WO2014133468A1 - Promoteurs chimères pour un niveau et une stabilité d'expression génique élevés dans des cellules mammifères - Google Patents

Promoteurs chimères pour un niveau et une stabilité d'expression génique élevés dans des cellules mammifères Download PDF

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WO2014133468A1
WO2014133468A1 PCT/SG2014/000100 SG2014000100W WO2014133468A1 WO 2014133468 A1 WO2014133468 A1 WO 2014133468A1 SG 2014000100 W SG2014000100 W SG 2014000100W WO 2014133468 A1 WO2014133468 A1 WO 2014133468A1
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nucleotide sequence
nucleic acid
acid molecule
isolated nucleic
seq
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Yuansheng Yang
Cheng Leong Steven HO
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Agency For Science, Technology And Research
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
    • C12N2710/22022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • C—CHEMISTRY; METALLURGY
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/60—Vector systems having a special element relevant for transcription from viruses

Definitions

  • 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.
  • 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.
  • Introns downstream of the promoter 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.
  • 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.
  • 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.
  • WT wild-type
  • hCMV human cytomegalovirus major immediate-early gene
  • mCMV murine cytomegalovirus major immediate-early gene
  • SV40 simian virus 40
  • the hCMV promoter is a commonly used strong promoter but is unstable.
  • 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.
  • the above functional chimeric gene regulatory units allow stable and high transgene expression in mammalian cell lines or mammalian tissue.
  • 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.
  • 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.
  • 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.
  • 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.
  • the enhancer sequence, the promoter sequence or both are derived from viruses.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the promoter comprises at least one binding site for a transcription factor.
  • the transcription factor is specificity protein 1 (Spl) transcription factor.
  • 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).
  • the isolated nucleic acid molecule of the invention further comprises at least one nucleotide sequence encoding for a recognition site of a restriction endonuclease.
  • 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.
  • the recognition site of the restriction endonuclease is a Spel recognition site, for example as set forth in SEQ ID NO:22.
  • 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.
  • this recognition site of the restriction endonuclease is a Notl recognition site, for example as set forth in SEQ ID NO:23.
  • 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.
  • the isolated nucleotide of the invention is set forth as SEQ ID NO:24.
  • 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').
  • the invention provides a vector comprising the isolated nucleic acid molecule of the invention.
  • 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.
  • 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.
  • 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.
  • Figure 1 shows the vectors used for recombinant protein expression
  • 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.
  • Figure 2 shows the enhancer sequence and promoter sequence combinations investigated in expression and stability experiments.
  • Figure 3 shows promoters tested for expression level and expression stability.
  • 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).
  • MFI mean fluorescence intensity
  • 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.
  • 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.
  • 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.
  • 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- ⁇ gene (SEQ ID NO: 18) (mCSfl), or the chicken /3-actin gene (SEQ ID NO: 19) (mCSal).
  • SEQ ID NO: 17 human cytomegalovirus immediate early gene
  • mCSfl human EFl- ⁇ gene
  • SEQ ID NO: 19 the chicken /3-actin gene
  • 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.
  • 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.
  • 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.
  • 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.
  • the enhancer sequence, the promoter sequence or both are derived from viruses.
  • the enhancer sequence, the promoter sequence or both ard derived from double-stranded DNA viruses.
  • the enhancer sequence, the promoter sequence or both are derived from viruses selected from the group consisting of Herpesviridae and Polyomaviridae.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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;
  • 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.
  • 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.
  • 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.
  • the promoter comprises at least one binding site for a transcription factor.
  • the transcription factor is specificity protein 1 (Spl) transcription factor.
  • 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).
  • 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').
  • the isolated nucleic acid molecule of the invention further comprises at least one nucleotide sequence encoding for a recognition site of a restriction endonuclease.
  • 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.
  • the recognition site of the restriction endonuclease is a Spel recognition site, for example as set forth in SEQ ID NO:22.
  • 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.
  • this recognition site of the restriction endonuclease is a Notl recognition site, for example as set forth in SEQ ID NO:23.
  • 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.
  • the isolated nucleotide of the invention is set forth as SEQ ID NO: 24.
  • 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.
  • linker region 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]-3 ', wherein [N] is a nucleotide that has a base selected from the group consisting of adenine, cytosine, guanine, thymine and uracile.
  • the invention provides a vector comprising the isolated nucleic acid molecule as defined herein.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • At least one relates to one or more, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.
  • sequence relates to the primary nucleotide sequence of nucleic acid molecules or the primary amino acid sequence of a protein.
  • 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.
  • 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.
  • 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.
  • polypeptide relates to one or more associated polypeptides, wherein the polypeptides consist of amino acids coupled by peptide (amide) bonds.
  • 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.
  • 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).
  • operably linked 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).
  • 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.
  • 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.
  • a species is defined as the largest group of organisms capable of interbreeding and producing fertile offspring.
  • virus species a virus species is a polythetic class of viruses that constitutes a replicating lineage and occupies a particular ecological niche.
  • Antibody also known as an immunoglobulin (Ig)
  • Ig immunoglobulin
  • Antibodies are typically made of basic structural units - each with two large heavy chains and two small light chains.
  • 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.
  • VH variable domain
  • 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.
  • fragment 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.
  • “Complement” 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • sequence comparison 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.
  • 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.
  • Transcriptional silencing relates to the epigenetic regulation of gene expression.
  • 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.
  • virus 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.
  • cytomegalovirus 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.
  • double-stranded DNA virus 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.
  • 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).
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • 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.
  • 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.
  • 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.
  • vectors are special plasmids, i.e. circular genetic elements.
  • a nucleic acid as contemplated herein is cloned into a vector.
  • 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.
  • 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.
  • 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.
  • 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.
  • the contained nucleic acid is not expressed in cloning vectors.
  • 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, Nde
  • host cell 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.
  • 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.
  • Preferred host cells are eukaryotic cells. More preferred are CHO cells.
  • 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.
  • CHO cells 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.
  • the mammalian host cell is a CHO-K1 cell or a CHO-DG44 cell.
  • 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
  • facilitating or enhancing expression 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.
  • “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.
  • 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.
  • macromolecular components RNA polymerase, 70S or 80S ribosomes, tR As, aminoacyl-tRNA synthetases, initiation, elongation and termination factors, etc.
  • 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
  • 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.
  • 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.
  • 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).
  • the invention relates to a vector comprising the isolated nucleic acid molecule according to the invention.
  • 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).
  • nucleic acids according to the present invention are used for facilitating or enhancing the expression of a polypeptide, peptide or RNA of interest.
  • 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).
  • 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.
  • the host is E. coli, Pichia pastoris, a HeLa cell, a CHO cell or a SF9 cell.
  • 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
  • Adherent CHO Kl cells (American Type Culture Collection, Manassas, VA) were grown in Dulbecco's modified Eagle's medium (DMEM) + GlutaMaxTM (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).
  • 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 protein free media
  • HT thymine
  • 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 ⁇ 10 5 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).
  • 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).
  • GFP green fluorescent protein
  • 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).
  • 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).
  • Transfections were all performed using Nucleofection kits from Lonza (Cologne, Germany) following the manufacturer's instructions using 1 * 10 7 cells and 5 ⁇ g 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.
  • the isolated clones were cultured in 6 well plates with 2 mL of cells at a density of 2 x 10 5 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.
  • week 1 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.
  • DMSO Sigma-Aldrich
  • Transfections were all performed using Nucleofection kits from Lonza (Cologne, Germany) following the manufacturer's instructions. Stable transfections were performed using 1* 10 7 cells and 5 ⁇ g 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*10 5 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.
  • MTX methotrexate
  • Example 1 Expression level of wild-type and chimeric promoters
  • 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.
  • MFI mean fluorescent intensity
  • Example 2 Expression stability of wild-type and chimeric promoters
  • Example 3 mAb expression level using mCS with introns
  • mCMV enhancer and SV40 promoter 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- ⁇ 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.
  • mAb titer obtained using the mCS promoter was 181 mg/L at 500 nM MTX ( Figure 7).
  • mCSvI was most effective with expression level doubling to 400 mg/L.
  • 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.

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Abstract

La présente invention concerne une molécule d'acide nucléique isolée comprenant une unité de régulation génique chimère fonctionnelle, comprenant une séquence nucléotidique d'amplification fonctionnelle et une séquence nucléotidique de promoteur fonctionnelle, l'utilisation de ladite molécule d'acide nucléique isolée pour faciliter ou amplifier l'expression, un vecteur ou une cellule hôte comprenant ladite molécule d'acide nucléique et un procédé de production d'un polypeptide, d'un peptide ou d'une molécule d'ARN d'intérêt.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015195049A1 (fr) * 2014-06-18 2015-12-23 Agency For Science, Technology And Research Nouveaux promoteurs pour une expression de haut niveau
WO2018093331A1 (fr) * 2016-11-16 2018-05-24 Agency For Science, Technology And Research Glutamine synthétase atténuée en tant que marqueur de sélection
JP2021510080A (ja) * 2017-12-29 2021-04-15 上海細胞治療集団有限公司Ｓｈａｎｇｈａｉ Ｃｅｌｌ Ｔｈｅｒａｐｙ Ｇｒｏｕｐ Ｃｏ．， Ｌｔｄ． Ｔ細胞に高い転写活性を有するキメラプロモーター

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987003905A1 (fr) * 1985-12-18 1987-07-02 Genetics Institute, Inc. Systeme promoteur
US7824907B2 (en) * 2003-03-11 2010-11-02 Merck Serono Sa Expression vectors comprising the mCMV IE2 promoter
US20110097798A1 (en) * 2008-06-30 2011-04-28 Atgcell Inc. Mammalian cell expression vectors and utilization
US20120301919A1 (en) * 2011-05-24 2012-11-29 Agency For Science, Technology And Research Ires mediated multicistronic vectors

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987003905A1 (fr) * 1985-12-18 1987-07-02 Genetics Institute, Inc. Systeme promoteur
US7824907B2 (en) * 2003-03-11 2010-11-02 Merck Serono Sa Expression vectors comprising the mCMV IE2 promoter
US20110097798A1 (en) * 2008-06-30 2011-04-28 Atgcell Inc. Mammalian cell expression vectors and utilization
US20120301919A1 (en) * 2011-05-24 2012-11-29 Agency For Science, Technology And Research Ires mediated multicistronic vectors

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ANDO, M. ET AL.: "Constant and steady transgene expression of interferon-y by optimization of plasmid construct for safe and effective interferon-y gene therapy", THE JOURNAL OF GENE MEDICINE, vol. 14, 2012, pages 288 - 295 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015195049A1 (fr) * 2014-06-18 2015-12-23 Agency For Science, Technology And Research Nouveaux promoteurs pour une expression de haut niveau
US10563222B2 (en) 2014-06-18 2020-02-18 Agency For Science, Technology And Research Promoters for high level expression
WO2018093331A1 (fr) * 2016-11-16 2018-05-24 Agency For Science, Technology And Research Glutamine synthétase atténuée en tant que marqueur de sélection
CN110023500A (zh) * 2016-11-16 2019-07-16 新加坡科技研究局 作为选择标记的减毒谷氨酰胺合成酶
JP2021510080A (ja) * 2017-12-29 2021-04-15 上海細胞治療集団有限公司Ｓｈａｎｇｈａｉ Ｃｅｌｌ Ｔｈｅｒａｐｙ Ｇｒｏｕｐ Ｃｏ．， Ｌｔｄ． Ｔ細胞に高い転写活性を有するキメラプロモーター
JP7399871B2 (ja) 2017-12-29 2023-12-18 上海細胞治療集団有限公司 Ｔ細胞に高い転写活性を有するキメラプロモーター

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