HK1100089B - Method for modulating gene expression by modifying the cpg content - Google Patents

Description

The present invention relates to modified polynucleotides derived from naturally occurring and synthetic genes or other coding sequences, which have a reduced or increased number of CpG dinucleotides in the coding region compared to the original sequence. These polynucleotides can be used to investigate, increase or decrease gene expression and, in particular, to improve the production of biomolecules, the efficiency of quality vaccines or gene therapies, and the quality of DNA from transgenic animals or plants.

Background to the invention

The provision of biomolecules in the form of peptides, proteins or RNA molecules is an important component in the biotechnology and pharmaceutical sectors.Recombinantly produced or in vivo expressed proteins and RNAs are used both for research into basic mechanisms and relationships and for the production of biotechnological reagents, the production of transgenic animals or plants or for medical applications in therapy and vaccine development.Depending on the application, the expression level of the corresponding molecules should be regulated.

In most cases, the aim is to increase the expression beyond the standard output. Each expression system or vector construct has limitations that determine the actual production output. The present invention relates to methods and applications that are capable of modulating the expression level of any gene in eukaryotic cells. In particular, the method is capable of modulating any gene so that the attainable gene expression is above the level achievable by previously known methods of increasing expression.

State of the art

CpG dinucleotides occupy a special position in the genome of eukaryotes. They are not statistically distributed like other dinucleotides, but are underrepresented in direct comparison over large areas of the genome.

An exception to this is areas with a much higher density of CpG dinucleotides, which are called CpG islands because of these properties.

The underrepresentation of CpG dinucleotides is explained by a chemical modification of the corresponding nucleotides. In the genome of vertebrates, about 60-90% of the cytosine is methylated into CpG dinucleotides and these methylated cytosines are often modified into thymines by deamination (Shen et al., 1994). This process results in the frequency of cytosines and guanosines being about 40% below the expected statistical distribution and the proportion of CpG dinucleotides being only about 20% of the expected frequency (Bird, 1980; Sved et al., 1990; Takai et al., 2002).

CpG islands are an exception to this unusual distribution of CpG dinucleotides (Antequera et al., 1993). CpG islands are usually located near promoters, may extend into the transcribed region or even lie within exons.

They are characterised by an approximately tenfold higher CpG frequency (about 60-70% C+G content) compared to average gene regions and, in particular, by the fact that they usually contain non-methylated CpGs (Wise et al., 1999). About 60% of all human genes, especially all household genes and about half of tissue-specific genes, are associated with CpG islands (Antequera et al., 1993; Larsen et al., 1992). CpG islands have been described and defined, among others, in the publications of Gardiner-Garden M. & Frommer M. (1997) J. Mol. Biol. 196, 261-282 and Takai D. & Jones P. A. (2002) PNAS 37,40-3745 99.

This uneven distribution and modification of CpG dinucleotides, i) underrepresented and methylated on the one hand and ii) concentrated and unmethylated in islands on the other, has an important control function in the regulation of gene expression (schematically shown in Figure I).

CpG dinucleotides are involved in the regulation of gene expression in early development, in connection with cell differentiation, genetic imprinting and other processes.In eukaryotes, a large number of studies have shown that the methylation of 5'CpG3' dinucleotides (mCpG) has a repressive effect on gene expression in vertebrates and flowering plants (Hsieh, 1994; Kudo, 1998; Jones et al., 1998; Deng et al., 2001; Hisano et al., 2003; Li et al., 2004) (Fig. IA).

There is also a large body of data in tumour research that shows that (i) the shutdown of expression of certain genes, often suppressors, is caused by hypermethylation of CpGs (Li et al., 2004; Kang et al., 2004; Ivanova et al., 2004; Wu et al., 2003) and that (ii) the uncontrolled expression of other genes is associated with hypothylaxis (Akiyama et al., 2003; Yoshida et al., 2003).

The process of gene shutdown by methylation is explained by a cascade of events that ultimately leads to a change in chromatin structure that creates a transcription-weak state. The methylation of 5'-CpG-3' dinucleotides within genes generates a potential binding site for protein complexes (primarily from the family of MeCP (methyl-CpG-binding proteins) and MBD (methyl-CpG binding protein domain) proteins) which bind methylated DNA sequences and simultaneously inhibit the repressure of histone deaclases (MBD-HDAC) and transcriptional essential proteins (Jone et al., 1998; Nan et al., 1998; Hendrich et al., 1999). This gene usually binds a complex of methylated proteins, which can also induce a methylation of the transcriptional activity of the methylated genes by blocking the activation of a methylated transcriptional group (Deng et al., 2001).

The above-described deregulation of expression in tumor cells is generally associated with a change in the methylation state in the CpG islets described above. In normal cells, actively expressed genes are usually associated with CpG islets that are not or only slightly methylated (Fig. IB). Methylation of CpG dinucleotides in these islets leads to a shutdown of expression of these genes (often tumor suppressor or cell cycle regulator genes) (Fig. IC) and consequently to uncontrolled proliferation of these cells.

The demethylation described in CpG islands results in a transcriptionally active state by changing the chromatin structure, similar to gene shutdown in the case of methylation. In addition to structural changes, activation of expression by activator proteins may occur. One such cellular activator protein is the human CpG binding protein (hCGBP). HCGBP specifically binds to non-methylated CpG dinucleotides in the promoter region, where it acts as a transactivator to increase transcription (Voo et al., 2000).

The knowledge that methylation of CpG sequences within a gene downregulates transcription has been used to prevent the expression of a gene that is either overexpressed or unwanted by methylation (Choi et al., 2004; Yao et al., 2003) (see Figure IA).

A further application of these findings is the targeted elimination of such CpG dinucleotides to enhance gene expression (Chevalier-Mariette et al., 2003).Elimination also prevents methylation and a consequent change in chromatin structure to a transcriptionally inactive state (Fig. ID).This publication examines the expression of a transgene with different CpG dinucleotide contents in operative linkage with a promoter located within a CpG islet in germ cells and the resulting embryos of transgenic males.In this particular case, elimination of CpG dinucleotides prevents transcriptional shutdown of a reporter (A.C.D.A. without transcription) while de novo it is removed from the embryo by another methylated DNA.For a reporter gene that did not have and was efficiently expressed, it was shown that the CpG gene was not methylated (Fig. ID, Transgen without CpG). The authors concluded that for continued in vivo expression of the CpG gene in the immediate vicinity of the promoter gene, the CpG gene must be reduced and the CpG gene must be removed.

An increase in gene expression can also be achieved by integrating complete CpG 5' islets of a promoter into corresponding vector constructs (WO 02081677) (see Figure IB).In identifying hCGBP, CpG dinucleotides were also integrated into the corresponding promoter region of a reporter gene and increased reporter activity was observed.However, in these transient cell culture studies, hCGBP was also overexpressed and therefore present at non-physiologically elevated concentrations (Voo, et al., 2000).

It is already known that the C/G content has an effect on mRNA stability. For example, Duan and Antezana (2003) show that the expression of three different variants of a human gene in CHO cells results in differences in mRNA concentration. In the first variant, the human gene sequence was altered to maximize the number of C/G dinucleotides. In one variant, the second number of TIA dinucleotides was instead maximized. The differences in Stevel-State Leady state, i.e. in the amount of mRNA, could be experimentally attributed to differences in the structure of the mRNA.

The results of the study showed that the HIV-I Gag gene was codon-optimized for expression in mammalian cells.

EP 156 112 describes codon-optimized nucleic acid sequences that code for gagpol.

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The purpose of the invention was to develop a method for the specific modulation of gene expression that would at least partially eliminate the disadvantages of the state of the art.

This task is solved by a method of targeted modulation of gene expression, which includes the following steps: i. Provide a target nucleic acid sequence to be expressed,ii. Modify the target nucleic acid sequence by increasing the number of CpG dinucleotides present in the target nucleic acid sequence using the degeneration of the genetic code to increase gene expression, by increasing the number of CpG dinucleotides compared to a codon-optimized wild-type sequence derived sequence using the degeneration of the genetic code, or by decreasing the number of CpG dinucleotides present to reduce gene expression,iii. Multiply the modified target nucleic acid sequence with a modified number of CpG dinucleotides into an appropriate number of appropriate expression in an operational vector with a suitable transcription control system for the expression of target nucleic acids in a target nucleic acid.

It was quite surprising that the method of the present invention could be used to achieve the exact opposite effect to that which would be expected in the present state of the art, i.e. that the method of the present invention can increase the expression of the target nucleic acid sequence by increasing the number of CpG dinucleotides in a target nucleic acid sequence, while decreasing the number of CpG dinucleotides in the target nucleic acid sequence prevents their expression.

The expression system may be a cell on the one hand, or a cellless system or an in vitro system on the other. A mammalian expression system is used. Preferably, human cells, especially somatic cells, are used and not germ cells. Particularly preferably, the expression system or cell is a system or a cell that is poor in methylation, i.e. that essentially no de novo methylation takes place. On the other hand, it is also possible to use this process - for the production of transgenic non-human organisms, especially from plants and animals.

The present invention thus relates in particular to a method for the purpose of altering the expression level of a transcript and/or for the purpose of altering protein production in mammalian cells, characterized by modifications of the reading frame of a DNA sequence to be transcribed.

The modifications concern a variation in the proportion of CpG dinucleotides correlated with a change in the expression level.

The technology of artificial gene synthesis enables the synthesis of any nucleotide sequence selected from these possibilities. By varying motifs within the coding region of a gene that correlate with the expression rate, this technology can be used to modulate protein production specifically by selecting the appropriate nucleic acid sequence.

Surprisingly, contrary to popular belief, the introduction of CpG dinucleotides in the manner of the invention has been shown to increase gene expression instead of reducing expression, and vice versa.

The term gene expression for the purposes of the present invention includes both transcription and translation, and in particular, this term includes protein production.

These changes at the nucleic acid level are introduced in the present invention preferably by the production of an artificial gene by de novo genosynthesis, whereby the amino acid sequence for which the corresponding gene is encoded preferably remains unchanged. De novo genosynthesis methods are known to the expert in the field. The change in CpG content is preferably by silent mutations or by mutations that do not destroy the activity of the gene product. The modified target nucleic acid sequences can, as shown in the example, e.g. be produced by a step-by-step PCR or ordered from common gene synthesis providers (e.g. Gene Genetics GmbH, Qiagen AG).

Surprisingly, the choice of the number of CpG dinucleotides can affect the expression of the corresponding gene negatively (lower number of CpG) or positively (increased number of CpG) and even exceed the expression rates that can be achieved with a codon-optimized gene. Expression can unexpectedly increase even if the increase in the number of CpG dinucleotides is at the expense of RNA and codon-optimization. Preferably, no CpG is added when the target nucleic acid sequence is modified, and preferably the modified target nucleic acid sequence is not associated with CpG islets.

For gene expression, these modifications are preferably introduced in such a way that the encoded amino acid sequence is not changed. Ideally, only the nucleic acid sequence of a corresponding gene should affect its expression level. Since the genetic code is degenerated, it is possible to choose a large number of corresponding nucleic acid sequences for a given amino acid sequence.

In contrast to the methods described above, the aim is to 1) modify the region encoding the transcript, so that this method can be used regardless of vectors and other genetic conditions, and 2) increase the number of CpG dinucleotides to increase expression.

Preferably, the number of CpG dinucleotides compared to the sequence of the target nucleic acid to be expressed shall be increased or decreased by at least 2, preferably at least 3, preferably at least 5, preferably at least 8, preferably at least 10, preferably at least 15, and up to 20 or more, in particular by 30-50 or even up to 100 or more, depending on the length of the target nucleic acid sequence to be expressed, depending on the desired expression rate.

Preferably, the number of CpG dinucleotides is increased by at least 10%, preferably 20%, preferably 50%, preferably 100%, preferably 200%, or five-fold or tenfold or more, compared to the sequence of the target nucleic acid to be expressed.

When CpGs are eliminated, it is preferable to eliminate all CpGs that can be eliminated within the genetic code, but it is also possible to eliminate fewer CpGs, e.g. 10%, 50% or 75%, again depending on the desired level of expression.

Surprisingly, the present invention shows that increasing or decreasing the number of CpG dinucleotides allows for a gradual modulation of gene expression. Surprisingly, a dose effect was observed, i.e. the degree of gene expression can be adjusted by adding or removing more or fewer CpG dinucleotides.

As mentioned above, it is possible and preferable to exploit the degeneration of the genetic code in such a way that preferably the maximum number of CpG dinucleotides is added or eliminated without changing the amino acid sequence of the target amino acid sequence to be expressed.

On the other hand, if necessary, the number of CpG dinucleotides can be increased even further, even if this changes the corresponding amino acid sequence, in which case care must be taken not to affect the function of the peptide or protein.

The CpG dinucleotides can be removed or added within a codon or across codons, depending on the type of degeneration of the genetic code.

In addition to the change in the number of CpG dinucleotides in the target nucleic acid to be expressed, the latter can be further altered depending on the desired degree of gene expression at the nucleic acid level. e.g. if an increase in gene expression is desired, the number of CpG dinucleotides is preferably increased in such a way that no adverse effects are produced by the addition of additional CpG dinucleotides. e.g. more pronounced secondary structures of mRNA that could directly affect RNA translation, other factors that negatively affect expression, e.g. B-stability factors, number of splice-forming, gene-specific endogenotic factors.

Of course, it is also possible and preferable to do nucleic acid optimization to either promote or inhibit or reduce gene expression in addition to increasing or decreasing the number of CpG dinucleotides.

Such optimizations are the insertion or removal of motifs that can affect gene expression, e.g. secondary structure-stabilizing sequence sequences, regions with increased self-homology, regions with increased homology to the natural gene, RNA instability motifs, splice-activating motifs, polyadenylation motifs, adenine-rich sequence segments, endonuclease detection sites, and the like.

The expression can be increased or decreased by optimizing or decreasing the codon selection in addition to the insertion of CpG dinucleotides. For example, expression-optimized constructs according to the invention can be generated by selecting the codon distribution as it occurs in the expression system used. The mammalian expression system is preferably a human system. Preferably, therefore, codon optimization is adapted to the human gene condon selection.

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Any suitable expression vector can be used as an expression vector. Such a vector is preferably suitable for expression in mammalian cells. The modified target nucleic acid to be expressed is cloned into the vector so that it is in operational linkage with an appropriate transcription control sequence and, if necessary, additional regulatory elements. Such a transcription control sequence can be a suitable promoter, which can be either constitutive or inducible.

Constitutively active promoters are preferably selected from, but not limited to, the CMV (cytomegalovirus) promoter and Simian Virus 40 (SV40). Inductible promoters include but are not limited to tetracycline-dependent promoters. The specialist in the field is readily able to select additional suitable promoters depending on the application, e.g. also promoters of cellular origin.

In principle, any inducible promoter system known to the state of the art is suitable, for example a natural or artificial inducible promoter, such as a tetracycline inducible promoter (Tet on / Tet off system), may be used, but an inducible viral promoter may also be used.

Preferably, the inducible promoter is inducible by a transactive factor. A viral inducible promoter inducible by a viral transactive factor may be derived from any virus. Preferably, sequences of retroviruses, HCV (hepatitis C virus), HBV (hepatitis B virus), HSV (herpes simplex virus), EBV (Epstein-Barr virus), SV 40 (simian virus 40), AAV (adenossociated virus ART), Adenovirus, papillomaviruses or Ebola virus are used. The transactive factors used herein are, but are not limited to, the following viruses according to the EAPB: HBV5 (HCV), HBV (HBV), XP/A16A, HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV (HBV), HBV

The inducible promoter inducible by a viral transactive factor is preferably a retroviral LTR promoter or a functional subsequence thereof. Preferably, therefore, the transactive factor is a retroviral act or tax protein. The LTR promoter can be selected from the LTRs of HIV-1, HIV-2, SIV, HTLV and other related retroviruses that have LTR promoters. In particular, lentiviral promoters, especially those of HIV, are preferred.

Preferably, the transcription control sequences used in the present invention, i.e. promoters and/or enhancers, etc., are not associated with CpG islets.

It is also possible to reduce the number of CpG dinucleotides in the remaining sequences or parts of the vector, in addition to increasing the number of CpG dinucleotides in the target nucleic acid to be expressed, eliminating the CpG dinucleotides entirely in these remaining vector sequences or parts of the vector, preferably by maintaining the amino acid sequence using genetic code degeneration, or only partially eliminating the CpG dinucleotides in these sequences, e.g. by at least 5%, preferably at least 10%, preferably at least 15%, preferably at least 25%, preferably at least 50%, preferably at least 75%, or preferably at least as far as possible, to eliminate all CpG dinucleotides.

Thus, depending on the application (silencing or expression enhancement), the number of CpG dinucleotides can be varied independently of the chosen codon optimisation.

In most cases, complete elimination of CpGs from the reading frame is possible.

The target nucleic acid sequence may code for an RNA, derivatives or mimetics thereof, a peptide or polypeptide, a modified peptide or polypeptide, a protein or a modified protein.

The target nucleic acid sequence may also be a chimeric and/or composite sequence of different wild-type sequences, for example, it may code for a fusion protein or mosaic-like polygenic constructs. The target nucleic acid sequence may also code for a synthetic sequence. It is also possible to design the nucleic acid sequence synthetically, for example using a computer model.

The peptide/protein may be used for e.g. (i) the manufacture of therapeutic products such as human enzymes (e.g. asparaginase, adenine deaminase, parasulin, tPA, clotting factors, vitamin K-polypeptide reductase), hormones (e.g. erythrin, follicular follicle-timing hormone, diethylamine, and other hormones of human origin (e.g. T-valine, T-valine, T-valine, and T-valine), which can be used as antibodies to HIV, influenza, hepatitis, hepatitis, hepatitis, hepatitis C, hepatitis C, hepatitis C, hepatitis C, hepatitis C, hepatitis C, hepatitis C, hepatitis C, hepatitis C, hepatitis C, hepatitis C, hepatitis C, hepatitis C, hepatitis C, hepatitis C, hepatitis C, hepatitis C, hepatitis C, hepatitis C, and other infectious diseases, and may be used as a test for the production of human proteins (e.g. human immunoglobulin, human follicular hormone, human immunoglobulin, and other hormones) or proteins (e.g.g. human immunoglobulin, human immunoglobulin, human immunoglobulin, human immunoglobulin, human immunoglobulin, human immunoglobulin, etc.) which can be used as antibodies against influenza, hepatitis C, hepatitis C, hepatitis C, hepatitis C, hepatitis C, hepatitis C, or hepatitis C, etc.) or hepatitis C, etc.

Alternatively, a gene may be selected that produces messengers (cytokines/chemokines) such as G-CSF, GM-CSF, interleukins, interferons, PDGF, TNF, RANTES or MIP1α or domains, fragments or variants thereof that are capable of inducing the natural defense mechanisms of neighboring cells or enhancing a specific immune response in combination with appropriate antigens.

Another possible application is the production of proteins, such as enzymes (polymerases, proteases, etc.) for biotechnological applications.

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Finally, the target nucleic acid to be expressed may be a functional RNA (e.g. ribozyme, decoy or siRNA) which may preferably be used for therapeutic or enzymatic purposes.

Furthermore, the present invention relates to a modified nucleic acid with a transcriptional region derived from a wild type sequence, where the transcriptional region is modified to increase the number of CpG dinucleotides compared to the wild type sequence using the degeneration of the genetic code. The modified nucleic acid can be expressed in an expression system as described above, and the transcriptional region is modified to be codonoptimized with respect to the mammalian expression system used, and the number of CpG dinonopreotides is increased compared to the condensed, wild type sequence derived from the genetic code using the degeneration of the sequence.

A wild-type sequence within the meaning of this invention is a naturally occurring nucleic acid sequence.

However, as noted above, it is also possible that the target nucleic acid sequence encodes a composite gene sequence that may be composed of different wild-type sequences, in which case wild-type sequence refers to the sequence that has not yet been modified in the sense of the present invention (increase or decrease in the number of CpG dinucleotides).

The number of CpG dinucleotides in the nucleic acid of the invention may be increased by several CpG dinucleotides, as mentioned above, preferably to the maximum number possible in the context of the degeneration of the genetic code.

The vector is preferably derived from known vectors. In the sequence ranges of the vector that differ from the modified nucleic acid sequence, the number of CpG dinucleotides is preferably reduced. Preferably, the number of CpG dinucleotides in these vectors is increased by at least 5%, preferably at least 10%, preferably at least 15%, preferably at least 75%, preferably at least 50%.

The reduction of CpGs is preferably achieved by artificial gene synthesis of the individual vector modules (antibiotic resistance genes, selection markers, multiple cloning site, etc.) as described above. The individual modules are assembled into a functional vector using singular restriction sites with corresponding DNA fragments of essential, non-modifiable modules (replication origin, polyadenylation site, viral promoter, etc.) which may be viral (e.g. derived from adenoviruses, retroviruses, herpesviruses, alphaviruses, etc.) or bacterial or naked DNA (expression drugs).

The modular structure of the vector also allows for a quick and easy change in the individual modules, the number of modules being variable and adaptable to the application.

For stable integration into cells, elements such as eukaryotic selection markers (e.g. resistance genes to hygromycin, zeocin, etc.; selection reporters, such as GFP, LNGFR, etc.; or recombination sequences for directed recombination) may be used, whereby the corresponding gene sequences may also be reduced in CpG content where possible. For applications in gene therapy, sequences that counteract immunostimulatory motifs (e.g. immunosuppressive CG motifs) may be introduced. Accordingly, for applications in immunizations, such as in vaccinations or in the manufacture of immuno-stimulating antibodies, sequences containing immunostimulating factors (e.g. CG motifs) may be integrated.

A vector of preference for this invention is the vector shown in SEQ ID No 27.

Another subject matter of the present invention is eukaryotic cells, preferably mammalian cells, most preferably human cells, which contain a target nucleic acid or vector (preferably in the form of a DNA construct) as described above, where the nucleic acid or vector is in a transcriptional form.

The DNA construct may be episomal or stable in the chromosome, for example, and may have one or more copies in the cell. Gene carriers of viral (e.g. adenoviruses, retroviruses, herpesviruses, alphaviruses, etc.) or bacterial origin or naked DNA (expression plasmids) may be used to insert the said DNA constructs.

A further subject of the present invention is a mammalian expression system, comprising a (a) a modified nucleic acid sequence with a transcription region derived from a wild-type sequence, where the modified nucleic acid sequence is modified to be codon-optimized with respect to the mammalian expression system used and has an increased or decreased number of CpG dinucleotides compared to the codon-optimized sequence, in operational linkage with a transcription control sequence, and (b) an expression environment selected from a mammalian cell and a cell-free expression environment, where (a) the modified nucleic acid expression system shows an increased number of DNA with a reduced number of modified DNA with an increased number of DNA with a modified expression.

The present invention can therefore be used to increase or decrease the expression of a target nucleic acid sequence. If the expression is increased, an increase in expression of at least 5%, preferably at least 10%, preferably at least 20%, more preferably at least 30%, more preferably at least 50%, more preferably at least 100-400% or more should be preferred. Depending on the length of the target nucleic acid sequence to be expressed and the number of CpG dinucleotides that can be inserted, an increase in expression of two, three, five or even ten to twenty or even one hundred to two hundred times can also be achieved.

If a reduction in expression is desired, it is preferable to reduce the expression, i.e. to reduce the amount of transcript by at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 50%, preferably at least 75%, and preferably to approach the detection limit.

As described in detail above, the level of transcription depends on the number of CpG dinucleotides in the gene. This means that a higher increase in expression can be achieved in longer genes or in genes with more possibilities for inserting CpG dinucleotides. Conversely, the present invention should allow the targeted elimination of all CpG dinucleotides to significantly reduce expression, up to the limit of detection, depending on the application.

The present invention also relates to medicinal products and diagnostic devices based on the modified nucleic acids and/or vectors of the invention, which can be used in diagnostic, therapeutic and/or gene therapy applications, in particular for the manufacture of vaccines.

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Description of the drawings Figure 1: The number of Regulation of gene expression by methylation (state of the art).

A: Methylation of CpG dinucleotides leads to shutdown of gene expression. B: CpG protects islands from methylation and the associated shutdown. C: Secondary hypothylation of the CpG islands leads to gene shutdown. D: Secondary hypomethylation can be prevented by reducing CpG dinucleotides in the reading frame.

Figure 2: The number of GFP expression analysis in cells that are stable in transfection.

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Figure 3: The number of

GFP protein detection in stable transfected cells. Expression analysis of GFP reading frames. Recombinant flp-in CHO cells that have integrated the huGFP or ΔCpG-GFP gene into the cell genome have been lysified and gene expression has been demonstrated by conventional immunoblot analysis. Order of huGFP, ΔCpG-GFP and mock samples are given. Both monoclonal cell lines have been established from poly (poly) cell cultures (mono. 14 and 7 for ΔCpG-GFP and mono. 10 and 9 for huGFP).

Figure 4: The number of

Quantitative determination of specific transcripts of stable cells. Real-time PCR analysis of specific hygromycin resistance genes and gfp RNAs from cytoplasmic RNA preparations. Real-time PCR evaluation of LC analyses are shown for CHO cells (hygromycin resistance A and gfp B) and for 293T cells (hygromycin resistance C and gfp D). The number of PCR cycles (X-axis) and fluorescence intensity (Y-axis) are shown. The specific kinetics are shown for huGFP and ΔCpG-GFP products and for the primer.

Figure 5: The number of

MIP1alpha expression analysis after transient transfection. Representative ELISA analysis of cell lysate and residues of transfected H1299 cells. H1299 cells were transfected with 15 μg of wild-type and optimized murine MIP1alpha constructs each. The respective protein concentration was quantified by conventional ELISA tests in cell culture residue and cell lysate using corresponding standard curves. The bars represent the mean total protein concentration for each of 2 independent approaches, the error bars corresponding to the standard deviation. The X axis shows the number of CPGp nucleotides in the open reading frame and the Y axis shows the total protein concentration in pg/ml. The axes correspond to the wild-type expression of each structure.

Figure 6: The number of

MIP1alpha and GM-CSF expression analysis after transient transfection. Representative ELISA analysis of transfected H1299 cell residues. H1299 cells were transfected with 15 μg of wild-type and optimized human MIP1alpha (A) and GM-CSF (B) constructs each. The respective protein concentration in the cell culture 48 h post-transfection was quantified by conventional ELISA tests of corresponding standard curves. The bars represent the mean value for 2 independent approaches each, the error bars corresponding to the standard deviation. On the X axis, the number of CGp dinucleotides in the reading frame is open and on the Y axis, the protein concentration in pg/ml is indicated. The bars correspond to the residual protein concentration in the wild-type structure.

Figure 7: The number of Schematic representation of the expression plasmids used.

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Figure 8: The number of

HIV-1 p24 Detection of transient transfection Expression analysis of P-smallsyn and Pc-ref vectors H1299 cells were transfected with the designated constructs and protein production was demonstrated by conventional immunoblot analysis Cell lysate analysis of HIV-1 p24 transfected H1299 cells Molecular weights (precision plus protein standard, pc-rad) and order of R/p24, s/p24 and mock transfected samples are indicated Mock transfection corresponds to transfection with original DNA3.1 plasmid

Figure 9: The

HIV-1 p24 expression analysis of different expression constructs. H1299 cells were translocated with 15 μg R/p24, R/p24ΔCpG, s/p24 and s/p24ΔCpG constructs and pcDNA3.1 (mock control) in two independent two-pronged approaches. The respective p24 protein concentration in the cell lysate was quantified by conventional immunoblot analysis (A) and ELISA tests (B) using corresponding standard curves. The bars represent the mean p24 concentration (in μg/ml) in the cell lysate for each of the 2 independent approaches.

Examples Example 1 Production of GFP reporter genes with different CpG content

Two variants of green fluorescent protein (GFP) genes have been produced that differ in the number of CpG dinucleotides. The huGFP gene had 60 CpGs, the ACpG-GFP gene had no CpGs. The CpG depleted gene ΔCpG-GFP was artificially constructed. Care was taken in designing the ΔCpG-GFP to avoid inserting rare codons or negatively-active elements such as cis-splice sites or poly-A signaling sites. The codon adaptation index (CAI), a measure of the codon's quality, was only slightly altered by the depletion of the CpG-GFP nodes (CAIQ) = 0.95; CAIQ = 0.94; and the codon's codon subquotient was not altered.

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Production of stable cell lines with the GFP gene variants

For rapid establishment and selection of stable recombinant cells, the Flp-In system of in vitro was used. Another major advantage of this system is the directed integration of a copy of the transgene into a defined locus of the target cell. This technology thus creates the best prerequisite for quantitative comparison of the expression of any transgene, as physiological and genetic factors of the target cell are largely identical. To provide additional assurance, two different mammalian cells were selected for these comparative analyses. The Flp-In CHO and Flp-In 293T cell lines were obtained from in vitro and were cultured at 37°C and 5 ml. The ZIPLINES were cultured in Eagle's Dulcofenic acid-containing glucose (ZEM) and high-glycemic acid (FAM) (100% to 10 g/ml) and in the ratio 1:10 to 10 μg/ml.

The establishment of stable transfected cells was carried out as specified by the manufacturer. 2.5 x 105 cells were sown in 6-well culture dishes and 24 h later transplanted by calcium phosphate coprecipitation (Graham and Eb, 1973) with 1.5 μg transfer plasmid and 13.5 μg pOG44.

Definition of the expression CFP

The expression of the reporter constructs was determined by regular measurements of GFP-mediated green autofluorescence in a flow cytometer (Becton-Dickinson) over a period of 16 months. The mean fluorescence intensity data are summarized in Figures 2A (293T cells) and 2B (CHO cells). HuGFP expression was detected constantly over the entire measurement period in both cell lines with a mean fluorescence intensity of 800 (293T) and 700 (CHO) respectively. The ΔCpG-GFP reporter construct reduced in CpG was also detected over the entire measurement period in contrasted fluorescence cells. However, the mean fluorescence intensity was reduced by a factor of 10-20 (293CHO-2G) compared to the mean fluorescence intensity after the measurement period.

Since a variety of causes can be considered for a decrease in GFP-mediated fluorescence (protein instability, reduced RNA chemical export, lower transcription rate, etc.), Western blot analysis and quantitative real-time PCR were also performed.

For protein detection by immunoblot, the stable transfected CHO cells were washed twice with ice-cold PBS (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCI), scraped in ice-cold PBS, decentrifuged for 10 min at 300 g and lysified on ice in lysis buffer (50 mM Tris-HCl, pH 8.0, 0.5 % Triton X-100 (w/v)) for 30 min. Insoluble components of the cellulose were decentrifuged for 30 min at 10000 g and 4 °C. The total protein content of the residue was confirmed by the Bio-Rad BFP protein antibody assay (Bio-Rad BFP, Munich). The secondary samples were measured at a volume equal to 2-Lactase (F-Lactase) and were determined by a single-spectrum test at 5 °C. Both the protein and the protein were detected at a temperature of 95 °C (F-C) and a total protein were detected at a temperature of 12 °C. The results were determined by a single-spectrum test at a temperature of 95 °C (F-C) and a total protein of 95 μM (F-C) per cellulose, both in the case of nitrogenoxy and HF-Rad BFP (F-Rad BFP, Munich).

To investigate transcription activity, a quantitative real-time light cycler (Roche) PCR was performed on the stably transcribed CHO cells, with subsequent PCR (RT-RNA oligoglycolic 1 and RT-RNA oligoglycolic 2), and cytoplasmic RNA was prepared from the cells (RNeasy, quiagen) and treated with DNase (500 U RNase-free DNase / 20 μg RNA). 1 μg of DNase-treated RNA was used as a matrix for a reverse transcription (random primed, p(dN6) 1st strand C-DNA synthesis kit for RT-PCR, Roche) with subsequent PCR (RT-RNA oligoglycolic 1 and RT-RNA oligoglycolic 2). The resulting PCR RNA was very diluted and used for a light cycler (SFPY, RNase-free DNase) analysis. The resulting data were corrected for a light cycler (SFPY, RNase-free) product. The concentration of the cyclooxygen in the internal control cell was also determined. The concentration of C4A (C3A) and C3A (C3A) cells in the C3A cell is a good comparison to the concentration of the other two proteins in the C3A cell. The results are very low in comparison to the C4A (C3A) and C4 (C3A) cells in the C3A) cells.

Example 2 Manufacture of murine Mip1alpha genes with different CpG content

In this example, the nucleic acid sequence of the murine MIP1alpha gene was altered to produce a number of constructs with different numbers of CpG dinucleotides, but without changing the coding amino acid sequence. To do this, the amino acid sequence of the murine MIP1alpha gene product was translated back into synthetic MIP1alpha coding reading frames, using human cell coding. In a first series of constructs, the randomly generated CpG dinucleotides were gradually removed from the sequence again, but without adding rare codons that would worsen expression as expected. In addition, a CIP1 Dinotype was developed to optimize the coding of this gene, which would be possible in the case of a deliberately coded gene, as in the case of a CIP1 Dinotype, which was designed to be a more sensitive codon than the one in the CIP1 Dinophone.

The manufactured MIP1 alpha vector variants were found to be completely different in the expression level of the (murine) MIP1alpha. Unpredictable to the expert, the variants with the lowest CpGs were found to be the worst expressed and with the increase in CpGs, an increase in MIP1alpha expression in mammalian cells was associated. In particular, it was unpredictable to the expert that the construct with the maximum possible number of CpG dinucleotides was introduced, but at the expense of a significantly greater increase in gene expression than on the insonophone coded.

Variants of the murine Mip1alpha gene that differ in the number of CpG dinucleotides were artificially constructed and subcloned into the expression vector pcDNA3.1 using the HindIII and Notl interfaces, as described in example 1. The artificially produced genes were adapted to the mammalian system in their coding selection. No rare mammalian codons were used to remove the CpG dinucleotides, no rare codons were used to insert CpG dinucleotides beyond the number of dinucleotides achieved with normal codonation, and rare codon adaptors were also deliberately used.

Err1:Expecting ',' delimiter: line 1 column 1209 (char 1208)

Verification of the Mip1alpha expression

Human H1299 cells with their respective expression constructs were translocated and the amount of protein in the cells and cell culture residue was measured by commercial ELISA kits to quantify the expression of the chemokine.

1.5 x 105 human lung cancer cells (H1299) were sown in 6-well cell culture dishes and 24 h later transfected by calcium phosphate precipitation with 15 μg of the corresponding expression plasmid. Cells and the cell culture residue were harvested 48 h after transfection. The transfected cells were lysified as described in example 1 and the total protein content of the cell lysate was determined with the Bio-Rad Protein Assay. The cell culture residue was removed by centrifugation at 10000 x g for 15 min at 4 °C.

From 1-5 μg of total protein from cell acids and from diluted cell culture residues, the expression of Mip1alpha was checked in a commercially available ELISA assay (R & D system) according to the manufacturer's specifications. Comparable to the data from the GFP and p24 expression constructs, the total amount of detectable Mip1alpha correlated with the number of CpGs in the reading frame. The data are summarised in Table 1.

A representative result of an evaluation by cytokine ELISA is shown in Figure 5.The bars represent the mean of two independent transfection approaches, the error bars represent the respective standard deviation.

Table 1 lists the relative protein levels of two independent transient transfection experiments (in two-pronged approaches) with respect to the wild-type construct, showing a significant reduction in protein expression with the reduction in CpG dinucleotides and a significant increase compared to the wild-type gene and the codon-optimized genes, correlating with the additional introduction of such motifs and despite a deterioration in codon adaptation. Other

pc-maMIP wt	15	100 %	4 %	8	0,76
pc-maMIP 0	5	2 %	9 %	0	0,92
pc-maMIP 2	7	8 %	27 %	2	0,93
pc-maMIP 4	9	7 %	33 %	4	0,93
pc-maMIP 13	11	146 %	5 %	13	0,97
pc-maMIP 42	13	246 %	4 %	42	0,72

* Prozentualer Mittelwert der Proteinmenge aus 2 Versuchen (in Doppelansätzen) im Verhältnis zur Gesamtproteinmenge des Wildtyp-Konstruktes (maMIP wt) ** Standardabweichung *** Codon-Adaptationsindex

Example 3 Production of human and murine cytogenes with different CpG content

Err1:Expecting ',' delimiter: line 1 column 1152 (char 1151)

Testing of cytokine expression

Human cells with the appropriate expression constructs were translocated and the amount of protein in the cell culture surplus was measured by commercial ELISA kits to quantify cytokine expression.

As described in example 2, H1299 cells were transiently transfected with 15 μg of the corresponding expression plasmid. The cell culture residue was harvested 48 h after transfection. Insoluble cell components were removed from the cell culture residue by centrifugation.

Compared to the data from the above-mentioned expression constructs, the total amount of detectable cytokines in the culture residue correlated with the number of CpGs in the reading frame. The data are summarized in Table 2. A representative result of an ELISA cytokine analysis is shown in Figure 6. The results correspond to the mean of two independent transfection assays representing errors in each. The standard deviation of this table 2 is significantly increased by introducing a transgenic protein (transgenic) in a transgenic protein (transgenic) type 2 in the wild, with a significant increase in the transgenic protein production in the wild. Other

pc-huMIP wt	21	100 %	8	0,76
pc-huMIP 43	17	393 %	43	0,72
pc-huGM wt	23	100 %	10	0,82
pc-huGM 63	19	327 %	63	0,70
pc-hulL wt	56	100 %	3	0,65
pc-huIL 21	52	313 %	21	0,98
pc-muGM wt	58	100 %	11	0,75
pc-muGM 62	54	410 %	62	0,75

* Prozentualer Mittelwert der Proteinmenge aus jeweils einem Versuch, in Doppelansätzen im Verhältnis zur Gesamtproteinmenge des entsprechenden wildtyp Konstruktes (bezeichnet wt) ** Codon-Aadaptationsindex

Example 4 Production of a plasmid with reduced number of CpG dinucleotides to increase expression

The DNA sequence encoded for the ampicillin resistance gene (bla) was synthesized using the restriction interfaces ClaI and BglII and subcloned using the restriction interfaces ClaI and BglII. The number of CpGs was reduced from 72 to 2. The multiple cloning site was redesigned, synthesized and subcloned using the restriction interfaces SacI and PmeI, reducing the number of CpGs from 11 to 1. The promoter (3V1 CpGs), the polygon (3pglG) and the replication site (3pglG) were integrated into the CpGs (45 CMG) unchanged.Err1:Expecting ',' delimiter: line 1 column 722 (char 721)25 is indicated.

Err1:Expecting ',' delimiter: line 1 column 515 (char 514)

Err1:Expecting ',' delimiter: line 1 column 478 (char 477)

Testing of HIV-1 p24 expression in different vector backgrounds

To test the influence of the CpG number in the vector from transcript expression, the R/p24 and s/p24 constructs were translocated transiently into human cells and the expression of p24 was analysed.

As described in example 2, H1299 cells were transiently transfected with 15 μg of the corresponding expression plasmid. Cells were harvested 48 h post-transfection. The transfected cells were lysed as described in example 1, and the total protein content of the residue was determined with the Bio-Rad Protein Assay. 50 μg of total protein from cell cultures were tested for p24 expression in a Western blot analysis with a monoclonal p24 specific antibody 13-5 (Wolf et al., 1990) as described in example 1 (Figure 8).

Production of HIV p24 Genes with different CpG content

Err1:Expecting ',' delimiter: line 1 column 469 (char 468)

Testing of HIV-1 p24 expression

To verify the influence of the CpG number in the vector and insert (transcript), the constructs R/p24, R/p24ΔCpG, s/p24 and s/p24ΔCpG were translocated transiently into human cells and the expression of p24 was analysed.

As described in example 2, H1299 cells were transiently transfected with 15 μg of the corresponding expression plasmid. Cells were harvested 48 h after transfection. The transfected cells were lysified as described in example 1 and the total protein content of the lysate was determined with the Bio-Rad Protein Assay.

50 μg total protein from cell acids was checked for p24 expression in a Western blot analysis with a monoclonal p24 specific antibody 13-5 as described in example 1 (Fig. 9A). As shown in example 4, the use of the CpG deleted vector P-smallsyn with identical transgene resulted in a visible increase in p24 production (comparison R/p24 and s/p24). Compared to the GFP and cytokine/chemokine expression constructs, the amount of detectable p24 in the cell acids correlated with the number of CpGs in the reading (comparison R/p24 and s/p24/p24/p24/p24/p24 without R/p24/p24 and s/p24/p24 without Cp24/p24 (comparison P/p24/p24/p24 and s/p24/p24 without Cp24/p24), respectively) using the identical vector background, and the results were confirmed as a cavity of 2.5 (or 9%) in the ELISA (see Figure 4).

The correlation of protein production with the number of CpG dinucleotides has been demonstrated in the examples above. The selected genes come from organisms as diverse as a jellyfish, a human pathogenic virus and mammals. It is therefore reasonable to consider this mechanism to be universal. The examples further demonstrate that this correlation holds in vitro for both transient transfection and stable recombinant cells. The method described here, which specifically alters gene expression in eukaryotes by modulating the CpG dinucleotide in both the coding region and the background, can therefore be used for the production of biomolecules for biotechnological, diagnostic or medical applications.

Description of the sequences The following is the list of active substances:

SEQ ID NO.	Bezeichnung	Sequenz 5' - 3'
28	huGFP-1
29	huGFP-2	AGTAGGATCCTATTACTTGTACAGCTCGT
30	RT-oligo1	CCCTGAAGTTCATCTGCACC
31	RT-oligo2	GATCTTGAAGTTCACCTTGATG
32	mamip-1	CAGGTACCAAGCTTATGAAGGTCTCCACCACTGC
33	mamip-2
34	hugm-1	CAGGTACCAAGCTTATGTGGCTGCAGAGCCTGC
35	hugm-2
36	humip-1	CAGTACCAAGCTTATGCAGGTCTCCACTGCTGC
37	humip-2
38	p24-1
39	p24-2
40	CMV-1
41	CMV-2	GAATGAGCTCTGCTTATATAGACC
42	ori-1
43	ori-2
44	pa-1
45	pa-2
46	ref-del-1
47	ref-del-2	AGTCATGCATCCATAGAGCCCACCGCATCCCCA
48	huil-1	CAGGTACCAAGCTTATGAGAATTTCGAAACCAC
49	huil-2
50	magm-1
51	magm-2

Polypeptide coding sequences and vector sequences

References

The following is a list of the most commonly reported cases of the disease: acute renal failure, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, hypertension, and hypertension, and hypertension, and hypertension.The study was conducted in the laboratory of the University of California, Berkeley, and was funded by the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), the National Institutes of Health (NIH), and the National Institutes of Health (NIH).The following is a list of the most commonly used methods of determining the molecular weight of a cell, including the number of cells that are involved in the process of DNA replication:The following is a list of the most commonly used methods for the determination of the concentration of a substance in a test chemical:The protein is also known as the CpG-binding protein MeCP2 and is a member of the CpG family of proteins. The CpG-binding protein MeCP2 represses Sp1-activated transcription of the human leukosialin gene when the promoter is methylated, Mol.Cell Biol. 18, 5492-5499.Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227, 680-685.Transcriptional silencing of the RUNX3 gene by CpG hypermethylation is associated with lung cancer, Biochem.Biophys.Res.Commun. 314, 223-228.Nan, X., Ng, H. H., Johnson, C. A., Laherty, C. D., Turner, B. M., Eisenman, R. N. and Bird, A. (1998) Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deaclase complex, Nature 393, 386-389.Shen, J. C., Rideout, W. M., III and Jones, P. A. (1994) The expected model of the hydrolytic rate of deamination of 5-methylcytosine in DNA, DNA-branded, and a double-stranded DNA, J. A., 97-97 (1996).The following is a list of the most commonly used methods for the determination of the C-terminal domain of a DNA molecule: Cloning of a mammalian transcriptional activator that binds unmethylated CpG motifs and shares a CXXC domain with DNA Mathyltransferase, Human Trithorax, and Methyl-CpG Binding Domain Protein I, Mol. And. Biol. Mar. 2000, 2108-212.and Pravtcheva, D. D. (1999) The undermethylated state of a CpG island region in igf2 transgenes is dependent on the H19 enhancers, Genomics 60, 258-271.Wolf, H., Modrow, S., Soutschek, E., Motz, M., Grunow, R. and Döbl, H. (1990) Production, mapping and biological characterisation of monoclonal antibodies to the core protein (p24) of the human immunodeficiency virus type 1., AIFO 1, 24-29.Wu, Q., Shi, H., Suo, Z. and Nesland, J. M. (2003) 5'-CpG island methylation of the FHIT gene is associated with reduced protein expression and higher clinical stage in cervical carcinomas, Ultrastructol.Path. 27, 417-422.Yao, X., J., F., Hu, Daniels, M., J. M., Shiran, H., Zhou, X., Yan, H., Lu, H., Zeng, Z., Wang, Q., Li, T. and Hoffman, A. R. (2003) A methylated oligonucleotide inhibits IGF2 expression and enhances survival in a model of hepatocellular carcinoma, J.Clin.lnvest 111, 265-273.Yoshida, M., Nosaka, K., Yasunaga, J. I., Nishikata, I., Morishita, K. and Matsuoka, M. (2003) Aberrant expression of the MEL1S gene identified in association with hypomethylation in adult T-cell leukemia cells, Blood.

The following shall be added:

< 110 > Geneart GmbH < 120 > Method for modulating gene expression by The following table shows the results of the analysis of the results of the analysis of the CpG content in the product: Other, including: The following are the types of products: The following information is provided for the purpose of the analysis: The following information is provided for the purpose of the analysis: The amount of the credit risk mitigation effect of the credit risk mitigation techniques shall be reported. The number of employees in the company is not known. The following are the main characteristics of the product: The following are the main categories of products: The following are the main characteristics of the product: < 213> Artificial sequence < 223> Description of the artificial sequence:deplete The following information shall be provided in the following cases: The following are the main factors: Other, including: The following are the types of products: The following is the list of the main components of the test chemical: < 223> Description of the artificial sequence:humanes The following information is provided for the purpose of the analysis: The amount of the credit risk mitigation effect of the credit risk mitigation techniques shall be reported. The number of employees in the company is not limited to the following: The following table shows the data: The following are the main categories of products: The following are the main characteristics of the product: < 213> Artificial sequence < 223> Description of the artificial sequence:humanes The following information is provided for the purpose of the analysis: The following are the main factors: Other The following are the types of products: The following is the list of the main components of the test chemical: < 223> Description of the artificial sequence:murine MIP1alpha-OCpG (murine MIPlalpha gene with 0 The following are the active substances: The amount of the credit risk mitigation effect of the credit risk mitigation techniques shall be reported. The number of employees in the company is not limited to the following: The following table shows the data: The number of employees The following are the main characteristics of the product: < 213> Artificial sequence < 223> Description of the artificial sequence:murine MIP1alpha-OCpG (murine MIP1alpha gene with 0 The total number of cells in the test chemical is calculated as follows: The following are the main factors: Other The following are the types of products: The following is the list of the main components of the test chemical: < 223> Description of the artificial sequence: murine MIP1alpha-2CpG (murine MIP1alpha gene with 2 The following are the active substances: The amount of the credit risk mitigation effect of the credit risk mitigation techniques shall be reported. The following table shows the data:.279) < 400> 7 The following are the main factors: The number of employees The following are the main characteristics of the product: < 213> Artificial sequence < 223> Description of the artificial sequence: murine MIPlalpha-2CpG (murine MIP1alpha gene with 2 The total number of cells in the test chemical is calculated as follows: The following are the main factors: Other The following are the types of products: The following is the list of the main components of the test chemical: < 223> Description of the artificial sequence:murine MIPalpha-4CpG (murine MIPalpha gene with 4 The following are the active substances: The amount of the credit risk mitigation effect of the credit risk mitigation techniques shall be reported. The number of employees in the company is not limited to the following: The number of employees The number of employees The following are the main characteristics of the product: < 213> Artificial sequence < 223> Description of the artificial sequence:murine MIPalpha-4CpG (murine MIPalpha gene with 4 The total number of cells in the test chemical is calculated as follows: The number of employees Other The following are the types of products: The following is the list of the main components of the test chemical: < 223> Description of the artificial sequence:murine MIPalpha-13CpG (murine MIPalpha gene with 13 The following are the active substances: The amount of the credit risk mitigation effect of the credit risk mitigation techniques shall be reported. The number of employees in the company is not limited to the following: The number of employees The number of employees The following are the main characteristics of the product: < 213> Artificial sequence < 223> Description of the artificial sequence:murine MIPalpha-13CpG (murine MIPalpha gene with 13 The total number of cells in the test chemical is calculated as follows: The number of employees Other The following are the types of products: The following is the list of the main components of the test chemical: < 223> Description of the artificial sequence:murine MIPalpha-42CpG (murine MIPalpha gene with 42 The following are the active substances: The amount of the credit risk mitigation effect of the credit risk mitigation techniques shall be reported. The number of employees in the company is not limited to the following: The number of employees The following are the main characteristics of the product: < 213> Artificial sequence < 223> Description of the artificial sequence:murine MIPalpha-42CpG (murine MIPalpha gene with 42 The following are the main components of the test chemical: The following are the main factors: Other The following are the types of products: The following are the main characteristics of the product: The test chemical is used to determine the concentration of the active substance in the test chemical. The amount of the credit risk mitigation effect of the credit risk mitigation techniques shall be reported. The following table shows the data:.279) < 400> 15 The following table shows the data: The number of employees The following are the main characteristics of the product: Other, including mixtures of animal or vegetable fats The test chemical is used to determine the concentration of the active substance in the test chemical. The following are the main factors: The number of employees The following are the types of products: The following is the list of the main components of the test chemical: < 223> Description of the artificial sequence:humanes The following information is provided for the purpose of the analysis: The amount of the credit risk mitigation effect of the credit risk mitigation techniques shall be reported. The number of employees in the company is not limited to the following: The following are the main factors: The number of employees The following are the main characteristics of the product: < 213> Artificial sequence < 223> Description of the artificial sequence:humanes The following information is provided for the purpose of the analysis: Other Other, including: The following are the types of products: The following is the list of the main components of the test chemical: < 223> Description of the artificial sequence:synthetic Other The following information shall be provided in the form of a summary of the results of the analysis: The amount of the credit risk mitigation effect of the credit risk mitigation techniques shall be reported. The number of employees in the company is not limited to the following: Other, including: The number of employees The following are the main characteristics of the product: < 213> Artificial sequence The following information is provided for the purpose of the analysis: The number of employees The number of employees The following are the types of products: The following are the main characteristics of the product: The following information is provided for the purpose of the analysis: The amount of the credit risk mitigation effect of the credit risk mitigation techniques shall be reported. The number of employees in the company is not limited to the following: The following are the main factors: The number of employees The following are the main characteristics of the product: < 213 > human The following information is provided for the purpose of the analysis: The following are the main factors: Other, including: The following are the types of products: The following are the main characteristics of the product: The following information shall be provided for the purpose of the analysis: The amount of the credit risk mitigation effect of the credit risk mitigation techniques shall be reported. The following table shows the data:.935) < 400> 23 The following are the main factors: The number of employees The following are the main characteristics of the product: < 213 > human The following information is provided for the purpose of the analysis: The following are the main factors: Other, including: The following are the types of products: The following is the list of the main components of the test chemical: < 223> Description of the artificial sequence:p smallsyn The following table shows the data: The following table shows the data: Other, including: The following are the types of products: The following is the list of the main components of the test chemical: The test chemical is used to determine the concentration of the active substance in the test chemical. Genetic information on the presence of HIV-1 in the human immunodeficiency virus (HIV-1) The test chemical is a chemical that is used to reduce the concentration of CpG-dinucleotides in the blood. The amount of the credit risk mitigation effect of the credit risk mitigation techniques shall be reported. The number of employees in the company is not limited to the following: The following are the main factors: The following are the main factors: The following are the main characteristics of the product: < 213> Artificial sequence The test chemical is used to determine the concentration of the active substance in the test chemical. Genetic information on the presence of HIV-1 in the human immunodeficiency virus (HIV-1) The following is a list of the active substances that may be used in the preparation of the test chemical: The number of employees The number of employees The following are the types of products: The following is the list of the main components of the test chemical: < 223> Description of the artificial sequence:Primer The following are the main components of the test: The following shall be added to the list of products: The number of employees The following are the types of products: The following is the list of the main components of the test chemical: < 223> Description of the artificial sequence:Primer The following are the main components of the test: The following shall be added to the list of products: The following are the main factors: The following are the types of products: The following is the list of the main components of the test chemical: < 223> Description of the artificial sequence:Primer RT other products The following is the list of the products which are to be classified in the product group: The following are the main factors: The following are the types of products: The following is the list of the main components of the test chemical: The following is a description of the sequence: The following are the main characteristics of the product: The number of employees The following are the types of products: The following is the list of the main components of the test chemical: < 223> Description of the artificial sequence:Primer The following table shows the results of the analysis: The following shall be added to the list of products: Other The following are the types of products: The following is the list of the main components of the test chemical: < 223> Description of the artificial sequence:Primer The following table shows the results of the analysis: The following shall be added to the list of products: The number of employees The following are the types of products: The following is the list of the main components of the test chemical: The following is a description of the artificial sequence:Primer hugm-1<400> 34 The following shall be added to the list of products: Other The following are the types of products: The following is the list of the main components of the test chemical: The following is a description of the artificial sequence:Primer hugm-2<400> 35 The following shall be added to the list of products: The number of employees The following are the types of products: The following is the list of the main components of the test chemical: < 223> Description of the artificial sequence:Primer Humip-1< 400> 36 The following shall be indicated in the table: The following are the main factors: The following are the types of products: The following is the list of the main components of the test chemical: < 223> Description of the artificial sequence:Primer Humip-2< 400> 37 The following shall be added to the list of products: Other The following are the types of products: The following is the list of the main components of the test chemical: The following is a description of the artificial sequence:Primary p24-1<400> 38 The following are the main characteristics of the product: The following are the main factors: The following are the types of products: The following is the list of the main components of the test chemical: The following is a description of the artificial sequence:Primary p24-2<400> 39 The following shall be added to the list of products: The number of employees The following are the types of products: The following is the list of the main components of the test chemical: The following is a description of the sequence:CMV-1 primate < 400> 40 The following shall be added to the list of products: The number of employees The following are the types of products: The following is the list of the main components of the test chemical: The following is a description of the sequence:CMV-2 primate < 400> 41 The following are the main characteristics of the product: The following are the main factors: The following are the types of products: The following is the list of the main components of the test chemical: The following is a description of the artificial sequence:Primer ori-1<400> 42 The following are the main categories of products: The following are the main factors: The following are the types of products: The following is the list of the main components of the test chemical: The following is a description of the artificial sequence:Primer ori-2<400> 43 The following shall be indicated in the table: The number of employees The following are the types of products: The following is the list of the main components of the test chemical: The following is a description of the artificial sequence:Primer pa-1<400> 44 The following shall be indicated in the table: The following are the main factors: The following are the types of products: The following is the list of the main components of the test chemical: The following is a description of the artificial sequence:Primer pa-2<400> 45 The following shall be indicated in the table: The number of employees The following are the types of products: The following is the list of the main components of the test chemical: < 223> Description of the artificial sequence:Primer The following table shows the results of the analysis: The following are the main components of the calculation: The number of employees The following are the types of products: The following is the list of the main components of the test chemical: < 223> Description of the artificial sequence:Primer The following table shows the results of the analysis: The following are the main characteristics of the product: The number of employees The following are the types of products: The following is the list of the main components of the test chemical: The following is a description of the artificial sequence:primary 5'-3<220> The number of patients with a history of infection is not known. The following shall be added to the list of products: The following are the main factors: The following are the types of products: The following is the list of the main components of the test chemical: The following is a description of the artificial sequence:primary 5-3<220> The number of cases of the disease is not known. The following shall be added to the list of products: The number of employees The following are the types of products: The following is the list of the main components of the test chemical: The following is a description of the artificial sequence:primary 5-3<220> The maximum value of the test substance shall be calculated as follows: The following shall be added to the list of products: Other The following are the types of products: The following is the list of the main components of the test chemical: The following is a description of the artificial sequence:primary 5' to 3' < 220> The following table shows the data for the following categories of products: The following shall be indicated in the table: Other The following are the types of products: The following is the list of the main components of the test chemical: < 223> Description of the artificial sequence:synthetic Other The following information is provided for the purpose of the analysis: The amount of the credit risk mitigation effect of the credit risk mitigation techniques shall be reported. The following table shows the data:.489) < 400> 52 The following are the main factors: The number of employees The following are the main characteristics of the product: < 213> Artificial sequence The following information is provided for the purpose of the assessment: The number of employees Other The following are the types of products: The following is the list of the main components of the test chemical: < 223> Description of the artificial sequence:synthetic Other The following information shall be provided in the form of a summary of the results of the analysis: The amount of the credit risk mitigation effect of the credit risk mitigation techniques shall be reported. The number of employees in the company is not limited to the following: The following table shows the data: The number of employees The following are the main characteristics of the product: < 213> Artificial sequence The following information is provided for the purpose of the assessment: The following table shows the data: Other The following are the types of products: The following are the main characteristics of the product: The following information shall be provided to the competent authority: The amount of the credit risk mitigation effect of the credit risk mitigation techniques shall be reported. The Commission has also adopted a proposal for a directive on the protection of workers from risks related to exposure to ionising radiation. The number of employees The number of employees The following are the main characteristics of the product: < 213 > human The following information is provided for the purpose of the analysis: The following are the main factors: Other The following are the types of products: The following are the main characteristics of the product: The following information shall be provided in the form of a summary of the results of the analysis: The amount of the credit risk mitigation effect of the credit risk mitigation techniques shall be reported. The following shall be added:

Claims (42)

1. Method for the targeted modulation of gene expression, comprising:

   (i) provision of a target nucleic acid sequence to be expressed,

   (ii) modification of the target nucleic acid sequence, the number of CpG dinucleotides present in the target nucleic acid sequence being increased using the degeneracy of the genetic code to increase gene expression, wherein the number of CpG dinucleotides is increased in comparison with a codon-optimised sequence derived from the wild-type sequence using the degeneracy of the genetic code, or is reduced to reduce gene expression,

   (iii) cloning of the thereby modified target nucleic acid sequence with a modified number of CpG dinucleotides into a suitable expression vector in operative linkage with a suitable transcriptional control sequence,

   (iv) expression of the modified target nucleic acid sequence in a suitable mammalian expression system.
2. Method according to claim 1, wherein in step (ii) the modification of the target nucleic acid sequence is carried out in such a manner that, in addition to increasing or reducing the number of CpG dinucleotides, one or more additional modifications is/are carried out at the nucleic acid level.
3. Method according to claim 2, wherein the additional modifications at the nucleic acid level are selected from the group consisting of: alteration of codon choice, insertion or elimination of sequence orders stabilising the RNA secondary structure, regions with elevated self-homology, regions with elevated homology to the natural gene, RNA-instability motifs, splice-activating motifs, polyadenylation motifs, adenine-rich motifs, endonuclease recognition sites.
4. Method according to one of the preceding claims, wherein modification of the target nucleic acid sequence by increasing or reducing the number of CpG dinucleotides is carried out having regard to a codon choice optimised for the expression system.
5. Method according to one of the preceding claims, wherein modification of the number of CpG dinucleotides is carried out having regard to a codon choice optimised for mammals.
6. Method according to one of claims 1 to 5, wherein gene expression is increased.
7. Method according to one of claims 1 to 5, wherein gene expression is reduced.
8. Method according to one of the preceding claims, wherein the target nucleic acid sequence to be expressed is heterologous to the mammalian expression system.
9. Method according to one of the preceding claims, wherein the mammalian expression system used is a cell selected from the group consisting of mammalian cells, human cells and somatic cells; or a cell-free expression environment is used.
10. Method according to one of the preceding claims, wherein the target nucleic acid to be expressed is of eukaryotic, prokaryotic, viral or synthetic origin.
11. Method according to one of the preceding claims, wherein a low methylation system is used as the expression system.
12. Method according to one of the preceding claims, wherein the modified target nucleic acid sequence and the transcriptional control sequence are not associated with CpG islands.
13. Method according to one of the preceding claims, wherein the number of CpG dinucleotides is increased or reduced by at least two.
14. Method according to one of the preceding claims, wherein the number of CpG dinucleotides is increased or reduced by at least 10%, preferably at least 50%, preferably at least 100%.
15. Method according to one of the preceding claims, wherein all CpG dinucleotides are removed using the degeneracy of the genetic code.
16. Method according to one of the preceding claims, wherein the target nucleic acid sequence encodes an RNA, derivatives or mimetics thereof, a peptide or polypeptide, a modified peptide or polypeptide, a protein or a modified protein.
17. Method according to claim 16, wherein the target nucleic acid sequence encodes a therapeutic and/or diagnostic protein.
18. Method according to claim 17, wherein the target nucleic acid sequence encodes a protein selected from human, parasitic, viral or bacterial proteins, enzymes, hormones, vaccines, messenger substances and regulator proteins.
19. Method according to claim 17 or 18, wherein the target nucleic acid sequence encodes a protein selected from asparaginase, adenosine deaminase, insulin, tPA, clotting factors, vitamin K epoxide reductase, erythropoietin, follicle-stimulating hormone, oestrogens, bone morphogenetic proteins, antithrombin, HIV-, HBV-, HCV-, influenza-, borrelia-, haemophilus-, meningococcus-, anthrax-derived proteins, botulinus toxoid, diphtheria toxoid, tetanus toxoid, plasmodium proteins, blood group antigens, HLA proteins, cytokines, chemokines, G-CSF, GM-CSF, interleukins, interferons, PDGF, TNF, RANTES, MIP1α and transcription factors.
20. Method according to claim 16, wherein the target nucleic acid sequence encodes a functional RNA.
21. Method according to claim 20, wherein the target nucleic acid sequence is an siRNA, a ribozyme, an antisense-RNA or a decoy.
22. Modified nucleic acid with a region capable of transcription that can be expressed in a mammalian expression system, and which is derived from a wild-type sequence, wherein the region capable of transcription is modified such that it is codon-optimised in relation to the mammalian expression system used, wherein a codon choice is used as is most or second-most commonly used in mammalian cells, and such that the number of CpG dinucleotides is increased compared to the codon-optimised sequence derived from the wild-type sequence using the degeneracy of the genetic code.
23. Nucleic acid according to claim 22, wherein the number of CpG dinucleotides is increased compared to the wild-type sequence by at least 10%, preferably at least 25%, preferably at least 50%, preferably at least 100%, preferably at least 200%, preferably by a factor of five, preferably by a factor of ten or more.
24. Nucleic acid according to one of the preceding claims 22 or 23, wherein the nucleic acid is not associated with a CpG island.
25. Nucleic acid according to one of claims 22 to 24, comprising a sequence selected from: SEQ ID Nos. 13, 17, 19, 52 and 54.
26. Vector comprising a nucleic acid according to one of claims 22 to 25 in operative linkage with a suitable transcriptional control sequence.
27. Vector according to claim 26, wherein the transcriptional control sequence comprises a promoter.
28. Vector according to claim 27, wherein the promoter is a constitutively active promoter.
29. Vector according to claim 28, wherein the constitutively active promoter is selected from (cytomegalovirus) CMV promoter and simian virus 40 (SV40) promoter.
30. Vector according to claim 27, wherein the promoter is an inducible promoter.
31. Vector according to claim 30, wherein the inducible promoter is a tetracycline-dependent promoter.
32. Vector according to one of claims 26 to 31, wherein the promoter is not associated with a CpG island.
33. Vector according to one of claims 26 to 32, wherein the sequences or parts thereof present on the vector and different from the nucleic acid according to one of claims 22 to 25 have a reduced number of CpG dinucleotides.
34. Vector according to one of claims 26 to 33, wherein the sequences or parts thereof different from the nucleic acid according to one of claims 22 to 25 have a number of CpG dinucleotides reduced by about 25%, preferably 50%, more preferably 75%, more preferably by 100%.
35. Vector according to one of claims 26 to 34, with the nucleic acid sequence shown in SEQ ID No. 25.
36. Cell containing a nucleic acid or a vector according to one of claims 22 to 35.
37. Mammalian expression system comprising:

    (a) a modified nucleic acid sequence with a region capable of transcription, which is derived from a wild-type sequence, wherein the modified nucleic acid sequence is modified such that it is codon-optimised in relation to the mammalian expression system used and has an increased or reduced number of CpG dinucleotides compared to the codon-optimised sequence, in operative linkage with a transcriptional control sequence,

    (b) an expression environment selected from a mammalian cell and a cell-free expression environment in which (a) can be expressed, wherein the mammalian expression system exhibits increased expression in the case of expression of a modified nucleic acid sequence with an increased number of CpG dinucleotides, and reduced expression in the case of expression of a modified nucleic acid sequence with a reduced number of CpG dinucleotides.
38. Medicament comprising as active substance a nucleic acid and/or a vector and/or a cell and/or a mammalian expression system according to one of claims 23 to 37.
39. Use of a nucleic acid and/or a vector and/or a cell and/or a mammalian expression system according to one of claims 22 to 37 for the production of a medicament for diagnostic and/or therapeutic treatment.
40. Use according to claim 39 for gene therapy treatment.
41. Use of a nucleic acid, a vector and/or a cell and/or a mammalian expression system according to one of claims 22 to 37 for the production of vaccines.
42. Nucleic acid with a sequence selected from SEQ ID Nos. 1, 5, 7, 9 and 26.