IL92803A - Synthetic antisense oligodeoxynucleotides or phosphorothioate oligodeoxynucleotides as modulators for selective bone marrow cells development and pharmaceutical compositions containing the same - Google Patents

Description

oay nin >NJI ¾w j i>upt?O mnnann!? o noN!?niaD οτηκ ο ηπ mnpin «Dm SYNTHETIC ANTISENSE OLIGODEOXYNUCLEOTIDES OR PHOSPHOROTHIOATE OLIGODEOXYNUCLEOTIDES AS MODULATORS FOR SELECTIVE BONE MARROW CELLS DEVELOPMENT AND PHARMACEUTICAL COMPOSITIONS CONTAINING THE SAME This invention was made with support by the U.S. Army Medical Research and Development Command under Contract No. DAMD-17-87-C-7169 to Hermona Soreq. The U.S. Army has certain rights in the invention.

FIELD OF THE INVENTION The invention relates to synthetic oligodeoxynucleotides and phosphorothioate oligodeoxynucleoties capable of selectively modulating bone marrow cells development. The invention also relates to pharmaceutical preparations for modulating bone marrow cells development comprising as active ingredient at least one oligodeoxynucleotide or phosphorothioate oligo-deoxynucleotide of the invention.

Various publications are referenced throughout this application by Arabic numerals in parantheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures in these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as at the date of the invention described and claimed herein.

BACKGROUND OF THE INVENTION The CHE and ACHE genes encoding the acetylcholine hydro-lyzing enzymes butyrylcholinesterase (BuChE , E.C. 3.1.1.8) and acetylcholinesterase (AChE, E.C. 3.1.1.7) are expreseed in various developing cell types , including embryonic (1), haematopoietic (2) and germ cells (3,4).

Biochemical and histochemical analyses indicate that both AChE and BuChE are expressed in high levels in various fetal tissues of multiple eukaryotic organisms (5), where cholin-esterases are coordinately regulated with respect to cell proliferation and differentiation (6). However, no specific role could be attributed to ChE in embryonic development and their biological function(s) in these tissues remained essentially unknown.

In addition to its presence in the membranes of mature erythrocytes, AChE is also intensively produced in developing blood cells in vivo (7) and in vitro (8) and its activity serves as an acceptable marker for developing mouse megakaryocytes (2). Furthermore, administration of acetylcholine analogues as well as ChE inhibitors has been shown to induce megakaryocytopoiesis and increased platelet counts in the mouse ( 9 ) , implicating this enzyme in the commitment and development of these haematopoietic cells.

Recently, the DNA coding for BuChE has been cloned (10) and the human CHE1 locus has been mapped (11) to the 3q21-ter chromosomal domain that is subject to aberrations in leu-kemias accompanied by abnormal megakaryocytopoiesis and platelet counts (12). Co-amplification of the ACHE and CHE genes was subsequently observed in leukemias and platelet disorders (13).

Cholinergic mechanisms have been implicated in megakaryocytopoiesis (18), and AChE is recognized as a specific marker of murine megakaryocytes (2). However, the involvement of BuChE in megakaryocyte development has never yet been demonstrated. The presence of this enzyme in bone marrow cells has now been directly revealed.

Antisense oligodeoxynucleotides to viral (20) or cellular (21) mRNAs have been shown to be capable of arresting translation of their corresponding mRNAs in cultured cells. When targeted against growth-related genes, such as c-myc, they also block cell proliferation in haematopoietic cell lines (22). Recently, a synthetic COOH-terminal PF4 peptide was demonstrated to inhibit growth of a human megakaryocytic cell line (23). However, selective modulation of a single haematopoietic lineage in a mixed population of primary bone marrow cells has now been first demonstrated.

While little is known about the molecular mechanisms regulating megakaryocytopoiesis in vivo , a variety of multi-lineage and lineage-specific native and recombinant growth factors have been shown to stimulate megakaryocytopoiesis (24-27). Nonetheless, no lineage-specific factor has yet been purified capable of driving this process. Abnormal megakaryocytopoiesis, resulting in platelet overproduction (thrombocytosis) or depletion (thrombocytopenia) is implicated in a number of debilitating bleeding disorders (28).

The inventors have now established selective modulation of bone marrow cells development by synthetic oligodeoxynucleotides .

SUMMARY OF THE INVENTION The invention relates to synthetic antisense oligodeoxy-nucleotides and phosphorothioate oligodeoxynucleoties capable of selectively modulating bone marrow cells development. The term modulating as used herein refers to selective inhibition and/or stimulation of megakaryocytopoiesis in bone marrow cells.

More particularly, the invention relates to oligodeoxy-nucleotides capable of inhibiting megakaryocytopoiesis in bone marrow cells, directed against human BuChEmRNA. Preferably, the invention relates to an antisense oligo-deoxynucleotide directed against the region spanning the initiator AUG in human BuChEmRNA, comprising at least the sequence: ( 3 ' ) TAC GTA TCG TTT CAG ( 5 ' ) The term oligodeoxynucleotides of the invention as used herein encompasses oligodeoxynucleotides having either phosphate bonds or phosphorothioate bonds linking between the nucleotide bases.

The invention also relates to pharmaceutical compositions for the modulation of bone marrow cells development comprising as active ingredient of at least one antisense oligodeoxynucleotides of the invention, in a pharmaceutically acceptable carrier, optionally also comprising additional pharmaceutically acceptable ingredients .

DESCRIPTION OF THE FIGURES Figure la shows the structure of the oligodeoxynucleotides of the invention in relation to the region spanning the initiator AUG in human BuChEmRNA.

Figure lb shows differential cell analysis of antisense oligodeoxynucleotide treated, semi-solid bone marrow cultures.

Inset shows colony counts.

Figure 2 shows BuChE-OCM augmentation of megakaryocyto- poiesis .

Inset shows colony counts.

Figure 3 shows the influence of BuChE-OCM on the morphology of haematopoietic cells.

DETAILED DESCRIPTION OF THE INVENTION The invention relates to synthetic antisense oligodeoxynucleotides capable of selectively modulating bone marrow development. The term modulating as used herein refers to selective inhibition and/or stimulation of megakaryocytopoiesis in bone marrow cells.

The invention more particularly relates to antisense oligodeoxynucleotides capable of inhibiting megakaryocytopoiesis in bone marrow cells, directed against a region spanning the initiator AUG in human BuChEmRNA. Still more specifically, the invention relates to an antisense oligodeoxynucleotide directed against a region spanning the initiator AUG in human BuChEmRNA, comprising at least the sequence : ( 3 ' ) TAC GTA TCG TTT CAG ( 5 ' ) Phosphorothioate- and phosphate-containing sense (S-ChE0) and antisense (AS-ChE<£) 15-mer oligodeoxynucleotides can be synthesized, for example, by using an Applied Biosystem 380B DNA synthesizer, as will be described in more detail in the following Examples.

To assess the etiological involvement of BuChE in megakaryo-cytopoiesis, cultured murine bone marrow cells were incubated with an antisense oligodeoxynucleotide (AS-ChE0) (Fig. la) targeted to BuChEmRNA (which may be obtained according to (10)) which was found to block translation of recombinant BuChEmRNA in microinjected Xenopus oocytes (14) up to 80% (not shown). Cells were incubated in methylcellulose supplemented with a low-protein medium (LPM), synthetic serum substitute and conditioned medium from WEHI-3 cells (WEHI), a rich source of the multilineage haematopoietic growth factor interleukin 3 (IL-3) (15). As will be shown in the Examples, following 4 days in culture, AS-ChE0 was found to reduce the total number of colonies (Fig. lb, inset) and the relative proportion of megakaryocytes in the culture population (Fig. lb). In addition, megakaryocyte colonies grown in the presence of AS-chE<#> appeared smaller than controls (not shown in the Figures). A stable phosphorothioate analogue of AS-ChE0 (16) elicited a similar depression of colony formation in the cultures (Fig. lb, inset), while both phosphate and phosphorothioate complementary sense oligodeoxynucleotides (S-ChE , Fig. la) had no effect on either colony counts or their cellular composition (Fig. lb).

When incubation medium from Xenopus oocytes microinjected with recombinant BuChEmRNA (BuChE-OCM) (14) was added to bone marrow cultures, colony formation in a variety of semi-solid and liquid culture conditions was significantly increased (Table I). Conditioned medium from control oocytes injected with Barth's saline solution (Brt-OCM) also elicited an enhancement of colony formation, However the Brt-OCM effect was significantly smaller than that observed with BuChE-OCM (P<0.025-0.001 ) (Table I). Furthermore, differential cell count attributed the increase in colony forming units in BuChE-OCM treated cultures to enhanced megakaryocyte proliferation, while the increase in Brt-OCM treated cultures appeared in the macrophage population (Fig. 2a). A substantial increase in the number of late as opposed to early megakaryocytes was observed in BuChE-OCM treated cultures as compared with both control and Brt-OCM treated cultures. This shift was accompanied by a significant increase in the average diameter of megakaryocytes (Fig. 3), suggesting that BuChE-OCM played a specific role in megakaryocyte maturation. In both horse serum (HS ) -containing and HS-free media, cultures deprived of WEHI-3 conditioned medium gave rise to colonies which were small, poorly developed and which disintegrated after day 3, indicating that the oocyte conditioned medium (OCM) affects megakaryocyte colony formation by synergizing with the IL-3 in WEHI. Substitution of recombinant murine purified IL-3 for WEHI confirmed this conclusion (Table I).

BuChE-OCM exerted a more pronounced enhancement effect in methylcellulose cultures supplemented with 15% horse serum, a rich source of tetrameric BuChE (17). Given the negligible BuChE enzymatic activity contributed by the oocyte conditioned medium in these cultures (P<.01% of serum contribution) (14), it appears unlikely that the stimulating factor is catalytically active BuChE.

In order to directly examine whether BuChE is expressed in murine megakaryocytes, cytochemical cholinesterase staining of megakaryocytes grown in LPM liquid cultures was performed. Activity staining was pronounced and appeared sensitive to inhibition by both the AChE-specific inhibitor BW284C51 (1,5-bis (allyldimethyl-airaioniumphenyl ) -pentan-3-one dibromide) and the BuChE-specific inhibitor iso-OMPA (tetra isopropylpyrophosphoramide) (Fig. 3, panel c), indicating the presence of both emzymes and directly revealing the previously undetected presence of BuChE in these cells.

As stated in the introduction, abnormal megakaryocytopoiesis, resulting in platelet overproduction (thrombocytosis) or depletion (thrombocytopenia) is implicated in a number of debilitating bleeding disorders. The invention is related to oligodeoxynucleotides capable of modulating megakaryocytopoiesis . AS-ChE0 oligonucletides, for example the oligodeoxynucleotide 31 TAC GTA TCG TTT CAG 5 ' , may be used to arrest thrombocytosis or the abnormal proliferation of other cell types, dependent on the ChE gene expression. BuChE-OCM as defined above, obtained, for example, with Xenopus oocytes, or other cultured cells, such as eukaryotic cells, may be used to stimulate megakaryocytopoiesis in cases of thrombocytopenia.

The invention also relates to pharmaceutical compositions for the modulation of bone marrow cell development comprising as active ingredient of at least one antisense oligodeoxynucleotides of the invention, in a pharmaceutically acceptable carrier, optionally also comprising additional pharmaceutically acceptable ingredients.

More specifically, the pharmaceutical preparation of the invention may comprise as active ingredient of at least one antisense oligodeoxynucleotides capable of inhibiting megakaryocytopoiesis according to the present invention. Such may be the antisense oligodeoxynucleotides directed against a region spanning the initiator AUG region in human BuChEmRNA, for example 3 ' TAC GTA TCG TTT CAG 5 ' .

The invention will now be described in more detail on hand of the following examples.

Passages in the description and following examples which are not within scope of the claims do not consitute part of the claimed invention.

EXAMPLES ' Example 1 Inhibition of megakaryocytopoiesis by antisense ChE oligo-deoxynucleotide (a) 01jgodeoxynuc1eotides Phosphorothioate (P ) and phosphate (P) containing sense (S-ChE0) and antisense (AS-ΟιΕφ) 15-mer oligodeoxynucleo-tides were synthesized using an Applied Biosystem 380B DNA synthesizer. The phosphate oligodeoxynucleotides were synthesized by the phosphoramidite method, the phosphoro-thioates by the H-phosphonate method. All were purified by reverse phase HPLC, the phosphorothioate oligodeoxy-nucleotide before and after removal of the dimethoxytrityl group. The region spanning the initiator AUG in BuChEmRNA (5) was confirmed to be potentially effective in the examined final concentration of 5 /xM (expressed as oligomer concentration) on the basis of its ability to block translation of synthetic BuChEmRNA when coinjected in this concentration into Xenopus oocytes. The oligodeoxynucleotides are shown in Fig. 1(a). (b) Differential cell analysis of antisense oliaodeoxy-nucleotide treated, semi-solid bone marrow cultures Bone marrow cells from the femore and tibia of 8-12 week-old endotoxin-resistant C3H/HeJ mice were cultured in LPM synthetic medium (Biological Industries, Beit HaEmek, Israel) containing 10% conditioned medium for WEHI-3 cells, 1% BSA, -4 5 10 M thioglycerol and 1% methylcellulose. 0.5-1.0 x 10 cells/ml were plated in 35 mm petri dishes (Nunc 1008) (Ρ_), or 24 well tissue culture Costar plates (P experiments), and incubated 4 days at 37° C under 5% C02 with high humidity.

Oligodeoxynucleotides, at a final concentration of 5 iM, were added upon initiation of cultures.

Colonies grown in serum-free methylcellulose cultures containing sense or anti-sense ChE oligodeoxynucleotide (P ) were picked with drawn-out Pasteur pipettes, concentrated (5 min at 500xg) by Cytospin (Shandon, 2) centrifugation in phosphate buffered saline (PBS), stained with May-Grunwald Giemsa and analyzed microscopically. The relative fraction of each cell type represented among the total cells recovered from two independent experiments are shown in Fig. 1(b). The distribution obtained with S-ChE0 was essentially identical to that observed in control (no oligo) cultures. At least 500 cells were counted for each data set.

The total number of colonies observed in a Zeiss Stereozoom binocular after 4 days in the presence of P_ or P oligo-deoxynucleotides are plotted, in the inset of Fig. 1, as percent of control (no oligo) cultures. Data represent average of 2 (P ) or 3 (P) independent experiments ± Standard Evaluation of the Mean (S.E.M.).

Example 2 Augmentation of megakaryocytopoiesis by BuChE-OCM (a) Preparation of BuChE-OCM Mature female Xenopus laevis frogs were fed commercially available frog brittle (Nasco, WI). Oocytes were surgically removed and manually teased from the ovarian connective tissues using fine-tipped forceps in Barth's medium (80 mM NaCl, 1 mM KCl, 0.3 mM Ca(N03)2.4H20, 1.6 mM MgS04.7H20, 2.4 mM NaHC03, 2 mM Tris, 400 mg/ml streptomycin sulphate, 400 mg/liter penicillin G; titrated to pH 8.5).

RNA was prepared as follows: The cDNA clone encoding human serum BuChE (10) and containing approximately 70 3' -terminal adenylate residues was subcloned into the pSP 64 transcription vector (Promega Biotek) essentially according to (30). Construct preparation is described in detail in (14). In vitro transcription and 5' capping were carried out using commercially available kits from Amersham Corp. and following the enclosed mannual. Both 5' capping and 3' polyadenylation were found to be essential for efficient translation of the synthetic mRNA. Poly(A)+RNA from fetal human brain and muscle was prepared as detailed in (31) and its in vitro and in vivo translatability verified as previously described in (32,33). mRNA microinjection was carried out according to (29), using 50 nl of 0.1 mg/ml synthetic mRNA. (b) Augmentation of meoakarvocvtopoiesis A representative differential cell analysis (1 of 3 experiments) of colonies picked from serum-fortified methyl-cellulose cultures is shown in Fig. 2. Cultures were grown with or without addition of 0.5% incubation medium from Xenopus oocytes injected with synthetic BuChEmRNA (BuChE-OCM), or Barth's saline solution (Brt-OCM) . Early megakaryocytes, defined as immature forms with one or two nuclei, were distinguished from late ones, characterized by their larger size and tendency to shed cytoplasmic fragments. There was a relative increase in late-stage megakaryocytes upon addition of BuChE-OCM, and increase in macrophages in Brt-OCM containing cultures.

Total number of colonies observed after 3 days incubation is plotted, in the inset of Fig. 2, as precentage of control (no OCM) cultures ± S.E.M. (n=8 independent experiments; triplicate cultures per experiment).

Example 3 Influence of BuChE-OCM on the morphology of haematopoietic cells.

Liquid cultures were grown in LPM ± BuChE-OCM and cells were picked and stained as in Example 1 and photographed in a Zeiss Axioplan microscope equipped with an HCIOO camera. Results are shown in Fig. 3.

Figs. 3a, 3b: Representative microscope fields. (a) - Control cells. Heterogeneity of cellular morphologies should be noted. (b) - Megakaryocyte-enriched BuChE-OCM treated culture. The presence of multicell megakaryocyte colony (bottom) and large megakaryocytes (top) should be noted.

Fia. 3c: Cytochemical staining of megakaryocytes for cholin-esterase activity.

Fixed cells were stained for cholinesterase activity by indolyl acetate (15) in the absence of inhibitors (top), in the presence of 10 M of 1,5-bis (4-allyldimethylammonium-phenyl)-pentan-3-one dibromide (BW284C51), a specific AChE inhibitor (second), or tetraisopropylpyrophsphoramide (iso-OMPA) , a selective BuChE inhibitor (third), or both inhibitors (bottom) . There was partial sensitivity of staining to both inhibitors, indicating the presence of both AChE and BuChE in murine megakaryocytes.

Fig. 3d; Stimulation of megakaryocyte growth by BuChE-OCM.

Percent of cells in each size range (5 /im intervals) are plotted for BuChE-OCM and Brt-OCM treated cultures. 60-70 cells were counted for each culture. Average diameter of megakaryocytes grown in the presence of BuChE-OCM (18.8 jm ± 0.63 S.E.M.) was found to be significantly larger (P<0.025) than those grown in the presence of Brt-OCM (14.4 /xm ± 0.85 S.E.M. ), an increase comparable to that observed in the in vivo response to the stimulus of thrombocytopenia on megakaryocyte growth (26).

The effect of oocyte conditioned media on haematopoietic colony formation in semi-solid methylcellulose cultures is summarized in Table I .

Average total number of colonies per culture dish ± S.E.M. (as a fraction of control culture on first day counted) is shown for various culture media with and without the addition of conditioned medium (0.5%, V:V) from Xenopus oocytes injected with synthetic BuChEmRNA (BuChE-OCM), or Barth's saline solution (Brt-OCM) and incubated for the indicated number of days at 37 "C. Bone marrow cells were cultured in 35 mm petri dishes as described in Example 1. OCM (1:200) was added upon initiation of culture. Oocyte microinjections were as described (9,13); HS/WEHI - 15% horse serum (Gibco), 10% conditioned medium from WEHI-3 cells, 10 M thioglycerol (Sigma), and 1% methylcellulose (DOW, A4M Premium) made up in Iscove's modification of Dulbecco's medium (I DM) (Gibco); HS/IL-3 - as above with 20 jLiml purified recombinant murine 11-3 replacing WEHI conditioned medium; LPM/WEHI - LPM synthetic serum substitute containing 10% conditioned medium from WEHI-3 cells, 1% deionized bovine serum albumin (BSA) (Sigma, Fraction V) , -4 10 M thioglycerol and 1% methylcellulose. Colonies cultured in HS-containing media were larger, developed faster and contained a higher portion of megakaryocytes than those grown on LPM. Therefore, each type of experiment was evaluated separately. Statistical significance (P) was calculated by Students t-test vs. no OCM controls. Numbers in parantheses represent range of colonies counted.

TABLE I Medium DAY Exots. No OCM •BuChE-OCM +Brt-OCM HS/WEHI 8 1.00+/O.03 1.81+/0-06 1.24±0.09 (59-276) P<0.001 p<0.001 4 11 1.16+/-0.07 2.03+/-O.12 1.50+/-0.08 (72-252) p<0.001 p<0.001 HS/IL-3 1.00+/0.10 2.02+/-0.06 1.25+/-0.05 (61-277) p<0.008 p<0.085 1.69+/0.05 2.34+/-0.17 1.90+/-0.30 (122-276) p<0.01 p<0.085 LPM/WEHI 1.00 +/0.07 1.43+/-0.04 1.28+/ (221-386) p<0.007 p<0.

REFERENCES : 1. Layer, P.G. and Sporns, 0., Proc. Natl. Acad. Sci. USA, 84:284-288 (1987). 2. Burstein, S.A. , et al., J. Cell Physiol., 122:159-165 (1985) . 3. Johnson, CD. , et al., Neuron, 1:165-173 (1988). 4. Malinger, G., et al., Mol. Neurosci., 1:77-84 (1989). 5. Rakonczay, Z., et al., Subcellular Biochemistry, 12:335- 378, Harris, J.R., Ed., Plenum Press, N.Y. (1988). 6. Layer, P.G., et al., J. Nerurochem. , 49:175-182 (1987). 7. Paulus, J. P., et al., Blood, 58:1100-1106 (1981). 8. Burstein, S.A., et al., J. Cell Physiol., 103:201-208 (1980). 9. Burstein, S.A., et al., Clin. Haematol., 12:3-27 (1983). 10. Prody, C, et al., Proc. Natl. Acad. Sci. USA, 84: 3555- 3559 (1987). 11. Soreq, H. , et al., Hum. Genet., 77:325-328 (1987). 12. Pintado, T., et al., Cancer, 55:535-541 (1985). 13. Lapidot-Lifson, Y., et al., Proc. Natl. Acad. Sci. USA, 86:4715-4717 (1989). 14. Soreq, H., et al., J. Biol. Chem. , 264:10608-10613 (1989) . 15. Stanley, E.R., et al., Cell, 45:667-674 (1986). 16. Eckstein, F., Ann. Rev. Biochem. , 54:367-402 (1985). 17. Whittaker, M. , Cholinesterases . Monographs in Human Genetics, Vol. 11, (Ed. R.S. Sparkes) (Karger , Bazel, 1986). 18. Burstein, S.A., et al., J. Cell Physiol., 54:201-208 (1980) . 19. Rotshenker, S. and Tal, M. , J. Physiol., 360:387-396 (1985) . 20. Matsukura, M. , et al., Proc. Natl. Acad. Sci. USA, 84: 7706-7710 (1987).

Zheng, H., et al., Proc. Natl. Acad. Sci. USA, 86:3758-3762 (1989).

Wickstrom, E.L., et al., Proc. Natl. Acad. Sci. USA, 85: 1028-1032 (1988).

Gewirtz, A.M.', et al., J. Clin. Invest., 83:1477-1486 (1989) .

Williams, N., et al., Blood Cells, 15:123-133 (1989). Hill, R.J. and Levin, J., Blood Cells, 15:141-166 (1989).

Bruno, E., et al., Exp. Hematol., 16:371-377 (1988). Ishibashi, . , et al., Proc. Natl. Acad. Sci. USA, 86: 5953-5957 (1989).

Gewirtz, A.M., Semin. Hematol., 23:27-42 (1986).

Soreq, H. , et al., Embo J. 3:1371-1375 (1984).

Krieg, P.A. and Melton, D.A., Nucleic Acids Res., 12: 7057-7070 (1984).

Prody, C, et al., J. Neurosci. Res., 16:25-35 (1986). Soreq, H. et al., Mol. Cell Neurobiol., 6:227-237 (1986) .

Mollgard, K . , et al. , Dev. Biol., 128:207-221 (1988).

Claims (7)

92803/4 CLAIMS:

1. Synthetic antisense oligodeoxynucleotide or phosphoro- thioate oligodeoxynucleotide to human BuChmRNA capable of selectively modulating bone marrow cells development.

2. Synthetic antisense oligodeoxynucleotides according to claim 1 being antisense oligodeoxynucleotides directed against a region spanning the initiator AUG in human BuChEmRNA, having either phosphate bonds or phosphoro- thioate bonds linking between the nucleotides bases.

3. A synthetic oligodeoxynucleotide according to claim 2 having the formula: ( 3 ' ) TAC GTA TCG TTT CAG ( 5 · ) .

4. Synthetic antisense oligodeoxynucleotides according to claims 1 to 3 as herein exemplified in Example 1.

5. Pharmaceutical compositions for modulating bone marrow cells development comprising as active ingredient a pharmaceutically effective amount of at least one anti- sense oligodeoxynucleotide according to any one of claim 1 to 4.

6. Pharmaceutical compositions according to claim 5 for inhibiting megakaryocytopoiesis .

7. Pharmaceutical compositions according to claim 6 wherein said active ingredient is an oligodeoxynucleotide according to claim 3.