United States Patent (19) 11 Patent Number: 5,891,725 Soreq Et Al

USOO5891725A United States Patent (19) 11 Patent Number: 5,891,725 Soreq et al. (45) Date of Patent: Apr. 6, 1999

54 SYNTHETIC ANTISENSE Gnatt et al., “Expression of alternatively terminated unusual OLIGODEOXYNUCLEOTIDES AND human butyrylcholinesterase meSSenger RNA PHARMACEUTICAL COMPOSITIONS transcripts. . . " Cancer Research, 50:1983–1987, (1990). CONTAINING THEM Iyer et al., “The automated Synthesis of Sulfur-containing oligodeoxyribonucletodies . . . . J. Org. Chem., 55: 75 Inventors: Hermona Soreq, Rishon Le Zion; 4693-4699 (1990). Haim Zakut, Savyon, both of Israel; Lapidot-Lifson et al., “Cloning and antisense oligodeoxy Fritz Eckstein, Gottingen, Germany nucleotide inhibition of a human homolog . .” Proc. Natl. Acad. Sci., USA, vol.89, pp. 579–583, (1992). 73 Assignee: Yissum Research Development Co. of Lapidot-Lifson et al., “Coamplification of human acetyl the Hebrew Univ. of Jerusalem, cholinesterase and butyrylcholinesterase genes in blood Jerusalem, Israel cells . . . . Proc. Natl. Acad. Sci., USA, Vol. 86, pp. 21 Appl. No.: 318,826 4715-4719 (1989). Layer et al., “Quantitative development and molecular forms 22 PCT Filed: Apr. 15, 1993 of acetyl-and butyrylcholinesterase . . 'Journal of Neuro 86 PCT No.: PCT/EP93/00911 chemistry, 49: 175-182 (1987). Lee and Nurse, “Complementation used to clone a human S371 Date: Dec. 1, 1994 homologue of the fission yeast cell cycle control gene cdc2” S 102(e) Date: Dec. 1, 1994 Nature, 327:31–35 (1987). Loke et al., “Characterization of oligonucleotide transport 87 PCT Pub. No.: WO93/21202 into living cells” Proc. Natl. Acad. Sci., USA, 86:3474-3478 PCT Pub. Date: Oct. 26, 1993 (1989). Malinger et al., “Cholinoceptive properties of human pri 30 Foreign Application Priority Data mordial, preantral, and antral oocytes. . 'J. Mol. NeurOSci., Apr. 15, 1992 IL Israel ...... 1.01600 1:77–84 (1989).

51 Int. CI. . ... C07H 21/04; C12O 1/68 (List continued on next page.) 52 U.S. Cl...... 435/372; 435/6; 435/91.1; 435/1723; 435/325; 435/372; 435/375; Primary Examiner John L. LeGuyader 536/23.1; 536/24.31; 536/24.5; 514/44 Attorney, Agent, or Firm-Kohn & ASSociates 58 Field of Search ...... 536/24.5, 23.1, 57 ABSTRACT 536/24.31, 24.33; 435/91.1, 6, 172.3, 240.2, 375, 377; 935/8, 34, 76, 78; 514/44 The invention relates to Synthetic phosphorothioated or partially phosphorothioated oligodeoxynucleotides capable 56) References Cited of Selectively modulating hemopoietic bone marrow cells development. More particularly, the invention relates to U.S. PATENT DOCUMENTS Synthetic antisense oligodeoxynucleotides directed against a 5,225,326 7/1993 Bresser et al...... 435/6 region Spanning the AUG initiation condon in human ACHE or 2HS genes, having phosphorothioate bonds linking OTHER PUBLICATIONS between the nucleotide bases, Synthetic antisense oligode B. Monig et al., JBC, vol. 267(28) (Oct. 5, 1992) 19954–62. Oxynucleotides directed against a region Spanning the AUG E. Uhlmann et al., Chem. Rev. 90 (4) (Jun. 1990) 543–84. initiation condon in human ACHE gene, 2HS gene or BCHE P. Westermann et al., Biomed Biochim. Acta 48(1) (89) gene or against a 5'-region in the human CHED (cdc2 85-93. homolog) gene, having phosphorothioate bonds linking W. James, Antiviral Chem. & Chemother. 2(4) (91) between the four 3'-terminus nucleotide bases. The inven 191-214. tion also relates to pharmaceutical compositions comprising J. Holt et al., Mol. Cell. Biol. 8(2) (Feb. 1988) 963–73. as active ingredient at least one Synthetic phosphorothioated D. Tidd, Anticancer Res. 10 (90) 1169-82. or partially phosphorothioated antisense oligodeoxynucle J. Milligan et al., J. Med. Chem. 36(14) (Jul. 9, 1993) otides according to the invention in physiologically accept 1923-37. able carrier in diluent. The antisense oligodeoxynucleotides C. Stein et al, Science 261 (Aug. 20 1993) 1004-1012. of the invention and the pharmaceutical compositions con B. T. Seang et al., Cancer Gene Therapy 1(1) (Mar. 1994) taining them are Suitable for inhibiting abnormal hemopoi 65-71. etic cells proliferation, inhibiting abnormal platelet J. Wetmer, Crit. Rev. in Biochem. Mol. Biol. 26(3/4) (91) proliferation, increasing Stem cell fraction in bone marrow 227-59. cell cultures, enhancing macrophage production and increas Baker et al., “Effects of oligo Sequence and chemistry on the ing Stem cells counts, reducing immune response of organs efficiency of oligodeoxyribonucleotide-mediated mRNA to be transplanted, Suppressing immune response of a recipi cleavage' Nucleic Acids Research, vol. 18, No. 12, p. 3537 ent of an allotransplanted organ, and arresting growth of (1990). malignant tumors. Caceres and Kosik, “Inhibition of neurite polarity . . . ss Nature, 343:461–463 (1990). 11 Claims, 15 Drawing Sheets 5,891,725 Page 2

OTHER PUBLICATIONS Calabretta et al., “Normal and leukemic hematopoietic cells manifest differential sensitivity to inhibitory effects of Agarwal et al., “Pharmacokinetics, biodistribution, and Sta bility of oligodeoxynucleotide phosphorothioates in mice’ c-myb antisense . * Proc. Natl. AcadSci, USA, Proc. Natl. Acad. Sci. USA, 88:7595–7599 (1991). 88:2351-2355 (1991). Akhtar et al., “Interactions of antisense DNA oligonucle Chiang et al., “AntiSense oligonucleotides inhibit intercel otide analogs with phospholipid membranes (liposomes)” lular adhesion molecule 1 expression . . . . J. Biol. Chem., Nucleic Acids Research, vol. 19, No. 20 pp. 5551-5559 (1991). 266:18162–18171 (1991). Moffat, “Making sense of antisense” Science, 253:510 Cooney et al.,"Site-specific oligonucleotide binding (1991). represses transcription of the human c-myc gene in vitro' Morrison, “Suppression of basic fibroblast growth factor Science, 241:456 (1988). expression by antisense oligodeoxynucleotides . . . J. Biol. Chem., 266:728 (1991). Eckstein, “Nucleoside phosphorothioates' Ann. Rev. Bio Owens and Bunge, “Schwann cells infected with a recom chem, 54:367-402 (1985). binant retrovirus expressing myelin-associated Eguchi, “Antisense RNA” Ann. Rev. Biochem., 60:631-52 glycoprotein . . .” Neuron, 7:565-575 (1991). (1991). Patinkin et al., “Manipulations of cholinesterase gene expression modulate murine megakaryocytopoiesis in vitro' Galileo et al., “Retrovirally introduced antisense integrin Molecular and Cellular Biology, 10:6046-6050 (1990). RNA inhibits neuroblast migration in Vivo”. Neuron, Pieken et al., “Kinetic characterization of ribonuclease-r- 9:1117–1131 (1992). esistant 2'-modified hammerhead ribozymes' Science, Haseloff and Gerlach, “Simple RNA enzymes with new and 253:314 (1991). activities' Prody et al., “Isolation and characterization of full-length highly specific endoribonuclease Nature, cDNA clones coding for cholinesterase from fetal human 334:585-591 (1988). tissues” Proc. Natl. Acad. Sci. USA, 84:3555–3559 (1987). Layer and Sporns, "Spatiotemperoal relationship of embry Rakonczay and Brimijoin, "Biochemistry and pathophysi onic cholinesterases with cell proliferation in chicken . . . ' ology of the molecular forms of cholinesterases” in Subcel lular Biochemistry, edited by J.R. Harris, Plenum Publish Proc. Natl. Acad.Sci. USA, 84:284–288 (1987). ing, vol. 12, p. 335 (1988). Leonetti et al., “Intracellular distribution of microinjected Shaw et al., “Modified deoxyoligonucleotides stable to antisense oligonucleotides' Proc. Natl Acad.Sci. USA, exonuclease degradation in Serum' Nucleic Acids Research, 88:2702–2706 (1991). 19:747–750 (1991). Soreq et al., “Molecular cloning and construction of the Moffat, “Making sense of antisense” Science, 253:510-511 coding region for human acetylcholinesterase . . . Proc. (1991). Natl. Acad. Sci. USA, 87:9688–9692 (1990). Moffat, “Triplex DNA finally comes of age” Science, Soreq et al., “Expression and tissue-specific assembly of 252:1374-1375 (1991). human butyrylcholine esterase . ” J. Biol. Chem., 264:10608–10613 (1989). Moreno and Nurse, “Substrates for p34°: in vivo veritas?” Weinstein et al., “Suppression by antisense mRNA demon Cell, 61:549–551 (1990). Strates a requirement for the glial fibrillary acidic protein' J. Cell. Biol., 112:1205 (1991). Nielsen et al., “Sequence-selective recognition of DNA by Whitesell et al., “Episome-generated n-myc antisense RNA Strand displacement with a thymine-Substituted polyamide' restricts the differentiation potential . . " Molecular and Science, 254:1497–1500 (1991). Cellular Biology, 11:1360–1371 (1991). Rangini et al., “CHox E, a chicken homeogene of the H2.0 Woolf et al., “The stability, toxicity and effectiveness of type exhibits dorso-ventral restriction . . . 'Mechanisms of unmodified and phosphorothioate antisense oligodeoxy Development, 35:13–24 (1991). nucleotides . .” Nucleic Acids Research, 18:1763 (1990). Yakubov et al., Mechanism of oligonucleotide uptake by Sarver et al., “Ribozymes as potential anti-HIV-1 therapeu cells: involvement of specific receptors? Proc. Natl. Acad. tic agents” Science, 247: 1222–1225 (1990). Sci. USA, 86:6454–6458 (1989). Blake et al..."Hybridization arrest of globin synthesis in Spitzer and Eckstein, “Inhibition of deoxyribonucleases by rabbit riticulocyte lysates and cells Biochem., phosphorothioate groups in oligodeoxyribonucleotides’ 24:6139–6145 (1985). Nucleic Acids Res., 16:11691–11704 (1988). Burstein et al., “Quantitation of megakaryocytopoiesis in Szczylik et al., “Selective inhibition of leukemia cell pro liquid culture by enzymatic determination of acetylcho liferation by BCR-ABL antisense oligodeoxynucleotides’ linesterase” J. Cell. Physiol., 122:159-165 (1985). Science, 253:562–565 (1991). Burstein et al., “Megakaryocytopoiesis in culture: modula tion by cholinergic mechanisms' J. Cell. Physiol., Wilkinson et al.,"Expression of the proto-oncogene int-lis 103:201–208 (1980). restricted to specific neural cells...” Cell, 50:79–88 (1987). U.S. Patent Apr. 6, 1999 Sheet 1 of 15 5,891,725

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5,891,725 1 2 SYNTHETIC ANTISENSE Mutation Research, in press (1992)). The hemopoietic sys OLIGODEOXYNUCLEOTIDES AND tem thus appears to be Subject to developmental control as PHARMACEUTICAL COMPOSITIONS affected by the expression of the ChEs. CONTAINING THEM Inhibition of the expression of developmentally important genes should, in principle, divert developmental processes to This application is a 371 of PCT/EP93/00911, filed Apr. directions which are not dependent on the expression of 15, 1993. these genes. One useful approach to affect Such develop mental processes is based on “antisense' technology. The BACKGROUND OF THE INVENTION use of oligonucleotides to intervene in the genetic processes The BCHE and ACHE genes encoding the acetylcholine of the cell bears an important therapeutic potential. Thus, hydrolyzing enzymes butyrylcholinesterase (BuChE, EC “informational drugs with possible clinical applications are 3.1.1.8) and actylcholinesterase (AChE, EC 3.1.1.7) are being developed which arrest the expression of cloned expressed in various developing cell types, including embry genes. The hematopoietic System may be the first logical onic Layer, P. G. and Sporns, O., Proc. Natl. Acad. Sci. USA target for novel therapy protocols based on the recent 15 achievement in genetic engineering, Since it includes pro 84.284-288 (1987), hematopoietic Burstein, S.A., et al., J. liferating Stem cells and because of its extreme Sensitivity to Cell Physiol. 122:159-165 (1985) and germ cells Johnson, external stimuli Wilson, J. D., et al., Harrison's Principles C. D., et al., Neuron 1:165-173 (1988); Malinger, G., et al., of Internal Medicine, 12th Ed., McGraw-Hill, Inc., New Mol. Neurosci. 1:77–84 (1989). York, Chapters 268-269; 285–288 (1991)). Stem cells may Both AChE and BuChE include the peptide motif S/T-P- be defined as cells which can replicate repeatedly and X-Z, which makes-them potential Substrates for phosphory differentiate into various kinds of committed cells. Commit lation by calc2 kinases, the general controllers of the cell ment will gradually limit the differentiation choices for cells cycle Lapidot-Lifson, Y., et al., Proc. Natl. Acad. Sci., USA in which it occurs, until precursor cells are formed with only 89: 579–583 (1992)). Most other substrates of cdc2 kinases perform biological functions necessary for cell cycle-related one choice (i.e. erythrocytes, megakaryocytes or 25 macrophages). The first stem cells can thus be defined as processes Moreno, S. and Nurse, P., Cell 61:549-551 totipotent, i.e. they may make all of the choices in the blood (1990)). Thus, interference with either CHE or cdc2 tran and immune System. The orientation of Such cells into Scription processes may be expected to divert and/or arrest desired direction should be most useful in overcoming cell division, and controlling these processes can be useful undesired changes, Such as depletion or excess of Specific for Several, medically important, procedures. Subpopulations of hemopoietic cells. LeSS, although also Biochemical and histochemical analyses indicate that useful is the redirection of the more limited pluripotent Stem both AChE and BuChE are expressed, in high levels, in cells. Stem cells account for s0.1% of cells in bone marrow. Various fetal tissues of multiple eukaryotic Species They can be detected either directly, by immunocytochemi Rakonczay, Z., et al., Subcellular Biochemistry cal methods, or retroactively, by cell culture growth and 12:335-378, Harris, J. R., Ed., Plenum Press, N.Y. (1988), 35 Subsequent evaluation of formed colonies. For therapeutic where cholinesterases (ChEs) are coordinately regulated purposes, it would be desirable to control stem cells differ with respect to cell proliferation and differentiation Layer, entiation and cause totipotent and pluripotent Stem cells to P. G., et al., Neurochem. 49:175-182 (1987). The specific replicate. role to be attributed to ChEs in embryonic development may Production rate of bone marrow cells in healthy individu hence be related with cell division, so that their biological 40 als may reach 10' platelets and differentiated blood cells function(s) in these tissues are tentatively implicated in the per hour. Life span of these cells varies from years for Some control of organogenesis. lymphocytes, 120 days for erythrocytes to 10 days for In addition to its presence in the membranes of mature platelets and 10 hours for neutrophils. Changes in the erythrocytes, AChE is also intensively produced in devel Subpopulations of hemopoietic Stem cells may be found in oping blood cells in vivo Paulus, J. P., et al., Blood 45 patients Suffering malignant myeloproliferative diseases, 58:1100–1106 (1981) and in vitro Burstein, S.A., et al., J. Such as various leukemias etc., in blood cells proliferative Cell Physiol. 103:201-208 (1980) and its activity serves as diseases Such as polycythemia Vera etc., and in autoimmune an acceptable marker for developing mouse megakaryocytes diseases like lupus erythomatosus etc., in which the blood Burs- tein (1985) ibid.). Furthermore, administration of production System is defective. Defective hemopoiesis is acetylcholine analogues as well as cholinesterase inhibitors 50 further observed in patients undergoing various commonly has been shown to induce megakaryocytopoiesis and used therapeutical treatments like chemotherapy and irra increased platelet counts in the mouse Burstein, S.A., et al., diation which impair the blood production system. Thus all Clin. Haematol. 12:3-27 (1983)), implicating this enzyme in cancer patients, individuals following tissue transplantation the commitment and development of these haematopoietic and others who have Suffered poisoning by chemicals and/or cells. 55 different drugs, display abnormal hemopoiesis with its Sub The DNAS coding for human BuChE and AChE have Sequent consequences. The therapeutical value of the ability been cloned Prody, C., et al., Proc. Natl. Acad. Sci., USA to modulate hemopoietic cell division in patients Suffering 86:3555–3559 (1987); Soreq et al., Proc. Natl. Acad. Sci., any of the above pathological conditions is Self-evident. USA87:9688–9692 (1990) and the human CHEl locus has Furthermore, modulation of hemopoietic cell division, and been mapped Gnatt, A., et al., Cancer Res. 50:1983-1987 60 especially increasing the number of hemopoietic Stem cells, (1990) to the 3q26-ter chromosomal domain that is subject may be of particular advantage for the clinical procedure of to aberrations in leukemias accompanied by abnormal mega bone marrow transplantation. Techniques are already avail karyocytopoiesis and platelet counts Pintado, T, et al., able for freezing bone marrow cells and for their Subsequent Cancer 55:535-541 (1985)). Co-amplification of the ACHE transplantation in patients Suffering any of the above patho and BCHE genes was Subsequently observed in leukemias 65 logical conditions. However, the only transplanted bone and platelet disorders Lapidot-Lifson, Y., et al., Proc. Natl. marrow cells which can Survive in the recipient and prolif Acad. Sci., USA 4715–4717 (1989); Zakut, H., et al., erate to improve his/hers condition are stem cells. The above 5,891,725 3 4 procedure may be autotransplantation, using the recipient's bose phosphate backbone of DNA) were shown to cause own bone marrow, or allotransplantation, using bone mar displacement of their complementary Strands from double row from a compatible donor. stranded DNA Nielsen et al., Science 254:1497 (1991)). AS mentioned above, cholinergic Signals are implicated in These newly designed drugs will Selectively interrupt gene the commitment and development of haematopoietic cells. function without affecting the transcript products. In Recently, preliminary evaluation of antisense oligonucle contrast, ribozyme Sequences were shown to Specifically otides incorporation in Vivo was performed which revealed interact with the mRNA transcripts. These are ribonucleic the short term therapeutic applicability of this approach acid Sequences, including RNase active sites flanked by Cohen, et al., Antisense Res. & Dev. 2:191 (1991)). Anti antisense oligonucleotides Haseloff and Gerlach, Nature Sense oligonucleotides of 15-20 bases are usually long 3:585 (1988). When targetted to the human immunodefi enough to ascertain that they will only have one comple ciency virus (HIV) they destroy HIV mRNA effectively mentary Sequence in the mammalian genome. In addition, Sarver et al., Science 247: 1222 (1990). However, oligori they hybridize well with their target mRNA Cohen et al., bonucleotides are more difficult to synthesize than ibid.). Modification of the phosphodiester backbone renders oligodeoxynucleotides, particularly in chemically modified these oligonucleotides resistant to degradation by nucleases 15 forms resistant to RNase attacks Pieken et al., Science Spitzer, F. and Eckstein, F., Nuc. Ac. Res. 16: 11691-11704 253:314 (1991)). (1988). Both methylphosphonate and phosphorothioate Phosphorothioate antisense oligonucleotides do not show groups were used for this purpose Baker, C., et al., Nuc. Ac. Significant toxicity and exhibit Sufficient pharmacodynamic Res. 18:3537 (1990)). Being nonionic, the methyl half-lives in animals Agrawal, S., et al., Proc. Natl. Acad. phosphonate analogs were predicted to exhibit increased Sci. USA 88:7595 (1991)). Antisense induced loss-of cellular uptake Blake, et al., Biochem. 24:6139 (1985)). function phenotypes related with cellular development were However, antisense methylphosphonate oligomers were shown for the glial fibrillary acidic protein (GFAP), impli shown to be incapable of inhibiting N-ras expression in vitro cated in astrocyte growth within astrocyte-neuron cocultures Tidd, et al., Anti-Cancer Drug Design 3:117 (1988) Winstein et al., J. Cell Biol., 112:1205 (1991)), for the whereas the in vitro translation of Several oncogene mRNAS 25 myelin-associated glycoprotein in Schwann cells, respon was Successfully blocked by phosphodiester and/or phoS sible for formation of the compact myelin sheath formation phorothioate antisense oligonucleotides c-myc: McMan surrounding these cell Owens and Bunge, Neuron 7:56 away et al., Lancet 335:808 (1990), Watson et al., Cancer (1991), for the microtubule-associated tau proteins impli Res. 51:3996 (1991);bcl-2: Reed et al., Cancer Res. 50:6565 cated with the polarity of hippocampal neurons and their (1990); myb: Calabrett et al., Proc. Natl. Acad. Sci. USA axon formation Caceres and Kosik, Nature 343:461 (1990) 88:2351 (1991); bcr-ab: Szczylik et al., Science 253:562 , for the f-integrin, important for neuronal migration along (1991). Both Sense and nonsense oligonucleotides served as radial glial cells, and for the establishment of tectal plate controls in these Studies and were shown to be non-effective, formation in chick Galileo et al., J. Cel. Biol. 112:1285 while antisense oligonucleotides Selectively inhibited their (1991) and for the N-myc protein, responsible for the target gene expression and phosphorothioate oligonucle 35 maintenance of cellular heterogeneity in neuroectodermal otides were more potent because of their greater Stability cultures (ephithelial vs. neuroblastic cells, which differ in Woolf, T. M., et al., Nuc. Ac. Res. 18:1763 (1990)3. their colony forming abilities, tumorigenicity and AntiSense oligonucleotides are able to interfere Specifi adherence) Rosolen et al., Cancer Res. 50:6316 (1990); cally with synthesis of the target protein of interest Moffat, Whitesell et al., Mol. Cell. Biol 11:1360 (1991)). Antisense Science 253:510 (1991)). This may occur by inhibition of 40 oligonucleotide inhibition of basic fibroblast growth factor polySome formation and/or functioning, according to the (bFgF), having mitogenic and angiogenic properties, Sup position of the antisense oligonucleotide within the target pressed 80% of growth in glioma cells Morrison, J. Biol. mRNA. Thus, the frequent choice of the Sequence Surround Chem. 266:728 (1991) in a saturable and specific manner. ing the translation initiation codon as target for antisense The antisense oligonucleotides were targetted against the oligonucleotide inhibition aims to prevent the formation of 45 initiation and splice sites in bFgFmRNA, they reduced the initiation complex. Indeed, antisense RNAS occur natu activity of the resulting protein and Sense oligomers rally as regulators of translation Eguchi et al., Ann. Rev. remained inactive. In Soft-agar cultures, antisense oligo Biochem. 60:631 (1991). Other mechanisms of antisense nucleotides reduced the size of glial colonies and induced oligonucleotide inhibition involve activation of ribonuclease appearance of larger cells within them R. Morrison, Neu H, which Subsequently performs digestion of the antisense 50 roscience Facts 3: 3 (1992): bFGF expression in human oligonucleotide-mRNA hybrids Chiang, M. Y., et al., J. glioma cells). Biol. Chem. 266:18162 (1991)), or interference with splic Being hydrophobic, antisense oligonucleotides interact ing through antisense oligonucleotides targeted to mRNA well with phospholipid membranes Akhtar, S., et al., Nuc. splice sites Kole et al., Adv. Drug Deliv. Rev. 6:271 (1991)). Res. 19:5551-5559 (1991)). Following their interaction with In addition to their mRNA targets, antisense oligonucle 55 the cellular plasma membrane, they are actively transported otides are also complementary to the genomic Sequences into living cells (Loke, S. L., et al., Proc. Natl. Acad. Sci. expressing these mRNAS. When injected into cultured cells, USA86:3474 (1989)), in a saturable mechanism predicted to they accumulate within nuclei Leonetti, J. P., et al., Proc. involve specific receptors Yakubov, L.A., et al., Proc. Natl. Natl. Acad. Sci. USA 88:2702 (1991)), suggesting that they Acad. Sci. USA 86:6454 (1989)). may also function by interfering with transcription through 60 AntiSense inhibition of key molecules involved in Signal formation of a third DNA strand, associated by Hoogsteen transduction processes may De expected to interfere also base pairing with the major groove of the B-form DNA with Secondary mechanisms depending on the targetted key duplex Moffat, Science 252:1374 (1991)). In vitro tran molecule. Thus, cholinergic signaling through the m mus Scriptional arrest of c-myc expression was shown to operate carinic acetylcholine receptor is coupled to pertussis toxin by this mechanism in a cell-free System cooney et al., 65 Sensitive G proteins and adenylyl cyclase activity. The Science 241: 456 (1988). Recent polyamide nucleic acid gamma-aminobutyric type B receptor (GABA) is similarly oligomers (with polyamide backbone replacing the deoxyri coupled to this signal transduction process, and both recep 5,891,725 S 6 tors are expressed in cerebellar granular neurons. AntiSense malignant tumors, for Selectively arresting the rapid cell oligonucleotides to the m receptor mRNA blocked com division characteristic of the tumor tissue, while not inter pletely the Synthesis of this receptor within 3 days, and fering with the benign process of cell division within cells reduced the GABA receptor by 40% within 6 days. It Surrounding the tumor. remains to be shown whether this latter effect was due to the In particular, the invention relates to Synthetic oligode conserved oligo Sequence being present also in the yet oxynucleotides being antisense oligodeoxynucleotides uncloned GABA receptor, or whether the delay effect was secondary to m inhibition Morrison R., ibid.). directed against a region Spanning the AUG initiation codon In view of the above-mentioned implication of cholin in human ACHE (acetylcholinesterase) or 2HS (cdc2kinase) ergic Signals in the commitment and development of hae genes, having phosphorothioate internucleotidic bonds matopoietic cells, it is an object of the present invention to between all of the nucleotides or between only the four provide for compounds and methods capable of diverting the 3'-terminus nucleotides and to Synthetic oligodeoxynucle process of cholinergic Signalling, which may direct bone otides according being antisense oligodeoxynucleotides marrow Stem cells into continued replication and re-orient directed against the region Spanning the AUG initiation their Subsequent differentiation, both in culture and in Vivo, codon in human BCHE (butyrylcholinesterase) gene or a into mononuclear cells of the hemopoietic and immune 15 5'-region in the CHED (cdc2 homolog) gene, having phos System. phorothioate internucleotidic bonds between the four Thus, the ChES related antisense oligodeoxynucleotides 3'-terminus nucleotides. of the present invention appear to be potent candidates for Still more particularly the invention relates to a Synthetic the modulation of bone marrow cells development described antisense oligodeoxynucleotide directed against a region above. This adds to the effects of already characterized Spanning the initiator AUG codon in the human 2HS gene growth factors, Such as the granulocyte colony Stimulating having the formula: (SEQ ID NO:1) factor (G-CSF), interleukin 3, 6 and 11, Lif (leukemia 5'-GGTATAATCTTCCAT3' inducing factor) and the recently described stem cell factor, which interacts with the receptor produced from the C-kit 25 having phosphorothioate internucleotidic bonds between all protoncogene. the nucleotides (AS2HS-T) or between the four 3'-terminus nucleotides (AS 2HS-S); SUMMARY OF THE INVENTION to a Synthetic antisense oligodeoxynucleotide directed The invention relates to Synthetic phosphorothioated or against a region spanning the initiator AUG codon in partially phosphorothioated oligodeoxynucleotides capable the human ACHE gene SEQ ID NOS: 5–7 having the of Selectively modulating hemopoietic bone marrow cells formula: (SEQ ID NO:2) development. The term modulating as used herein refers to Selective inhibition and/or Stimulation of megakaryocyto poiesis and/or erythropoiesis in bone marrow cells and additionally to Selective diversion of hemopoietic bone having phosphorothioate internucleotidic bonds between all marrow Stem cells development from megakaryocytes and/ 35 the nucleotides (AS ACHE-T) or between the four or erythrocytes to macrophages and mononuclear cells. 3'-terminus nucleotides (AS ACHE-S); More particularly, the invention relates to Synthetic phos to a Synthetic antisense oligodeoxynucleotide directed phorothioated or partially phosphorothioated oligodeoxy against a region spanning the initiator AUG codon in nucleotides capable of inhibiting or Stimulating megakaryo the human BCHE gene SEQ ID NOS: 8–9 having the cytopoiesis and/or erythropoiesis and of diverting 40 formula: (SEQ ID NO:3) hemopoietic bone marrow Stem cells development from megakaryocytes and erythrocytes to macrophages and other 5'-GACTTTGCTATGCAT3' mononuclear hemopoietic cells. having phosphorothioate internucleotidic bonds between the The oligodeoxynucleotides of the invention are capable of four 3'-terminus nucleotides (AS BCHE-S); and modulating hemopoietic bone marrow Stem cells develop 45 to a Synthetic antisense oligodeoxynucleotide directed ment in vitro. Such cells can be cells extracted from patients against a 5'-region in the human CHED gene having the in need of transplantation. The oligodeoxynucleotides of the formula: (SEQ ID NO:4) invention can effectively influence the cell composition of 5'-TTTTCCCCAGTCAAT-3' the culture until the desired cell composition is reached, 50 and/or can increase the number of viable Stem cells in the having phosphorothioate internucleotidic bonds between the culture. four 3'-terminus nucleotides (AS CHED-S). The oligodeoxynucleotides of the invention can also The invention also relates to pharmaceutical or medical modulate cell division properties in organs or tissues to be compositions comprising as active ingredient at least one of transplanted, in order to improve the procedure and decrease 55 the oligonucleotides of the invention, in a physiologically or tissue rejection following the transplantation procedure. medically acceptable carrier, optionally also comprising The oligodeoxynucleotides of the invention can also be additional physiologically acceptable additives. The active administered to potential donors of bone marrow or organs, ingredient may consist of one of the oligodeoxynucleotides prior to the donation procedure, in order to enrich the or of mixture?s thereof. hemopoietic bone marrow fraction of Specific Stem cells of 60 More particularly the compositions of the invention may the hemopoiectic System. be suitable for the modulation of hemopoietic bone marrow The oligodeoxynucleotides of the invention can also be Stem cells development. These compositions may inhibit applied to embryonic or fetal bone marrow cells, prior to abnormal hemopoietic cells proliferation. Also, the compo their Storage in cell banks in order to retain Such cells in Sitions may be used to enhance macrophage production and viable forms devoid of tissue compatibility antigens. 65 increase Stem cell counts. Furthermore, the oligodeoxynucleotides of the invention Still more particularly the compositions of the inventions can also be effective in the treatment of patients with certain may be Suitable for in vitro modulating cell division prop 5,891,725 7 8 erties in organs or tissues to be transplanted in order to DESCRIPTION OF THE FIGURES decrease immune response and resulting tissue rejection following the transplantation procedure. The compositions FIG. 1 Transcription vectors for ChERNAS. may also be used for in vitro increasing Stem cell fraction in S-labeled RNA transcripts composed of the “sense” bone marrow cells to be transplanted. (mRNA) or “antisense” (cRNA) strands complemen Furthermore, the compositions of the invention may be tary to the cDNAs encoding AChE and BuChE were used for treating embryonic or fetal bone marrow cells prior prepared as detailed under Material and Methods (4) to their storage in cell banks in viable forms devoid of tissue from the pGEM-7Z(+) and Bluescript SK(+) plasmids, compatibility antigens, comprising as active ingredient the using T7, SP6 or T3 RNA polymerases. Informative oligodeoxynucleotides of the invention, in a pharmaceuti restriction sites are noted for the cDNA inserts and their cally acceptable carrier, optionally also comprising addi boundary polylinker domains. The labeled RNA prod tional physiologically acceptable agents. The active ingre ucts were further subjected to controlled alkaline dient may consist of one of the oligodeoxynucleotides or of hydrolysis to produce sufficiently shortened probes for mixture?s thereof. in situ hybridization. Additionally, the compositions of the invention may be 15 FIG. 2A Titration curves of IL-treated murine bone used for treatment of organ donor and recipient, prior to the marrow cells cultures with S or Ts oligodeoxynucleotides donation procedure, to decrease the immune response in (Antisense-AS; Sense-S). procedures of allotransplantation, and for the treatment of Bone marrow cells were grown in methyl cellulose/LPM potential bone marrow donors, prior to the extraction of the (Beit Haemek), 1% BSA (Sigma) and 10% WEHI-CM, bone marrow, in order to enrich the hemopoietic bone a source of IL-3. They were incubated at 37 C. and 5% marrow fraction of Specific Stem cells of the hemopoietic CO at a cell concentration of 10 cells/ml. AS- and System. S-oligodeoxynucleotides were prepared at Stock con Still further, the compositions of the invention may be centrations of 2-4 mM made in 10 mM Tris +1 mM used for treating patients with malignant tumors, Selectively EDTA, pH 7.5, and were kept frozen at -20° C. until arresting cell division in the tumor tissue, but not in the 25 use. They were subsequently diluted in PBS to 100 uM benign cells Surrounding the tumor. These compositions and added to cultures at time 0 to give final concen may be particularly Suitable for the treatment of chondro trations of 2.5-10 uM. The oligodeoxynucleotides were SCO.S. retained in the culture throughout the experiment. The invention also relates to methods of modulating Colonies were scored on Day 4 with a Zeiss stereo hemopoietic bone marrow Stem cells development in Zoom microscope equipped with an optic fiber dark patients in need of Such treatment by administering to the field device. patient a therapeutically effective amount of the oligode FIG. 2B Representative fields for cultured bone marrow oxynucleotides or compositions of the present invention. For cells treated with sense and antisense BCHE oligodeoxy example, abnormal hemopoietic cells proliferation may be nucleotides of the Ts and S types at 5 uM concentrations inhibited, macrophage production enhanced and Stem cell 35 and 4 days incubation. counts increased by the methods of the present invention. FIG. 3 Cell composition of sense and antisense The invention also relates to methods of in vitro modul oligodeoxynucleotides-treated megakaryocyte colonies, lating cell division properties in organs or tissues to be grown in the presence of IL3 only (megakaryocytopoietic transplanted, in order to decrease immune response and conditions). resulting tissue rejection following the transplantation 40 Total contents of plates were Scraped and collected on procedure, by contacting the organ with an effective amount Day 4, washed once with PBS and cytocentrifuged. of the oligodeoxynucleotides or compositions of the inven Cells were stained with May-Grunwald Giemsa and at tion under conditions Suitable culture conditions appropriate least 1,000 cells counted per given experimental con for allotransplatations procedures. These methods may also dition. Cells were classified according to the same be used for in vitro increasing Stem cell fraction in bone 45 criteria as in Patinkin et al., Mol. Cell Biol., 10:6046, marrow cells Samples to be transplanted. (1990). Furthermore, the invention also relates to methods of FIG. 4 The Table presents data of several molecular treating embryonic or fetal bone marrow cells prior to their parameters of the various oligodeoxynucleotides and their Storage in cell banks in viable forms devoid of tissue variable effects in inhibiting colony formation under mega compatibility antigens, by contacting a Sample with the 50 karyocytopoietic conditions. oligodeoxynucleotides or compositions of the invention FIG. 5 Titration curves of colony formation following under Suitable culture conditions. administration of oligodeoxynucleotides under erythropoi Additionally, the methods of the invention encompass etic conditions, with also transferrin and erythropoietin treatment of organ donor and recipient, prior to the donation (EPO) added (CFU-GEMM). procedure, to decrease the immune response in procedures 55 For the CFU-GEMM conditions bone marrow cells were of allotransplantation, and of potential bone marrow donors, grown in methyl cellulose/LPM (Beit Haemek) con prior to the extraction of the bone marrow, in order to enrich taining 10"M thioglycerol (Sigma), 1% BSA (Sigma), the hemopoietic bone marrow fraction of Specific Stem cells 10% WEHI-CM (containing IL-3), 2.8x10"M iron of the hemopoietic System. Saturated human transferrin (Behring, Marburg) and 2 Still further, the invention relates to methods of treating 60 units erythropoietin (EPO, 1,000u/mg)(Terry Fox Labs, patients with malignant tumors, Selectively arresting cell Vancouver, B.C). Colonies were scored after 8 days division in the tumor tissue, but not in the benign cells incubation at 37 C. and 5% CO, using a Zeiss Surrounding the tumor by administering to the patient in StereoZoom micro Scope equipped with an optic fiber need of Such treatment a therapeutically effective amount of dark field device. the oligodeoxynucleotides or compositions of the invention. 65 FIG. 6A1-2: 6A1 Differential cell counts of hematopoi These methods may be particularly suitable for the treatment etic cells grown in culture in the presence of oligodeoxy of chondrosarcomas. nucleotides. Mouse primary bone marrow cells were grown 5,891,725 10 in culture as in Patinkin et al., 1990 in the presence of 2 uM ate oligodeoxynucleotides or with PBS as detailed AS-ACHE or S-ACHE oligomers or of 4 uMAS-BCHE or under methods, 3 weeks after the treatment. In Situ S-BCHE oligomers. Growth conditions were in the presence hybridization and emulsion autoradiography were per of interleukin 3 and erythropoietin (top panel) or interleukin formed in parallel. The presented photographs display 3 alone (bottom panel). Columns present percent fractions of smears hybridized with antisense AChEcRNA or macrophages (MO), polymorphonuclear cells (PMN), early BChEcRNA from a single mouse out of each treatment and late megakaryocytes (E,L/MEG) and early and late group. Note the lower intensity labeling with BuChE erythroid cells (E.L/Ery) (upper left corner key). cRNA as compared with AChEcRNA in all mice, and 6A2 Dose-dependent changes in the differential Sub the reduction in labeling intensity in the treated as classes of cultured hematopoietic cells Subjected to compared with PBS injected mice. AS-ACHE treatment. Presented are cell types the fractions FIG. 11 Modulation of ChEmRNA levels in megakaryo of which increase under AS-ACHE treatment in a dose cytes from control and AS-CHE treated mice. dependent manner (macrophages, early and late ChEmRNA levels in immature, pro-, intermediary and megakaryocytes, top A panel) and those cell types the mature megakaryocytes were determined in average fractions of which decrease under Such treatment (early and no. of grains per cell as detailed in text for mice treated late red blood cells, RBC, and polymorphonuclear cells, 15 with PBS, AS-ACHE and AS-BCHE. “Sense” AChEm PMNs, bottom B panel). RNA and BuChEmRNA probes served for controls and FIG. 7 Representative micrographs of bone marrow bone marrow Smears hybridized with them remained Smears of mice injected once, intraperitoneally, with 25 ug/g practically unlabeled (see empty Symbols in top body weight AS-ACHE, 20 days post-injection or with drawing). Note differences in the developmental pat phosphate buffered saline (PBS) for control. terns of labeling with the two cRNA probes and reduc FIG. 8 ChEcRNA labelings increase with megakaryocytes tions in these labelings in mice treated with AS-CHES. development. FIG. 12 Cell count alterations in megakaryocyte popula Bone marrow Smears from untreated female mice were tions from AS-ACHE and AS-BCHE treated mice. subjected to in situ hybridization with the noted S Total counts of megakaryocytes for each bone marrow -labeled ChERNA probes. Labeled megakaryocytes 25 Smear were averaged within each treatment group (MK) were photographed following emulsion autora (N=4) and are shown with their standard deviations. diography as detailed under Methods. Columns represent average % fractions of Specific A: Promegakaryocytes, megakaryocyte Subtypes within these groups. Devia tions are marked at column tops. Note variations in B: Intermediary cells; total megakaryocyte number and altered differential C: Mature, polynuclear megakaryocytes. cell counts for AS-ACHE, but not AS-BCHE treated Note the increase in grain no./cell which accompanies mice as compared with the PBS-injected controls. megakaryocytes development, the difference between FIG. 13 Suppression of ChEmRNA levels is selective as AChEmRNA and BuChEmRNA labelings and the observed by normal levels of actin mRNA transcription in absence of grains over cells hybridized with the control 35 AS-ACHE-treated mice. (“sense”) probes. 13A: Selective PCR amplification of B-actine mRNA FIG. 9 Variability in AChEmRNA levels during mega from AS-ACHE-treated and untreated (PBS) mice. karyocyte maturation. Total RNA was extracted from bone marrow of A: The average no. of grains per cell was determined for AS-ACHE phosphorothioate oligodeoxynucleotide the noted no. of megakaryocytes at the pro (-A-), 40 or PBS injected mice using the RNasol method intermediary (-O-) and mature ( ) stage. Cells according to manufacturers instructions. 1 ug RNA were divided to groups according to their developmen was reverse transcribed using random hexamers as tal stage and the no. of grains over them (from 0 to 20, primers. The resultant cDNA was PCR amplified from 20 to 40 etc.). Curves represent the distribution of with the mouse f-actine specific primerS 822(+) and grain density for each of the megakaryocyte Subtypes. 45 996(-) Lapidot-Lifson, Y., et al., Proc. Natl. Acad. Note the wider variability of grains over mature as Sci. USA89: 579–583 (1992) using the RNA-PCR compared with intermediary and promegakaryocytes kit (Perkin Elmer/Cetus), according to manufactur and the larger no. of intermediary cells as compared er's instructions. PCR conditions were: denaturation: with the other groups. 94 C., 1 min. (1st cycle 3 min.); annealing: 55° C., Inset: Average no. of grains per cell type is shown in a 50 1 min., elongation: 72 C., 1 min. (last cycle 5 min.), histogram. Background labeling over Slide areas 35 cycles. PCR products (10%) were analyzed on uncovered by cells, representing Spontaneous grain ethidium bromide stained 1.6% agarose gel. Note the formation in the photographic emulsion did not exceed presence of B-actin PCR fragment (175 bp) in appar 5% of the signal and was subtracted from the experi ently similar amounts in AS- ACHE and PBS mental results. 55 injected mice in the presence of reverse transcriptase B: Variable labeling over intermediary and mature mega (+RT) and its absence where reverse transcriptase karyocytes is shown in emulsion autoradiography was not included in the reaction mixture (-RT), M, micrographs. Note that various nuclei and cells are molecular weight marker (marker VI, Boehringer labeled with different intensities. The empty space Mannhiem); bp, base pairs. around nuclear clusters is probably due to receding 60 13B: Schematic presentation of the mouse actin gene. cytoplasm at the preparation Steps prior to the in Situ Positions of the 822(+) and 966(-) primers within the hybridization procedure Courtney, M., et al., Blood coding region of actin cDNA as related to the exon/ 77:560-568 (1991)). intron Structure of the mouse B-actine gene is shown. N' FIG. 10 Labeling variations in mice administered in vivo and C denote the amino and carboxyl termini of the with AS-CHE oligodeoxynucleotides. 65 mature protein. P(A), the polyadenylation signal; a V Bone marrow Smears were prepared from adult female denotes the position of intron. The length is shown in mice treated once with “antisense' phosphorothiothio kb. 5,891,725 11 12 FIG. 14 Cytoplasmic accumulation of 5'-GACTTTGCTATGCAT-3'(SEQ ID NO:3) AS-oligodeoxynucleotides. having phosphorothioate internucleotidic bonds between the AS-CHED covalently bound to a fluorescent FITC tag four 3'-terminus nucleotides (AS BCHE-S); and was administered at final concentration of 4 uM to an antisense oligodeoxynucleotide directed against a CFU-MEG cultures. 2 hr. and 4 days after, cells were 5'-region in the human CHED gene having the formula: cytospinned and Stained as detailed under Methods. Fluorescent photography using a Zeiss Axioplan micro 5'-TTTTCCCCAGTCAAT-3' (SEQ ID NO:4) scope and a x100 Plan neofluar lense was then employed to reveal subcellular localization (S) of the having phosphorothioate internucleotidic bonds between the AS-oligodeoxynucleotide within treated cells. Ana 1O four 3'-terminus nucleotides (AS CHED-S). lyzed cell types included mature megakaryocytes (A), The partially phosphorothioated oligodeoxynucleotides mixed colonies containing young, dividing megakaryo are preferred. cytes and mature polymorphonuclear cells (B), and yet The phosphorothioated and partially phosphorothioated Smaller polymorphonuclear cells, early in their devel 15-mer oligodeoxynucleotides can be Synthesized, for opment (C). 15 example, by using an Applied Biosystems 380B DNA synthesizer, as will be described in more detail in the DETAILED DESCRIPTION OF THE following Examples. INVENTION To examine the concentration dependence of the antisense oligodeoxynucleotides and to determine their toxicity levels, The invention relates to Synthetic phosphorothioated or titration curves were obtained for each of the present partially phosphorothioated oligodeoxynucleotides capable oligodeoxynucleotides, using megakaryocytopoietic bone of Selectively modulating hemopoietic bone marrow cells marrow cultures. AS may be seen from Experimental development. Example 1, both the fully (T)and partially (S) phospho More particularly the invention relates to Synthetic phos rothioated oligodeoxynucleotieds AS-BCHE, AS-2HS and phorothioated or partially phosphorothioated oligodeoxy 25 AS-CHED reduced colony counts specifically. However, the nucleotides capable of inhibiting or Stimulating megakaryo S. oligodeoxynucleotides were considerably leSS toxic than cytopoiesis and of diverting hemopoietic bone marrow stem their Ts equivalents. AS may be seen from this Example and cells development from megakaryocytes and erythrocytes to FIGS. 1 and 2, the fraction of megakaryocytes was signifi dividing Stem cells, macrophages and other mononuclear cantly reduced and a matching increase in macrophages was cells. observed for AS-BCHE and AS-CHED, but not for AS-2HS In particular, the invention relates to Synthetic oligode or S-BCHE treatments. Thus, the antisense BCHE and oxynucleotides being antisense oligodeoxynucleotides CHED oligodeoxynucleotides of the invention are capable directed against a region Spanning the AUG initiation codon of reducing megakaryocytopoiesis and Stimulating mac in human ACHE (acetylcholinesterase) or 2HS (cdc2kinase) rophages formation. genes, having phosphorothioate internucleotidic bonds 35 In addition, Several molecular parameters of the various between all of the nucleotides or between only the four oligodeoxynucleotides, Such as thermodynamic properties, 3'-terminus nucleotides and to Synthetic oligodeoxynucle dimer formation, hydrogen bonds, intramolecular otides being antisense oligodeoxynucleotides directed hybridization, as well as their potential for inhibiting colony against a region Spanning the AUG initiation codon in formation were determined. Results are given in Experimen human BCHE (butyrylcholinesterase) or a 5'-region in 40 tal Example 2. CHED (cdc2 homolog) genes, having phosphorothioate The effects of the present oligodeoxynucleotides in cell internucleotidic bonds between the four 3'-terminus nucle cultures under megakaryocytopoietic conditions, were com otides. pared with those in cell cultures under erythropoietic con Specific Synthetic antisense oligodeoxynucleotides ditions. Remarkably, megakaryocytopoiesis was efficiently according to the present invention are the following: 45 blocked by both AS-ACHE and AS-BCHE under mega karyocytopoietic conditions, while Stimulation of cell pro an antisense oligodeoxynucleotide directed against a liferation was observed for AS-ACHE under erythropoietic region Spanning the initiator AUG codon in the human conditions, with 10-fold increase in colony counts and an 2HS gene having the formula: accompanying increase in megakaryocyte fractions. To the 50 best of the inventors knowledge, induction of cell prolif 5'-GGTATAATCTTCCAT-3' (SEQ ID NO:1) eration by in vitro oligodeoxynucleotide treatment has not been demonstrated at the date of the application. Results are having phosphorothioate internucleotidic bonds between all given in Experimental Example 3. nucleotides (AS 2HS-T) or between the four 3'-terminus Furthermore, the different oligodeoxynucleotides of the nucleotides (AS 2HS-S); 55 invention exhibit specific effects which were examined by an antisense oligodeoxynucleotide directed against a comparable colony counts. Treatment, in culture, with region Spanning the initiator AUG codon in the human ASACHE, at low concentration (2 uM) was shown to be ACHE gene having the formula: accompanied by a Selective decrease in megakaryocytes under CFU-MEG (megakaryocytopoietic) conditions and 5'-CTGCGGGGGCCTCAT-3 (SEQ ID NO:2) 60 reduction in erythrocytes under CF U- GEMM (erythropoietic) conditions. At relatively high concentra having phosphorothioate internucleotidic bonds between all tions (12 uM), treatment, in culture, with AS-ACHE further nucleotides (ASACHE-T) or between the four 3'-terminus increases the number of megakaryocytes, under erythropoi nucleotides (AS ACHE-S); etic conditions, as observed in cultures. Treatment, in an antisense oligodeoxynucleotide directed against a 65 culture, with AS-BCHE, but not with S-BCHE, at 4 uM region Spanning the initiator AUG codon in the human concentration, reduced megakaryocytopoiesis under both BCHE gene having the formula: megakaryocytopoietic and erythropoietic conditions. 5,891,725 13 14 Results are given in Experimental Example 4. sheds more light on the variability in AChEmRNA labeling Experiments in Vivo Show an increase in lymphocytes, in what the inventors defined as mature megakaryocytes. including the desired Stem cells, following treatment with Thus, cells seem to accumulate AChEmRNA molecules with ASACHE. The effect is specific and concentration aging, perhaps to ensure that nuclear division and develop dependent, and was observed 3 and 6 weeks following a ment will cease in these cells. single injection of the AS-ACHE, but not the S-ACHE It is a purpose of the invention to provide compounds and oligodeoxynucleotides. Parallel decrease in erythrocytes methods which would improve treatment protocols for blood accompanied this drastic change in bone marrow composi and immune System diseases. Increasing the fraction of Stem cells in bone marrow to be transplanted may shorten the tion. These important results are given in Experimental delay period through which the transplanted bone marrow Examples 5 and 8. does not yet proliferate efficiently enough and the recipient The above effects were further substantiated by the is in need of receiving fresh blood transfusions. Treating the increased ChEmRNA levels through megakaryocytes matu recipient's own prefrozen bone marrow (for example bone ration in untreated mice, which were altered following marrow may be extracted from patients before commencing treatment with the oligodeoxynucleotides of the invention. chemotherapy or radiotherapy) with the oligodeoxynucle Results are given in Experimental Examples 6 and 7. 15 otides of the invention will avoid the current need for The present findings Suggest that in Vivo treatment with immune Suppression following the transplantation proce low amounts of the Synthetic phosphorothioated or partially dure. phosphorotioated antisense oligodeoxynucleotides of the According to current procedures, 2x10 bone marrow present invention can modulate platelet production. The cells per kg body weight are needed for transplantation. A present findings demonstrate prolonged hematopoietic single extraction involves 10-15 ml bone marrow tissue. effects of in trap e rito neally injected Increasing the number of proliferating Stem cells in Such AS-oligodeoxynucleotides on the mRNA levels of their Samples will improve the results of the transplantation. target Sequences as well as on the composition and/or total Furthermore, Small Seed Samples can thus be grown into number of megakaryocytes on the other hand. AS can be Sizable cell populations. This can assist in Storing a prospec seen in Table 2, the fractions of other hemopoietic bone 25 tive recipient's own cells Since Such Seed cultures may be marrow cells are also influenced by these anti-Sense oli prepared, for example from the umbilical cord and can be godeoxynucleotides. kept frozen, HLA typed, until needed. The inventors have used in situ hybridization followed by In addition, the antisense-oligodeoxynucleotides of the image analysis and Statistical management of labeling present invention are potentially important for use in immu results. This high resolution approach demonstrated a noSuppression procedures. Modification of immune function 20-fold increase in AChEmRNA levels and a more modest by pharmacological agents is emerging as a major area of 4-fold increase in BuChEmRNA from promegakaryocytes therapeutics, particularly in clinical procedures required for to mature, platelet-producing megakaryocytes. It may be allotransplantation. Activation or Suppression of the immune noted that the enzymatic activities of AChE and BuChE are response are both believed to involve processing of the both visible at all these developmental Stages, and even in 35 “Self” or “non-Self antigens by phagocytic cells Such as the apparently unlabeled immature cells Burstein et al., macrophages for a comprehensive review of this topic See 1980); Patinkin et al., (1990). However, the histochemical Paul, W. E., Fundamental Immunology, 2nd Ed., Raven analysis did not allow for quantification. The results pre Press, N.Y. 1989). Therefore, rationalized modulation of sented in the following Examples therefore imply that the hemocytopoiesis should be of value for transplantation various CHE genes undergo a considerable transcriptional 40 procedures. Thus, Suppression of macrophage production, activation during the megakaryocytopoietic process. Such as the effect resulting from the administration of The complex regulation of CHE genes in mammalian AS-2HS, may Selectively decrease rejection responses. megakaryocytopoiesis Suggests distinct roles for AChE and Moreover, induction of stem cells production by AS-ACHE BuChE in the development of these cells. This notion is will increase the fraction of undifferentiated cells, among reinforced by the AS-CHE treatments and their effects on the 45 those cells are those which do not yet present the tissue number and composition of megakaryocytes in the treated compatibility antigens responsible for the rejection mice, as shown in the Examples. Furthermore, these findings response. Thus, combined treatment with AS-2HS, which present the first Steps towards the development of an in vivo Suppresses hemopoiesis in general, and AS-ACHE can be paradigm for the Selective inhibition of CHE genes expres useful at the levels of the recipient, the transplanted organ or Sion using oligodeoxynucleotide-based therapeutical prepa 50 tissue and, in cases where this is known in advance, also at rations. Finally, the present observations indicate that plate the donor level. In the latter case, the Suggested treatment let production in Vivo is controlled by cholinergic Signalling. may create in advance, within the donor, macrophages The development-suppressing effect of AS-BCHE both in devoid of the phenotype which is responsible for the rejec IL-treated cell cultures and in Vivo further Suggests that tion proceSS. Precisely modulated quantities of the various erythropoietin, which was absent from the cultures, does not 55 antisense oligodeoxynucleotides will be required for these principally alter the effect of AS-BCHE, although the extent mixture treatment, all according to the therapeutic of Suppression may not be similar in culture and in Vivo. objectives, the route of administration, and the condition of AS-ACHE suppresses selectively its target mRNA while the patient, when used in Vivo. Thus it may be necessary for moderately effecting BuChEmRNA levels. In contrast, the attending physician to titer the dosage and modify the AS-BCHE decreases both ChEmRNAS, though with higher 60 route of administration as required to obtain the optimal efficiency for BuChEmRNA. Previous culture studies dem therapeutic effect. The progreSS of the therapy can be easily onstrated a general inhibition of megakaryocytopoiesis by monitored by conventional hematology assays or laboratory AS-BCHE Patinkin et al., (1990). However, its effect on cell counts. Suitable starting therapeutic doses can be AChEmRNA is significant: the AChEmRNA decrease may extrapolated from the in vitro efficacy studies described not be attributed to the general slowdown in 65 herein. megakaryocytopoiesis, as it appeared in cells measured An important feature of the present invention is that the precisely as belonging to Specific Subgroups. This, in turn, oligodeoxynucleotide can be administered by perfusion (for 5,891,725 15 16 organs) or by Simple Subcutaneous, intramuscular, intrave The antisense oligodeoxynucleotides were kept in 4 mM nous or intraperitoneal injection (for patients) and that their concentration at -20° C. and were diluted in phosphate effects last for at least several weeks. The limited toxicity of buffered saline (PBS) prior to their administration to mice. the S antisense oligodeoxynucleotides is of particular (2) In vivo administration importance for their therapeutical uses. Intraperitoneal injection of AS-CHE and AS-cdc2 oli This invention provides pharmaceutical compositions godeoxynucleotides was performed using a Hamilton comprising at least one of the antisense oligodeoxynucle Syringe. Amounts were calculated according to previous otides of this invention, or mixtures of at least two of Said experiments in cell cultures Patinkin et al., (1990); Lapidot oligodeoxynucleotides, and physiologically acceptable Lifson et al., (1992) so that the final concentration of the carriers, eXipients or Stabilizers, usually in the form of AS-CHES reached 5 lug?gr. weight and the injected Volume Solutions. Acceptable carriers, eXipients are nontoxic to did not exceed 10 ul/gr. weight. No toxicity effects were recipients at the dosages and concentrations employed, and observed on the injected mice, all of which appeared to include buffers, Such as phosphate buffered Saline and like behave normally and displayed no weight losses. Whenever physiologically acceptable buffers, and more generally all higher concentrations were used, these are mentioned and Suitable carriers known in the art. The compositions may 15 the administration procedure remained the Same. further optionally contain physiologically acceptable addi (3) Transcription vectors for in vitro synthesis of human tives Such as anioxidants, mono- and disaccharides; Salt AChEcRNA and BuChEcRNA. forming counterions Such as Sodium and/or nonionic Sur Human AChEcDNA (1.5 kb long; clone no. hFEL, Soreq factants. Sustained release compositions are also et al., 1990) was subcloned at EcoRI sites into the contemplated within the Scope of this application. These pGEM7ZF(+) plasmid (Promega) containing the RNA poly may include Semipermeable polymeric matrices in the form merase binding sites from T7 and SP6 bacteriophages. In of shaped articles Such as films or microcapsules. The vitro RNA transcription with SP6 or T7 RNA polymerases antisense oligodeoxynucleotides and compositions of the was used to produce antisense AChEcRNA or sense invention must be Sterile. AChEmRNA, respectively. Human BuChEcDNA (2.2 kb 25 long; Soreq et al., 1989) was subcloned in PstI/EcoRV EXAMPLES restriction sites into the Bluescript SK(+) plasmid (I) Materials and Methods (Stratagene) carrying the RNA polymerase binding sites (1) Synthesis of Antisense olicodeoxVnucleotides from T7 and T3 bacteriophages. In vitro RNA transcription Oligodeoxynucleotides were Synthesized on an Applied with T7 and T3 RNA polymerase was employed to produce Biosystems 380B DNA synthesizer using phosphoramidites from this vector “antisense’ BuOhEcRNA and “sense” from the same company according to the manufacturer's BuChEmRNA, respectively. instructions. They were purified by reverse phase HPLC on (4) Preparation of riboprobes for in situ hybridization. a Waters dual pump 6000A System in combination with a Slabeled in vitro RNA transcripts (107 cpm/ug) were Waters automated gradient controller and a model 481 UV produced using the Amersham RPN 2006 kit and RNA Spectrophotometer operated at 260 nm with the 5'-protecting 35 polymerases from Boehringer (Mannheim), using linearized dimethoxytrityl group Still attached to the oligodeoxynucle plasmids digested with Hind III, Xho I for the sense and otides. This was removed by standard treatment with 80% antisense AChE RNA probes and with Apa I, Sma I for the aqueous acetic acid. The oligodeoxynucleotides obtained Sense and antisense BuChE RNA transcripts, respectively, were checked for purity again by HPLC. To incorporate the all according to the manufacturers instructions. Radiola phosphorothioate groups, the oxidation Step employing 40 beled probes were subjected to alkaline hydrolysis for 20 iodine was replaced by reaction with 3H-1,2-benzodithiol minutes. The resultant 200-400 bases long fragments were 3-one 1,1-dioxide Iyer, R. P., et al., J. Org. Chem. Separated from unincorporated nucleotides by SepahdeX 55:4693-4699 (1990)). This treatment protects the oligode column chromatography according to Wilkinson, D. G., et oxynucleotides against nuclease (Eckstein, F., Ann. Rev. al., Cell 50:79–88 (1987)). See FIG. 1. Biochem., 54:367-402 (1985); Spitzer, F. and Eckstein, F., 45 (5) Tissue preparation and in Situ hybridization Nucleic Acids Res., 18:11691-11704 (1988) and prolongs Bone marrow was Squeezed from dissected femur bones their duration in vivo Woolf, T. M., et al., Nucleic Acids of Sacrificed 6 weeks old female mice anesthesized with Res., 18:1763–1769 (1990); Shaw, J. P., et al., Nucleic Acids ether and was Smeared as Single cell layers on microscope Res., 19:747–750 (1991). Wherever partial protection was slides coated with 3-aminopropyltriethozysilane (TESPA, required, reaction with 3H-1,2-benzodithiol-3-one 1,1- 50 Sigma), to prevent loss of cells during the experimental dioxide was performed for the first three Steps only, after procedure Rentrop, M., et al., Histochemical Journal which regular Synthesis was continued. The resultant par 18:271-276 (1986)). Slides were dried at room temperature tially protected oligodeoxynucleotides were therefore for 2 hours, fixed in 4% paraformaldehyde (20 min. at room blocked by phosphorothioate groups only in the last three temperature), washed with 3x and twice 1x phosphate internucleotidic bonds at their 3'-terminus. The antisense 55 buffered Saline, dehydrated through ethanol Series, air dried oligodeoxynucleotides employed were AS-ACHE (5'- and stored at -20° C. for up to 2 weeks Hogan, B., et al., CTGCGGGGCCTCAT-3'; SEQ ID NO:2) and AS-BCHE A Laboratory Manual, Cold Spring Harbor Laboratory (5'-GACTTTGCTATGCAT-3'; SEQ ID NO:3), designed to (1986)). For in situ hybridization slides went through complement the initiator AUG domain in AChEmRNA refixation, 0.2M HCl incubation for 20 min. to reduce Soreq et al., (1990) ibid.) and BuChEmRNA Prody et al., 60 non-specific probe binding, proteinase K treatment, acety (1987) ibid.), respectively. Also employed were AS-CHED lation and dehydration as described Rangini, Z., et al., (5'-TTTTCCCCAGTCAAT-3'; SEQ ID NO:4), directed Mechanisms of Development, 35:13–24 (1991). Hybridiza against a 5'-region in the human CHED gene Lapidot tion was performed in the presence of S-CHERNA Lifson, Y., et al., Proc. Natl. Acad. Sci. USA, 89:579–583 (approx. 1x10 cpm per slide) according to Rangini et al., (1992) and AS-2HS (5'-GGTATAATCTTCCAT-3'; SEQ ID 65 (1991) except that no thio-ATP was added. Exposure was for NO: 1) designed to complement the initiator AUG codon in 6 weeks. Counter-staining was made with May- Grunwald the human cdc2 HS kinase mRNANurse, Nature (1986)). Giemsa. 5,891,725 17 18 (6) microscopal image analysis and Statistical data manage Also, for each experiment the total number of CFU-GEM ment. or CFU-MEGG was determined in a Zeiss Stereozoom The in Situ hybridization results were analysed using a binocular after 4 or 8 days in the presence of the examined Nikon Microphot microScope equipped with a Single slide oligodeoxynucleotides. Colony counts were plotted, either automatic Stage, automatic focus and 60x plan apo oil directly or as percent of the total number of colonies in immersion objective and connected through an interface to control, untreated cultures. Data represent average of at least a Magiscan Image Analysis microscope controller (Applied three independent experiments it Standard Evaluation of the Imaging International Ltd, Dukesway, U.K.). Image analysis Mean (S.E.M.) in each case, unless otherwise noted. was carried out by the Software package “GENIAS', which counts Silver grains, measures cell parameters, and evaluates (II) Experiments and Results asSociation of grains with cells. Data management was then 1O (1) Concentration dependence of the performed using the “RESULTS’ program, which examines AS-oliaodeoxynucleotides and their toxicity levels. the statistical significance of data obtained from “GENIAS” All pink or red CFU-GEMM colonies, 20.5 mm in and Subjects area and perimeter measurements to various diameter, of a given plate are picked with a micropipette, Statistical hypothesis tests (Applied Imaging, U.K.). Briefly, washed in PBS and cytocentrifuged. The CFU-GEMM monochromatic images of the examined megakaryocytes 15 colonies constitute about 90% of the total colonies present. were captured using a red filter to improve the contrast. Cells are stained with May-Grunwald Giemsa and at least Since the Silver grains and cells were at different focal 1,000 cells counted per a given experimental condition. Both planes, the grains were focussed accurately as their best early erythroblasts and megakaryocytoblasts are Small resolution was required. Grain counts were detected auto deeply staining cells with very large nuclei and very narrow matically based on their darkness, and high frequency noise rims of cytoplasm. They are distinguished from one another information included in the grain image was automatically by indirect immunocytochemical staining with 1:1,000 dilu deleted. Cell borders to be measured were manually tion of human anti-GPIIb/IIIa Ab (a gift from Barry S. delineated, after which grains were counted and measured Coller, Stonybrook, N.Y.), followed by a 1:100 dilution of Separately for each cell. Background grain density was anti-mouse Ig fluoresce in-linked Ab (Amersham measured in parallel and Subtracted from the experimental 25 International) and direct staining with anti-human results. Collected data included cell counts and parameters Glycophorin-alpha Ab (Immunotech, Marseille). Late eryth (measured cell areas), number of grains per cell and per unit roblasts were characterized by a white or very light blue area and the Statistical Significance of variations between cytoplasm Surrounding a Small dark nucleus. these parameters. Presented data are average results for To examine the concentration dependence of the Separate in Vivo treatments, with approximately 40 mega AS-oligodeoxynucleotide effects and determine their toxic karyocytes at different developmental Stages analysed with ity levels, titration curves were derived for each of the each of the ChEcRNA probes in bone marrow preparations employed oligodeoxynucleotides, using megakaryocytopoi from four different mice/treatment. etic bone marrow cultures. For this purpose, IL treated (7) Differential cell analysis of antisense oligonucleotide murine bone marrow cell cultures were grown for 4 days in treated, Semi-Solid bone marrow cultures. 35 the presence of increasing concentrations of either fully To grow CFU-MEG colonies, bone marrow cells from the phosphorothioated (Ts) or partially protected at the last 3' femur and tibia of 8-12 week-old endotoxin-resistant C3H/ internucleotidic bonds (S) oligodeoxynucleotides up to 10 He J mice were cultured in LPM synthetic medium tiM final concentration and colony numbers were recorded. (Biological Industries, Beit HaEmek, Israel) containing All of the Ts oligodeoxynucleotides, blocking CHED, 10% conditioned medium for WEHI-3 cells as a Source for 40 BCHE or 2HS, were found to reduce the colony counts interleukin 3 (IL3), 1% BSA, 10"M thioglycerol and 1% significantly. However, the S-BCHE oligodeoxynucleotide, methylcellulose (megakaryocytopoietic conditions -CFU with no counterpart hybridizable chain in any cell, also MEG). reduced colony counts at concentration above 5 uM, dem To obtain erythropoietic conditions and grow CFU onstrating a non-specific inhibitory effect of Such oligode GEMM colonies, 2.8x10"Miron-saturated human transfer 45 Oxynucleotides on colony formation in culture. This, in turn, rin and 2 units of erythropoietin (EPO, 1,000U/mg/ml) were Suggested that the inhibitory effects of the anti-Sense oli added (CFU-GEMM conditions). godeoxynucleotides could also include a non-specific inhibi For both culture types, 0.5-1.0x10 cells/ml were plated tory component. This hypothesis was reinforced by the in 35 mm petridishes (Nunc 1008), or 24 well tissue culture observation that the S. oligodeoxynucleotides, having the Costar plates, and incubated 4 or 8 days at 37 C. under 5% 50 Same nucleotide Sequences, were, in general, far less toxic CO with high humidity for CFU-MEG and CFU-GEMM then their Ts counterparts in each of the cultures. The conditions, respectively. Oligodeoxynucleotides, at the non-specific was thus indicated to be related with the denoted final concentrations of 1-20 uM, were added upon phosphorothioate composition of these oligodeoxynucle initiation of cultures. otides. Colonies grown in Serum-free methylcellulose cultures 55 To compare the efficacy of S and Ts containing Sense or antisense oligonucleotides were picked oligodeoxynucleotides, differential cell counts were taken with drawn-out Pasteur pipettes, concentrated (5 min at for the S and Ts AS-BCHE oligodeoxynucleotides at the 500xg) by Cytospin (Shandon, 2) centrifugation in phos final concentration of 5 uM. Significant reductions in the phate buffered saline (PBS), stained with May-Grunwald fraction of megakaryocytes and a matching increase in Giemsa and analyzed microscopically. The relative fraction 60 macrophages were observed with both oligodeoxynucle of each cell type represented among the total cells recovered otides to comparable extents. FIG. 2B demonstrates repre from the noted number of independent experiments were sentative fields from such cultures, and FIG. 3 presents the then determined. Satisfactory control experiments revealed differential cell fractions for 5uM T BCHE, CHED or 2Hs distributions which were essentially identical to that in the form of a histogram. The differential count analysis observed in control (no oligo) cultures, indicating that there 65 demonstrated that each of the examined anti-Sense was no nonspecific toxicity. At least 500 cells were counted oligodeoxynucleotides, whether Ts or S, retained its ability for each data Set. to modulate the megakaryocytopoietic process. Thus, the 5,891,725 19 20 non-specific toxicity effect appeared to be non-Selective, at 4 uM concentration, blocking ca. 50% of colonies. In affecting all cell lineages. This, in turn, further Strengthened contrast, AS-BCHE blocked only 30% of colony formation the assumption that part of the reduction effect on colony at 4 uM and higher concentrations. In both cases, the Sense numbers was due to the non-specific inhibition because of S, oligodeoxynucleotides were-totally non-toxic, Suggest the phosphorothioate nature of the anti-Sense oligodeoxy 5 ing that the inhibition effects exerted by the A.S.- nucleotides. oligodeoxynucleotides were Selective and Specific. (2) Molecular parameters and toxicity of the examined In CFU-GEMM cultures, opposing effects were observed oligodeoxynucleotides. for AS-ACHE, which induced a dramatic 3-fold increase in colony counts at the final concentration of 10 uM. In To Search for correlations between the primary nucleotide contrast, AS-BCHE was ineffective at this concentration. Sequence of the examined oligodeoxynucleotides and their Interstingly, CFU-GEMM colonies grown in the presence of variable toxicity (potency of inhibiting bone marrow colony AS-ACHE appeared Smaller in Size and composed of many formation under megakaryocytopoietic conditions) effects, more Small cells than the parallel colonies grown under Several molecular parameters were determined. These control conditions. Total cell counts for these colonies included their melting temperature (Tm), nucleotide com further revealed 2-fold increase in cell numbers for position in percentage, their tendency to form dimers under 15 AS-ACHE colonies as compared with controls. physiological conditions and the optional loop Structures FIG. 5 presents the titration effects of the ChE-related which these oligodeoxynucleotides may form within them examined oligodeoxynucleotides under CFU-GEMM form Selves. In particular, inventors examined the possibility of ing conditions. the four 3'-terminal nucleotides (and hence the last 3 inter (4) Determination of the specificity of the examined oli nucleotidic bonds) in these oligodeoxynucleotides to be godeoxynucleotides by differential colony counts. involved in intramolecular structures. This analysis (FIG. 4) The Specificity of the oligodeoxynucleotide effects was revealed an apparent direct relationship between the ability further examined by comparable differential counts. The of the three 3'-terminal internucleotidic bonds to form change in colony counts observed for AS-ACHE as com intramolecular loops and the relative toxicity values of the pared with S-ACHE was thus shown to be accompanied by corresponding AS-oligodeoxynucleotides at 5-10 uM con 25 a selective decrease in megakaryocytes under CFU-MEG centrations. Thus, AS-oligodeoxynucleotides with no ten conditions and reduction in both megakaryocytes and eryth dency for intramolecular looping at their 3' terminus would rocytes under CFU-GEMM conditions, all at the very low 2 tend to be less toxic yet similarly effective in cultured cells. luM final concentration. In parallel, AS-BCHE but not It is important to note at this point that Studies of others have S-BCHE at 4 uM concentration reduced megakaryocyto shown extension in Vivo of AS-oligodeoxynucleotides poiesis under both Sets of experimental conditions. Cultures Agarwal, et al., (1991) ibid. This process, presumably grown in the absence of any oligodeoxynucleotide (none) useful as a Scavenging mechanism to remove alien served for controls. FIGS. 6 (A1, A2) present these cell AS-oligonucleotides from the circulation depends on the fractions in the form of histograms. It should also be noted existence of free 3'-termini in the attached oligonucleotides. that under erythropoietic conditions and at a concentration Partial phosphorothioate protection at this important posi 35 of 12 uM, AS-ACHE further decreases the number of tion may hence be sufficient to ensure stability of these erythrocytes and megakaryocytes, as observed in the cell AS-oligodeoxynucleotides and, in addition, can reduce their culture experiments. non-specific toxicity effects by assisting in natural Scaveng (5) Effect of AS-ACHE on bone marrow cells in vivo. ing of these compounds when not involved in DNA-mRNA To extend these studies into the in vivo situation, labo hybrids. 40 ratory grown wild mice were injected intraperitoneally 48 (3) Effects of examined oligodeoxynucleotides under mega hr. postnatal with 5 or 25ug?.gr of the Ts oligodeoxynucle karyocytopietic and erythropoietic conditions. otides dissolved in PBS. 3 weeks after, bone marrow was ASSuming that the above detailed Specific examined. FIG. 7 depicts a representative micrograph of the AS-oligodeoxynucleotides effects are caused due to damage bone marrow Smears. in cholinergic Signalling, parallel oligodeoxynucleotides 45 Apparently, erythrocyte fractions were reduced also in were prepared according to the 15 nucleotides Spanning the Vivo, whereas lymphocytes (including stem cells) increased region of the AUG codon in the AChE coding Sequence considerably. S-ACHE remained ineffective. Soreq et al., (1990) ibid.). Both sense and antisense oli Megakaryocytes could not be counted because of the godeoxynucleotides were designed to include three Small in Vivo counts, however photography revealed an in 3'-terminal blocked internucleotidic bonds (i.e. in their S. 50 vivo reduction in their numbers as well. However, while the form) and their molecular properties were similarly ana in Vivo effects appeared to be relatively moderate as com lyzed. The absence of 3'-involvement in intramolecular pared with those measured under culture conditions, these loops was ensured by computer analysis of the designed findings demonstrate that the AS-ACHE effects measured in oligodeoxynucleotides and prompted further experiments, vitro were indeed true effects, reflecting the effect of this using these AChE oligodeoxynucleotides in cell cultures, in 55 oligodeoxynucleotide under in Vivo conditions as well. parallel with the BuChE ones described above. It should be noted that the fraction defined as “lympho To deepen the cell culture approach, bone marrow cells cytes' in the in Vivo analyses of bone marrow Smears were grown either with IL alone, (i.e. megakaryocytopoi includes proliferating Stem cells. However, the in vivo etic conditions, inducing only CFU-MEG colonies) or with approach does not allow for counting of dividing Stem cells. the addition of transferrin and erythropoietin as well (i.e. 60 In contrast, cell counts and differential cell compositions in erythropoietic conditions, in which CFU-GEMM colonies culture experiments clearly showed increased fractions of may also develop, GEMM implying granulocytes, young, apparently dividing cells. Thus, the culture experi erythrocytes, macrophages and megakaryocytes). ments and the in Vivo ones are complementary to each other This composite experiment revealed that in the CFU in providing important information on the duration of the MEG cultures, both AS-ACHE and AS-BCHE blocked 65 treatment, its effectiveness and its Specificity. In particular, megakaryocytopoiesis efficiently, however at various effi the ability of the AS-oligodeoxynucleotides to enhance cell cacies. AS-ACHE (but not S-ACHE) was effective already division is important. 5,891,725 21 22 (6) Increased ChEmRNA levels through megakaryocytes only be detected in intermediate and mature megakaryocytes maturation in untreated mice and remained unaltered in these two stages of megakaryo In situ hybridization was performed with bone marrow cytes development (FIG. 11). smears from Sabra mice using antisense ChEcRNA and Computerized image analysis and processing of the Silver sense ChEmRNA probes transcribed from the pGEM-7ZF grain counts (at least 40 cells/sample for 4 mice and two (+) AChEcDNA plasmid and the Bluescript SK(+) BuOh probes per treatment), demonstrated differently Suppressed EcDNA plasmid (FIG. 1). The Smeared bone marrow cells developmental patterns for the two ChEmRNA in the included lymphoid and erythroid cells, as well as mega AS-CHE injected animals as compared with control, PBS karyocytes in different developmental Stages. Significant injected mice. Three weeks following the treatment, labeling with the antisense probe appeared only over AChEmRNA and BuChEmRNA levels were reduced by megakaryocytes, Suggesting intensive expression of the approximately 62 and 30%, respectively, in mature mega CHE genes in these cells. Labeling with the control, Sense karyocytes from AS-ACHE-treated mice. In contrast, probes, was negligible throughout the Smears, demonstrat AS-BCHE treatment reduced both AChEmRNA and ing the Specificity of the hybridization reactions. Immature BuChEmRNA levels by about 66% (FIG. 11). Statistical megakaryocytes, with up to 4 nuclei, appeared generally 15 analysis revealed for these values a bidirectoinal variance at unlabeled. Distribution of silver grains reflecting ChEm a significance level of 5%. Hoel, P. G., Elementary RNA levels in mouse megakaryocytes at three later differ Statistics, 4th Ed., John Wiley & Sons, Inc. New York entiation Stages was determined following Magiscan London (1976)). Furthermore, the suppressed levels of classification, into: mRNAs within megakaryocytes at different developmental (A) Promegakaryocytes of 14–20 um cell diameter with Stages appeared to Significantly dependent on the nature of 4-8 nuclei and Smaller cytoplasm than nucleus, the AS-oligodeoxynucleotides treatment in different mice as (B) >20 um diameter intermediate cells, with 8-16 nuclei shown by the averaged slopes of the curves in FIG. 11. Thus, and cytoplasm equal in size to nucleus, AChEmRNA was more significantly suppressed when (C) Mature >20 um cell diameter megakarcyocytes with AS-ACHE was injected than with AS-BCHE, and the effect 16-64 nuclei and abundant cytoplasm Courtney, M., et 25 of AS-BCHE upon BuChEmRNA levels was more con al., Blood, 77: 560-568 (1991)). spicuous after injection of AS-BCHE than with AS-ACHE. FIG. 8 demonstrates high intensity AChEcRNA labeling Altogether, these data demonstrate the in Vivo effectiveness, as compared with lower density BuChEcRNA labeling and the long term duration and the interrelationship between the no labeling with sense AChEmRNA or BuChEmRNA in effects of the two AS-CHE drugs. untreated animals. It should be noted that RNase treatment (8) AS-CHES modulate megakaryocytes development in prior to the in situ hybridization abolishes all labeling in this vivo. procedure (not shown), providing evidence for the RNA The total no. of megakaryocytes in each of the bone dependence of these reactions. marrow Smears from the different mice was found to be Megakaryocytes belonging to each of the above Sub-types somewhat higher in AS-ACHE treated mice (N=78.2+18.6) were further divided into Sub-groups according to their 35 as compared with controls (N=66.5+4.9). In contrast, the no. radiolabel intensities (up to 20 grains, between 20 and 40, of megakaryocytes per Smear in AS-BCHE treated mice was etc). The variability in AChEcRNA grain no./cell was found significantly lower, with very limited variability between the to increase with megakaryocytes development (FIG. 9A-B), different mice (N=57.7+1.1). Differential cell counts of bone in accordance with the longer life time of mature megakary marrow megakaryocyte populations were further performed ocites as compared with their progenitors (Mazur, 1987). 40 using the Magiscan image analysis System (Materials and (7) Altered CHEmRNA levels in mice infected with anti Methods (6)). Intermedial cells represented approximately Sense oligodeoxynucleotides. 85% of the total no. of megakaryocytes in control mice, with To selectively interfere with the expression of the CHE mature cells accounting for about 10% and immature and genes, 4 different mice were injected once with 5 lug/gr promegakaryocytes composing the remaining minor frac weight of 15-mer antisense phosphorothioate oligodeoxy 45 tions (FIG. 12A). Reduced numbers of intermedial cells and nucleotides complementary to the initiator AUG domains in enlarged fractions of both immature and mature megakaryo AChEmRNA or BuChEmRNA, respectively, or with PBS cytes were observed in AS-ACHE treated mice (FIG. 12B), for controls. Three weeks following the injection, bone while AS-BCHE treated mice displayed apparently normal marrow Smears were prepared from all of these mice. Each composition of megakaryocyte Subtypes (FIG. 12C). Thus, Smear was divided into Separate parts which were hybridized 50 the AS-ACHE treatment induced a general enhancement in with one of the two ChEcRNA probes. Following 6 weeks megakaryocytopoiesis, accompanied by distortion of the of exposure to emulsion autoradiography, Slides were devel megakaryocytopoietic process in Vivo. In contrast, oped to create Silver grains over cells containing ChEcR AS-BCHE Suppressed megakaryocytopoiesis without NAS. Labeling decreased in bone marrow Smears prepared affecting the differential cell counts, Suggesting that it either from AS-ACHE and, more effectively, in AS-BuCHE treated 55 blocks the early Stages of megakaryocytopoiesis or that it mice. FIG. 10 displayS representative photographs demon Similarly Suppresses all Stages. strating the variable ChEmRNA labeling in the differently (9) Primary AS-Oligodeoxynucleotides Suppression of One treated mice. ChEmRNA Selectively Leads to Secondary Inhibition of Its Computerized image analysis and Statistical processing of Counterpart ChEmRNA Type. the silver grain counts (at least 40 cells/sample for 4 mice 60 Bone marrow cDNA from AS-ACHE-treated and control, and two probes per treatment), revealed different accumu PBS-injected mice was subjected to direct amplification lation patterns for the two ChEmRNAS in the AS-CHE using polymerase chain reaction (PCR) with mouse actin injected animals as compared with control, PBS-injected primers. This analysis, displayed in FIG. 13, revealed appar mice. ently similar levels of the amplified actin cDNA fragments In control mice, AChEmRNA levels per cell increased by 65 in treated and control mice, demonstrating that the 20-fold from the promegakaryocyte Stage to mature AS-oligodeoxynucleotide treatment did not generally abol megakaryocytes, while the lower BuChEmRNA levels could ish transcription in the bone marrow. Differential cell counts 5,891,725 23 24 in the analyzed bone marrow Smears further revealed appar capable of taking up the FITC-AS-CHED with apparently ently normal composition of ca. 40% erythroid cells, 27% Similar efficiencies, Suggesting that the presumed receptor granulocytes, 17% lymphocytes and stem cells, 13-14% Sites enabling this uptake process appear early in the devel myeloid cells and 2-3% eosinophils for several of the opment of both these cell types and are expressed through AS-ACHE-treated and control animals. Peripheral auto out their life span. Most importantly, this analysis demon mated blood profiles of these animals displayed ca. 75% strated nuclear exclusion of the accumulated FITC-AS lymphocytes, 18% neutrophils, 5% monocytes and 2% CHED (which was S. in its phosphorothioate composition). eosinophils, a normal composition characteristic of healthy Thus, most of the uptaken compound accumulated in the animals, for both treated and control mice. Thus, the AS-oligodeoxynucleotide treatment was apparently harm cytoplasm of the treated cells, where it could hybridized less for hemopoietic composition at the time of this analysis, with the mature CHED mRNA species but not with the suggesting that the indirect suppression of BuChEmRNA by CHED gene and/or with its nascent, non-processed nuclear AS-ACHE and the yet more efficient suppression of mRNA transcript. Moreover, cytoplasmic FITC fluores AChEmRNA by AS-BCHE reflects selectively induced sec cence could also be detected in megakaryocytes after 4 days ondary effects of each of these AS-oligodeoxynucleotide 15 in culture (D), indicating that the AS-oligodeoxynucleotides treatmentS. remain in this Subcellular Site for at least Several days. The (10) cytoplasmic accumulation of Specificity of the fluorescence Signals, in turn, was proved by AS-oligodeoxynucleotides indicates post-transcriptional the absence of Such signals in young and developed regulation as their primary role. megakaryocytes, polymorphonuclear cells and macrophages Referring to FIG. 14, comparison of bright field and in culture to which no FITC-oligodeoxynucleotides were fluorescence photography revealed an apparent relationship added (E.F). between the abundance of cytoplasm and the brightness of In addition to the cytoplasmic signals, the FITC-labeled the fluorescent Signal. Thus, mature megakaryocytes accu AS-oligodeoxynucleotide created fluorescent Signals larger mulated relatively large amounts of the FITC-labeled over the cell Surface, So that the cell diameter appeared AS-CHED (A), whereas young megakaryocytes displayed 25 larger under fluorescent than with bright field photography Significantly lower intensities of fluorescence (B) and poly (compare A-D in FIG. 14). This could imply a continuous morphonuclear cells yet lower Signals. Both young and occupation of all available receptor Sites over the Surface of mature megakaryocytes and polymorphonuclear cells were treated cells for the entire duration of the experiment.

SEQUENCE LISTING

( 1) GENERAL INFORMATION: ( i i i ) NUMBER OF SEQUENCES: 9

( 2) INFORMATION FOR SEQ ID NO:1: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 15 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: other nucleic acid ( i v ) ANTI-SENSE: YES ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:1:

G GTATAAT CT T C CAT 1 5

( 2) INFORMATION FOR SEQ ID NO:2: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 15 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULETYPE: other nucleic acid ( i v ) ANTI-SENSE: YES ( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:2:

C T G C G G G G GC CT CAT 1 5

( 2) INFORMATION FOR SEQ ID NO:3:

5,891,725 37 38 -continued

T CAGAA T G G G GAAATAA T G C CTTTTT CTAC TATTTT GAA C A C C GAT C C T C CAAA CT T C C G

T G GCCA GAAT GGA T G G GA GT GA T G CAT G. GC TAT GAAA TT G AATTT GT CTT TGGTTTAC CT

C T G GAA A GAA GAGAT AAT TA CA CAA AA GCC GAG GAAA TTT T GA GT AGAT C CAT AG T GAAA

C G G T G G G CAA ATTTT G CAAA ATA T G G GAAT C CAAATGA, GA C T C A GAA CAA TAG CA CAA. GC

T G G C C T G T CT T CAA AA G CAC T GAA CAA AAA TAT CT AAC CT T GAA TACA GA GT CAA CAA GA

ATAATGA CGA AACTA C G T G C T CAA CAA T GT C GATT C T G GA CAT CATTTTT T C CAAAA GT C

TT G GAAA T GA CA. GG AAA. TAT T GAT GAA G CA GAA T G G GA GT G GAA A G CA. GG ATT C CAT C GC

T G GAA CAATT A CAT G A T G GA C T G GAAAAAT CAATT TAACG ATTACA CT AG CAAGAAA GAA

A GT T G T G T G G GT C T C T AATT AAT AGATT TA C C C TT TAT AG AA CAT ATTTT CCTTT AGAT C

AAG G CAAAAA TAT CAG GA. GC TTTTTT ACAC A C C T ACT AAA AAA GT TAT TA T G TAG CT GAA

A CAAA AA T G C CA GAA G GATA ATATTGATT C CT CA CAT CTT TAACTTAGTA TTTTAC CTAG

CAT TT CAAAA C C CAAA T G GC TAGAA CAT GT T TAATT AAAT TT CACAATAT AAA GT T CTAC

A GT TAATTAT G T G CAT ATTA AAA CAA T G GC C T G GTT CAAT T T C T TT C T TT CCTT AATAAA

TT TAA GT TTT TT C C C C C CAA AAT TAT CA GT G C T C T G CTTT TA GT CAC GT G TATTTT CATT

A CCA C T C GTA AAAAG G T ATC TTTTTT AAAT GAA TT AAA. TA TT GAA ACA CT GT ACA C CATA

GTTT ACAATA TTA T G T TT C C TAAT TAAAAT AAGAA TT GAA T GT CAAT ATG A GATA TT AAA

ATA A G CA CAG AAAATC

( 2) INFORMATION FOR SEQ ID NO:9: ( i) SEQUENCE CHARACTERISTICS: ( A ) LENGTH: 2381 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: cDNA to mRNA ( i i i ) HYPOTHETICAL: NO ( i v ). ANTI-SENSE: NO ( v i ) ORIGINAL SOURCE: ( A ) ORGANISM: Homo sapiens (D) DEVELOPMENTAL STAGE: fetal ( F ) TISSUE TYPE: Brain, Liver ( v i i i ) POSITION IN GENOME: (B) MAP POSITION: 3g26 ( i x ) FEATURE: (A) NAMEKEY: mat peptide (B) LOCATION: 160.188 ( C ) IDENTIFICATION METHOD: experimental ( D.) OTHER INFORMATION: EC number=3.1.1.8 evidence=EXPERIMENTAL f gene=“BCHE” f note= “butyrylcholinesterase mature peptide' ( i x ) FEATURE: (A) NAMEKEY: sig peptide (B) LOCATION: 76.159 ( i x ) FEATURE: ( A ) NAME/KEY: mRNA (B) LOCATION: 1.2381 ( i x ) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 76.1884 ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO:9:

5,891,725 41 42 -continued

TT CATTAC CA C T C GT AAAAA G GTAT CTTTT TT AAA T GAA G T TAAA TATTG AAA CA CT G T A 2 28 O

CA C CATA GT T TA CAATAATT A G T G T TT C CT AA GT TAA AAT AA GAA TT GAA T G T CAATAAT 2 3 4 O

G A GAATAATT AA AATAAG CA CAGAAAAT CA CAAAAAAAAA C 23 8 1

We claim: 7. A composition for increasing hemopoietic Stem cell 1. A Synthetic partially phosphorothioated antisense oli fraction in bone marrow cells Samples in Vitro wherein the godeoxynucleotide directed against a region spanning the composition comprises a partially phosphorothioated oli initiator AUG codon in the human ACHE gene, said oli godeoxynucleotide according to claim 1. godeoxynucleotide having the formula: 5'-CTGCGGGGGCCTCAT-3 (SEQ ID NO:2) 8. A composition for enhancing macrophage production wherein Said oligodeoxynucleotide has phosphorotioate 15 and increasing hemopoietic Stem cell counts wherein the bonds linking between the four 3'-terminus nucleotide bases. composition comprises a partially phosphorothioated oli 2. The Synthetic partially phosphorothioated oligodeoxy godeoxynucleotide according to claim 1. nucleotide of claim 1 comprising SEQ ID No:2 which Stimulates megakaryocytopoiesis. 9. A composition for ex vivo reducing the immune 3. The Synthetic partially phosphorothioated oligodeoxy response of organs to be transplanted wherein the compo nucleotide of claim 1 comprising SEQ ID No:2 which Sition comprises a Synthetic partially phosphorothioated inhibits erythropoiesis during the first four days in vitro and oligodeoxynucleotide according to claim 1. ten days in Vivo. 4. The Synthetic partially phosphorothioated oligodeoxy 10. A method of increasing stem cell fraction in bone nucleotides of claim 1 comprising SEQ ID No.2 which 25 marrow cells Samples eX Vivo to be transplanted by contact diverts hemopoietic bone marrow cells development from ing a Sample of extracted bone marrow with an effective megakaryocytes and erythrocytes to macrophages and amount of an oligodeoxynucleotide according to claim 1. mononuclear cells. 5. The Synthetic partially phosphorothioated oligodeoxy 11. A method of treating embryonic or fetal bone marrow nucleotide of claim 1 comprising SEQ ID No:2 which cells ex vivo prior to their Storage in cell banks in viable increases in vitro hemopoietic Stem cell fraction in bone forms devoid of tissue compatibility antigens, by contacting marrow cells Samples. a Sample with an effective amount of an oligodeoxynucle 6. The Synthetic partially phosphorothioated oligodeoxy otide according to claim 1. nucleotide according to claim 1 which enhances in vivo macrophage production and increasing hemopoietic Stem cell counts.