID NO:4; SEQ ID NO:6; or a complement thereof; (II) a nucleotide sequence that shares at least 75% sequence identity with a nucleotide sequence of (I) or a complement thereof.

[00044] In various embodiments, the chimeric gene regulatory unit comprises, consists essentially of or consists of (I) a nucleotide sequence as set forth in any one of SEQ ID NO:7;

SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO: 10; SEQ ID NO: l I ; SEQ ID NO: 12; or a complement thereof; (II) a nucleotide sequence that shares at least 75% sequence identity with a nucleotide sequence of (I) or a complement thereof, preferably the chimeric gene regulatory unit comprises, consists essentially of or consists of (I) a nucleotide sequence as set forth in SEQ ID NO:7; (II) a nucleotide sequence that shares at least 75% sequence identity with a nucleotide sequence of (I) or a complement thereof.

[00045] In various embodiments, the isolated nucleic acid molecule further comprises at least one nucleotide sequence encoding for an intron. Said intron nucleotide sequence preferably lies 3' to the promoter nucleotide sequence and 5' to the nucleotide sequence encoding for a polypeptide, peptide or RNA molecule of interest.

[00046] In various embodiments, the intron sequence comprises, consists essentially of or consists of (I) a nucleotide sequence as set forth in any one of SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; or a complement thereof; (II) a nucleotide sequence that shares at least 75% sequence identity with a nucleotide sequence of (I) or a complement thereof, preferably said nucleotide sequence comprises, consists essentially of or consists of (I) a nucleotide sequence as set forth in any one of SEQ ID NO: 17; or a complement thereof; (II) a nucleotide sequence that shares at least 75% sequence identity with a nucleotide sequence of (I) or a complement thereof.

[00047] In various embodiments, the promoter comprises at least one binding site for a transcription factor. In various embodiments, the transcription factor is specificity protein 1 (Spl) transcription factor. In some embodiments, the Spl transcription factor may be of human origin as set forth in SEQ ID NO:20 (NCBI Reference Sequence NP 001238754; Version: NP_001238754.1 GI:352962149) or mouse origin as set forth in SEQ ID NO:21 (NCBI Reference Sequence: NP_038700.2; Version: NP_038700.2 GI: 119226255).

[00048] In various embodiments the at least one binding site for a transcription factor, preferably Spl transcription factor, comprises, consists essentially of or consists of the nucleotide sequence set forth in SEQ ID NO: 13 (5'-(G/T)GGGCGG(G/A)(G/A)(C/T)-3').

[00049] In various embodiments the isolated nucleic acid molecule of the invention further comprises at least one nucleotide sequence encoding for a recognition site of a restriction endonuclease. In various embodiments, said at least one nucleotide sequence encodes for a recognition site of a restriction endonuclease that is 3' to the enhancer nucleotide sequence and 5' to the promoter nucleotide sequence. In various embodiments, the recognition site of the restriction endonuclease is a Spel recognition site, for example as set forth in SEQ ID NO:22.

[00050] In various embodiments, the isolated nucleic acid molecule of the invention comprises a nucleotide sequence encoding for a recognition site of a restriction endonuclease, wherein said nucleotide sequence encoding for a recognition site of a restriction endonuclease is 3' to the promoter nucleotide sequence and 5' to the at least one nucleotide sequence encoding for an intron. In various embodiments, this recognition site of the restriction endonuclease is a Notl recognition site, for example as set forth in SEQ ID NO:23.

[00051] In various embodiments, the isolated nucleic acid molecule of the invention comprises at least one nucleotide sequence encoding for a recognition site of a restriction endonuclease that is 3' to the enhancer nucleotide sequence and 5' to the promoter nucleotide sequence, preferably a Spel site, and a nucleotide sequence encoding for a recognition site of a restriction endonucleases that is 3' to the promoter nucleotide sequence and 5' to the at least one nucleotide sequence encoding for an intron, preferably a Notl site. In various embodiments, the isolated nucleotide of the invention is set forth as SEQ ID NO: 24.

[00052] In various embodiments, the enhancer nucleotide sequence and the promoter nucleotide sequence and/or the promoter nucleotide sequence and the at least one nucleotide sequence encoding for an intron are separated by a linker region. The term "linker region" as used herein refers to a non-coding nucleotide sequence that does not recruit transcription factors to said nucleotide and does not influence transcription of the gene of interest. Said "linker region" may be defined by the sequence 5'-[N]-[N]-[N]-[N]-[N]-[N]-3 ', wherein [N] is a nucleotide that has a base selected from the group consisting of adenine, cytosine, guanine, thymine and uracile.

[00053] In a further aspect, the invention provides a vector comprising the isolated nucleic acid molecule as defined herein.

[000541 hi a third aspect, the invention provides a host cell comprising the isolated nucleic acid molecule or vector as defined herein. The host cell may be a prokaryotic or eukaryotic cell, preferably a eukaryotic cell. More preferably the host cell is a Chinese hamster ovary (CHO) cell. [00055] In a still further aspect, the invention provides for the use of the isolated nucleic acid molecule as defined herein for facilitating or enhancing the expression of a polypeptide, peptide or RNA of interest, wherein the isolated nucleic acid molecule comprises the chimeric gene regulatory unit as defined above and a nucleotide sequence encoding the polypeptide, peptide or RNA of interest, said nucleotide sequence encoding the polypeptide, peptide or RNA of interest being operably linked to the chimeric gene regulatory unit, as defined above.

[00056] In another aspect, the invention also provides for a method of producing a polypeptide, peptide or RNA of interest. The method includes providing an isolated nucleic acid molecule including a nucleotide sequence encoding for the polypeptide, peptide or RNA of interest as defined herein, and allowing expression of the desired polypeptide. The expression may be in vitro transcription and translation or may be carried out in a suitable host organism that is cultivated under conditions that allow production of the polypeptide, peptide or RNA of interest. The host cell may be a prokaryotic or eukaryotic cell, preferably a eukaryotic cell. More preferably the host cell is a Chinese hamster ovary (CHO) cell.

[00057] The term "nucleic acid molecule" or "nucleic acid sequence", as used herein, relates to DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) molecules. Said molecules may appear independent of their natural genetic context and/or background. The term "nucleic acid molecule/sequence" further refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA- RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms.

[00058] "At least one", as used herein, relates to one or more, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[00059] The term "sequence", as used herein, relates to the primary nucleotide sequence of nucleic acid molecules or the primary amino acid sequence of a protein. [00060] The term "isolated", as used herein in the context with the term "nucleic acid molecule", relates to the purified form of a nucleic acid molecule that is not bound by biological molecules such as proteins or sugars that can interact with nucleic acid molecules under physiological conditions.

[00061] The term "functional chimeric gene regulatory unit", as used herein, relates to a portion of a nucleic acid molecule that comprises at least a functional enhancer nucleotide sequence and a functional promoter nucleotide sequence. The term "enhancer nucleotide sequence" is a short region of DNA or RNA that can be bound with proteins (namely, the transacting factors, much like a set of transcription factors) to enhance transcription levels of a single gene or genes in a gene cluster. While enhancers are usually cis-acting, an enhancer does not need to be particularly close to the genes it acts on. The term "promoter nucleotide sequence" is a region of DNA or RNA that initiates transcription of a particular gene. Promoters are located near the Transcription Start Sites of genes, on the same strand and upstream on the nucleotide (towards the 3' region of the anti-sense strand, also called template strand and non-coding strand). Promoters can be about 100-1000 base pairs long.

[00062] The term "protein" or "polypeptide", as used herein, relates to one or more associated polypeptides, wherein the polypeptides consist of amino acids coupled by peptide (amide) bonds. The term polypeptide refers to a polymeric compound comprised of covalently linked amino acid residues. The amino acids are preferably the 20 naturally occurring amino acids glycine, alanine, valine, leucine, isoleucine, phenylalanine, cysteine, methionine, proline, serine, threonine, glutamine, asparagine, aspartic acid, glutamic acid, histidine, lysine, arginine, tyrosine and tryptophan. "Polypeptide", as used herein, relates to polymers made from amino acids connected by peptide bonds. The polypeptides, as defined herein, can comprise 15 or more amino acids, preferably 20 or more amino acids. "Peptides", as used herein, relates to polymers made from amino acids connected by peptide bonds. The peptides, as defined herein, can comprise 2 or more amino acids, preferably 5 or more amino acids, more preferably 10 to 50 amino acids.

[00063] "RNA" or "ribonucleic acid" as interchangeably used herein relates to a chain of nucleotides wherein the nucleotides contain the sugar ribose and bases selected from the group of adenine (A), cytosine (C), guanine (G), or uracil (U). "DNA" or "deoxyribonucleic acid" as interchangeably used herein relates to a chain of nucleotides wherein the nucleotides contain the sugar 2'-deoxyribose and bases selected from adenine (A), guanine (G), cytosine (C) and thymine (T).

[00064] "Operably linked", as used herein, is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

[00065] The term "3 'end" or "3'hydroxyl end" as interchangeably used herein relates to the termination at the hydroxyl group of the third carbon in the sugar-ring of a nucleic acid molecule, and is also known as the tail end. The term "5'end" or "5'phosphate end" as interchangeably used herein designates the end of the DNA or RNA strand that has a phosphate group at the fifth carbon in the sugar-ring of the deoxyribose or ribose at its terminus.

[00066J "Species", as used herein, relates to the basic units of biological classification and a taxonomic rank. A species is defined as the largest group of organisms capable of interbreeding and producing fertile offspring. As regards the term "virus species", a virus species is a polythetic class of viruses that constitutes a replicating lineage and occupies a particular ecological niche.

[00067] "Antibody", also known as an immunoglobulin (Ig), as used herein relates to a large Y-shaped protein that is used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. Antibodies are typically made of basic structural units - each with two large heavy chains and two small light chains. There are several different types of antibody heavy chains, and several different kinds of antibodies, which are grouped into different immunoglobulin isotypes based on which heavy chain they possess. Five different antibody isotypes are known in mammals, which perform different roles, and help direct the appropriate immune response for each different type of foreign object they encounter. In a preferred embodiment of the present invention, the protein of interest is an IgG. Antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains. "Humanized antibodies", as used herein, relate to antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans.

[00068] The term "fragment", as used herein, relates to a polypeptide, peptide, DNA or RNA that comprises or consists of an amino acid or nucleotide sequence that is at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.25%, or 99.5% identical or homologous to its reference amino acid sequence or nucleotide sequence.

[00069] "Complement", as used herein, relates to a nucleic acid molecule which is complementary to another nucleic acid molecule when both nucleic acid molecules are aligned antiparallel to each other in that one or more or all nucleotides of either of the nucleic acid molecules forms Watson-Crick base pairs with their corresponding counterparts. In various embodiments, the complements are full complements in that each nucleotide of the respective molecule or sequence forms a Watson-Crick base pair with a corresponding nucleotide on the other strand.

[00070] In various embodiments, the isolated nucleotide acid molecule of the present invention may comprise, consist essentially of or consist of the nucleotide sequence set forth in SEQ ID Nos. 1-12. Also encompassed are nucleotide sequences that are at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.25%, or 99.5% identical or homologous to the nucleotide sequences set forth in SEQ ID Nos. 1 -12 over their entire length. [00071] In various embodiments, the intron sequence comprises, consists essentially of or consists of (I) a nucleotide sequence as set forth in any one of SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; or a complement thereof. Also encompassed are nucleotide sequences that are at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%., 98.5%, 99%, 99.25%., or 99.5% identical or homologous to the nucleotide sequences set forth in SEQ ID Nos. 17-19 over their entire length.

[00072J The identity of nucleic acid sequences or amino acid sequences is generally determined by means of a sequence comparison. This sequence comparison is based on the BLAST algorithm that is established in the existing art and commonly used and is effected in principle by mutually associating similar successions of nucleotides or amino acids in the nucleic acid sequences and amino acid sequences, respectively. A tabular association of the relevant positions is referred to as an "alignment." Sequence comparisons (alignments), in particular multiple sequence comparisons, are commonly prepared using computer programs which are available and known to those skilled in the art.

[00073] A comparison of this kind also allows a statement as to the similarity to one another of the sequences that are being compared. This is usually indicated as a percentage identity, i.e. the proportion of identical nucleotides or amino acid residues at the same positions resp. in positions corresponding to one another in an alignment. The more broadly construed term "homology" also, in the context of amino acid sequences, incorporates consideration of the conserved amino acid exchanges, i.e. amino acids having a similar chemical activity, since these usually perform similar chemical activities within the protein. The similarity of the compared sequences can therefore also be indicated as a "percentage homology" or "percentage similarity." Indications of identity and/or homology can be encountered over entire polypeptides or genes, or only over individual regions. Homologous resp. identical regions of various nucleic acid sequences or amino acid sequences are therefore defined by way of matches in the sequences. Such regions often exhibit identical functions. They can be small, and can encompass only a few nucleotides or amino acids. Small regions of this kind often perform functions that are essential to the overall activity of the protein. It may therefore be useful to refer sequence matches only to individual, and optionally small, regions. Unless otherwise indicated, however, indications of identity and homology herein refer to the full length of the respectively indicated nucleic acid sequence or amino acid sequence.

[00074] "Transcriptional silencing", as used herein, relates to the epigenetic regulation of gene expression. In particular, this term refers to the ability of a cell to prevent the expression of a certain gene that is, for example, down regulated by methylation of CpG-islands or histone modifications. Resistance to "transcriptional silencing" can be achieved by avoiding the above modifications of DNA or histones that decrease gene expression. -

[00075] The term "virus", as used herein, relates to a small infectious agent that replicates only inside the living cells of other organisms. Viruses can infect all types of life forms, from animals and plants to bacteria and archaea. "Human cytomegalovirus" is a species of the cytomegalovirus family of viruses, which in turn is a member of the viral family known as Herpesviridae or herpesviruses. It is typically abbreviated as HCMV and is alternatively known as human herpesvirus-5 (HHV-5). Within Herpesviridae, HCMV belongs to the Betaherpesvirinae subfamily, which also includes cytomegaloviruses from other mammals. One of these other mammalian cytomegalovirus species is the "murine cytomegalovirus" wherein enhancer nucleotide sequences of this species are preferred sequences of the present invention. The term "simian virus 40" or "SV40", as used herein, relates to a polyomavirus that is found in both monkeys and humans. Like other polyomaviruses, SV40 is a DNA virus that has the potential to cause tumors, but most often persists as a latent infection.

[00076] The term "double-stranded DNA virus", as used herein, relates to non-enveloped or enveloped viruses having double-stranded DNA genomes. Virus families that belong to enveloped double-stranded DNA viruses are Herpesviridae, Poxviridae and Hepadnaviridae. Virus families that belong to non-enveloped double-stranded DNA viruses are Polyomaviridae, Adenoviridae and Papillomaviridae.

[00077] The term "intron nucleotide sequence" relates to a nucleotide sequence within a gene that is removed by RNA splicing while the final mature RNA product of a gene is being generated: The term intron refers to both the DNA sequence within a gene and the corresponding sequence in RNA transcripts. Sequences that are joined together in the final mature RNA after RNA splicing are exons. Introns are found in the genes of most organisms and many viruses, and can be located in a wide range of genes, including those that generate proteins, ribosomal RNA (rRNA), and transfer RNA (tRNA).

[00078] "Transcription factor binding site" or "binding site for a transcription factor", as interchangeably used herein, relates to a nucleotide sequence to which a transcription factor can attach on DNA. The binding site of each transcription factor is different from the other ones by comprising a specific base sequences, so called "motifs", that allow binding of the specific transcription factor. A preferred transcription factor binding site of the present invention is the Spl binding site. Spl is a zinc finger transcription factor that binds to GpC-rich motifs of many promoters. The encoded protein is involved in many cellular processes, including cell differentiation, cell growth, apoptosis, immune responses, response to DNA damage, and chromatin remodeling. Post-translational modifications such as phosphorylation, acetylation, glycosylation, and proteolytic processing significantly affect the activity of this protein, which can be an activator or a repressor. In the SV40 virus, Spl binds to the GpC boxes in the regulatory region (RR) of the genome. The sequence of human Spl is set forth in SEQ ID NO:20. The sequence of mouse Spl is set forth in SEQ ID NO:21.

[00079J "Vectors" are understood for purposes herein as elements, made up of nucleic acids, that contain a nucleic acid contemplated herein as a characterizing nucleic acid region. They enable said nucleic acid to be established as a stable genetic element in a species or a cell line over multiple generations or cell divisions. In particular when used in bacteria, vectors are special plasmids, i.e. circular genetic elements. In the context herein, a nucleic acid as contemplated herein is cloned into a vector. Included among the vectors are, for example, those whose origins are bacterial plasmids, viruses, or bacteriophages, or predominantly synthetic vectors or plasmids having elements of widely differing derivations. Using the further genetic elements present in each case, vectors are capable of establishing themselves as stable units in the relevant host cells over multiple generations. They can be present extrachromosomally as separate units, or can be integrated into a chromosome resp. into chromosomal DNA.

[00080] Expression vectors encompass nucleic acid sequences which are capable of replicating in the host cells that contain them, and expressing therein a contained nucleic acid. In various embodiments, the vectors described herein thus also contain regulatory elements that control expression of the nucleic acids encoding a polypeptide, peptide or RNA of interest. Expression is influenced in particular by the promoter or promoters that regulate transcription. Expression vectors can furthermore be regulated, for example by way of a change in culture conditions or when the host cells containing them reach a specific cell density, or by the addition of specific substances, in particular activators of gene expression. In contrast to expression vectors, the contained nucleic acid is not expressed in cloning vectors.

[00081] The term "restriction endonuclease" or "restriction enzyme", as interchangeably used herein, refers to an enzyme that cleaves DNA at or near specific recognition nucleotide sequences known as restriction sites. Restriction enzymes are commonly classified into three types, which differ in their structure and in whether they cleave their DNA substrate at the recognition site or the recognition and cleavage sites are separate from one another. To cleave DNA, all restriction enzymes hydrolyze two covalent bonds, namely one bond in the sugar- phosphate backbone of each strand of the DNA double helix. Restriction endonucleases may comprise but are not limited to Aatll, Acc65I, Accl, Acll, Afel, Aflll, Agel, Apal, ApaLI, Apol, Ascl, Asel, AsiSI, Avrll, BamHI, Bell, Bglll, Bmel580I, Bmtl, BsaHI, BsiEI, BsiWI, BspEI, BspHI, BsrGI, BssHII, BstBI, BstZ17I, Btgl, Clal, Dral, Eael, Eagl, EcoRI, EcoRV, Fsel, Fspl, Haell, Hindi, Hindlll, Hpal, Kasl, pnl, Mfei, Mhil, Mscl, MspAl I, Mfel, Mlul, Mscl, MspAlI, Nael, Narl, Ncol, Ndel, NgoMIV, Nhel, Notl, Nrul, Nsil, Nspl, Pad, Pcil, Pmel, Pmll, Psil, PspOMI, Pstl, Pvul, PvuII, Sad, SacII, Sail, Sbfl, Seal, Sfcl, Sfol, SgrAI, Smal, Smll, SnaBI, Spel, Sphl, Sspl, Stul, Swal, Xbal, Xhol and Xmal. Preferably, the recognition site of the restriction endonuclease used in accordance with the present invention is a Spel and/or Notl recognition site, as set forth in SEQ ID Nos. 22 and 23, respectively.

[00082] The term "host cell", as used herein, is intended to means in principle all cells, i.e. prokaryotic or eukaryotic cells. Those host cells that can be manipulated in genetically advantageous fashion, e.g. as regards transformation using the nucleic acid or vector and stable establishment thereof. In addition, preferred host cells are notable for being readily manipulated in microbiological and biotechnological terms. This refers, for example, to easy culturability, high growth rates, low demands in terms of fermentation media, and good production and secretion rates for foreign proteins or R A. The polypeptides or K A can furthermore be modified, after their manufacture, by the cells producing them, for example by the addition of sugar molecules, formylation, amination, etc. Post-translation modifications of this kind can functionally influence the polypeptide or RNA.

[00083] Further embodiments are represented by those host cells whose activity can be regulated on the basis of genetic regulation elements that are made available, for example, on the vector, but can also be present a priori in those cells. They can be stimulated to expression, for example, by controlled addition of chemical compounds that serve as activators, by modifying the culture conditions, or when a specific cell density is reached. This makes possible economical production of the proteins or RNAs contemplated herein.

[00084] Host cells contemplated herein can be modified in terms of their requirements for culture conditions, can comprise other or additional selection markers, or can also express other or additional proteins. They can, in particular, be those host cells that transgenically express multiple proteins or enzymes.

[00085] Preferred host cells are eukaryotic cells. More preferred are CHO cells. "CHO cell" or "Chinese hamster ovary cell", as used interchangeably, relate to a cell line derived from the ovary of the Chinese hamster (Cricetulus griseus). CHO cells are epithelial cells which grow as an adherent monolayer or in suspension. They, characteristically, require the amino acid proline in their culture medium. Different subgroups of CHO cells are CHO DP- 12 cells, CHO- Kl cells, CHO/dhfr- cells, CHO-S cells, CHO-GS cells CHO-K1 DUX B 1 1 cells (Simonsen and Levinson (1983), PNAS, 80, 2495-2499), dpl2.CHO cells (EP 307,247), CHO pro3- cells and CHO-DG44 cells. In a preferred embodiment of the present invention the mammalian host cell is a CHO-K1 cell or a CHO-DG44 cell. In other preferred embodiments of the invention the CHO cell is CHO pro-, CHO S, CHO WTT (WT- 1, 2, 3, 4 or 5), CHO pro-3, CHO pro-3 MtxRI, RII or RIII, CHO UA21, CHO DG21 or DG22, CHO UA41, CHO DG41, 42, 43, 44 or 45, CHO DR1000L-4N, CHO DG44 suspension, CHO GAT-, CHO SCI , CHO AA8, CHO Kl , CHO K1 SV, CHO UKB25 (d+/d-), CHO DUK-B 1 1 (d+/d-), CHO DUK22(d-/d-), CHO DUK51(d-/d-), CHO DXA1 1 , DXB 1 1, DXC1 1, DXE 1 1, DXF 1 1, DXG1 1, DXHU, DXI1 1 or DXJU, CHO-T, CHO 3E7 or freestyle CHO-S.

[00086] The term "facilitating or enhancing expression", as used herein, means that the amount of a polypeptide, peptide or RNA of interest expressed from a nucleic acid molecule comprising the functional chimeric gene regulatory unit as described herein is increased compared to the amount of the same polypeptide, peptide or RNA expressed from a nucleic acid molecule that comprises a naturally occurring gene regulatory unit.

[00087] "Culruring", "cultivating" or "cultivation", as used herein, relates to the growth of cells in a specially prepared culture medium under supervised conditions. The term "conditions suitable for recombinant expression" relates to conditions that allow for production of the polypeptide, peptide or RNA of interest in cells using methods known in the art, wherein the cells are cultivated under defined media and temperature.

[00088] "IVTT reaction" or "in vitro transcription translation reaction" as interchangeably used herein relates to cell-free systems that allow for specific transcription and translation by comprising macromolecular components (RNA polymerase, 70S or 80S ribosomes, tR As, aminoacyl-tRNA synthetases, initiation, elongation and termination factors, etc.) required for transcription and translation. To ensure efficient translation, the system may also be supplemented with amino acids, energy sources (ATP, GTP), energy regenerating systems, and other co-factors (Mg , K , etc.). Such systems or extracts are also known as "coupled" and "linked" systems as they start with DNA templates, which are subsequently transcribed into RNA and then translated. Preferred IVTT reactions comprise the rabbit reticulocyte lysate, the wheat germ extract and the E. coli cell-free system, in a more preferred embodiment the IVTT reaction is the rabbit reticulocyte lysate.

[00089] In specific embodiments of the invention, the isolated nucleic acid molecule according to the invention additionally comprises, for example, a nucleic acid sequence encoding for a gene of interest and/or sequences that allow its insertion into a vector, and can be cloned in a known host organism. Several cloning techniques, including amplification of nucleic acids, their restriction by according enzymes, purification and ligation, and transformation techniques, are known in the art and described in more detail by Sambrook et al.(Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). The produced nucleic acid constructs are verified by sequencing. Sequencing of the nucleic acid constructs can be done by the chain termination method, Sanger sequencing or Maxam-Gilbert sequencing or any other technique known in the art. Alternatively, high-throughput sequencing, like pyrosequencing, SOLiD sequencing or DNA nanoball sequencing, is used to determine the sequence of the nucleic acid molecules of the present invention (Alphey, L. (1997) DNA Sequencing: From Experimental Methods to Bioinformatics, 1st Ed., Bios Scientific Pub Ltd., Oxford, UK).

[00090] In another aspect, the invention relates to a vector comprising the isolated nucleic acid molecule according to the invention. In specific embodiments, a host cell is transformed with the vector comprising the nucleic acid molecules according to the present invention. Methods for the transformation of host cells include, but are not limited to competent cell based techniques, electroporation, gold particle gun based techniques, liposome-based transfection reagents and bacterial or viral infections. The host cells comprise bacterial cells, yeast cells, plant cells, nematode cells, insect cells and mammalian cells. The transfection method of choice can vary dependent on the host cell (The QIAGEN Transfection Resource Book (2002), 2nd Ed., QIAGEN GmbH, Hilden, Germany).

[00091] In a further aspect of the present invention, nucleic acids according to the present invention are used for facilitating or enhancing the expression of a polypeptide, peptide or RNA of interest. In these methods the polypeptide, peptide or RNA can be synthesized in cell-free in vitro systems like the rabbit reticulocyte lysate, the wheat germ extract and the E. coli cell-free system or in a host cell. Protocols including conditions for polypeptide, peptide or RNA synthesis and subsequent purification and storage conditions are described in the prior art, for example by Krieg (Krieg, P. (1996) A Laboratory Guide to RNA: Isolation, Analysis, and Synthesis, 1st Ed., Wiley- Liss, Hoboken, New Jersey).

[00092] In specific embodiments, the present invention relates to methods of producing a polypeptide, peptide or RNA of interest using cell-free in vitro transcription/translation systems or host cells and the nucleic acid molecules and vectors according to the present invention. Host cells for the synthesis of protein comprise bacterial, yeasty plant, nematode hosts as well as insect or mammalian cells. In specific embodiments, the host is E. coli, Pichia pastoris, a HeLa cell, a CHO cell or a SF9 cell. Preferably, the host cell is a CHO cell. Detailed protocols including system specific synthesis conditions and purification methods can be found for recombinant cell- based expression in Merten et aj. (Merten et al. (2001) Recombinant Protein Production with Prokaryotic and Eukaryotic Cells: A Comparitive View on Host Physiology, 1st Ed., Kluwer

Academic Publishers, Dordrecht, The Netherlands) and for cell-free expression in Spirin and Swartz (Spirin, A.S. and Swartz, J.R. (2008) Cell-Free Protein Synthesis: Methods and Protocols, 1st Ed., Wiley- VCH Verlag GmbH, Weinheim, Germany).

EXAMPLES

Materials and Methods

Cell culture

[00093] Adherent CHO Kl cells (American Type Culture Collection, Manassas, VA) were grown in Dulbecco's modified Eagle's medium (DMEM) + GlutaMax™ (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Sigma- Aldrich, St. Louis, MO), referred to as serum media, in T-flasks. Routine subculture of cells was performed every 3 to 4 days. Cell density and viability were measured using the trypan blue exclusion method on a Vi-Cell XR cell viability analyzer (Beckman Coulter, CA).

[00094] DHFR deficient CHO DG44 cells (Life Technologies, Carlsbad, CA) were grown in protein free media (PFM) supplemented with O. mM sodium hypoxanthine and 0.016mM thymine (HT; Life Technologies). PFM was prepared from a 1 :1 mixture of HyQ PF (Hyclone, Logan, UT) and CD CHO (Life Technologies), supplemented with 1 g/L sodium bicarbonate (Sigma-Aldrich, St-Louis, MO), 6mM Glutamine (Sigma-AIdrich) and 0.05% Pluronic F-68 (Life Technologies). Cells were passaged every 3 to 4 days by diluting the cultures to 2^χ 10⁵ cells/mL in fresh media. Cell viability and density was determined by trypan blue exclusion method using a Vi-Cell XR cell viability analyzer (Beckman Coulter, CA).

Plasmid construction

[00095] The vector used for comparison of chimeric promoters is similar to the one as previously described using an attenuated internal ribosome entry site (IRES) to link the product gene and a mutant neomycin phosphotransferase selection marker (Ho et al. 2012). The antibody genes are replaced with a green fluorescent protein (GFP) transgene (Figure la). The promoters used are constructed from three commonly used wild-type (WT) promoters, the promoter of the human cytomegalovirus major immediate-early gene (hCMV), the promoter of the murine cytomegalovirus major immediate-early gene (mCMV) and a promoter from the simian virus 40 (SV40). Sequences are set forth in SEQ ID Nos. 1-6 and 14- 16. WT promoters were inserted using Mlul and Notl restriction sites. The tested chimeric promoters are a combination of the enhancer and mini promoter segments of the WT promoters and are listed in Figure 3. Enhancers were inserted using Mlul and Spel sites and mini promoters were inserted using Spel and NotI sites (Figure la).

[00096] The design of the vectors used for enhancement of the chimeric gene regulatory unit by addition of intron is shown in Figure lb. The neomycin phosphotransferase gene on a previously described IRES-mediated tricistronic vector (Ho et al. 2012) was replaced with a dihydrofolate reductase (DHFR) gene. The CMV promoter was replaced with the best performing chimeric promoter and introns inserted directly downstream of the promoter. First introns from the human CMV, the human EF- la gene and the chicken /3-actin genes were tested for improving the mAb expression level of the best performing promoter. AH restriction enzymes were supplied by New England Biolabs (Ipswich, MA).

Transfecting and generating GFP clones

[00097] Transfections were all performed using Nucleofection kits from Lonza (Cologne, Germany) following the manufacturer's instructions using 1 * 10⁷ cells and 5 μg of each linearized plasmid performed in triplicates. Transfected cells were transferred to 6 well plates containing 2mL serum media for recovery. Upon confirmation of GFP expression under the microscope after 24 hours of recovery, cultures were refreshed with serum media containing 800 g/mL G418 (Sigma-Aldrich) to start selection. Media was changed every 3 to 4 days. Selection and recovery was completed in four to five weeks. Six clones were isolated from each transfected pool by limiting dilution to obtain a total of 18 clones for each promoter tested.

Stability testing

[00098] The isolated clones were cultured in 6 well plates with 2 mL of cells at a density of 2 x 10⁵ cells/mL. After 72h, cells were detached using trypsin (Gibco, Life Technologies) and GFP expression was measured in terms of the mean fluorescent intensity (MFI) using a FACS Calibur system (Becton Dickinson, Franklin Lakes, NJ). Non-transfected cells were measured in parallel for all flow cytometry measurements as a control to determine the proportion of cells expressing GFP. Care was taken to ensure there were no non-expressing cells at the start of the experiment. One set of each clone at the start of stability testing, designated as week 0, was cryopreserved using serum media with 10% DMSO (Sigma-Aldrich) in a nitrogen vapor cryotank. The media for clones in culture were switched to G418-free serum media for the start of stability testing. After 8 weeks, the cryopreserved clones from week 0 were thawed. GFP expression of both the thawed clones from week 0 and the clones passaged in G418-free serum media were measured together by flow cytometry. Expression stability for these GFP clones were measured by determining the percentage of MFI still retained in the clones passaged for 8 weeks without G418 compared to the thawed week 0 clones in G418 media.

Generating monoclonal antibody (mAb) producing pools

[00099] Transfections were all performed using Nucleofection kits from Lonza (Cologne, Germany) following the manufacturer's instructions. Stable transfections were performed using 1* 10⁷ cells and 5 μg of each linearized plasmid. Cells were transferred to 6-well plates containing 2 mL of HT supplemented PFM for 24 hours of recovery. Following that, cells were inoculated to shake flasks and the media was replaced with 25 mL of PFM for selection. Cells were seeded at densities of 4*10⁵ cells/mL during the selection process. Selection and recovery was complete for each selection step when viability was above 95%. Stepwise methotrexate (MTX) amplification was then carried out with concentrations of 50 nM and 500 nM. Batch cultures were seeded at 2x l0⁵ cells/mL and maintained till viability was around 50% for supernatant collection for product quantification. Titers were measured using an IMMAGE 800 immunochemistry system (Beckman Coulter, Buckinghamshire, England) and any cell counts were carried out using the Vi-Cell XR (Beckman Coulter).

Example 1: Expression level of wild-type and chimeric promoters

[000100] A series of wild-type promoters, namely hCMV, mCMV, SV40, were used to express GFP in CHO cells. 18 clones were selected from 3 separately transfected pools using each vector. GFP expression was measured using flow cytometry (Figure 4). hCMV clones averaged the highest mean fluorescent intensity (MFI) of 392, mCMV was the next highest at 346 and SV40 had the lowest expression among WT promoters at 288.

[000101] The series of chimeric promoters averaged MFI ranging from 364 for SmC to 231 for hCS (Figure 4). The series of 18 clones isolated for SmC, mChC and mCS had similar expression to the clones isolated from the stronger WT promoters from hCMV and mCMV. Clones from hCS, ShC and hCmC had significantly lower GFP expression.

Example 2: Expression stability of wild-type and chimeric promoters

{000102] All clones were cultured for 8 weeks without any selection drug. MFI after 8 weeks was compared to the expression at the start of stability testing (week 0) and the percentage of expression retained was determined. GFP expression for the WT hCMV and mCMV clones dropped significantly to only 21% and 38% respectively (Figure 4). Clones from SV40 promoter retained GFP expression better with an average of 79% still present after 8 weeks. Although using the SV40 promoter allowed most of the expression to be retained, expression from the promoter was the weakest among the WT promoters.

[000103] Several of the chimeric promoters generated GFP transgene expression levels comparable to WT hCMV and mCMV promoters at week 0. Expression stability for the chimeric promoter clones were also determined (Figure 5). The clones from hCmC retained 87% of GFP expression on average after 8 weeks, outperforming the most stable WT promoter, SV40. The average expressions retained by mCS, ShC and hCS clones were similar to that of the SV40 promoter. Clones from SmC and mChC only retained 54% and 50% of expression. It was interesting to observe that on average all the chimeric promoters retained expression better than hCMV and mCMV.

Example 3: mAb expression level using mCS with introns

[000104] As the combination of mCMV enhancer and SV40 promoter (mCS) shows the best performence, giving both high expression level and stability (Figure 6), it was further optimized by addition of either the first introns from hCMV (mCSvI), or the human EF-Ια gene (mCSfi), or the chicken ?-actin gene (mCSal) (Figure lb) for generation of a therapeutic recombinant protein, namely monoclonal antibody (mAb), expressing CHO DG44 cell lines. CHO DG44 cells were transfected separately with vectors driven by each of the four promoters. mAb titer obtained using the mCS promoter was 181 mg/L at 500 nM MTX (Figure 7). Addition of all three introns improved mAb expression level. mCSvI was most effective with expression level doubling to 400 mg/L. Using mCSfi and mCSal also exhibited improved mAb titers, raising expression to 322 mg/L and 308 mg/L respectively compared to pools generated using mCS promoter only.

[000105] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[000106] One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Further, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The compositions, methods, procedures, treatments, molecules and specific compounds described herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims. The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[000107] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

Thus, for example, the terms "comprising", "including," containing", etc. shall be read expansively and without limitation. The word, "comprise" or variations such as "comprises" or

"comprising" will accordingly be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or group of integers. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has. been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[000108] The content of all documents and patent documents cited herein is incorporated by reference in their entirety.

References

Barnes LM, Bentley CM, Dickson AJ. 2004. Molecular Definition of Predictive Indicators of Stable Protein Expression in Recombinant NSO Myeloma Cells. Biotechnology and Bioengineering 85(2): 1 15- 121.

Brandeis M, Frank D, Keshet I, Siegfried Z, Mendelsohn M, Nemes A, Temper V, Razin_. A, Cedar H. 1994. Spl elements protect a CpG island from de novo methylation. Nature 371(6496):435-8.

Chusainow J, Yang YS, Yeo JH, Toh PC, Asvadi P, Wong NS, Yap MG. 2009. A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer? Biotechnology and Bioengineering 102(4): 1182-96.

Hastings PJ, Lupski JR, Rosenberg SM, Ira G. 2009. Mechanisms of change in gene copy number. Nature Reviews Genetics 10(8):551-564.

Ho SCL, Bardor M, Feng H, Mariati, Tong YW, Song Z, Yap MGS, Yang Y. 2012. IRES- mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines. Journal of Biotechnology 157(1): 130- 139.

Jun SC, Kim MS, Hong HJ, Lee GM. 2006. Limitations to the development of humanized antibody producing Chinese hamster ovary cells using glutamine synthetase-mediated gene amplification. Biotechnol Prog 22(3):770-80.

Kang M, Kim SY, Lee S, Lee YK, Lee J, Shin HS, Kim YS. 2005. Human 3-globin second intron highly enhances expression of foreign genes from murine cytomegalovirus immediate- early promoter. Journal of Microbiology and Biotechnology 15(3):544-550.

Kim SY, Lee JH, Shin HS, Kang HJ, Kim YS. 2002. The human elongation factor 1 alpha (EF-1 alpha) first intron highly enhances expression of foreign genes from the murine cytomegalovirus promoter. Journal Of Biotechnology 93(2): 183-187. \

Klose RJ, Bird AP. 2006. Genomic DNA methylation: The mark and its mediators. Trends in Biochemical Sciences 31 (2):89-97.

Le Hir H, Nott A, Moore MJ. 2003. How introns influence and enhance eukaryotic gene expression. Trends in Biochemical Sciences 28(4):215-220.

Magnusson T, Haase R, Schleef M, Wagner E, Ogris M. 201 1. Sustained, high transgene expression in liver with plasmid vectors using optimized promoter-enhancer combinations. Journal of Gene Medicine 13(7-8):382-391.

Mariati, Ng YK, Chao SH, Yap MG, Yang Y. 2010. Evaluating regulatory elements of human cytomegalovirus major immediate early gene for enhancing transgene expression levels in CHO Kl and HEK293 cells. Journal of Biotechnology 147(3-4): 160-163.

Osterlehner A, Simmeth S, Gopfert U. 201 1. Promoter methylation and transgene copy numbers predict unstable protein production in recombinant Chinese hamster ovary cell lines. Biotechnology and Bioengineering 108(1 1 ):2670-2681. Rees S, Coote J, Stables J, Goodson S, Harris S, Lee MG. 1996. Bicistronic vector for the creation of stable mammalian cell lines that predisposes all antibiotic-resistant cells to express recombinant protein. Biotechniques 20(1): 102 -4, 106, 108-10.

Sautter K, Enenkel B. 2005. Selection of high-producing CHO cells using NPT selection marker with reduced enzyme activity. Biotechnology and Bioengineering 89(5):530-8.

Senigl F, Plachy J, Hejnar J. 2008. The core element of a CpG island protects avian sarcoma and leukosis virus-derived vectors from transcriptional silencing. J Virol 82(16):7818-27.

Yang Y, Mariati, Chusainow J, Yap MG. 2010. DNA methylation contributes to loss in productivity of monoclonal antibody-producing CHO cell lines. J Biotechnol 147(3-4): 180-5.

Claims

1. An isolated nucleic acid molecule comprising a functional chimeric gene regulatory unit comprising a functional enhancer nucleotide sequence and a functional promoter nucleotide sequence, wherein the enhancer nucleotide sequence is 5' to the promoter nucleotide sequence and derived from a first species of organisms and the promoter nucleotide sequence is derived from a second species of organisms, wherein the first species and the second species are not the same species.

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

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

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

5. The isolated nucleic acid molecule according to claims 2 to 4 wherein the at least one nucleotide sequence encoding for a polypeptide of interest, wherein said polypeptide is a polypeptide chain of a naturally occurring or artificial immunoglobulin.

6. The isolated nucleic acid molecule according to claims 2 to 5 wherein the polypeptide of interest is an antibody.

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

8. The isolated nucleic acid molecule according to claims 1 to 7 wherein the enhancer sequence, the promoter sequence or both are derived from viruses.

9. The isolated nucleic acid molecule according to claim 8 wherein the enhancer sequence, the promoter sequence or both are derived from double-stranded DNA viruses.

10. The isolated nucleic acid molecule according to claims 8 and 9 wherein the enhancer sequence, the promoter sequence or both are derived from viruses consisting of the group of Herpesviridae and Polyomaviridae.

11. The isolated nucleic acid molecule according to claims 1 to 10 wherein the chimeric gene regulation unit has an increased resistance to transcriptional silencing.

12. The isolated nucleic acid molecule according to claims 8 to 11 wherein the virus from which the promoter sequence, the enhancer sequence or both are derived is selected from the group consisting of human cytomegalovirus; murine cytomegalovirus; and simian virus 40.

13. The isolated nucleic acid molecule according to claims 1 to 12 further comprising at least one nucleotide sequence encoding for a recognition site of a restriction endonuclease.

14. The isolated nucleic acid molecule according to claim 13 wherein said at least one nucleotide sequence encoding for a recognition site of a restriction endonuclease is 3' to the enhancer nucleotide sequence and 5' to the promoter nucleotide sequence.

15. The isolated nucleic acid molecule according to claim 13 or 14, wherein the recognition site of the restriction endonuclease is a Spel recognition site as set forth in SEQ ID NO:22.

16. The isolated nucleic acid molecule according to claims 1 to 15 wherein the promoter sequence comprises, consists essentially of or consists of (I) a nucleotide sequence as set forth in any one of SEQ ID NO:l ; SEQ ID NO:3; SEQ ID NO:5; or a complement thereof; (II) a nucleotide sequence that shares at least 75% sequence identity with a nucleotide sequence of (I) or a complement thereof.

17. The isolated nucleic acid molecule according to claims 1 to 16 wherein the enhancer sequence comprises, consists essentially of or consists of (I) a nucleotide sequence as set forth in any one of SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; or a complement thereof; (II) a nucleotide sequence that shares at least 75% sequence identity with a nucleotide sequence of (I) or a complement thereof.

18. The isolated nucleic acid molecule according to claims 1 to 17 wherein the chimeric gene regulatory unit comprises, consists essentially of or consists of (I) a nucleotide sequence as set forth in any one of SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; or a complement thereof; (II) a nucleotide sequence that shares at least 75% sequence identity with a nucleotide sequence of (I) or a complement thereof.

19. The isolated nucleic acid molecule according to claims 1 to 18 wherein the chimeric gene regulatory unit comprises, consists essentially of or consists of (I) a nucleotide sequence as set forth in SEQ ID NO: 7 or a complement thereof; (II) a nucleotide sequence that shares at least 75% sequence identity with a nucleotide sequence of (I) or a complement thereof.

20. The isolated nucleic acid molecule according to claims 1 to 19 further comprising at least one nucleotide sequence encoding for an intron.

21. The isolated nucleic acid molecule according to claim 20 wherein the intron nucleotide sequence lies 3' to the promoter sequence and 5' to the nucleotide sequence encoding for a polypeptide, peptide or N A molecule of interest.

22. The isolated nucleic acid molecule according to claims 20 and 21 wherein the intron nucleotide sequence comprises, consists essentially of or consists of (I) a nucleotide sequence as set forth in any one of SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; or a complement thereof; (II) a nucleotide sequence that shares at least 75% sequence identity with a nucleotide sequence of (I) or a complement thereof.

23. The isolated nucleic acid molecule according to claims 20 to 22 wherein the intron nucleotide sequence comprises, consists essentially of or consists of (I) a nucleotide sequence as set forth in any one of SEQ ID NO: 17; or a complement thereof; (II) a nucleotide sequence that shares at least 75% sequence identity with a nucleotide sequence of (I) or a complement thereof.

24. The isolated nucleic acid molecule according to claims 20 to 23 further comprising a nucleotide sequence encoding for a recognition site of a restriction endonuclease, wherein said nucleotide sequence encoding for a recognition site of a restriction endonuclease is 3' to the promoter nucleotide sequence and 5' to the at least one nucleotide sequence encoding for an intron.

25. The isolated nucleic acid molecule according to claim 24 wherein the recognition site of the restriction endonuclease 3' to the promoter nucleotide sequence and 5' to the at least one nucleotide sequence encoding for an intron is a Notl recognition site as set forth in SEQ ID NO:23.

26. The isolated nucleic acid molecule according to claims 1 to 25 wherenrthe nucleotide sequence is set forth as SEQ ID NO:24.

27. The isolated nucleic acid molecule according to claims 1 to 26 wherein the promoter comprises at least one binding site for a transcription factor.

28. The isolated nucleic acid molecule according to claim 27 wherein the transcription factor is specificity protein 1 (Spl) transcription factor.

29. The isolated nucleic acid molecule according to claims 27 and 28 wherein the S l transcription factor comprises, consists essentially of or consists of a polypeptide sequence as set forth in any one of SEQ ID NOj20; SEQ ID NO:2l ; or a fragment thereof.

30. The isolated nucleic acid molecule according to claims 27 to 29 wherein the at least one binding site for a transcription factor comprises, consists essentially of or consists of the nucleotide sequence set forth in SEQ ID NO:13 (5'-(G/T)GGGCGG(G/A)(G/A)(C/T)-3').

31. A vector comprising the isolated nucleic acid molecule according to claims 1 to 30.

32. A host cell comprising the isolated nucleic acid molecule according to claims 1 to 30 or the vector according to cl im 31.

33. The host cell according to claim 32 wherein said host cell is a eukaryotic cell.

34. The host cell according to claims 32 and 33 wherein said host cell is a Chinese hamster ovary (CHO) cell.

35. Use of the isolated nucleic acid molecule according to claims 1 to 30 for facilitating or enhancing the expression of a polypeptide, peptide or RNA of interest, wherein said isolated nucleic acid molecule comprises a nucleotide sequence encoding the polypeptide, peptide₁ or RNA of interest, wherein said nucleotide sequence encoding the polypeptide, peptide or RNA of interest being operably linked to the chimeric gene regulatory unit of the isolated nucleic acid molecule.

36. A method of producing a polypeptide, peptide or RNA of interest comprising:

providing the isolated nucleic acid molecule according to claims 1 to 30, wherein said isolated nucleic acid molecule comprises a nucleotide sequence encoding the polypeptide, peptide or RNA of interest, wherein said nucleotide sequence encoding the polypeptide, peptide or RNA of interest being operably linked to the chimeric gene regulatory unit of the isolated nucleic acid molecule; and

producing the polypeptide, peptide or RNA of interest by in vitro transcription and translation or in a suitable host cell under conditions that allow production of the polypeptide, peptide or RNA of interest.

37. The method according to claim 36 wherein the suitable the host cell is a eukaryotic cell.

38. The method according to claims 36 and 37 wherein the suitable the host cell is a Chinese hamster ovary (CHO) cell.