WO2012099540A1 - Cmv promoter variants - Google Patents

Abstract

The present invention relates to CMV promoter variants comprising one or more mutations corresponding to cytosine residues within CpG motifs. Said promoter variants are capable of driving strong and sustained heterologous gene expression in in vitro and in vivo applications such as gene therapy and recombinant protein expression.

Description

CMV PROMOTER VARIANTS

CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims the benefit of priority of US provisional application

No. 61/433,291 filed January 17, 2011, the contents of it being hereby incorporated by reference in its entirety for all purposes.

FIELD OF INVENTION

[0002] The present invention relates to CMV promoter variants capable of driving strong and sustained heterologous gene expression in in vitro and in vivo applications such as gene therapy and recombinant protein expression.

BACKGROUND OF THE INVENTION

[0003] The human cytomegalovirus (CMV) major immediate-early enhancer/promoter

(CMV promoter) is one of the most commonly used promoters to drive the expression of heterologous genes. However, many reports have shown that gene expression driven by CMV gets silenced after prolonged culturing, thereby compromising heterologous gene expression (Mehta et al. 2009). In human and mouse embryonic stem cells, it has been reported that silencing of CMV occur after short period of culture (in a few days), thereby limiting the use of CMV in these important cell lines (Liew et al. 2007). For the production of recombinant biotherapeutics, whereby cells such as Chinese Hamster Ovary (CHO) cells need to be cultured for even longer periods of time (for months), gene silencing can severely hamper protein production capabilities (Wurm, 2004). Indeed, Yang et al. (2010) have reported that gene silencing contributes to loss in productivity in monoclonal antibody-producing CHO cell lines.

[0004] Several mechanisms have been reported to be the cause for gene silencing including chromatin condensation, histone modifications and DNA methylation (Bird 2007; Miranda et al. 2007; Mellor et al. 2008). The methylation of CpG and non-CpG sequences of the CMV promoter has been associated with its silencing (Brooks et al. 2004). Although the overall increase in CpG methylation has been correlated with gene silencing, studies have not been able to determine the critical CpG sites that are responsible for initiation of silencing. Furthermore, it has been reported that for foreign DNA integrated into mammalian genome, the initiation of methylation is not exclusively targeted by nucleotide sequence but is also determined by site of integration, conformation of the integrated DNA, mode of cell selection and chromatin structure (Orend et al 1995).

[0005] Several strategies have been proposed to prevent promoter silencing. The use of chemicals that are DNA methylation inhibitors such as 5-Aza-2'-deoxytidine and butyrate is not possible in cell culture due to its side effects such as toxicity that inhibits cell growth and can result in cell death (Choi et al 2005; Yang et al 2010). Other strategies involve the use of insulators, matrix attachment regions and additional CpG island elements to prevent DNA methylation (Pikaart et al. 1998; Dang et al. 2000; Hejnar et al. 2001 ; Williams et al. 2005). However, these strategies involve appending additional DNA sequences, thereby enlarging the size of the vector or plasmid. With cloning, larger plasmid sizes often is associated with some issues in transformation, stable propagation and ligation. SUMMARY OF THE INVENTION

[0006] The present invention is based on the identification of CpG sequences in the CMV that are prone to methylation leading to silencing of heterologous gene expression and suitable substitutions of these cytosine nucleotides that prevent methylation without decreasing promoter activity. The present invention thus provides novel modified CMV promoters that provide for the stable and sustained expression of heterologous genes without compromising strength of expression.

[0007] In a first aspect the present invention is directed to a nucleic acid molecule including a cytomegalovirus major immediate-early enhancer/promoter (CMV promoter) or fragment thereof, wherein the CMV promoter or fragment thereof comprises one or more mutations in at least one of the sequence- regions 1-4, wherein sequence region 1 comprises nucleotide positions 38 to 167, sequence region 2 comprises nucleotide positions 226 to 276, sequence region 3 comprises nucleotide positions 368 to 629, and sequence region 4 comprises nucleotide positions 1149 to 1221. The one or more mutations are at any one of the sequence positions selected from the group consisting of:

(a) Region 1 positions: 38, 67, 82, 86, 132,

(b) Region 2 positions: 167, 226, 228, 238, 244, 253, 259, 276,

(c) Region 3 positions: 368, 400, 490, 629,

(d) Region 4 positions: 1 149, 1 176, 1203, 1209, 1221, wherein the sequence position numbering corresponds to that of the linear wildtype sequence of the human CMV promoter as set forth in SEQ ID NO: 1.

[0008] In another aspect, the invention features a vector comprising the nucleic acid molecule of the invention.

[0009] In a further aspect, the invention relates to a host cell comprising the nucleic acid molecule or vector of the invention.

[0010] In a still further aspect, the invention is directed to a method for the production of a polypeptide, comprising providing a nucleic acid molecule of the invention, wherein the nucleic acid molecule comprises a nucleotide sequence encoding the polypeptide that is operably linked to the CMV promoter or fragment thereof; and expressing the nucleotide sequence encoding the polypeptide to produce the polypeptide.

[0011] In another aspect, the invention relates to the use of the inventive nucleic acid molecule for enhancing the expression of a nucleotide sequence encoding a polypeptide, wherein the nucleic acid molecule comprising the CMV promoter or fragment thereof is operably linked to the nucleotide sequence encoding the polypeptide.

[0012] The invention also features a method of using the nucleic acid molecule of the invention to maintain or increase expression of a nucleotide sequence encoding a polypeptide, wherein the nucleic acid molecule comprising the CMV promoter or fragment thereof is operably linked to the nucleotide sequence encoding the polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] 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, in which:

[0014] Figure 1 shows: (A) Positions of methylated cytosines on the CMV driving the expression of Heavy Chain in pHCMV-VHRhd-ylC-neo. (B) Position of methylated cytosines on the CMV driving the expression of Light Chain in pHCMV-V_LRhd-ylC-dhfr. (C) The positions of the 4 regions and 25 cytosines on CMV that are prone to methylation.

[0015] Figure 2 shows the 25 cytosine position on CMV and the replacement of 22 of the cytosine residues with alternative nucleotides to remove the possibility of methylation.

[0016] Figure 3 shows a diagram of CMV constructs illustrating the position of methylation prone cytosines and their replacement with alternative nucleotides (A), which are in bold and underlined. (A) Wild-type CMV without any cytosine replacement. The CMV enhancer region spans from position 629 to 1033, the minimal promoter spans from 1101 to 1223. The TATA box is in bold while the translational ATG start site is underlined. (B) BTI CMV promoter Region IV with cytosines at positions 1149, 1176, 1203, 1209 and 1221 replaced with alternative nucleotides. (C) BTI CMV promoter Region III, IV with cytosines at positions 368, 400, 490, 629, 1149, 1176, 1203, 1209 and 1221 replaced with alternative nucleotides. (D) BTI CMV promoter Region II, III, IV with cytosines at positions 226, 228, 238, 244, 253, 259, 276, 368, 400, 490, 629, 1149, 1176, 1203, 1209 and 1221 replaced with alternative nucleotides. (E) BTI CMV promoter Region I, II, III, IV with cytosines at positions 38, 67, 82, 86, 132, 167, 226, 228, 238, 244, 253, 259, 276, 368, 400, 490, 629, 1149, 1176, 1203, 1209 and 1221 replaced with alternative nucleotides. (F) BTI minimal CMV promoter, the minimal promoter sequence capable of driving gene expression with cytosines at positions 1149, 1176, 1203, 1209 and 1221 replaced with alternative nucleotides. The shown sequences correspond to the nucleotide sequence of cytomegalovirus major immediate-early enhancer/promoter (CMV).

[0017] Figure 4 shows the maintenance of promoter strength upon removal of CpG sequences in CMV promoters after transfection into hESC cells. Compared to the wild- type CMV promoter, all full length BTI CMV constructs could maintain the strength of heterologous gene expression of luciferase.

[0018] Figure 5 shows sustained and stable expression of heterologous gene expression using BTI CMV Region I, II, III, IV, BTI CMV Region II, III, IV and BTI CMV Region III, IV. As expected Wild-type CMV which showed a significant drop in gene expression at 16 days post- transfection (dark grey bars) compared to 2 days post- transfection (light grey bars). In contrast, the BTI CMV constructs (with the exception of BTI CMV Region IV) continue to show high gene expression. Similarly, using minimal BTI CMV promoter, there was no decrease in the luciferase expression of 16 days versus 2 days post-transfection.

[0019] Figure 6 shows the instability of heterologous gene expression driven by wild- type CMV in CHO cells. With the presence of selection pressure (+G418), most clones show a significant decrease in heterologous gene expression after +4 passages. With no selective pressure (-G418), the decrease in heterologous gene expression was more pronounced, with most clones showing additional decreases in expression with prolonged culturing at +4, +8 and +16 passages. This showed that for wild-type CMV, stability of heterologous gene expression was compromised from +4 passages onwards.

[0020] Figure 7 shows the stability of heterologous gene expression driven by BTI CMV promoter Region I, II, III, IV. In CHO cells, with the presence of selection pressure (+G418), most clones continue to show comparable heterologous gene expression after +4, +8 and +16 passages. With no selective pressure (-G418), most clones showed comparable heterologous gene expression with prolonged culturing at passage +4, +8 and +16. This showed that for BTI CMV promoter Region I, II, III, IV, stability of heterologous gene expression could be maintained throughout +16 passages or longer.

[0021] Figure 8 shows the stability of heterologous gene expression driven by BTI

CMV promoter Region II, III, IV. In CHO cells, with the presence of selection pressure (+G418), most clones continue to show comparable heterologous gene expression after +4, +8 and +16 passages. With no selective pressure (-G418), most clones showed comparable heterologous gene expression with prolonged culturing at passage +4, +8 and +16. This showed that for BTI CMV promoter Region II, III, IV, stability of heterologous gene expression could be maintained throughout +16 passages or longer.

[0022] Figure 9 shows the stability of heterologous gene expression driven by BTI CMV promoter Region III, IV. In CHO cells, with the presence of selection pressure (+G418), most clones continue to show comparable heterologous gene expression after +4, +8 and +16 passages. With no selective pressure (-G418), most clones showed comparable heterologous gene expression with prolonged culturing at passage +4, +8 and +16. This showed that for BTI CMV promoter Region III, IV, stability of heterologous gene expression could be maintained throughout +16 passages or longer.

[0023] Figure 10 shows the stability of heterologous gene expression driven by BTI CMV promoter Region IV. In CHO cells, with the presence of selection pressure (+G418), most clones continue to show comparable heterologous gene expression after +4, +8 and +16 passages. With no selective pressure (-G418), most clones showed comparable heterologous gene expression with prolonged culturing at passage +4 and +8. However at +16 passages, there was a decrease in heterologous gene expression indicating instability beginning at +16 passages for this construct. This showed that for BTI CMV promoter Region IV, stability of heterologous gene expression could be maintained up to passage +16 or longer with the presence of selective pressure. DETAILED DESCRIPTION OF THE INVENTION

[0024] Although the CMV promoter has been used in DNA vectors to drive the expression of heterologous genes in many different mammalian cell types, the expression levels often gets silenced after prolonged culturing. Stable gene expression for prolonged periods either in vitro or in vivo is highly desired for many different applications such as gene therapy or recombinant protein production.

[0025] The cytomegalovirus (CMV) major immediate-early enhancer/promoter (CMV promoter) is methylation sensitive and both CpG and non-CpG methylation has been reported to lead to its inactivation in transfected cells. Interestingly, many transcription factors target to CpG-containing sequences as well. Therefore, the removal of CpG sequences from CMV promoter is highly complex. On one hand, even though removal of CpG sequences might remove the risk of methylation/silencing, on the other hand, removal of CpG could destroy the transcription factor binding sites, thereby significantly reducing the strength of the CMV promoter or in the worst case scenario completely destroying the CMV promoter activity. It is thus imperative to identify the critical CpG positions that are responsible for silencing the promoter rather than randomly targeting the CpG positions.

[0026] The inventor of the present invention has now identified cytosine residues that are prone to methylation and thus leading to inactivation of the promoter but that can be mutated to be insensitive to promoter inactivation, for example by methylation, with mutations of these residues at the same time retaining the strong promoter activity of the thus modified CMV promoter.

[0027] In a first aspect, the present invention thus features a nucleic acid molecule comprising a cytomegalovirus major immediate-early enhancer/promoter (CMV promoter) or fragment thereof, wherein the CMV promoter comprises one or more mutations in at least one of the sequence regions 1-4, wherein sequence region 1 comprises nucleotide positions 38 to 167, sequence region 2 comprises nucleotide positions 226 to 276, sequence region 3 comprises nucleotide positions 368 to 629, and sequence region 4 comprises nucleotide positions 1149 to 1221. The mutations are at any one of the sequence positions selected from the group consisting of:

(e) Region 1 positions: 38, 67, 82, 86, 132,

(f) Region 2 positions: 167, 226, 228, 238, 244, 253, 259, 276,

(g) Region 3 positions: 368, 400, 490, 629, (h) Region 4 positions: 1149, 1176, 1203, 1209, 1221,

with these sequence positions corresponding to those of the linear wildtype sequence of the human CMV promoter as set forth in SEQ ID NO:l . The nucleic acid molecule can also consist of the cytomegalovirus major immediate-early enhancer/promoter (CMV promoter) as set forth in SEQ ID NO:l with the aforementioned mutations or a fragment thereof.

[0028] The terms "polynucleotide" and "nucleic acid (molecule)" are used interchangeably to refer to polymeric forms of nucleotides of any length, including naturally occurring and non-naturally occurring nucleic acids. The polynucleotides may contain deoxyribonucleo tides, ribonucleotides and/or their analogs. Methods for selection and preparation of nucleic acids are diverse and well described in standard biomolecular protocols. A typical way would be preparative PCR and chromatographic purification starting from existing template DNAs or stepwise synthesis of artificial nucleic acids. Nucleotides may have any three-dimensional structure. The term "nucleic acid molecule" includes single-, double-stranded and triple helical molecules. The following are non- limiting embodiments of nucleic acids: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A nucleic acid molecule may also comprise modified nucleic acid molecules, such as nucleic acid molecule analogs, i.e. nucleic acid molecules that include analogs of the natural bases. Analogs of purines and pyrimidines are known in the art, and include, but are not limited to, inosine, aziridinylcytosine, 4-acetylcytosine, 5- fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl- aminomethyluracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1- methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethyl guanine, 2- methyladenine, 2-methylguanine,3-methylcvtosine,5-methylcytosine, pseudouracil, 5- pentylnyluracil and 2,6-diaminopurine. The use of uracil as a substitute for thymine in a deoxyribonucleic acid is also considered an analogous form of pyrimidine.

[0029] "CMV" or "cytomegalovirus" is a double-strand DNA virus of the Herpesviruses group. The term as used herein includes all cytomegaloviruses, including human CMV (HCMV) also known as Human Herpesvirus 5 (HHV-5).

[0030] "Wildtype" as used herein relates to the naturally occurring form of a nucleic acid that is fully functional, i.e. does not include any known mutations that impair its functionality. With respect to the human CMV promoter sequence, the wildtype sequence as referred to herein is set forth in SEQ ID NO: 1.

[0031] The term "fragment" as used herein with regard to a nucleic acid molecule, in particular the CMV promoter, relates to nucleic acid molecules, including polynucleotides and oligonucleotides, that are derived from the full length CMV promoter corresponding to that set forth in SEQ ID NO:l and that are terminally shortened, i.e. lacking at least one of the 3 '-terminal and/or 5 '-terminal nucleotides. Such fragments comprise preferably at least 100, more preferably 200, most preferably 300 or more consecutive nucleotides of the sequence of the full length CMV promoter. In specific embodiments, the fragments of the invention comprise or consist of at least one, two or three of the sequence regions 1-4, as set forth above. In various embodiments, the fragments comprise at least one, two or three of the sequence regions 1-4, wherein at least one of these regions comprises a mutation as set forth above. When the fragments comprise or consist of two or three of the identified sequence regions, they usually also comprise the nucleotides between these regions, i.e. the linking sequences. In one embodiment, a fragment of the invention comprises or consists of: sequence region 4, sequence regions 3 and 4 or sequence regions 2-4. In various embodiments, the promoter fragments of the invention are fully or partially functional, i.e. retain at least part of the promoter activity of the full length sequence. In one embodiment, the fragment comprises or consists of a nucleotide sequence that corresponds to positions 1101 to 1232 of the linear wildtype sequence of the CMV promoter as set forth in SEQ ID NO: 1.

[0032] "Mutation" as used herein in relation to the nucleic acid molecules of the invention, relates to a modification of the natural nucleotide sequence. In various embodiments of the invention, the mutation is a point mutation, i.e. the substitution, deletion or insertion of a single nucleotide. In preferred embodiments of the invention, the mutation is a substitution of a single nucleotide in a given position by another nucleotide. For example, in the nucleotide sequence a cytosine base may be substituted by another D A base, such as thymine, adenine or guanine, or uracil. Possible is also the substitution with a base analog or non-naturally occurring base, such as inosine or any one of the other analogs listed above. The present invention also encompasses embodiments, wherein the nucleic acid molecule comprises further mutations at positions other than those indicated above. These additional mutations preferably do not impair promoter activity. [0033] The terms "one or more" or "at least one", as interchangeably used herein, relate to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more of a given species. Similarly, "two or more" or "at least two" relates to 2, 3, 4, 5, 6, 7, 8, 9,

10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more of a given species.

[0034] In various embodiments, the nucleic acid molecule of the invention comprises a CMV promoter or fragment thereof that comprises mutations at 2, 3, 4, 5, 6, 7, 8, 9, 10,

1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or all 22 of the sequence positions.

[0035] In various embodiments, the mutated CMV promoter or fragment thereof may comprise one or more mutations in only one of the indicated sequence regions. For example, the CMV promoter may comprise mutations at all of the indicated positions in only one of the sequence regions 1-4. However, in preferred embodiments, the mutated promoter comprises one or more mutations at positions within 2, 3 or all 4 of the given regions. In one embodiment of the invention, the nucleic acid molecule comprises one or more, for example 1, 2, 3, 4 or 5, mutations at positions 1 149, 1176, 1203, 1209, and 1221. In another embodiment, the nucleic acid molecule comprises one or more, for example 1, 2, 3 or 4, mutations at positions 368, 400, 490, and 629. In still another embodiment, the nucleic acid molecule comprises one or more, for example 1, 2, 3, 4, 5, 6, 7, or 8, mutations at positions 167, 226, 228, 238, 244, 253, 259, and 276. In a still further embodiment, the nucleic acid molecule comprises one or more, for example 1, 2, 3, 4 or 5, mutations at positions 38, 67, 82, 86, and 132. In various embodiments, the above embodiments can be combined such that the nucleic acid molecule comprises one or more of the given mutations in more than one sequence region. In preferred embodiments, the nucleic acid molecule comprises one or more mutations in each of regions 3 and 4 or regions 2-4. In further preferred embodiments, all of the indicated positions in (i) sequence region 3, (ii) sequence region 4, (iii) sequence regions 3 and 4, or (iv) sequence regions 2- 4 are mutated.

[0036] In various embodiments of the invention, the one or more mutations are nucleotide substitutions selected from the group consisting of C38T, C67A, C82G, C86G, C132T, C167A, C167T, C167G, C226A, C228T, C238A, C244A, C253T, C259T, C276A, C276T, C276G, C368T, C400T, C490A, C629T, C1 149T, C1 176G, C1203A, C1203T, C1203G, C1209A, C1209T, C1209G, and C1221A. As indicated above, the position numbering corresponds to the numbering of the human CMV promoter as set forth in SEQ ID NO: l . The nucleic acid molecules of the invention can comprise one or more of at least two, three or four of the following groups of mutations:

(a) C38T, C67A, C82G, C86G, C132T,

(b) C167A, C167T, C167G, C226A, C228T, C238A, C244A, C253T, C259T, C276A, C276T, C276G,

(c) C368T, C400T, C490A, C629T, and

(d) C1149T, C1176G, C1203A, C1203T, C1203G, C1209A, C1209T, C1209G, and C1221A.

[0037] In various embodiments, one or more or all positions of groups (c) and (d) are point-mutated to the given nucleotides. In other embodiments, one or more or all positions of groups (b), (c) and (d) are point-mutated to the given nucleotides. -

[0038] In specific embodiments of the invented nucleic acid molecules, the nucleic acid molecule comprises or consists of the CMV promoter sequence set forth in any one of SEQ ID Nos. 2-6.

[0039] The nucleic acid molecule of the invention may further comprise a nucleotide sequence encoding a peptide or polypeptide of interest. In such an embodiment, it is preferred that the CMV promoter or fragment thereof and the nucleotide sequence encoding the peptide or polypeptide of interest are operably linked.

[0040] "Peptide" generally refers to a short chain of amino acids linked by peptide bonds. Typically peptides comprise amino acid chains of about 2-100, more typically about 4-50, and most commonly about 6-20 amino acids. "Polypeptide" generally refers to individual straight or branched chain sequences of amino acids that are typically longer than peptides. "Polypeptides" usually comprise at least about 100 to 1000 amino acids in length, more typically at least about 150 to 600 amino acids, and frequently at least about 200 to about 500 amino acids. "Proteins" include single polypeptides as well as complexes of multiple polypeptide chains, which may be the same or different. Multiple chains in a protein may be characterized by secondary, tertiary and quaternary structure as well as the primary amino acid sequence structure, may be held together, for example, by disulfide bonds, and may include post-synthetic modifications such as, without limitation, glycosylation, phosphorylation, truncations or other processing.

[0041] The nucleic acid molecule, such as DNA, comprising the CMV promoter or fragment thereof and a nucleotide sequence encoding a polypeptide of interest is referred to as "capable of expressing a nucleic acid molecule" or capable "to allow expression of a nucleotide sequence" if the promoter sequence and the polypeptide-encoding sequence are "operably linked". An operable linkage is a linkage in which the regulatory sequence elements and the sequence to be expressed are connected in a way that enables gene expression. The precise nature of the regulatory regions necessary for gene expression may vary among species, but in general these regions comprise a promoter, such as the invented CMV promoter or fragment thereof. The nucleic acid molecule can, in addition to the promoter, also include further enhancer or repressor elements as well as translated signal and leader sequences for targeting the native polypeptide to a specific compartment of a host cell.

[0042] A "nucleotide" generally refers to native (naturally occurring) nucleotides, which include a nitrogenous base selected from the group consisting of adenine, thymidine, cytosine, guanine and uracil, a sugar selected from the group of ribose, arabinose, xylose, and pyranose, and deoxyribose (the combination of the base and sugar generally referred to as a "nucleoside"), and one to three phosphate groups, and which can form phosphodiester internucleosidyl linkages. The "nucleotide" also refers to nucleotide analogs. Such analogs can have a sugar analog, a base analog and/or an internucleosidyl linkage analog. Additionally, analogs exhibiting non-standard base pairing are also included (see for example U.S. Pat. No. 5,432,272). Such nucleotide analogs include nucleotides that are chemically modified in the natural base ("base analogs"), chemically modified in the natural sugar ("sugar analogs"), and/or chemically modified in-the natural phosphodiester or any other internucleosidyl linkage ("internucleosidyl linkage analogs"). In certain embodiments, the aromatic ring or rings contain at least one nitrogen atom. In certain embodiments, the nucleotide base is capable of forming Watson-Crick and/or Hoogsteen hydrogen bonds with an appropriately complementary nucleotide base. Exemplary nucleotide bases and analogs thereof include, but are not limited to, naturally occurring nucleotide bases, e.g., adenine, guanine, cytosine, uracil, and thymine, and analogs of the naturally occurring nucleotide bases, e.g., 7-deazaadenine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaadenine, N662-isopentenyladenine, N2- dimethylguanine (dmG), 7-methylguanine (7mG), inosine, nebularine, 2-aminopurine, 2- amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudouridine, pseudocytosine, pseudoisocytosine, 5-propynylcytosine, isocytosine, isoguanine, 7-deazaguanine, 2thiopyrimidine, 6-thioguanine, 4-thiothymine, 4-thiouracil, 0.sup.6-methylguanine, N.sup.6-methyladenine, 0.sup.4-methylthymine, 5,6-dihydrothymine, 5,6-dihydrouracil, pyrazolo[3,4-D]pyrimidines (see for example U.S. Pat. Nos. 6,143,877 and 6,127,121), ethenoadenine, indoles such as nitroindole and 4-methylindole, and pyrroles such as nitropyrrole.

[0043] In addition, the nucleic acid molecule of the invention may comprise, 3'- terminal to the polypeptide-encoding sequence, non-coding sequences that may contain regulatory elements involved in transcriptional termination, polyadenylation or the like. If, however, these termination sequences are not satisfactory functional in a particular host cell, then they may be substituted with signals functional in that cell.

(0044] In various embodiments, the nucleotide sequence encoding a polypeptide of interest is a heterologous nucleotide sequence. In specific embodiments, the nucleotide sequence encoding the polypeptide is a mammalian, preferably human sequence.

[0045] The term "heterologous" when used in reference to a nucleotide sequence or nucleic acid molecule, means a nucleotide sequence not naturally occurring in the respective cell into which the nucleic acid molecule has been (or is being) introduced. A heterologous nucleic acid sequence thus originates from a source other than the respective cell and can occur naturally or non-naturally. A respective heterologous nucleic acid sequence may for example be integrated into the nucleic acid molecule of the present invention.

[0046] The nucleic acid molecule of the invention may be a DNA molecule.

[0047] In various embodiments, the nucleic acid molecule of the invention is an isolated nucleic acid molecule. This includes nucleic acid molecules that have been separated from other components, such as cellular components, for example by suitable purification methods. An "isolated" nucleic molecule of the present invention can also refer to one that is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. An isolated molecule, for example a DNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In other words, an isolated nucleic acid molecule can be free or substantially free of sequences (for example protein-encoding sequences) which flank the nucleic acid (i.e., sequences located at the 5' and 3's ends of the nucleic acid) in the genomic DNA of a cell or organism from which the nucleic acid is derived

[0048] In various embodiments, the mutated cytomegalovirus major immediate-early enhancer/promoter (CMV promoter) or fragment thereof is a mutated human cytomegalovirus major immediate-early enhancer/promoter (CMV promoter) or fragment thereof.

[0049] The nucleic acid molecules of the invention can also be part of a vector or any other kind of cloning vehicle, such as a plasmid, a phagemid, a phage, a baculovirus, a cosmid or an artificial chromosome. The term "vector" generally refers to a single or double-stranded circular nucleic acid molecule that can be introduced, e.g. transfected, into cells and replicated within or independently of a cell genome. A circular double- stranded nucleic acid molecule can be cut and thereby linearized upon treatment with restriction enzymes. An assortment of nucleic acid vectors, restriction enzymes, and the knowledge of the nucleotide sequences cut by restriction enzymes are readily available to those skilled in the art. A nucleic acid molecule according to the present invention can be inserted into a vector by cutting the vector with restriction enzymes and ligating the two pieces together. Commercially available plasmids such as pBR322, puC19, pBluescript and the like may be used. Typically, mammalian expression vectors can be used and are commercially available. Examples of mammalian expression vectors can include but are not limited to pCDM8 (Seed, B. Nature, 1987, 329: 840), pMT2PC (Kaufman et al, 1987, EMBO J. 6: 187-195), pCI and pSI mammalian vectors.

[0050] Cloning vehicles can include, aside from the regulatory sequences described above and a nucleic acid sequence encoding a polypeptide of interest, replication and control sequences derived from a species compatible with the host cell that is used for expression as well as selection markers conferring a selectable phenotype on transformed or transfected cells. Large numbers of suitable cloning vectors are known in the art, and are commercially available.

[0051] The nucleic acid molecules of the invention, and in particular a cloning vector containing the coding sequence of a polypeptide of interest and the mutated CMV promoter or fragment thereof wherein both are operably linked can be transformed into a host cell capable of expressing the gene. Transformation can be performed using standard techniques (Sambrook, J. et al. (1989)).

[0052] In various embodiments, the invention thus also features a vector comprising the nucleic acid molecule of the invention. The vector may be a prokaryotic or eukaryotic vector. In one embodiment, the vector is a plasmid.

[0053] Further, the invention is also directed to a host cell containing a nucleic acid molecule as disclosed herein. The transformed host cells can be cultured under conditions suitable for expression of the nucleotide sequence encoding a polypeptide of interest. Host cells can be established, adapted and completely cultivated under serum free conditions, and optionally in media which are free of any protein/peptide of animal origin. Commercially available media such as RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium (DMEM; Sigma), Minimal Essential Medium (MEM; Sigma), CHO-S-SFMII (Invitrogen), serum free-CHO Medium (Sigma), and protein-free CHO Medium (Sigma) are exemplary appropriate nutrient solutions. Any of the media may be supplemented as necessary with a variety of compounds, examples of which are hormones and/or other growth factors (such as insulin, transferrin, epidermal growth factor, insulin like growth factor), salts (such as sodium chloride, calcium, magnesium, phosphate), buffers (such as HEPES), nucleosides (such as adenosine, thymidine), glutamine,. glucose or other equivalent energy sources, antibiotics, trace elements. Any other necessary supplements may also be included at appropriate concentrations that are known to those skilled in the art.

[0054] The host cell may be any suitable cell, such as a eukaryotic cell. The eukaryotic cell can, for example be an animal cell, a plant cell (for e.g. monocots, dicots, algae), a fungus, a yeast cell, flagellum, microsporidia or protist. An animal cell can be derived from a mammal such as a primate, human, murine, bovine, rodent, human, insect, reptile, or a bird, to mention only a few. Examples of an animal cell such as a (immortalized) mammalian cell can include HeLa cells, Chinese hamster ovary cells (for e.g. CHO-K1), COS cells (e.g., COS-1, COS-7), baby hamster kidney cells (BHK), human embryonic kidney (HEK) (e.g. HEK 293), Bowes melanoma cells, rat myeloma cells, mouse myeloma cells, antibody producing-hybridoma cells, human leukemia cells and the like. In some embodiments, the host cell is a neuron. In other embodiments, the host cell is a stem cell. A yeast cell can for example be S. cerevisiae, S. pombe, C. albicans, or Saccharomycetale cell.

[0055] The invention also relates to a method for the production of a polypeptide of interest, the method including

- providing a nucleic acid molecule of the invention, wherein the nucleic acid molecule comprises a nucleotide sequence encoding the polypeptide of interest that is operably linked to the CMV promoter or fragment thereof; and

- expressing the nucleotide sequence encoding the polypeptide to produce the polypeptide. [0056] The method can be carried out in vivo, the polypeptide can for example be produced in a host organism and then isolated from this host organism or its culture. It is also possible to produce the polypeptide in vitro, for example by use of an in vitro translation system.

[0057] When producing the polypeptide in vivo the nucleic acid encoding the polypeptide is introduced into a suitable host organism, for example a eukaryotic organism, preferably mammalian cells, by means of recombinant DNA technology. For this purpose, the host cell is first transformed with a cloning vector comprising a nucleic acid molecule of the invention encoding the polypeptide using established standard methods (Sambrook, J. et al. (1989), supra). The host cell is then cultured under conditions, which allow expression of the heterologous DNA and thus, the synthesis of the corresponding polypeptide. Subsequently, the polypeptide is recovered either from the cell or from the cultivation medium.

[0058] As mentioned, in one embodiment of the method the nucleotide sequence encoding the polypeptide is a heterologous sequence. In the described method, the nucleic acid molecule may be comprised in a vector, for example a plasmid.

[0059] The expression may be carried out in a suitable host cell under conditions that allow expression of the nucleotide sequence encoding the polypeptide in said host cell. The produced polypeptide may then be isolated from the host cells, for example by lysing the cells and purifying the polypeptide by suitable means, for example chromatographic means. Alternatively, the nucleic acid molecule of the invention may comprise a sequence that, when expressed with the polypeptide of interest, leads to the secretion of the produced polypeptide into the cultivating medium and thus can be isolated from the medium.

[0060] The nucleic acid molecules of the invention can be used for enhancing the expression of a nucleotide sequence encoding a polypeptide, wherein the nucleic acid molecule comprising the CMV promoter or fragment thereof is operably linked to the nucleotide sequence encoding the polypeptide. The nucleotide sequence encoding the polypeptide may be expressed in a suitable host cell.

[0061] Also contemplated is a method for maintaining or increasing the expression of a nucleotide sequence encoding a polypeptide, wherein the nucleotide sequence encoding a polypeptide is operably linked to a nucleic acid molecule comprising a mutated CMV promoter or fragment thereof according to the invention. Such a method includes methods of therapy, such as gene therapy, wherein the expression of a gene encoding for a polypeptide of therapeutic value is increased by use of the nucleic acids of the invention in an organism, such as a mammal, preferably a human being. Methods of introducing such nucleic acids into the organism to be treated and optionally for integrating the nucleic acid molecule in the organism's genomic DNA are known to those skilled in the art. The organism can be a mammal, for example a human being. In various embodiments, the nucleic acid molecule for therapeutic purposes, such as gene therapy, is administered by use of a suitable carrier, such as a viral vector. Alternatively, the method may be directed to the production of the polypeptide in a host cell.

[0062] The nucleic acids of the invention may also be used to increase transcription of nucleic acid sequences that do not encode a polypeptide, but rather code for an RNA molecule. The RNA molecule may have therapeutic benefit and may, for example, be a siRNA, microRNA or other therapeutic RNA molecule, such as an aptamer or ribozyme.

[0063] Other embodiments are within the following claims and non-limiting examples.

EXAMPLES MATERIALS & METHODS

Cell Lines and Cell Culture

hESC

[0064] hESC lines were cultured according to methods described by Chan et al (2008).

HES-3 from ES Cell International were cultured in medium conditioned by mitomycin-C inactivated immortalized mouse embryonic fibroblast (ΔΕ-MEF) feeder supplemented with 4 ng/ml of basic fibroblast growth factor (bFGF) (Invitrogen) on Matrigel (BD Bioscience) coated plates. ΔΕ-MEF conditioned medium contained Knockout-DMEM (KO-DMEM) supplemented with 15% Knockout serum replacer (KO-SR), 1% nonessential amino acids, 4ng/ml bFGF, 1 mM L-glutamine, and 1% penicillin- streptomycin (all from Invitrogen) and 0.1 mM /3-mercaptoethanol (Sigma). Medium was changed daily. Cells were kept in a 5% C02 incubator at 37°C and passaged by mechanical dissociation of colonies following collagenase IV treatment every 7 days. Chinese Hamster Ovary (CHO) cell lines

[0065] Antibody-producing CHO cell lines were cultured in suspension in a protein- free medium consisting of a 1 :1 ratio of HyQ PF-CHO (Hyclone) and CD CHO (Gibco- Invitrogen) supplemented with lg L^"1 sodium bicarbonate (Sigma), 4mM L-glutamine (Sigma), 0.05% Pluronic F-68 (Gibco) and O. lmM sodium hypoxanthine/0.016 mM thymidine (Gibco). These cells were previously transfected with two vectors, phCMV- V_HRhD-YlC-neo and phCMV-VLRhD-K_R-DHFR, encoding an IgG against RhD antigen as described in Chusainow et al (2008). CHO-S (ATCC) cells were cultured in suspension in CD CHO (Gibco-Invitrogen) supplemented with 8mM _L-glutamine (Sigma) and O. lmM sodium hypoxanthine/0.016 mM thymidine (Gibco-Invitrogen). The cells were sub- cultured every 3-4 days by diluting into fresh medium at 1.5 * 10⁵ cells per ml. Luciferase expressing clones were maintained as 1ml suspension culture in medium containing ΊΟΟμ^πύ G418 (Sigma), splitting at 1 :5 in a 24-well plate every 3-4 days.

Analyzing CMV promoter methylation by bisulfite sequencing

[0066] To analyze for CMV promoter methylation, the genomic DNA from four different IgG-producing clones (Chusainow et al (2008)) was extracted from cells harvested at exponential growth phase using Gentra Puregene Cell Kit (Qiagen). Bisulfite conversion was then carried out using EpiTect Bisulfite Kit (Qiagen) according to manufacturers' protocols. The purified bisulfite converted genomic DNA was used as template for PCR to amplify the CMV promoter sequences using Clontech Advantage 2 Polymerase Mix (Clontech). Specific amplification of CMV promoter controlling the expression of the antibody Heavy Chain were performed with either primers VH Pair #1 , #2 or #3 (VH Pair #1 : 5 -GGTGA GTAAA AATAG GAAGG TAAA-3' (SEQ ID NO:7) and 5'-ACATA AATAC CACCC ACTCC-3' (SEQ ID NO:8); VH Pair #2: 5 -TTTGG GTGAG TAAAA ATAGG AA-3' (SEQ ID NO:9) and 5'-AACTC CACCA ACTAC ACCTA AA-3' (SEQ ID NO: 10), VH Pair #3: 5 -GGGTG AGTAA AAATA GGAAG GT-3' (SEQ ID NO: l l) and 5'-AACTC CACCA ACTAC ACCTA AA-3' (SEQ ID NO: 12)). Specific amplification of CMV promoter controlling the expression of the antibody Light Chain were perform with either primers VL Pair #1 or #2 (VL Pair #1 : 5'- AAGAA TTTAG TTTGG TTGTA GTGA-3' (SEQ ID NO: 13) and 5'-ACTAA TACCA AACCA ACCAA TT-3' (SEQ ID NO: 14), VL Primers #2: 5'-GGGTG AGTAA AAATA GGAAG GT-3' (SEQ ID NO: 15) and 5 -AATAA CTCTA TCTCC TACAA ATACA AA- 3' (SEQ ID NO: 16)). These primer pairs were designed at non-CpG sites using Methyl Primer Express Software VI .0 (Applied Biosystems). The PCR products were gel purified using QIAQuick gel extraction kit (Qiagen) followed by ligation into pCR®2.1-TOPO TA cloning vectors (Invitrogen). At least 10 cloned PCR products were analysed for cytosine methylation on the CMV promoter for each cell line. Mutation and Replacement of Cytosines in CpG sites

[0067] Transcription factor sites on the CMV promoter were predicted and alternative nucleotides for methylation prone cytosines were determined based on the consensus sequences reported in literature. The methylation prone cytosines were replaced by alternative nucleotides using QuikChange Multi Site-directed Mutagenesis Kit (Strategene) according to manufacturer's protocol.

Plasmid Vector Construction

[0068] The luciferase vector (luciferase cloned into the multiple cloning site of pcDNA3.1(+); Invitrogen) served as the reporter system for examination of transgene silencing. After replacement of methylation prone cytosines, the BTI CMV promoter regions were recloned into the same luciferase expressing backbone with T4 ligase following restriction digestion with various restriction enzymes. BTI CMV promoter Region IV was recloned into Wild type CMV vector at Sacl/Aflll restriction sites. The BTI CMV promoter Region IV vector was subsequently employed as the vector backbone for insertion of BTI CMV promoter Region (I, II, III) at Bglll/Ndel restriction sites, and BTI CMV promoter Region (II, III) and BTI CMV promoter Region III at Mlul/Nde I restriction sites. BTI minimal CMV construct was cloned from BTI CMV promoter Region IV vector by PCR with primers containing Bglll or Aflll linker (5'-CAAAT GAGAT CTAGG CGTGT A-3' (SEQ ID NO: 17), 5'-CGCTG CCTTA AGTCT TCCAT- 3' (SEQ ID NO: 18)). The PCR fragment was subsequently ligated with luciferase expressing backbone of BTI CMV promoter Region IV vector at Bgl II/Afl II restriction sites to obtain the BTI minimal CMV vector.

Transfection & Selection

hESC

[0069] For each BTI CMV promoter construct, 1 μg of the vector was co-transfected with 0.02 μg pRL-RSV (Promega) as control plasmid into HESC using Lipofectamine (Invitrogen). Transfection was performed according to manufacturer's protocol with DNA and lipofectamine diluted in OptiMEM at the ratio of 1:2. For transient transfection, the cells were harvested for assays 48hrs post-transfection. For stable transfection, selection media comprising 50 μg/ml Geneticin antibiotic (Invitrogen) were used. Cells were harvested 16 days post-transfection for assays.

CHO cells [0070] Suspension CHO-S cells were transfected with BTI CMV promoter vectors using Fugene 6 Transfection Reagent (Roche) according to manufacturer's protocol. For each construct, 1 μg DNA was transfected into 3x l0⁵ cells in CD CHO culture medium, at a DNA to Fugene ratio of 1 :3. The culture medium was replaced with Geneticin containing medium 48 hrs post-transfection for selection of positively transfected cells. Thereafter, the cells were sub-cultured in selection medium for 4 weeks to achieve growth stability before single-cell cloning was performed.

Luciferase reporter assay

hESC

[0071] Luciferase assay was performed using Dual Luciferase Reporter Assay System

(Promega). Medium was removed and the transfected cells were washed once with phosphate buffered saline. 60μ1 of cell lysis buffer was added to each well and the culture was incubated at -20°C for 3-4 hours before the cells were harvested by scrapping from the culture plate surface. Cell lysis from technical replicates was combined into 1 sample prior to aliquoting into a 96-well white culture plate (Corning) at 60μ1 per well for the assay. ΙΟΟμΙ of Dual-Glo luciferase reagent was added to each well, and firefly luminescence was measured on a spectrophotomer. ΙΟΟμΙ of Stop & Glo reagent was subsequently added to each well for measurement of Renilla luminescence. Firefly luminescence was normalized to renilla luminescence to account for variation in transfection efficiency between samples.

CHO cells

[0072] Luciferase assay was performed using Bright-Glo Luciferase Assay System (Promega). Luciferase producing clones were seeded in triplicate at ΙΟΟμΙ suspension culture per well into a 96-well white culture plate (Corning). ΙΟΟμΙ of assay reagent was added to each well, and luminescence was measure on a spectrophotomer.

Stability Assay

[0073] Luciferase expressing clones were sub-cultured in medium with or without selection pressure in separate 24-well culture plates, splitting at 1 :5 every 3-4 days. Every 4 passages, Luciferase expression were assayed on a day 4 culture.

EXAMPLE 1: CMV Promoter Methylation Analysis and Design of CMV Mutants

[0074] Using bisulphate sequencing (Grunau et al. 2001), the frequency of CpG DNA methylation on the entire CMV enhancer/promoter spanning a total of 1232 nucleotides of which there are more than seventy CpG sites was examined. Antibody-producing CHO cell lines were generated by transfection with two separate vectors, the first with a CMV driving the expression of antibody heavy chain and the second with a CMV driving the expression of antibody light chain (Chusainow et al 2008) and the CMV promoters from both vectors of four different clones were analysed to identify the CpG sites that are prone to methylation.

[0075] The results shown in Figure 1 showed that for the CMV promoter driving the expression of antibody heavy chain (vector pHCMV-V_HRhD-ylC-neo), there are 45 CpG sites that are consistently methylated (Figure 1A). In the case of the CMV promoter driving the antibody light chain (vector pHCMV-V_LRhD-K_R-dhfr), there are 56 CpG sites that are consistently methylated (Figure IB). It is known that the site of chromosomal integration can alter the pattern of transgene methylation. This could potentially lead to false positive CMV CpG sites that were influenced by the site of integration. Thus, the methylation status of CMV promoters in two separate vectors was examined, since the two vectors are unlikely to be inserted into the exact same chromosome site at the same time. Furthermore, the four cell lines would probably have different insertion sites as well due to the phenomenon of random integration. CpG sites which were found to be methylated across all four cell lines and the two different promoters are shown in Figure lC. There are 25 CpG sites that are consistently methylated and clustered over 4 regions of the CMV.

[0076] Surprisingly the enhancer region upstream of the CMV promoter (Position 628 to 1033), which is a key determinant of promoter strength, was not consistently prone to methylation (Figure 1C). This enhancer has one of the strongest known RNA polymerase II promoter activities in mammalian cells and contains many transcriptional regulatory elements. These results suggested that the CpG sites responsible for gene silencing occur not on the enhancer region but outside of it instead. In general, the CpG sites prone to methylation are clustered around 4 regions of the CMV promoter/enhancer, Region I (position 38 to 167), Region II (Position 226 to 276), Region III (Position 368 to 629) and Region IV Position 1149 to 1221 ).

[0077] The CMV promoter sequence contains reiterative sequences of 17-, 18-, 19- and 21-bp repeat elements (Meier and Stinksi, 1996; Mehta et al. 2009) which are binding sites for eukaryotic transcription factors such as CREB/ATF, NFKB, SPl and YY1. The strength and activation of CMV promoter also depends strongly on the balance between the levels of these transcription regulators. Of the 25 CpG sites that are prone to methylation, 22 CpG sites are predicted to be involved in the binding of different transcription factors (Figure 2). By comparing the consensus recognition sequences of these transcription factors across various species (human, simian, murine and rat), the cytosines at CpG sites could be replaced with alternative nucleotides to remove methylation potential and thus, prevent silencing (Figure 2). The methylation potential of 22 CpG sites could be removed through nucleotide replacement. The exceptions to this are cytosines at position 64, 84 and 269 that needed to be conserved for predicted transcription factors KIV, E2F and TAX/CREB binding respectively.

[0078] Illustrated in Figure 3 are novel CMV constructs showing the position of methylation prone cytosines and their replacement with alternative nucleotides. The constructs include Wild-type CMV without any cytosine replacement (Figure 3 A; SEQ ID NO:l), BTI CMV promoter Region IV with cytosines at positions 1149, 1176, 1203, 1209 and 1221 replaced with alternative nucleotides (Figure 3B; SEQ ID NO:2), BTI CMV promoter Region III, IV with cytosines at positions 368, 400, 490, 629, 1149, 1176, 1203, 1209 and 1221 replaced with alternative nucleotides (Figure 3C; SEQ ID NO:3), BTI CMV promoter Region II, III, IV with cytosines at positions 226, 228, 238, 244, 253, 259, 276, 368, 400, 490, 629, 1149, 1176, 1203, 1209 and 1221 replaced with alternative nucleotides (Figure 3D; SEQ ID NO:4), BTI CMV promoter Region I, II, III, IV with cytosines at positions 38, 67, 82, 86, 132, 167, 226, 228, 238, 244, 253, 259, 276, 368, 400, 490, 629, 1149, 1176, 1203, 1209 and 1221 replaced with alternative nucleotides (Figure 3E; SEQ ID NO:5) and BTI minimal CMV promoter, the minimal promoter sequence capable of driving gene expression with cytosines at positions 1149, 1176, 1203, 1209 and 1221 replaced with alternative nucleotides (Figure 3F; SEQ ID NO:6).

EXAMPLE 2: Recombinant Protein Expression using modified CMV promoters

[0079] One of the key reasons for the popularity in using CMV promoter is that it is one of the strongest known promoters. Therefore, it is critical to ensure that any modification of the CMV promoter does not compromise promoter strength. For example, although a strategy to use a hybrid CMV enhance/Ubiquitin promoter enabled persistence gene expression, the strength of expression was dropped by more than 70 per cent (US Pat. No. 7,452,716). In another report where CpG sequences of CMV were modified to reduce immuno-stimulatory responses in gene therapy, it was found that CMV containing fewer CpG sites also showed a decrease in transgene expression by more than 60 per cent (WO 00/14262). [0080] To ensure that the targeted removal of CpG sites on the CMV promoter does not compromise the promoter strength, human embryonic stem cells (hESC) were transfected with modified CMV promoters (refer to Figure 3) driving the expression of reporter Luciferase transgene. On Day 2 (24 hours post-transfection), the hESC were assayed for Luciferase transgene expression. In Figure 4, compared to the wild-type full length CMV, all the full length BTI CMV constructs showed comparable high levels of luciferase transgene expression. This showed that the targeted removal of specific CpG sequences allowed for the promoter strength to be maintained at high levels.

[0081] In embryonic stem cells, it has been reported that silencing of CMV promoter occur preceding DNA methylation and can occur by mechanisms that are independent of de novo methylation (Meilinger et al 2009). This suggested that methylation prevention would not be an effective strategy to prevent CMV silencing in embryonic stem cells. In addition, Swindle et al (2004) had previously attempted mutation of CpGs in long terminal repeats of murine stem cell virus retroviral vector. Despite being able to prevent complete silencing, a major proportion of the cells still showed transgene silencing ranging from 25 to 95% (Swindle et al 2004), a term the authors refer to as variegated expression.

[0082] In contrast, it has been found that silencing of CMV promoter could be prevented through targeted removal of CpG sequences (Figure 5). In the control wild-type CMV promoter, luciferase expression was silenced by more than 70% after 16 days post- transfection. In the designed constructs, silencing of transgene was significantly suppressed. This was most obvious with BTI CMV Region III, IV promoter whereby the expression of reporter Luciferase expression did not show any significant silencing even after 16 days post-transfection. Interestingly, BTI CMV Region IV that only removed the CpG sequences at Region IV (position 1149, 1176, 1203, 1209, 1221), could not prevent transgene silencing. This suggests that removal of CpG sequences from Region IV as well as Region I, II and/or III is needed for sustained high level of transgene expression.

[0083] Figure 5 also shows that there was no further silencing of the minimal BTI CMV promoter after 16 days post-transfection. Even though the strength of the minimal promoter was 20 times lower than full length CMV, the low level expression does not decrease even after prolonged passaging in hESC. This suggested minimal BTI CMV promoter could be used in applications whereby sustained transgene expression is only required at low levels. [0084] Chinese Hamster Ovary (CHO) cells are one of the main mammalian cell lines used for recombinant protein or antibody production in industry. For a typical production process, the cells are usually cultured for extended periods of time (2-3 months). Consequently, production stability can be adversely affected by transgene silencing. Furthermore, selection pressure is usually removed during process scale-up, leading to the survival of sub-population of cells, which are non-producers, thereby further decreasing overall transgene productivity.

[0085] To examine the effectiveness of our modified CMV constructs in enabling sustained transgene expression, CHO-S cells were transfected with the CMV constructs (refer to Figure 3) driving expression of Luciferase as a reporter transgene. Each of the constructs also contains neomycin resistance gene for selection. After transfection, the cell populations were cultured for 4 weeks in the presence of neomycin to select for stable pools of cells. Subsequently single cell cloning was carried out. For each construct, at least 20 single cell clones were generated. Each of these clones was then cultured for prolonged periods of time either in the presence or absence of selection pressure. The start day of expression monitoring was defined as Passage 0. After every subsequent 4 passages, transgene expression was assayed to determine stability of expression. In terms of days in culture, Passage +4, +8, +16 are equivalent to 14 days, 28 days and 56 days respectively.

[0086] Figure 6A shows the transgene expression levels for clones transfected with wild-type CMV. At Passage 0, different clones showed variable starting expression levels of luciferase expression (ranging up to 7,000,000 luminescence_Max)- There are clones that showed very low transgene expression (clones 16, 10, 4, 46, 38, 7), intermediate transgene expression (clones 34, 31 , 5, 40, 47, 33, 19, 6, 17, 1, 35, 50, 8, 48, 13, 24) and high transgene expression (clones 27, 39, 14). The variability of expression levels in these clones indicated random integration sites and/or clonal variations.

[0087] Even in the presence of selection pressure (+G418), the majority of the clones utilizing wild-type CMV showed significant decrease of luciferase expression at Passage +4 (Figure 6A). With further extended culturing (Passage +8 and +16) the presence of selection pressure (+G418) allowed for the luciferase expression levels to stabilized albeit at lower levels (3,600,000 luminescence_Ma_x)- In the absence of selection pressure (-G418), accelerated silencing of transgene expression occurred at Passage +8 and +16 as indicated by significant decrease in luciferase expression (~2,000,000 luminescence_Ma_x). These results demonstrated the susceptibility of wild-type CMV promoter to silencing both in the presence and absence of selection pressure.

[0088] BTI CMV promoter Region I, II, III, IV has 22 CpG sequences removed (refer to Figure 3E). With BTI CMV promoter Region I, II, III, IV, compared to Passage +0, the transgene expression at Passage +4, +8 and +16 showed significantly lessened drop in luciferase expression (Figure 7). In the absence of selection pressure (-G418), some of the clones do showed decreases in transgene expression at Passage +8 and +16 (ranging up to 3,000,000 luminescenceMax)-

[0089] BTI CMV promoter Region II, III, IV has 16 CpG sequences removed (refer to Figure 3D). With BTI CMV promoter Region II, III, IV, compared to Passage +0, the transgene expression at Passage +4, +8 and +16 showed minimal decrease in luciferase expression (Figure 8). The sustained expression of transgene with this construct can be observed across all clones both in the presence and absence of selection. These results clearly demonstrate the effectiveness of the modified promoter in preventing transgene silencing for prolonged periods of culturing.

[0090] BTI CMV promoter Region III, IV has 9 CpG sequences removed (refer to Figure 3C). With BTI CMV promoter Region III, IV (Figure 9), compared to Passage +0, the transgene expression at Passage +4, +8 and +16 also showed minimal decrease in luciferase expression (Figure 9). The sustained expression of transgene with this construct can be observed across all clones both in the presence and absence of selection. These results clearly demonstrate the effectiveness of the modified promoter in preventing transgene silencing for prolonged periods of culturing.

[0091] With BTI CMV promoter Region IV (Figure 10), with selection pressure (+G418), the transgene expression at Passage +4, +8 and +16 were very comparable to that from Passage +0. This indicated that the construct allowed for sustainable levels of transgene expression even after prolonged culturing. However, in the absence of selection pressure (-G418), significant decrease in transgene expression could be observed at Passage +14. This suggested that removal of CpG sequences located at Region IV allowed for the silencing of transgene to be delayed until Passage +16.

[0092] The results from Figure 7 to 10 demonstrate the effectiveness of the modified

CMV promoters in delaying/preventing the silencing of transgene expression both in the presence or absence of selection pressure. The strategy was effective for sustaining the transgene expression in both intermediate and high transgene expressing clones. Furthermore, the results suggested that CpG sites on Region II, III and IV are important determinants of sustained transgene expression (Positions 226, 228, 238, 244, 253, 259, 276, 368, 400, 490, 629, 1149, 1176, 1203, 1209 and 1221).

[0093] 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. 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 preferred 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.

[0094] 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.

[0095] 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.

[0096] The content of all documents cited herein is hereby incorporated by reference in its entirety including any tables, drawings and figures.

REFERENCES

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Brooks AR, Harkins RN, Wang P, Qian HS, Liu P, Rubanyu GM. 2004. Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle. J Gen Med. 6: 395-404

Chan KKK, Wu Sm, Nissom PM, Oh SKW, Choo ABH. 2008. Generation of high-level stable transgene expressing human embryonic stem cell lines using Chinese Hamster Elongation Factor- la promoter system. Stem Cells Dev. 17: 825-836

Choi KH, BAsma H, Sing J, Cheng PW. 2005. Activation of CMV promoter-controlled glycosyltransferase and β-galactosidase glycogenes by butyrate, thrichostatin A and 5'- Aza-2'-deoxycytidine. Glyco conjugate J. 22: 63-69

Chusainow J, Yang YS, Yeo JHM, Toh PC, Asvadi P, Wong NSC, Yap MGS. 2008. A study of monoclonal antibody-producing CHO cell lines: What makes a stable high producer? Biotechnol Bioeng. 102: 1182-1196

Dang Q, Auten J, Plavec I. 2000. Human beta interferon scaffold attachment region inhibits de novo methlation and confers long-term, copy number-dependent expression to a retroviral vector. J Virol. 74: 2671-2678

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Liew CG, Draper JS, Walsh J, Moore H, Andrews PW. 2007. Transient and stable transgene expression in human embryonic stem cells. Stem cells. 25: 1521-1528 Mellor J, Dudek P, Clynes D. 2008. A glimpse into the epigenetic landscape of gene regulation. Curr Opinion Gene Dev. 18: 116-122

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Claims

What is claimed is: Claims

1. A nucleic acid molecule comprising a cytomegalovirus major immediate-early enhancer/promoter (CMV promoter) or fragment thereof, wherein the CMV promoter or fragment thereof comprises one or more mutations in at least one of the sequence regions 1-4, wherein sequence region 1 comprises nucleotide positions 38 to 167, sequence region 2 comprises nucleotide positions 226 to 276, sequence region 3 comprises nucleotide positions 368 to 629, and sequence region 4 comprises nucleotide positions 1149 to 1221, wherein the one or more mutations are at any one of the sequence positions selected from the group consisting of:

(i) Region 1 positions: 38, 67, 82, 86, 132,

0) Region 2 positions: 167, 226, 228, 238, 244, 253, 259, 276,

(k) Region 3 positions: 368, 400, 490, 629,

(1) Region 4 positions: 1149, 1 176, 1203, 1209, 1221,

wherein said sequence positions correspond to those of the linear wildtype sequence of the CMV promoter as set forth in SEQ ID NO: 1.

2. The nucleic acid molecule of claim 1, wherein the CMV promoter or fragment thereof comprises mutations at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or all 22 of the sequence positions.

3. The nucleic acid molecule of claim 1 or 2, wherein the CMV promoter or fragment thereof comprises one or more mutations at positions 1149, 1176, 1203, 1209, and 1221.

4. The nucleic acid molecule of claim 3, wherein the CMV promoter or fragment thereof comprises mutations at positions 1149, 1176, 1203, 1209 and 1221.

5. The nucleic acid molecule of any one of claims 1-4, wherein CMV promoter or fragment thereof comprises one or more mutations at positions 368, 400, 490, and 629.

6. The nucleic acid molecule of claim 5, wherein the CMV promoter or fragment thereof comprises mutations at positions 368, 400, 490 and 629.

7. The nucleic acid molecule of any one of claims 1-6, wherein the CMV promoter or fragment thereof comprises one or more mutations at positions 167, 226, 228, 238, 244, 253, 259, and 276.

8. The nucleic acid molecule of claim 7, wherein the CMV promoter or fragment thereof comprises mutations at positions 167, 226, 228, 238, 244, 253, 259 and 276.

9. The nucleic acid molecule of any one of claims 1-8, wherein the CMV promoter or fragment thereof comprises one or more mutations at positions 38, 67, 82, 86, and 132.

10. The nucleic acid molecule of claim 9, wherein the CMV promoter or fragment thereof comprises mutations at positions 38, 67, 82, 86 and 132.

1 1. The nucleic acid molecule of any one of claims 1-10, wherein the one or more mutations are nucleotide substitutions.

12. The nucleic acid molecule of claim 11, wherein the one or more mutations are nucleotide substitutions selected from the group consisting of C38T, C67A, C82G, C86G, C132T, C167A, C167T, C167G, C226A, C228T, C238A, C244A, C253T, C259T, C276A, C276T, C276G, C368T, C400T, C490A, C629T, C1149T, C1176G, C1203A, C1203T, C1203G, C1209A, C1209T, C1209G, and C1221A.

13. The nucleic acid molecule of any one of claims 1-12, wherein the fragment of the CMV promoter comprises or consists of a nucleotide sequence corresponding to positions 1 101 to 1232 of the linear wildtype sequence of the CMV promoter as set forth in SEQ ID NO:! .

14. The nucleic acid molecule of any one of claims 1-13, wherein the nucleic acid molecule comprises the CMV promoter sequence set forth in any one of SEQ ID Nos. 2-6.

15. The nucleic acid molecule of any one of claims 1-14, wherein the nucleic acid molecule further comprises a nucleotide sequence encoding a peptide or polypeptide of interest, wherein the CMV promoter or fragment thereof and the peptide- or polypeptide-encoding sequence are operably linked.

16. The nucleic acid molecule of claim 15, wherein the nucleotide sequence encoding a peptide or polypeptide of interest is a heterologous nucleotide sequence.

17. The nucleic acid molecule of any one of claims 1-16, wherein the nucleic acid molecule is a DNA molecule.

18. The nucleic acid molecule of any one of claims 1-17, wherein the nucleic acid molecule is an isolated nucleic acid molecule.

19. The nucleic acid molecule of any one of claims 1-18, wherein the cytomegalovirus major immediate-early enhancer/promoter (CMV promoter) or fragment thereof is the human cytomegalovirus major immediate-early enhancer/promoter (CMV promoter) or fragment thereof.

20. Vector comprising the nucleic acid molecule of any one of claims 1-19.

21. The vector of claim 20, wherein the vector is a prokaryotic or eukaryotic vector.

22. The vector of claim 20 or 21 , wherein the vector is a plasmid.

23. Host cell comprising the vector of any one of claims 20-22.

24. Method for the production of a polypeptide, comprising providing a nucleic acid molecule of any one of claims 1-19, wherein the nucleic acid molecule comprises a nucleotide sequence encoding the polypeptide that is operably linked to the CMV promoter or fragment thereof;

expressing the nucleotide sequence encoding the polypeptide to produce the polypeptide.

25. The method of claim 24, wherein the nucleotide sequence encoding the polypeptide is a heterologous sequence.

26. The method of claim 24 or 25, wherein the nucleic acid molecule is comprised in a vector.

27. The method of any one of claims 24-26, wherein expression is carried out in a suitable host cell under conditions that allow expression of the nucleotide sequence encoding the polypeptide in said host cell.

28. The method of claim 27, wherein the method further comprises the step of isolating the produced polypeptide.

29. Use of the nucleic acid molecule of any one of claims 1-19 for enhancing the expression of a nucleotide sequence encoding a peptide or polypeptide, wherein the nucleic acid molecule comprising the CMV promoter or fragment thereof is operably linked to the nucleotide sequence encoding the peptide or polypeptide.

30. The use of claim 29, wherein the nucleotide sequence encoding the peptide or polypeptide is expressed in a suitable host cell.

31. Method for maintaining or increasing the expression of a nucleotide sequence encoding a peptide or polypeptide, wherein the nucleotide sequence encoding a peptide or polypeptide is operably linked to a nucleic acid molecule according to any one of claims 1-19.