METHOD FOR MODULATING GENE EXPRESSION BY MODIFYING THE CPG CONTENT

Abstract

The invention relates to nucleic acid modifications for a directed expression modulation by the targeted insertion or removal of CpG dinucleotides. The invention also relates to modified nucleic acids and expression vectors.

Claims

1. Method for the targeted modulation of the gene expression, comprising the steps: (i) Provision of a target nucleic acid sequence to be expressed, (ii) Modification of the target nucleic acid sequence, in which the number of CpG dinucleotides present in the target nucleic acid sequence is raised using the degeneracy of the genetic code to increase the gene expression, or is lowered to reduce the gene expression, (iii) Cloning of the thereby modified target nucleic acid sequence with a modified number of the CpG dinucleotides in a suitable expression vector in operative coupling with a suitable transcription control sequence, (iv) Expression of the modified target nucleic acid sequence in a suitable expression system.

2. Method according to claim 1, in which in step (ii) the modification of the target nucleic acid sequence is carried out so 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. (canceled)

4. Method according to claim 1, in which the 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. (canceled)

6. Method according to claim 1, in which the gene expression is raised.

7. Method according to claim 1, in which the gene expression is reduced.

8. Method according to claim 1, in which the target nucleic acid sequence to be expressed is heterologous to the expression system.

9. Method according to claim 1, in which a eukaryotic or prokaryotic expression system is used as the expression system.

10.-12. (canceled)

13. Method according to claim 1, in which the modified target nucleic acid sequence and the transcription control sequence are not associated with CpG islands.

14. Method according to claim 1, in which the number of CpG dinucleotides is increased or reduced by at least two.

15. Method according to claim 1, in which the number of CpG dinucleotides is increased or reduced by at least 10%, preferably at least 50%, more preferably at least 100%.

16. Method according to claim 1, in which all CpG dinucleotides are removed using the degeneracy of the genetic code.

17.-20. (canceled)

21. Method according to claim 1, in which the target nucleic acid sequence codes for a functional RNA.

22. (canceled)

23. Modified nucleic acid with a region capable of transcription that can be expressed in an expression system, and which is derived from a wild-type sequence, in which the region capable of transcription is modified so that it is codon-optimised in relation to the employed expression system, and so that the number of CpG dinucleotides is increased compared to the codon-optimised sequence derived from the wild-type sequence, by using the degeneracy of the genetic code.

24. Nucleic acid according to claim 23, in which the number of CpG dinucleotides is increased compared to the wild-type sequence by at least 10%, preferably at least 25%, more preferably at least 50%, particularly preferably at least 100%, more particularly preferably at least 200%, especially by a factor of 5 and most especially by a factor of 10 or more.

25.-26. (canceled)

27. Vector comprising a nucleic acid according to claim 23 in operative coupling with a suitable transcription control sequence.

28. Vector according to claim 27, in which the transcription control sequence comprises a promoter.

29.-36. (canceled)

37. Cell containing a nucleic acid or a vector according to claim 23.

38. 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 has an increased or reduced number of CpG dinucleotides compared to the wild-type sequence, in operative coupling with a transcription control sequence, and b) an expression environment selected from a cell and a cell-free expression environment wherein a) can be expressed, in which the expression system in the case of expression of a modified nucleic acid sequence with an increased number of CpG dinucleotides exhibits an increased expression, and in the case of expression of a modified nucleic acid sequence with a reduced number of CpG dinucleotides exhibits a reduced expression.

39. (canceled)

40. Use of a nucleic acid and/or a vector and/or a cell and/or an expression system according to claim 23 for the production of a medicament for a diagnostic and/or therapeutic treatment.

41.-42. (canceled)

Description

DESCRIPTION OF THE DIAGRAMS

[0091] FIG. 1:

[0092] Regulation of the gene expression by methylation (prior art).

[0093] FIG. 1A: Methylation of CpG dinucleotides leads to the switching off of the gene expression.

[0094] FIG. 1B: CpG islands protect against a methylation and the switching off associated therewith.

[0095] FIG. 1C: Secondary hypomethylation of the CpG islands leads to a gene switching off.

[0096] FIG. 1D: Secondary hypomethylation may be prevented by reducing the CpG dinucleotides in the reading frame.

[0097] FIG. 2: GFP expression analysis in stably transfected cells.

[0098] FIG. 2A and FIG. 2B: Long-time flow cytometry analysis of stably transfected Flp-In 293T and CHO cells. The Y axis gives the GFP-conditioned fluorescence intensity (MFI mean fluorescence intensity) and the X axis gives the measurement times in weeks after transfection.

[0099] FIG. 2A: FACS analysis of huGFP and CpG-GFP recombinant 293T cells.

[0100] FIG. 2B: FACS analysis of huGFP and CpG-GFP recombinant CHO cells.

[0101] FIG. 2C: Fluorescence microscopy image of stable cell lines.

[0102] FIG. 3: GFP protein detection in stably transfected cells.

[0103] Expression analysis of the GFP reading frame.

[0104] Recombinant Flp-In CHO cells that have integrated the huGFP or the CpG-GFP gene stably into the cell genome were lysed, and the expression of the genes was detected by conventional immunoblot analyses. Plots of the huGHF, CpG-GFP and mock samples are given. Monoclonal cell lines were established from both polyclonal cell cultures (poly.) (mono. 14 and 7 for CpG-GFP and mono. 10 and 9 for huGFP). Mock cells correspond to an unchanged initial cell population.

[0105] FIG. 4:

[0106] Quantitative determination of specific transcripts of stable cells. Real-time PCR analysis of specific hygromycin-resistance gene and gfp RNAs from cytoplasmic RNA preparations. The real-time PCR evaluation of the LC analyses are shown for CHO cells (hygromycin-resistance FIG. 4A and gfp FIG. 4B) as well as for 293T cells (hygromycin-resistance FIG. 4C and gfp FIG. 4D). The number of PCRcycles (X axis) and the fluorescence intensity (Y axis) are shown. The specific kinetics are shown for huGFP products and CpG-GFP products, as well as for the primer dimers.

[0107] FIG. 5: MIP1alpha expression analysis after transient transfection.

[0108] Representative ELISA analysis of the cell lysates and supernatants of transfected H1299 cells. H1299 cells were transfected with in each case 15 g of wild-type and optimised murine MIP1alpha constructs. The respective protein concentration was quantified by conventional ELISA tests in the cell supernatant and in the cell lysate with the aid of corresponding standard curves. The shaded bars represent the mean value of the total protein concentration for in each case two independent batches, while the empty bars correspond to the standard deviation. The number of CpG dinucleotides in the open reading frame is plotted on the X axis and the total protein concentration in g/ml is plotted on the Y axis. Wt corresponds to the expression construct of the respective wild-type gene.

[0109] FIG. 6:

[0110] MIP1alpha and GM-CSF expression analysis after transient transfection. Representative ELISA analysis of the supernatants of transfected H1299 cells. H1299 cells were transfected with in each case 15 g of wild-type and optimised human MIP1alpha (FIG. 6A) and GM-CSF (FIG. 6B) constructs. The respective protein concentration in the supernatant of the cell culture 48 hours after transfection was quantified by conventional ELISA tests with the aid of corresponding standard curves. The shaded bars represent the mean value for in each case two independent batches, while the empty bars correspond to the standard deviation. The number of CpG dinucleotides in the open reading frame is plotted on the X axis and the protein concentration in the supernatant in g/ml is plotted on the Y axis. Wt corresponds to the expression construct of the respective wild-type gene.

[0111] FIG. 7: Diagrammatic illustration of the used expression plasmids.

[0112] FIG. 7A: Plasmid map of the P-smallsyn plasmid.

[0113] FIG. 7B: Plasmid map of the PC-ref. module and origin of the sequences (wild-type Wt in black, and synthetic in grey) are shown.

[0114] FIG. 8:

[0115] HIV-1 p24 detection after transient transfection.

[0116] Expression analysis of the P-smallsyn and Pc-ref vectors. H1299 cells were transfected with the specified constructs and the protein production was detected by conventional immunoblot analyses. Analysis of the cell lysates of HIV-1 p24 transfected H1299 cells. Molecular weights (precision plus protein standard, Bio-Rad) as well as the plot of the R/p24, s/p24 and mock-transfected samples are shown. Mock transfection corresponds to a transfection with the original pcDNA3.1 plasmid.

[0117] FIG. 9:

[0118] HIV-1 p24 expression analysis of various expression constructs. H1299 cells were transfected with in each case 15 g R/p24, R/24CpG, s/p24 and s/p24 CpG constructs, as well as with pcDNA3.1 (mock control) in independent double batches. The respective p24 protein concentration in the cell lysate was quantified by conventional immunoblot analyses (FIG. 9A) and by ELISA tests (FIG. 9B) with the aid of corresponding standard curves. The shaded bars represent the mean value of the p24 concentration (in g/ml) in the cell lysate for in each case 2 independent batches.

EXAMPLES

Example 1

[0119] Production of GFP Reporter Genes with Different CpG Content

[0120] Two variants of green fluorescence protein (GFP) genes, which differ in the number of CpG dinucleotides, were produced. The huGFP gene had 60 CpGs, the CpG-GFP gene had no CpGs. The CpG-depleted gene CpG-GFP was constructed artificially. In the design of the &CpG-GFP care was taken to ensure that no rare codons or negatively acting cis-active elements such as splicing sites or poly(A) signal sites were introduced. The codon adaptation index (CAI), which is a measure of the quality of the codon choice, was altered only slightly by the deletion of the CpGs (CaI(huGFP)=0.95; CAI(CpG-GFP)=0.94). The coding amino acid sequence of the GFP was in this connection not altered. Further interfaces were inserted for the sub-cloning. The nucleotide and amino acid sequences are given in SEQ ID NO. 1/2.

[0121] The sequence was produced as a fully synthetic gene (Geneart GmbH), cloned into the expression vector pcDNA/5FRT (Invitrogen) using the interfaces HindIII and Bam HI, and placed under the transcription control of the cytomegalovirus (CMV) early promoter/enhancer (pc CpG-GFP).

[0122] For the production of a similar expression plasmid, though unchanged in its CpG distribution, the coding region of the humanised GFP gene (huGFP) was amplified by means of a polymerase chain reaction (PCR) using the oligonucleotides huGFP-1 and huGFP-2 from a commercially obtainable vector, and likewise cloned into the expression vector pcDNA/5FRT (pc-huGFP, SEQ ID NO. 3/4) using the interfaces HindIII and Bam HI.

Production of stable Cell Lines with the GGP Gene Variants

[0123] The Flp-In system of Invitrogen was used for a rapid establishment and selection of stable, recombinant cells.

[0124] A further, major advantage of this system is a directed integration of a copy of the transgene into a defined locus of the target cell. This technology thus provides the best conditions for the quantitative comparison of the expression of an arbitrary transgene, since physiological and genetic factors of the target cell are largely identical. In order to achieve an additional certainty, two different mammalian cells were selected for these comparative analyses. The cell lilnes Fip-In CHO and Fip-In 293T were obtained from Invitrogen and cultured at 37 C. and 5% CO.sub.2. The cell lines were cultured in Dulbecco's modified eagle medium high glucose (DMEM) (293T) and HAMs F12 (CHO) with L-glutamine, 10% inactivated fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 g/ml). The cells were sub-cultured in a ratio of 1:10 after confluence was achieved.

[0125] The establishment of stably transfected cells was carried out according to the manufacturer's instructions. 2.510.sup.5 cells were seeded out in 6-well culture dishes and transfected 24 hours later by calcium phosphate co-precipitation (Graham and Eb, 1973) with 1.5 g transfer plasmid and 13.5 g pOG44. Cells were selected up to a ratio of >90% GFP positive cells with 100 g/ml hygromycin for 293T and 500 g/ml for CHO cells. The number of GFP positive cells was determined for all cell lines by means of conventional flow cytometry analysis.

[0126] Determination of the GFP expression

[0127] The expression of the reporter constructs was determined over a period of 16 months by regular measurement of the GFP-mediated green autofluorescence in a flow cytometer (Becton-Dickinson). The data of the mean fluorescence intensities are summarised in FIG. 2A (293T cells) and 2B (CHO cells). The huGFP expression was found to e relatively constant in both cell lines over the whole measurement period, with a mean fluorescence intensity of 800 (293T) and 700 (CHO). The CpG-GFP reporter construct, with a reduced number of CpGs, likewise exhibited a constant fluorescence intensity over the whole measurement period. The mean fluorescence intensity was however reduced by a factor of 10-20 (293T) and 6-9 (CHO) compared to the huGFP. The reduction of the GFP-mediated fluorescence could also be detected by fluorescence microscopy (FIG. 2C).

[0128] Since various causes may be involved in a decrease of the GFP-mediated fluorescence (instability of the protein, reduced nuclear export of RNA, lower transcription rate, etc.) additional western blot analyses and quantitative real-time PCRs were carried out.

[0129] For the protein detection by immunoblot, the stable transfected CHO cells were washed twice with ice-cold PBS (10 mM Na.sub.2HP4, 1.8 mM KH.sub.2PO.sub.4, 137 mM NaCl, 2.7 mM KCl), scraped off in ice-cold PBS, centrifuged for 10 minutes at 300 g, and lysed for 30 minutes in lysis buffer on ice (50 mM Tris-HCl, pH 8.0, 0.5% Triton X-100 (w/v)). Insoluble constituents of the cell lysate were centrifuged for 30 minutes at 10000 g and 4 C. The total amount of protein in the supernatant was determined by the Bio-Rad protein assay (Bio-Rad, Munich) according to the manufacturer's instructions. An equal volume of two-fold sample buffer (Laemmli, 1970) was added to the samples, and heated for 5 minutes at 95 C. 40 g of total protein from cell lysates were separated through a 12.5% SDS/polyacrylamide gel (Laemmlie, 1970), electrotransferred to a nitrocellulose membrane and detected with a monoclonal GFP-specific antibody (BD-Bioscience) and a secondary, HRP (horseradish peroxidase) coupled antibody, and identified by means of chromogenic staining. Protein detection by western blot confirmed the data from the FACS measurement. For both gene variants the full-length GFP protein was detected in stably transfected CHO cells; no differences could be detected in the processing or proteolytic degradation (FIG. 3).

[0130] In order to clarify the transcription activity, a quantitative real-time PCR (Light Cycler, Roche) was carried out for the stably transfected CHO cells. Cytoplasmic RNA was prepared from the cells (RNeasy, Quiagen) and treated with DNase (500 U Rnase-free DNase/20 g RNA). 1 g of the DNase-treated RNA was used as a matrix for a reverse transcription (Random Primed, p(dN).sub.6, 1.sup.st strand C-DNA synthesis kit for RT-PCR, Roche) followed by PCR (RT-oligol and RT-oligo2). The resulting PCR product was diluted and used for a light cycler (LC) analysis (SYBR, Roche). As internal control, the RNA amount of the hygromycin-resistance gene similarly integrated into the cell genome was measured. The results are summarised in FIG. 4. The RNA amounts of the hygromycin-resistance showed no difference in all the measured constructs (FIG. 4A for CHO cells and 4C for 293T cells). The results of the GFP RNA however correlated very well with the results of the protein expression (GFP fluorescence intensity). For the CpG-deleted construct, after quantification of the light cycler data an approximately seven times smaller cytoplasmic RNA amount was detected in CHO cells (FIG. 4B) and an approximately thirty times smaller RNA amount was detected in 293T cells (FIG. 4D), compared to the initial construct.

Example 2

[0131] Production of Murine Mip1alpha Genes with Different CpG Contents

[0132] In this example the nucleic acid sequence of the murine MiP1alpha gene was altered so as to form a series of constructs with different numbers of CpG dinucleotides, but without altering the coding amino acid sequence. For this purpose the amino acid sequence of the murine MIP1alpha gene product was translated back into synthetic MIP1alpha-coding reading frames, using the codon choice of human cells. In a first series of constructs the accidentally formed CpG dinucleotides were removed stepwise from the sequence, without however introducing rare codons that would be expected to adversely affect the expression. In addition a CpG dinucleotide-optimised Mip1alpha gene construct was produced, which contained twice as many CpG dinucleotides as the codon-optimised construct. In this case a deterioration of the codon choice was intentionally taken into account, in order to introduce as many CpG dinucleotides as possible.

[0133] According to the prior art it would be expected that this gene construct would have a lower expression than the codon-optimised gene construct on account of its poorer codon choice.

[0134] These gene variants were constructed as fully synthetic reading frames, using long oligonucleotides and a stepwise PCR, and cloned into an expression vector. The produced MIp1alpha vector variants differed completely as regards the level of expression of murine MIP1alpha. For the person skilled in the art it could not be foreseen that the variants with the lowest CpGs would be expressed worst, and an increase in the CpGs would be accompanied by an increase of the MiP1alpha expression in mammalian cells. In particular it could not be foreseen by the person skilled in the art that the construct with the maximum possible number of CpG dinucleotides, which however were introduced at the expense of a deterioration of the codon choice, exhibited a significantly stronger expression than the codon-optimised gene.

[0135] Variants of the murine Mip1alpha gene that differ in the number of CpG dinucleotides were synthetically constructed as described in Example 1 and sub-cloned into the expression vector pcDNA3.1 using the interfaces HindIII and NotI. The artificially produced genes were in each case matched as regards their codon choice to the mammalian system. When removing the CpG dinucleotides no rare mammalian codons were used, whereas when inserting CpG dinucleotides above the number of dinucleotides that are achieved with a normal codon adaptation, rare codons were intentionally also employed.

[0136] The constructs that are codon optimised but provided with different numbers of CpG dinucleotides, have throughout a CAI value of more than 0.9 and differ only slightly. The CAI values of the wild-type gene, as well as of the CpG dinucleotide optimised gene (42 CpGs) have on the other hand very low CAI values (below 0.8). According to the prior art a comparable expression of the codon-optimised genes would therefore be expected, though a significantly lower expression of the wild-type gene and of the CpG dinucleotide optimised gene. The identification of the constructs, the number of CpGs as well as the CAI values are given in Table 1. The nucleotide and amino acid sequences are given in SEQ ID NO. 5/6 to SEQ ID NO. 13/14. The analogous expression construct (wild-type reference construct) corresponding to the wild-type sequence was unchanged as regards its CpG distribution.

[0137] The coding region was amplified by means of a polymerase chain reaction (PCR) using the oligonucleotides mamip-1 and mamip-2 from a cDNA clone (obtained from RZPD) and likewise cloned into the expression vector pcDNA3.1 using the interfaces HindIII and NotI (pc-mamip-wt, SEQ ID NO. 15, GenBank Accession Number AA071899).

Checking the Mip1alpha Expression

[0138] In order to quantify the chemokine expression, human H1299 cells were transfected with the respective expression constructs and the amount of protein in the cells and in the cell culture supernatant was measured by means of commercial ELISA test kits.

[0139] 1.510.sup.5 human lung carcinoma cells (H1299) were seeded out in 6-well cell culture dishes and transfected 24 hours later by calcium phosphate precipitation with 15 g of the corresponding expression plasmid. The cells and cell culture supernatant were harvested 48 hours after the transfection. The transfected cells were lysed as described in Example 1 and the total amount of protein of the cell lysate was determined with the Bio-Rad protein assay. Insoluble cell constituents were removed from the cell culture supernatant by centrifugation at 10000 g for 15 minutes at 4 C.

[0140] From 1-5 g total protein from cell lysates as well as from diluted cell culture supernatants, the expression of MiP1alpha was checked in each case in a commercially obtainable ELISA assay (R & D Systems) according to the manufacturer's instructions. The total amount of detectable MiP1alpha correlated with the number of CpGs in the reading frame, in a comparable manner to the data of the GFP expression constructs and p24 expression constructs. The data are summarised in Table 1. The number of constructs permitted for the first time a detailed evaluation of the connection of the level of expression with the number of CpGs within the coding region.

[0141] A representative result of an evaluation by means of cytokine ELISA is shown in FIG. 5. The shaded bars correspond to the mean value of two independent transfection batches, while the empty bars represent the respective standard deviations.

[0142] The relative protein amounts of two independent transient transfection experiments (in double batches) referred to the wild-type construct are listed in Table 1. These results demonstrate a marked reduction of the protein expression with the decrease in CpG dinucleotides and a marked increase compared to the wild-type gene and to the codon-optimised genes, correlating with the additional introduction of such motifs and despite a deterioration of the codon matching.

TABLE-US-00001 TABLE 1 Expression comparison of murine MIP1alpha genes SEQ ID Construct NO. Expression* St. Dev.** CpG No. CAI*** 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 *Percentage mean value of the amount of protein from 2 tests (in double batches) in relation to the total amount of protein of the wild-type construct (maMIP wt) **Standard deviation ***Codon adaptation index

Example 3

[0143] Production of Human and Murine Cytokine Genes with Different CpG Contents

[0144] In order to be able to further confirm the hitherto obtained results and interpretations, variants of the human MIP1alpha gene, of the human GM-CSF gene, of the human IL-15 gene and of the murine GM-CSF gene, which differ in the number of CpG dinucleotides from the wild-type gene, were artificially constructed similarly to Example 2 and sub-cloned into the expression vector pcDNA3.1 using the interfaces HindIII and NotI. The identification of the constructs, number of CpGs as well as the CAI values are given in Table 2. The nucleotide and amino acid sequences of the wild-type sequences (wt) and of the sequences with an altered number of CpG dinucleotides are given in SEQ ID NO. 17/18 to SEQ ID NO. 23/24 and SEQ ID NO. 48/49 to SEQ ID NO. 54/55. The expression constructs were amplified by means of a polymerase chain reaction (PCR) using the oligonucleotides humip-1 and humip-2, hugm-1 and hugm-2, huil-1 and huil-2, magm-1 and magm-2 from corresponding cDNA clones (obtained from RZPD) and were cloned into the expression vector pcDNA3.1, likewise using the interfaces HindIII and NotI (pc-huMiP-wt, GenBank Accession Number NM_021006, pc-huGM-wt, GenBank Accession Number M11220, pc-huIL-wt, GenBank Accession Number BC018149, pc-muGM-wt, GenBank Accession Number NM_049969 with a deviation).

Checking the Cytokine Expression

[0145] In order to quantify the cytokine expression human cells were transfected with the respective expression constructs and the amount of protein in the cell culture supernatant was measured by means of commercial ELISA test kits.

[0146] As described in Example 2, H1299 cells were transfected transiently with 15 g of the corresponding expression plasmid. The cell culture supernatant was harvested for 48 hours after the transfection. Insoluble cell constituents were removed from the cell culture supernatant by centrifugation.

[0147] From dilute cell culture supernatants the expression of human MIP1alpha, human GM-CSF and IL-15 and murine GM-CSF was checked in each case in a commercially obtainable ELISA assay (R & D Systems for MIP1alpha; BD Pharmingen for GM-CSF and IL-15). In a comparable way to the data of the aforementioned expression constructs, the total amount of detectable cytokines in the culture supernatant correlated with the number of CpGs in the reading frame. The data are summarised in Table 2. A representative result of an evaluation by means of cytokine ELISA is shown in FIG. 6. The shaded bars correspond to the mean value of two independent transfection batches, while the empty bars represent the respective standard deviation.

[0148] The relative amounts of protein in each case from a transient transfection experiment (in double batches) referred to the wild-type construct are listed in Table 2. Similarly to the results from Example 2, these results too-confirm a marked increase in protein production, correlating with the additional introduction of such motifs, compared with the wild-type genes.

TABLE-US-00002 TABLE 2 Expression comparison of human cytokine/chemokine genes SEQ ID Construct NO. Expression* CpG No. CAI** 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-huIL 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 *Percentage mean value of the amount of protein from in each case one experiment, in double batches, in relation to the total amount of protein of the corresponding wild-type construct (denoted wt). **Codon adaptation index

Example 4

[0149] Production of a Plasmid with a Reduced Number of CpG Dinucleotides to Increase the Expression

[0150] The nucleic acid sequence of the plasmid pcDNA5 (Invitrogen) was used as a basis for the production of a modularly constructed plasmid in which the number of CpG dinucleotides had been reduced as far as possible. The DNA sequence which codes for the ampicillin resistance gene (bla) was synthetically produced as described in Example 1, and sub-cloned using the restriction interfaces ClaI and BglII. The number of CpGs was in this connection reduced from 72 to 2. Likewise, the multiple cloning site was redesigned, synthetically constructed, and sub-cloned using the restriction interfaces SacI and PmeI, whereby the number of CpGs was reduced from 11 to 1. The CMV promoter (31 CpGs), the BGH polyadenylation site (3 CPGs) and the pUC replication origin (45 CpGs) were integrated unchanged into the plasmid. The hygromycin-resistance cassette was deleted. The CMV promoter was cloned by PCR amplification with the oligonucleotides CMV-1 and CMV-2, which in addition added a ClaI and a SacI restriction interface 3 and 5. In a similar way pUC ori-1 was amplified with the oligonucleotides ori-1 (contains XmaI interface) and ori-2 (contains BglII interface), and the BGH polyadenylation site was amplified with the oligonucleotides pa-1 (PmeI) and pa-2 (XmaI) by PCR, and sub-cloned using the corresponding restriction enzymes. The plasmid pcDNA5 was used as a template in all PCR reactions. The structure of this plasmid is shown diagrammatically in FIG. 7A (P-smallsyn), and the complete sequence is given in SEQ ID NO. 25.

[0151] In order to investigate the influence of the number of CpGs in the vector on the level of expression of a transcript, the reference vector was modified so that it could be used as control. By PCR amplification using the oligonucleotides ref-del-1 and ref-del-2, which in each case introduced a NsiI restriction interface at the 5 end, cleavage with NsiI and ligation, the hygromycin-resistance cassette was removed from the plasmid pcDNA5 (see diagram 6B, Pc-ref).

[0152] The p24 capsid protein derived from HIV-1 was used as test transcript. The coding region of p24 already previously optimised for expression in human cells (Graf et al., 2000) was amplified by means of PCR using the oligonucleotides p24-1 and p24-2 from an HIV-1 syngag construct (Graf et al., 2000) and cloned into the two comparison vectors using the interface HindIII and Bam HI (R/p24 and s/p24).

[0153] Checking the HIV-1 p24 expression in different vector backgrounds

[0154] In order to check the influence of the CpG number in the vector from the expression of the transcript, the constructs R/p24 and s/p24 were transiently transfected into human cells and the expression of p24 was analysed.

[0155] As described in Example 2, H1299 cells were transfected transiently with 15 g of the corresponding expression plasmid. Cells were harvested 48 hours after the transfection. The transfected cells were lysed as described in Example 1 and the total amount of protein in the supernatant was determined with the Bio-Rad protein assay. 50 g of total protein from cell lysates were tested as described in Example 1 in a western blot analysis with a monoclonal p24-specific antibody, 13-5 (Wolf et al., 1990) (FIG. 8). In two independent transfection batches a markedly higher p24 expression was detected after transfection of the smallsyn construct (s/p24).

[0156] Production of HIV p24 genes with different CpG contents Two variants of the capsid protein gene p24 derived from HIV-1, which differ in the number of CpG dinucleotides, were produced. The syn p24 gene had 38 CpGs, whereas the p24CpG gene had no CpGs. The CpG-depleted gene p24CpG was artificially constructed as described in Example 1 and cloned into the expression vector P-smallsyn (described in Example 4) (s/p24CpG) and into the reference vector Pc-ref (R/p24&CpG) using the interfaces HindIII and Bam HI. The nucleotide and amino acid sequences of p24&CpG are given in SEQ ID NO. 26/27. The plasmids R/p24 and s/p24, which are described in Example 4, were used as reference constructs.

Checking the HIV-1 p24 Expression

[0157] In order to check the influence of the CpG number in the vector and in the insert (transcript), the constructs R/p24, R/p24CpG, s/p24 and s/p24CpG were transfected transiently into human cells and the expression of p24 was analysed.

[0158] As described in Example 2, H1299 cells were transiently transfected with 15 g of the corresponding expression plasmid. Cells were harvested 48 hours after the transfection. The transfected cells were lysed as described in Example 1 and the total amount of protein in the lysate was determined with the Bio-Rad protein assay. 50 g of total protein from cell lysates were checked as described in Example 1 in a western blot analysis with a monoclonal p.sup.24-specific antibody 13-5 for the expression of p24 (FIG. 9A). As was already shown in Example 4, the use of the CpG-deleted vector P-smallsyn in the identical transgene led to a visible increase in p24 production (comparison R/p24 and s/p24). Comparably to the data of the GFP and cytokine/chemokine expression constructs, the amount of detectable p24 in the cell lysate, using the identical vector background, correlated with the number of CpGs in the reading frame (comparison R/24 and R/p24CpG as well as s/p24 and s/p24CpG). The data were confirmed in a p24-specific ELISA test (FIG. 9B). The construct with 38 CpGs (p.sup.24) had a ca. 2.5 times (Pc-ref) or ca. 25% (P/smallsyn) larger amount of p24 than the construct without CpGs (p24 DcpG). The results are illustrated in FIG. 9.

[0159] The correlation of the protein production with the number of CpG dinucleotides could be demonstrated in the Examples mentioned here. The selected genes are derived from such different organisms as a jellyfish, a human pathogenic virus, and mammals. It is therefore obvious to regard this mechanism as generally valid. The examples demonstrate furthermore that this correlation in vitro is valid both in the case of a transient transfection as well as in stable recombinant cells. The method described here, namely to alter in a targeted manner the gene expression in eukaryotes by targeted modulation of the CpG dinucleotides, both in the coding region as well as in the vector background, may consequently be used for the production of biomolecules for biotechnological, diagnostic or medical applications. Description of the sequences

TABLE-US-00003 1. Oligonucleotides SEQ ID NO. Identification Sequence 5-3 28 huGFP-1 CAATAAGCTTGCCACCATGGTGAGCAAGGGCG AG 29 huGFP-2 AGTAGGATCCTATTACTTGTACAGCTCGT 30 RT-oligo1 CCCTGAAGTTCATCTGCACC 31 RT-oligo2 GATCTTGAAGTTCACCTTGATG 32 mamip-1 CAGGTACCAAGCTTATGAAGGTCTCCACCACT GC 33 mamip-2 CAGAGCTGGAGTCATGAAGACTAGGCATTCAG TTCCAGGTCAG 34 hugm-1 CAGGTACCAAGCTTATGTGGCTGCAGAGCCT GC 35 hugm-2 CAGAGCTCGAGTCATGAAGACTACTCCTGGAC TGGCTCCCAGC 36 humip-1 CAGTACCAAGCTTATGCAGGTCTCCACTGCT GC 37 humip-2 CAGAGCTCGAGTCATGAAGACTAGGCACTCAG CTCCAGGTCACTG 38 p24-1 ACTAGGTACCATCTAAGCTTATGCCCATCGTG CAGAACATCCA 39 p24-2 TCAAGAGCTCGACTGGATCCTATTACAGCACC CTGGCCTTGTGGC 40 CMV-1 CAAAGGTACCGTTAATCGATGTTGACATTGA TTATTGACTA 41 CMV-2 GAATGAGCTCTGCTTATATAGACC 42 ori-1 GTCACCCGGGTAGTGAATTCATGTGAGCAAA AGGC 43 ori-2 GATCTTTTCTACGGGAGATCTGTCAATCGAT AGCT 44 pa-1 GTTAGAGCTCCAGTGTTTAAACCTGTGCCTTC TAGTTGCCAG 45 pa-2 CAAACCTACCGATACCCGGGCCATAGAGCCC ACCGCATC 46 ref-del-1 TCAGATGCATCCGTACGTTAACATGTGAGCAA AAGGCCAGCA 47 ref-del-2 AGTCATGCATCCATAGAGCCCACCGCATCCC CA 48 hull-1 CAGGTACCAAGCTTATGAGAATTTCGAAACC AC 49 hull-2 CAGAGCTCGAGTCATGAAGACTAAGAAGTGTT GATGAACATTTGG 50 magm-1 CAGGTACCAAGCTTATGGCCCACGAGAGAAAG GC 51 magm-2 CAGAGCTCGAGTCATGAAGACTATTTTTGGCC TGGTTTTTTGC 2. Polypeptide-coding sequences and vector sequences SEQ ID NO. 1 +2: CpG-GFP (nucleic acid +polypeptide) ATGGTGTCCAAGGGGGAGGAGCTGTTCAGAGGGGTGGTGCCCATCCT GGTGGAGCTGGATGGGGATGTGAATGGCCACAAGTTCTCTGTGTGTGG GGAGGGGGAGGGGGATGCCAGCTATGGCAAGCTCACCCTGAAGTTCA TCTGCACCAGAGGCAAGCTGCCAGTGCCCTGGCCCACCCTGGTGACCA CCTTCACCTATGGGGTGCAGTGCTTCAGCAGATACCCAGACCACATGA AGCAGCATGACTTCTTCAAGTCTGCCATGCCTGAGGGCTATGTGCAGG AGAGGACCATCTTCTTCAAGGATGATGGCAACTACAAGACCAGGGCTG AGGTGAAGTTTGAGGGGGATAGCGTGGTGAACAGGATTGAGCTGAAGG GCATTGACTTTAAGGAGGATGGCAATATCCTGGGCCACAAGCTGGAGT ACAACTACAACAGGCACAATGTGTACATCATGGCAGACAAGCAGAAGA ATGGCATCAAGGTGAACTTGAAGATCAGGCACAACATTGAGGATGGCT CTGTGCAGGTGGCAGACCACTACCAGCAGAACACCCCCATTGGAGATG GCCCTGTCCTGGTGCCAGACAACCAGTACCTGAGCAGCGAGTGTGCCC TGAGCAAGGACCCCAATGAGAAGAGGGACCACATGGTGCTGCTGGAG TTTGTGACAGCTGCTGGCATCACCCTGGGCATGGATGAGCTGTACAAG TGA SEQ ID NO. 3 +4: huGFP (nucleic acid +polypeptide) ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCT GGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCG GCGAGGGCGAGGGCGATGCCACCTACGGGAAGCTGACCCTGAAGTTC ATCTGCAGGAGCGGGAAGCTGCCCGTGGCGTGGCCCACCGTCGTGACC ACCTTCACCTACGGCGTGCAGTGGTTCAGCCGCTACCCCGACCACATG AAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAG GAGCGCACCATCTTCTTGAAGGACGACGGCAACTACAAGACCCGCGCC GAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAA GGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGG AGTACAAGTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGA AGAACGGCATGAAGGTGAACTTCAAGATGCGCCACAACATCGAGGACG GCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGC GACGGCCGGGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTC CGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCGTGCT GGAGTTCGTGACCGCCGCCGGGATCACTGTCGGCATGGACGAGCTGT ACAAGTAA SEQ ID NO. 5 +6: murine MIP1alpha-0 CpG (nucleic acid +polypeptide) ATGAAGGTGAGCACAAGAGGTCTGGGTGTGGTGCTGTGTACCATGACC CTGTGCAACCAGGTGTTCTCTGCCCGTTATGGAGCAGATACCCGTACA GCCTGCTGTTTCAGCTAGAGCAGGAAGATCCCCAGGCAGTTCATTGTG GACTACTTTGAGAGGAGGAGCCTGTGTTCTCAGCCTGGGGTGATCTTTC TGACCAAGAGGAACAGGCAGATCTGTGCAGACAGGAAGGAGACATGG GTGCAGGAGTACATCACAGACCTGGAGCTGAATGCCTAG SEQ ID NO. 7 +8: murine MIP1alpha-2 CpG (nucleic acid +polypeptide) ATGAAGGTGAGCAGAACAGCTCTGGCCGTGCTGCTGTGTACCATGACC CTGTGCAACCAGGTGTTCTCTGCCCGTTATGGAGCAGATACGGCTACA GCCTGCTGTTTCAGGTACAGGAGGAAGATGGCGAGGGAGTTGATCGTG GACTACTTTGAGACCAGCAGCCTGTGTTCTCAGCCTGGGGTGATCTTTC TGACCAAGAGGAACAGGCAGATCTGTGCAGACAGCAAGGAGACATGG GTGCAGGAGTACATCACAGACCTGGAGCTGAATGCCTAG SEQ ID NO. 9 +10: murine MIP1alpha-4CpG (nucleic acid +polypeptide) ATGAAGGTGAGCACAACAGCTCTGGCCGTGCTGCTGTGTACCATGACC CTGTGCAACCAGGTGTTCTCTGCCCCTTACGGAGCAGATACCCCTACA GCCTGCTGTTTCAGCTACAGCAGGAAGATCCCCAGGCAGTTCATCGTG GACTACTTTGAGACCAGCAGGCTGTGTTCTCAGCCTGGGGTGATCTTTC TGACCAAGAGGAACCGCCAGATCTGTGCAGACAGCAAGGAGACATGG GTGCAGGAGTACATCACAGACCTGGAGCTGAATGCCTAG SEQ ID NO. 11 +12: murine MIP1alpha-13CpG (nucleic acid +polypeptide) ATGAAGGTGAGCACCACAGCTCTGGCTGTGCTGCTGTGCACCATGACC CTGTGCAACCAGGTGTTCAGCGCTCCTTACGGCGCCGATACCCCTACA GCCTGCTGCTTCAGCTACAGCAGGAAGATCCCCAGGCAGTTCATCGTG GACTACTTCGAGACCAGCAGCCTGTGTTCTCAGCCCGGCGTGATCTTC CTGACCAAGCGGAACAGACAGATCTGCGCCGACAGCAAGGAGACATG GGTGCAGGAGTACATCACCGACCTGGAGCTGAACGCCTAG SEQ ID NO. 13 +14: murine MIP1alpha-42 CpG (nucleic acid +polypeptide) ATGAAGGTGTCGACGACCGCGCTCGCCGTGCTGCTGTGCACGATGAC GCTGTGCAACCAGGTGTTCAGCGCCCCGTACGGCGCCGACACGCCGA CCGCGTGCTGCTTCTCGTACTCGCGGAAGATCCCGCGGCAGTTCATCG TCGACTACTTCGAAACGTCGTCGCTGTGCTCGCAGCCCGGCGTGATCT TCCTCACGAAGCGGAACCGGCAGATCTGCGCCGACTCGAAGGAAACG TGGGTGCAGGAGTACATCACCGACCTCGAACTGAACGCGTAG SEQ ID NO. 15 +16: murine MIP1alpha wild-type (7 CpG) (nucleic acid +polypeptide) ATGAAGGTCTCCACCACTGCCCTTGCTGTTCTTCTCTGTACCATGACAC TCTGCAACCAAGTCTTCTCAGCGCCATATGGAGCTGACACCCCGACTG CCTGCTGCTTCTCCTACAGCCGGAAGATTCCACGCCAATTCATCGTTGA CTATTTTGAAACCAGCAGCCTTTGCTCCCAGCCAGGTGTCATTTTCCTG ACTAAGAGAAACCGGCAGATCTGCGCTGACTCCAAAGAGACCTGGGTC CAAGAATACATCACTGACCTGGAACTGAATGCCTAG SEQ ID NO. 17 +18: human MIP1alpha-43CpG (nucleic acid +polypeptide) ATGCAAGTGTCGACCGCCGCTCTCGCCGTGCTGCTGTGCAGGATGGC GCTGTGCAACCAAGTGCTGAGCGCGCCTCTCGCCGCCGACACGCCGA CCGCGTGCTGCTTCTCGTACACGTCGCGGCAGATCCCGCAGAACTTCA TCGCCGACTACTTCGAGACGTCGTCGCAGTGCTCGAAGCCGAGCGTGA TCTTCCTGACGAAGGGCGGACGGCAAGTGTGCGCCGACCCGAGCGAG GAGTGGGTGCAGAAGTACGTGAGCGACCTCGAACTGAGCGCGTAG SEQ ID NO 19 +20: human GM-CSF-63CpG (nucleic acid +polypeptide) ATGTGGCTGCAGTCGCTGCTGCTGCTCGGAACCGTCGCGTGTTCGATC AGCGCGCCTGCGCGGTCGCCGTCGCCGTCGACGCAGCCGTGGGAGC ACGTGAACGCGATCCAGGAGGCGCGACGGCTGCTGAACCTGTCGCGC GATACAGCCGCCGAGATGAACGAGACCGTCGAGGTGATCAGCGAGAT GTTCGACCTGCAGGAGCCGACGTGCCTGCAGACGCGGCTCGAACTGT ATAAGCAGGGCCTCCGCGGCTCGCTCACGAAGCTGAAGGGCCCGCTC ACGATGATGGCGTCGCACTACAAGCAGCACTGCCCGCCGACGCCCGA AACGTCGTGCGCGACGCAGATCATCACGTTCGAGTCGTTCAAGGAGAA CCTGAAGGACTTCCTGCTCGTGATCCCGTTCGATTGCTGGGAGCCCGT GCAGGAGTAG SEQ ID NO. 21 +22: human MIP1alpha wild-type (8CpG) (nucleic acid +polypeptide) ATGCAGGTCTCCACTGCTGCCCTTGCCGTCCTCCTCTGCACCATGGCT CTCTGCAACCAGGTCCTCTCTGCACCACTTGCTGCTGACACGCCGACC GCCTGCTGCTTCAGCTACACCTCCCGACAGATTCCACAGAATTTCATAG CTGACTACTTTGAGACGAGCAGCCAGTGCTCCAAGCCCAGTGTCATCT TCCTAACCAAGAGAGGCCGGCAGGTCTGTGCTGACCCCAGTGAGGAG TGGGTCCAGAAATACGTCAGTGACCTGGAGCTGAGTGCCTAG SEQ ID NO. 23 +24: human GM-CSF wild-type (10CpG) (nucleic acid +polypeptide) ATGTGGCTGCAGAGCCTGCTGCTCTTGGGCACTGTGGCCTGCAGCATC TCTGCACCCGCCCGCTCGCCCAGCCCCAGCACGCAGCCCTGGGAGCA TGTGAATGCCATCCAGGAGGCCCGGCGTCTCCTGAACCTGAGTAGAGA CACTGCTGCTGAGATGAATGAAACAGTAGAAGTCATCTCAGAAATGTTT GACCTCCAGGAGCCGACCTGCCTACAGACCCGCCTGGAGCTGTACAA GCAGGGCCTGCGGGGCAGCCTCACCAAGCTCAAGGGCCCCTTGACCA TGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACCCCGGAAACTT CCTGTGCAACCCAGATTATCACCTTTGAAAGTTTCAAAGAGAACCTGAA GGACTTTCTGCTTGTCATCCCCTTTGACTGCTGGGAGCCAGTCCAGGA GTAG SEQ ID NO. 25: P-smallsyn (nucleic acid sequence of the plasmid) ATCGATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACG GGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTA CGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTG ACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCC ATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGT ACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGAC GGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGAC TTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGT GATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTC ACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTT TGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCC CATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAA GCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCTA AATTAATACGACTCACTATAGGGAGACCCAAGCTGTTAAGCTTGGTAGA TATCAGGGATCCACTCAGCTGATCAGCCTCCAGTTTAAACCTGTGCCTT CTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGAC CCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATT GCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTG GGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGC TGGGGATGCGGTGGGCTCTATGGCCCGGGTAGTGAATTCATGTGAGCA AAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGC GTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACG CTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGC GTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCGTGCC GCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCT TTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGC TCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTG CGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGA CTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAG GTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGG CTACACTAGAAGGACAGTATTTGGTATCTGCTCTCTGCTGAAGCCAGTT ACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACC GCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGA AAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGAGATCTGT CTGACTCTGAGTGGAACCAAAACTCATGTTAAGGGATTTTGGTCATGAG ATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTT TTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGGTTAACT TACCAATGCTTAATCAATGAGGCACCAATCTCTGCAATCTGCCTATTTCT CTCATCCATGGTTGCCTGACTGCCTGTGGTGTAGATAACTACAATCCTG GAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCTCTAGACCCT CTCTCACCTGCTCCAGATTTATCTGCAATGAACCAGCCAGCTGGAAGG GCAGACCTGAGAAGTGGTCCTGCAACTTTATCTGCCTCCATCCAGTCTA TTAATTGTTGTCTGGAAGGTAGAGTAAGCAGTTCACCAGTTAATAGTTT CCTCAAGGTTGTTGCCATTGCTACAGGCATGGTGGTGTCCCTCTCATCA TTTGGTATGGCTTCATTCAGCTCTGGTTCCCATCTATCAAGCCTAGTTA CATGATCACCCATGTTGTGCAAAAAAGCAGTCAACTCCTTTGGTCCTCC AATGGTTGTCAAAAGTAAGTTGGCAGCAGTGTTATCACTCATGGTTATG GCAGCACTGCATAATTCTCTTACTGTCATGCCATCTGTAAGATGCTTTTC TGTGACTGGACTGTACTCAACCAAGTCATTCTGAGAATAGTGTATTCTT CTACCCAGTTGCTCTTGCCCAGCATCAATTCTGGATAATACTGCACCAC ATAGCAGAACTTTAAAGGTGCTCATCATTGGAAATCTTTCTTCTGGTCTA AAACTCTCAAGGATCTTACCAGAGTTGAGATCCAGTTCAATGTAACCCA CTCTTGGACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGGGTTTCT GGGTGAGCAAAAACAGGAAGGCAAAAGGCAGCAAAAAAGGGAATAAG GGCAACTCTGAAATGTTGAATACTCATAGTACTACTCTTCCTTTTTCAAT ATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTT GAATGTATTTAGAAAAATAAACAAATAGGGGTATGCATTCAGCTCACAT TTCCCTGAAAAGTGCCACCTGAAATTGACTGATAGGGAGTTCTCCCAAT CCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCTCATAGTTAA GCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCACTGAGTAGTGGG CTAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCTACAATTGCAT GAAGAATCTGCTTAGGGTTAGGCCTTTTGCACTGCTTGGAGATGTACTG GCCAGATATACTA SEQ ID NO. 26 +27: p24CpG (nucleic acid +polypeptide) ATGGTGCACCAGGCCATCAGCCCCAGGACCCTGAATGCCTGGGTGAA GGTGGTGGAGGAGAAGGCCTTCAGCCCTGAGGTGATCCCCATGTTCTC TGCCCTGTCTGAGGGGGCCACCCCCCAGGACCTGAACACCATGCTGAA CACAGTGGGGGGCCACCAGGCTGCCATGCAGATGCTGAAGGAAACCA TCAATGAGGAGGCTGCTGAGTGGGACAGAGTGCACCCTGTGCATGCTG GCCCCATTGCCCCTGGCCAGATGAGGGAGCCCAGGGGCTCTGACATT GCTGGCACCACCTCCACCCTGCAGGAGCAGATTGGCTGGATGACCAAC AACCCCCCCATCCCTGTGGGGGAGATCTACAAGAGATGGATCATCCTG GGCCTGAACAAGATTGTGAGGATGTACAGCCCCACCTCCATCCTGGAC ATCAGGCAGGGCCCCAAGGAGCCCTTCAGGGACTATGTGGACAGGTT CTACAAGACCGTGAGGGCTGAGCAGGCCAGCCAGGAGGTGAAGAACT GGATGACAGAGACCCTGCTGGTGCAGAATGCCAACCCTGACTGCAAGA CCATCCTGAAGGCCCTGGGCCCAGCTGCCACCCTGGAGGAGATGATG ACAGCCTGCCAGGGGGTGGGAGGCCCTGGCCACAAGGCCAGGGTGCT GTAA SEQ ID NO. 52 +53: human IL-15-21CpG ATGCGGATCAGCAAGCCCCACCTGAGGAGCATCAGCATCCAGTGCTAC CTGTGCCTGCTGCTGAACAGCCACTTCCTGACAGAGGCCGGCATCCAC GTGTTTATCCTGGGCTGCTTCTCTGCCGGCCTGCCTAAGAGAGAGGCC AACTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATC CAGAGCATGCACATCGACGCCACCCTGTACACAGAGAGCGACGTGCAC CCTAGCTGTAAGGTGACCGCCATGAAGTGCTTCCTGCTGGAGCTGCAG GTGATCAGCCTGGAGAGCGGCGATGCCAGCATCCACGACACCGTGGA GAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAATGT GACCGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGGAGAAGAACA TCAAGGAGTTCCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAA CACCAGCTAG SEQ ID NO. 54 +55: murine GM-CSF-62CpG ATGTGGCTGCAGAACCTGCTGTTCCTCGGCATCGTCGTGTACTCGCTG AGCGCGCCGACGCGCTCGCCGATCACCGTGACGCGGCCGTGGAAGCA CGTCGAGGCGATCAAGGAGGCGCTGAACCTGCTCGACGACATGCCCG TGACGCTGAACGAGGAGGTCGAGGTCGTGTCGAACGAGTTCTCGTTCA AGAAGCTGACGTGCGTGCAGACGCGGCTGAAGATCTTCGAGCAGGGC CTGCGCGGCAACTTCACGAAGCTGAAGGGCGCGCTGAACATGACCGC GTCGTACTACCAGACGTACTGCCCGCCGACGCCCGAGACCGATTGCGA GACGCAGGTGACGACGTACGCCGACTTCATCGACTCGCTGAAGACGTT CCTGACCGACATCCCGTTCGAGTGCAAGAAGCCCGGCCAGAAGTAG SEQ ID NO. 56 +57: human IL-15 wild-type (3CpG) ATGAGAATTTCGAAACCACATTTGAGAAGTATTTCCATCCAGTGCTACTT GTGTTACTTCTAAACAGTCATTTTCTAACTGAAGCTGGCATTCATGTCT TCATTTTGGGCTGTTTCAGTGCAGGGCTTCCTAAAACAGAAGCCAACTG GGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTA TGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTG CAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCA CTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCA TCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGG ATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTG CAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCTTAG SEQ ID NO. 58 +59: murine GM-CSF-wild-type (11CpG) ATGTGGCTGCAGAATTTACTTTTCCTGGGCATTGTGGTCTACAGCCTCT CAGCACCCACCCGCTCACCCATCACTGTCACCCGGCCTTGGAAGCATG TAGAGGCCATCAAAGAAGCCCTGAACCTCCTGGATGACATGCCTGTCA CATTGAATGAAGAGGTAGAAGTCGTCTCTAACGAGTTCTCCTTCAAGAA GCTAACATGTGTGCAGACCCGCCTGAAGATATTCGAGCAGGGTCTACG GGGCAATTTCACCAAACTCAAGGGCGCCTTGAACATGACAGCCAGCTA CTACCAGACATACTGCCCCCCAACTCCGGAAACGGACTGTGAAACACA AGTTACCACCTATGCGGATTTCATAGACAGCCTTAAAACCTTTCTGACT GATATCCCCTTTGAATGCAAAAAACCAGGCCAAAAATAG

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