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United States Patent (10) Patent No.: US 8,759,307 B2 Stein Et Al USOO87593 07B2 (12) United States Patent (10) Patent No.: US 8,759,307 B2 Stein et al. (45) Date of Patent: Jun. 24, 2014 (54) OLIGONUCLEOTIDE COMPOUND AND 2006/0287268 A1 12/2006 Iversen et al. ................... 514,44 METHOD FOR TREATING NIDOVIRUS 2007/0021362 A1 1/2007 Geller et al. .. 514,44 2007/0265214 A1 11/2007 Stein et al. .... ... 514/44 INFECTIONS 2009 OO88562 A1 4/2009 Weller et al. ................. 536,245 (75) Inventors: David A. Stein, Corvallis, OR (US); FOREIGN PATENT DOCUMENTS Richard K. Bestwick, Corvallis, OR (US); Patrick L. Iversen, Corvallis, OR WO WO2005/OOO234 A1 1, 2005 (US); Benjamin Neuman, Encinitas, CA WO WO2005/O13905 A1 2, 2005 (US); Michael Buchmeier, Encinitas, OTHER PUBLICATIONS CA (US); Dwight D. Weller, Corvallis, OR (US) Moulton etal Bioconjug Chem. Mar.-Apr. 2004:15(2): 290-9. Cellu lar uptake of antisense morpholino oligomers conjugated to arginine (73) Assignees: Sarepta Therapeutics, Inc., Corvallis, rich peptides.* OR (US); The Scripps Research Moulton etal AntisenseNucleic Acid Drug Dev. Feb. 2003: 13(1): 31 Institute, La Jolla, CA (US) 43. HIV Tat peptide enhances cellular delivery of antisense morpholino oligomers. (*) Notice: Subject to any disclaimer, the term of this Geller et al., Inhibition of Gene Expression in Escherichia coli by patent is extended or adjusted under 35 Antisense Phosphorodiamidate Morpholino Oligomers Antimicro U.S.C. 154(b) by 1101 days. bial Agents and Chemotherapy, Oct. 2003, p. 3233-3239, vol. 47, No. 1O.* Agrawal, S., S. H. Mayrand, et al. (1990). “Site-specific excision (21) Appl. No.: 12/109,856 from RNA by RNase H and mixed-phosphate-backbone oligodeoxynucleotides.” Proc Natl AcadSci USA, 87(4): 1401-5. (22) Filed: Apr. 25, 2008 Agrawal et al., “Antisense therapeutics: is it as simple as complemen tary base recognition?”. Molecular Med. Today, 6:72-81 (2000). (65) Prior Publication Data Allende, R., T. L. Lewis, et al. (1999). "North American and Euro US 2009/OO 12280 A1 Jan. 8, 2009 pean porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions.”.J Gen Virol. 80(Pt.2):307-15. Bailey, C. P. J. M. Dagle et al., (1998). “Cationic oligonucleotides Related U.S. Application Data can mediate specific inhibition of gene expression in Xenopus oocytes.” Nucleic Acids Res., 26(21):4860-7. (60) Division of application No. 1 1/432,155, filed on May Barawkar, D. A. and T. C. Bruice, (1998). “Synthesis, biophysical 10, 2006, now abandoned, which is a properties, and nuclease resistance properties of mixed backbone continuation-in-part of application No. 1 1/022,358, oligodeoxynucleotides containing cationic internucleoside filed on Dec. 22, 2004, now abandoned. guanidinium linkages: deoxynucleic guanidine/DNA chimeras.” Proc Nat AcadSci USA,95(19): 11047-52. (60) Provisional application No. 60/532,701, filed on Dec. Blommers, M.J., U. Pieles, et al. (1994). “An approach to the struc 24, 2003. ture determination of nucleic acid analogues hybridized to RNA. NMR studies of a duplex between 2'-OMe RNA and an (51) Int. C. oligonucleotide containing a single amide backbone modification.” A6 IK 4.8/00 (2006.01) Nucleic Acids Res., 22(20):4187-94. C7H 2L/04 (2006.01) Bonham, M. A. S. Brown, et al. (1995). "An assessment of the C7H 2L/02 (2006.01) antisense properties of RNase H-competent and steric-blocking CI2N 7/00 (2006.01) oligomers.” Nucleic Acids Res. 23(7): 1197-203. U.S. C. Boudvillain, M., M. Guerin, et al. (1997). “Transplatin-modified (52) oligo(2'-O-methyl ribonucleotide)s: a new tool for selective modula USPC ............................ 514/44; 536/23.1: 536/24.5 tion of gene expression.” Biochemistry, 36(10): 2925-31. (58) Field of Classification Search CPC ....................................................... C12N 15/11 (Continued) USPC ........................ 514/44; 536/23.1, 24.5, 235.1 See application file for complete search history. Primary Examiner — Maria Leavitt (74) Attorney, Agent, or Firm — Seed IP Law Group PLLC (56) References Cited (57) ABSTRACT U.S. PATENT DOCUMENTS A method and oligonucleotide compound for inhibiting rep 5,185,444 A 2, 1993 Summerton et al. lication of a nidovirus in virus-infected animal cells are dis 5,892,023 A * 4/1999 Pirotzky et al. .............. 536, 24.5 closed. The compound (i) has a nuclease-resistant backbone, 6,365,351 B1 4/2002 Iversen (ii) is capable of uptake by the infected cells, (iii) contains 6,677,153 B2 1/2004 Iversen 6,784,291 B2 8, 2004 Iversen et al. between 8-25 nucleotide bases, and (iv) has a sequence 6,828,105 B2 12/2004 Stein et al. capable of disrupting base pairing between the transcriptional 6,841,542 B2 1/2005 Bartelmez et al. regulatory sequences in the 5' leader region of the positive 7,049,431 B2 5, 2006 Iversen et al. Strand viral genome and negative-strand 3' subgenomic 7,094,765 B1 8, 2006 Iversen et al. region. In practicing the method, infected cells are exposed to 7.468,418 B2 * 12/2008 Iversen et al. ... 530/300 7,943,762 B2 5/2011 Weller et al. .................... 536, 31 the compound in an amount effective to inhibit viral replica 2003,0224353 A1 12, 2003 Stein et al. tion. 2006/0257852 A1 1 1/2006 Rappuoli et al. 2006/02699.11 A1 11/2006 Iversen et al. ..................... 435.5 8 Claims, 11 Drawing Sheets US 8,759,307 B2 Page 2 (56) References Cited Moulton, H.M. and J. D. Moulton (2003). “Peptide-assisted delivery of steric-blocking antisense oligomers.” Curr Opin Mol Ther. OTHER PUBLICATIONS 5(2): 123-32. Neuman et al., (2004). “Antisense morpholino-oligomers directed Dagle, J. M. J. L. Littig, et al. (2000). “Targeted elimination of against the 5' end of the genome inhibit coronavirus proliferation and Zygotic messages in Xenopus laevis embryos by modified growth”, Journal of Virology, 48(11):5891-5899. oligonucleotides possessing terminal cationic linkages.” Nucleic Pasternak, A. O. E. van den Born, et al. (2001). “Sequence require Acids Res., 28(10): 2153-7. ments for RNA strand transfer during nidovirus discontinuous de Vries, A. A. F., M. C. Horzinek, et al. (1997). “The Genome Subgenomic RNA synthesis.” Embo J 20024):7220-8. Organization of the Nidovirales: Similarities and Differences Pasternak, A.O., E. van den Born, et al. (2003). “The Stability of the between Arteri-Toro-, and Coronaviruses* 1.” Seminars in Virology, Duplex between Sense and Antisense Transcription-Regulating 8(1): 33-47. Sequences Is a Crucial Factor in Arterivirus Subgenomic mRNA Ding, D., S. M. Grayaznov, et al. (1996). “An Synthesis.” J. Virol. 77(2): 1175-1183. oligodeoxyribonucleotide N3'-fwdarw.P5" phosphoramidate duplex Pasternak, A. O. W. J. Spaan, et al. (2004). “Regulation of relative forms an A-type helix in solution.” Nucleic Acids Res., 24(2):354-60. abundance of arterivirus Subgenomic mRNAs.” J Virol. Felgner, P. L., T. R. Gadek, et al. (1987). "Lipofection: a highly 78(15): 8102-13. efficient, lipid-mediated DNA-transfection procedure.” Proc Natl Peiris, J. S. K. Y. Yuen, et al. (2003). “The severe acute respiratory AcadSci USA, 84(21):7413-7. syndrome.” N Engl J Med., 349(25): 2431-41. Gait, M. J. A. S. Jones, et al. (1974). “Synthetic-analogues of Rota, P. A., M. S. Oberste, et al. (2003). “Characterization of a novel polynucleotides XII. Synthesis of thymidine derivatives containing coronavirus associated with severe acute respiratory syndrome.” Sci an oxyacetamido- or an oxyformamido-linkage instead of a ence, 300(5624): 1394-9. phosphodiester group.”J ChemSoc. Perkin 10(14): 1684-6. Savarinio et al., (2006). “Potential Therapies for Coronaviruses'. Gee, J. E. I. Robbins, et al. (1998). "Assessment of high-affinity Expert. Opin. Ther. 1269-1288. hybridization, RNase H cleavage, and covalent linkage in translation Sawicki, S. G. and D. L. Sawicki (1998). "A new model for arrest by antisense oligonucleotides.' Antisense Nucleic Acid Drug coronavirus transcription.” Adv Exp Med Biol. 440:215-9. Dev. 8(2):103-11. Schiavone, N., et al., (2004). “Antisense oligonucleotide drug Guan.Y., B.J. Zheng, etal. (2003). “Isolation and Characterization of design”. Current Pharmaceutical Design, 10(7):769-784. Viruses Related to the SARS Coronavirus from Animals in Southern Stein, D., E. Foster, et al. (1997). "A specificity comparison of four China. Science, 302:276-278. antisense types: morpholino. 2'-O-methyl RNA, DNA, and Hedges et al., (2004). J. Gen. Virol. 85(Part 2):379-390. phosphorothioate DNA” Antisense Nucleic Acid Drug Dev., Homes et al., (2003). “The SARS Coronavirus: A Postgnomic Era', 7(3):151-7. Science, pp. 1377-1378. Summerton, J. and D. Weller (1997). “Morpholino antisense Lai. M. M. and D. Cavanagh (1997). "The molecular biology of oligomers: design, preparation, and properties.” Antisense Nucleic coronaviruses.” Adv. Virus Res., 48: 1-100. Acid Drug Dev. 7(3): 187-95. Lesnikowski, Z. J. M. Jaworska, et al. (1990). "Octa(thymidine Thiel, V. et al., (Jun. 11, 1993), Genbank Accession No. AY291315. methanephosphonates) of partially defined stereochemistry: synthe “SARS Coronavirus Frankfurt 1 Complete Genome', retrievedMar. sis and effect of chirality at phosphorus on binding to 23, 2005). pentadecadeoxyriboadenylic acid.” Nucleic Acids Res., 18(8):2109 Thiel, V. K. A. Ivanov, et al. (2003). “Mechanisms and enzymes 15. involved in SARS coronavirus genome expression.” J Gen Virol. Linkletter, B. A. and Bruice, T.C., “Solid-phase synthesis of posi 84(Pt 9): 2305-15. tively charged deoxynucleic guanidine (DNG) modified Toulme, J. J. R. L. Tinevez, et al. (1996). “Targeting RNA structures oligonucleotides containing neutral urea linkages: Effect of charge by antisense oligonucleotides.” Biochimie, 78(7): 663-73. deletions on binding and fidelity.” Bioorg. Med Chem. 8(11): 1893 Van Den Born, etal. (2004). “Secondary structure and function of the 1901 (2000).
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