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.