USOO8759307B2

(12) United States Patent (10) Patent N0.: US 8,759,307 B2 Stein et al. (45) Date of Patent: Jun. 24, 2014

(54) 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 INFECTIONS 2007/0265214 A1 ll/2007 Stein et al. 514/44 2009/0088562 A1 4/2009 Weller et al...... 536/24.5

(75) Inventors: David A. Stein, Corvallis, OR (US); FOREIGN PATENT DOCUMENTS Richard K. Bestwick, Corvallis, OR (US); Patrick L. Iversen, Corvallis, OR WO WO2005/000234 A1 1/2005 (US); Benjamin Neuman, Encinitas, CA WO WO2005/013905 A1 2/2005 (US); Michael Buchmeier, Encinitas, OTHER PUBLICATIONS CA (US); Dwight D. Weller, Corvallis, OR (US) Moulton et al Bioconjug Chem. Mar. -Apr. 2004;15(2):290-9. Cellu lar uptake ofantisense oligomers conjugated to arginine (73) Assignees: Sarepta Therapeutics, Inc., Corvallis, rich peptides.* OR (US); The Scripps Research Moulton et al Antisense 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, N0. 10.* Appl. No.: 12/109,856 Agrawal, S., S. H. Mayrand, et al. (1990). “Site-speci?c excision (21) from RNA by RNase H and mixed-phosphate-backbone oligodeoxynucleotides.” Pr0c Nat/Acad Sci 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 2009/0012280 A1 Jan. 8, 2009 pean porcine reproductive and respiratory syndrome differ in non-structural coding regions.”JGen ViroL, 80( Pt 2):307-15. Bailey, C. P., J. M. Dagle et al., (1998). “Cationic Related US. Application Data can mediate speci?c inhibition of gene expression in Xenopus oocytes.” Nucleic Acids Res, 26(21):4860-7. (60) Division of application No. 11/432,155, ?led 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. 11/022,358, oligodeoxynucleotides containing cationic internucleoside ?led on Dec. 22, 2004, now abandoned. guanidinium linkages: deoxynucleic guanidine/DNA chimeras.” Provisional application No. 60/532,701, ?led on Dec. Pr0c NatAcad Sci USA, 95(19): 1 1047-52. (60) 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. Cl. oligonucleotide containing a single amide backbone modi?cation.” A61K 48/00 (2006.01) Nucleic Acids Res, 22(20):4187-94. C07H 21/04 (2006.01) Bonham, M. A., S. Brown, et al. (1995). “An assessment of the C07H 21/02 (2006.01) antisense properties of RNase H-competent and steric-blocking C12N 7/00 (2006.01) oligomers.” Nucleic Acids Res. 23(7): 1197-203. US. Cl. Boudvillain, M., M. Guerin, et al. (1997). “Transplatin-modi?ed (52) olig0(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 Classi?cation Search CPC ...... C12N 15/11 (Continued) USPC ...... 514/44; 536/23.1, 24.5, 235.1 See application ?le 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 -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 between 8-25 bases, and (iv) has a sequence 6,784,291 B2 8/2004 Iversen et al. 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 the compound in an amount effective to inhibit viral replica 7,943,762 B2 5/2011 Weller et al...... 536/31 2003/0224353 A1 12/2003 Stein et al. tion. 2006/0257852 A1 11/2006 Rappuoli et al. 2006/0269911 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 M0l 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 proliferation and zygotic messages in Xenopus laevis embryos by modi?ed growth”, Journal ofVlrology, 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. E, M. C. Horzinek, et al. (1997). “The Genome subgenomic RNA synthesis.” Embo J20(24):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 * 1.” Seminars in Virology, Duplex between Sense and Antisense -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): 1175-1 183. oligodeoxyribonucleotide N3‘.fwdarw.P5‘ phosphoramidate duplex Pasternak, A. O., W. J. Spaan, et al. (2004). “Regulation of relative forms anA-type helix in solution.”NucleicAcids 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. ef?cient, lipid-mediated DNA-transfection procedure.” Proc Natl Peiris, J. S., K. Y. Yuen, et al. (2003). “The severe acute respiratory Acad Sci USA, 84(21):7413-7. syndrome.” NEngl JMed., 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 Chem Soc [Perkin 1] 0(14):1684-6. Savarinio et al., (2006). “Potential Therapies for Coronaviruses”, Gee, J. E., I. Robbins, et al. (1998). “Assessment of high-af?nity 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, et al. (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 speci?city 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): 15 1-7. 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 de?ned stereochemistry: synthe “SARS Coronavirus Frankqu 1 Complete Genome”, [retrieved Mar. 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) modi?ed 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 ?delity.” Bioorg. Med Chem. 8(11):1893 Van Den Born, et al. (2004). “Secondary structure and function of the 1901 (2000). 5‘-proximal region of the equine arteritis virus RNA genome.” RNA, Lu et al., (2004). “Attenuation of SARS coronavirus by a short 10(3):424-437. hairpin RNA expression targeting RNA-dependent RNA Wang et al., (2004). “Inhibition of severe acute respiratory syndrome polymerase”, Virology, 324(1):84-89. virus replication by small interfering in mammalian cells”, Mickle?eld, J ., “Backbone modi?cation of nucleic acids: synthesis, 78(14):7523-7527. structure and therapeutic applications.” CurrMed Chem, 8(10): 1157 Zeng, L., E. K. Godeny, et al. (1995). “Analysis of simian hemor 79 (2001). rhagic fever virus (SHFV) subgenomic RNAs, junction sequences, NCBI Accession No. AY2741 19, SARS Coronavirus Tor2, complete and 5‘ leader.” Virology, 207(2): 543-8. genome, p. 1-2. Zhang et al., (2003). Inhibiting severe acute respiratory syndrome Marra, M. A., S. J. M. Jones, et al. (2003). “The Genome Sequence of associated coronavirus by small interfering RNA, Chinese Medical the SARS-Associated Coronavirus.” Science, 300(5624):1399-1404. Journal, 116(8):1262-1264. McCaffrey A.P. et al. (2003). “A potent and Speci?c Morpholino Zheng et al., (2004). “Prophylactic and therapeutic effects of small Antisense Inhibitor of Hepatitis C Translation in Mice”, Hepatology, interfering RNA targeting SARS-coronavirus”, Antiviral Therapy, 38(2):503-508. 9(3):365-374. Mertes, M. P. and E. A. Coats (1969). “Synthesis ofcarbonate analogs Ziebuhr, J ., E. J. Snijder, et al. (2000). “Virus-encoded proteinases of dinucleosides. 3‘-Thymidinyl 5‘-thymidinyl carbonate, and proteolytic processing in the Nidovirales.” J Gen Virol, 3‘-thymidinyl 5‘-(5-?uoro-2‘-deoxyuridinyl)carbonate, and 3‘-(5 81(4):853-879. ?uoro-2‘-deoxyuridinyl) 5‘-thymidinyl carbonate.” J Med Chem., 12(1):154-7. * cited by examiner

US. Patent Jun. 24, 2014 Sheet 2 0f 11 US 8,759,307 B2

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US 8,759,307 B2 1 2 OLIGONUCLEOTIDE COMPOUND AND containing an oxyacetamido- or an oxyformamido -linkage METHOD FOR TREATING NIDOVIRUS instead of a phosphodiester group.” J Chem Soc [Perkin 1] INFECTIONS 0(14): 1684-6. Gee, J. E., 1. Robbins, et al. (1998). “Assessment of high This application is a divisional of US. application Ser. No. 01 a?inity hybridization, RNase H cleavage, and covalent 11/432,155 ?led May 10, 2006, now abandoned, which is a linkage in translation arrest by antisense oligonucleotides .” continuation-in-part of US. application Ser. No. 11/022,358 Antisense Nucleic Acid Drug Dev 8(2): 103-11. ?led Dec. 22, 2004, now abandoned, which claims the bene?t Guan, Y., B. J. Zheng, et al. (2003). “Isolation and Character ofpriority ofU.S. Application No. 60/532,701 ?led Dec. 24, ization of Viruses Related to the SARS Coronavirus from 2003, now abandoned. All applications are incorporated in Animals in Southern China.” Science: 1087139. their entirety herein by reference. Lai, M. M. and D. Cavanagh (1997). “The molecular biology A portion of the work described herein was supported by of coronaviruses.” Adv Virus Res 48: 1-100. grant numbers NS 41219, Al 43103, Al 25913 from the Lesnikowski, Z. J ., M. Jaworska, et al. (1990). “Octa(thymi National Institutes of Health, and HHSN266200400058C dine methanephosphonates) of partially de?ned stere from Health and Human Services, The United States Govem ochemistry: synthesis and effect of chirality at phosphorus ment has certain rights in this invention. on binding to pentadecadeoxyriboadenylic acid.” Nucleic FIELD OF THE INVENTION Acids Res 18(8): 2109-15. Marra, M. A., S. J. M. Jones, et al. (2003). “The Genome This invention relates to an oligonucleotide analog for use 20 Sequence of the SARS-Associated Coronavirus.” Science in treating in animals a coronavirus infection or, more gener 300(5624): 1399-1404. ally, an infection by a member of the Nidovirales order, to an Mertes, M. P. and E. A. Coats (1969). “Synthesis of carbonate antiviral method employing the analog, and to a method for analogs of dinucleosides. 3'-Thymidinyl 5'-thymidinyl car monitoring binding of the analog to a viral genome target site. bonate, 3'-thymidinyl 5'-(5-?uoro-2'-deoxyuridinyl) car 25 bonate, and 3'-(5-?uoro-2'-deoxyuridinyl) 5'-thymidinyl REFERENCES carbonate.” JMed Chem 12(1): 154-7. Moulton, H. M. and J. D. Moulton (2003). “Peptide-assisted Agrawal, S., S. H. Mayrand, et al. (1990). “Site-speci?c exci delivery of steric-blocking antisense oligomers.” Curr sion from RNA by RNase H and mixed-phosphate-back Opin M0l Ther 5(2): 123-32. bone oligodeoxynucleotides.” Proc Natl Acad Sci USA 30 Pasternak, A. O., W. J. Spaan, et al. (2004). “Regulation of 87(4): 1401-5. relative abundance of arterivirus subgenomic mRNAs.” J Allende, R., T. L. Lewis, et al. (1999). “North American and European porcine reproductive and respiratory syndrome Virol 78(15): 8102-13. viruses differ in non-structural protein coding regions.” J Pasternak, A. O., E. van den Born, et al. (2001). “Sequence requirements for RNA strand transfer during nidovirus Gen Virol 80 (Pt 2): 307-15. 35 Balasuriya, U. B., J. F. Hedges, et al. (2004). “Genetic char discontinuous subgenomic RNA synthesis.” Embo J acterization of equine arteritis virus during persistent 20(24): 7220-8. infection of stallions.” J Gen Virol 85(Pt 2): 379-90. Pasternak, A. O., E. van den Born, et al. (2003). “The Stability Blommers, M. J., U. Pieles, et al. (1994). “An approach to the of the Duplex between Sense and Antisense Transcription structure determination of nucleic acid analogues hybrid 40 Regulating Sequences Is a Crucial Factor in Arterivirus ized to RNA. NMR studies of a duplex between 2'-OMe Subgenomic mRNA Synthesis.” J. Virol. 77(2): 1175 RNA and an oligonucleotide containing a single amide 1 183. backbone modi?cation.” Nucleic Acids Res 22(20): 4187 Peiris, J. S., K. Y. Yuen, et al. (2003). “The severe acute 94. respiratory syndrome.” NEnglJMed 349(25): 2431-41. Bonham, M. A., S. Brown, et al. (1995). “An assessment of 45 Rota, P. A., M. S. Oberste, et al. (2003). “Characterization of the antisense properties of RNase H-competent and steric a novel coronavirus associated with severe acute respira blocking oligomers.” Nucleic Acids Res 23(7): 1197-203. tory syndrome.” Science 300(5624): 1394-9. Boudvillain, M., M. Guerin, et al. (1997). “Transplatin-modi Sawicki, S. G. and D. L. Sawicki (1998). “A new model for ?ed oligo(2'-O-methyl ribonucleotide)s: a new tool for coronavirus transcription.” Adv Exp Med Biol 440: 215-9. selective modulation of gene expression.” Biochemistry 50 Siddell, S. G. (1995). The Coronaviridae. New York, Plenum 36(10): 2925-31. Press. Dagle, J. M., J. L. Littig, et al. (2000). “Targeted elimination of zygotic messages in Xenopus laevis embryos by modi Stein, D., E. Foster, et al. (1997). “A speci?city comparison of ?ed oligonucleotides possessing terminal cationic link four antisense types: morpholino, 2'-O-methyl RNA, DNA, and phosphorothioate DNA.” Antisense Nucleic ages.” Nucleic Acids Res 28(10): 2153-7. 55 deVries, A. A. F., M. C. Horzinek, et al. (1997). “The Genome Acid Drug Dev 7(3): 151-7. Organization of the Nidovirales: Similarities and Differ Strauss, J. H. and E. G. Strauss (2002). Viruses and Human ences betweenArteri-, Toro-, and Coronaviruses*1.”Semi Disease. San Diego, Academic Press. nars in Virology 8(1): 33-47. Summerton, J. and D. Weller (1997). “Morpholino antisense Ding, D., S. M. Grayaznov, et al. (1996). “An oligodeoxyri 60 oligomers: design, preparation, and properties.” Antisense bonucleotide N3'—>P5' phosphoramidate duplex forms an Nucleic Acid Drug Dev 7(3): 187-95. A-type helix in solution.” NucleicAcids Res 24(2): 354-60. Thiel, V., K. A. lvanov, et al. (2003). “Mechanisms and Felgner, P. L., T. R. Gadek, et al. (1987). “Lipofection: a enzymes involved in SARS coronavirus genome expres highly ef?cient, lipid-mediated DNA-transfection proce sion.” J Gen Virol 84(Pt 9): 2305-15. dure.” Proc NazlAcad Sci USA 84(21): 7413-7. 65 Toulme, J. J ., R. L. Tinevez, et al. (1996). “Targeting RNA Gait, M. J., A. S. Jones, et al. (1974). “Synthetic-analogues of structures by antisense oligonucleotides.” Biochimie polynucleotides XII. Synthesis of thymidine derivatives 78(7): 663-73. US 8,759,307 B2 3 4 VAN DEN BORN, E., A. P. GULTYAEV, et al. (2004). “Sec otide bases, and (iv) having a sequence that is complementary ondary structure and function of the 5'-proximal region of to at least 8 bases contained in one of: the equine arteritis virus RNA genome.” RNA 10(3): 424 (1) a sequence in a 5' leader sequence of the nidovirus’ 437. positive-strand genomic RNA from the group SEQ ID NOS: Zeng, L., E. K. Godeny, et al. (1995). “Analysis of simian 1-9, each sequence of which includes an internal leader tran hemorrhagic fever virus (SHFV) subgenomic RNAs, junc scriptional regulatory sequence; and, tion sequences, and 5' leader.” Vzrology 207(2): 543-8. (2) a sequence in a negative-strand 3' subgenomic region of Ziebuhr, 1., E. J. Snijder, et al. (2000). “Virus-encoded pro the virus from the group exempli?ed by SEQ ID NOS: 10-19, teinases and proteolytic processing in the Nidovirales.” J each sequence of which includes an internal body transcrip Gen V1r0l81(4): 853-879. tional regulatory sequence that is substantially complemen tary to the corresponding leader transcriptional regulatory BACKGROUND OF THE INVENTION sequence. The Nidovirales is a recently established order comprising The compound is capable of forming with the nidovirus (1) positive-strand genomic RNA or (2) the negative-strand 3' the families Arteriviridae (genus Arterivirus) and Coronaviri 5 dae (genera Coronavirus and Torovirus). Despite noteworthy sub genomic region, a heteroduplex structure characterized by differences in genome size, complexity and virion architec (1) a Tm of dissociation of at least 45° C., and (2) a disrupted ture, coronaviruses, toroviruses and arteriviruses are remark base pairing between the transcriptional regulatory sequences ably similar in genome organization and replication strategy in the 5' leader region of the positive-strand viral genome and (de Vries, Horzinek et al 1997). The name Nidovirales is 20 negative-strand 3' subgenomic region. derived from the Latin nidus, to nest, and refers to the 3' The compound may be composed of morpholino subunits coterminal nested set of subgenomic (sg) viral mRNAs pro and phosphorus-containing intersubunit linkages joining a duced during infection. Sequence comparisons of the repli morpholino nitrogen of one subunit to a 5' exocyclic carbon of case genes suggest that the Nidovirales have evolved from a an adjacent subunit. Exemplary intersubunit linkages are common ancestor despite their substantial differences. 25 phosphorodiamidate linkages in accordance with the struc Coronaviruses cause about 30% of common colds in ture: humans and, unlike rhinoviruses, cause both upper and lower respiratory infections, the latter being a more serious af?ic tion. In addition, coronaviruses cause gastroenteritis and diar rhea in humans and many other serious diseases in non 30 human animals including mice, chickens, pigs and cats. Although no known human arteriviruses exist, arteriviruses Y1 cause a number of diseases in horses, pigs, mice and mon keys. 35 ‘ :Oj/ Pj The most well-known human coronavirus has only recently appeared and is responsible for severe acute respira tory syndrome (SARS), a life threatening form of pneumonia T (Peiris, Yuen et al 2003). SARS is caused by a previously unknown coronavirus named SARS coronavirus (SARS CoV). First appearing in November 2002, an epidemic 40 where Y1:O, Z:O, Pj is a purine or pyrimidine base emerged that spread from its zoonotic origin in Guangdong pairing moiety effective to bind, by base-speci?c hydrogen Province, China, to 26 countries on ?ve continents. By bonding, to a base in a polynucleotide, and X is alkyl, alkoxy, August 2003, a cumulative total of 8422 cases and 774 deaths thioalkoxy, amino or alkyl amino, including dialkylamino. had been recorded by the World Health Organization. The In one general embodiment, the compound has a sequence rapid transmission by aerosols and the fecal-oral route and the 45 complementary to at least 8 bases contained in the 5' leader high mortality rate (11%) make SARS a potential global sequence of the nidovirus’ positive-strand genomic RNA threat for which no ef?cacious therapy is available. from the group SEQ ID NOS: 1-9. The compound sequence is No vaccines for coronaviruses or arteriviruses (Nidovi preferably complementary to at least a portion of the tran ruses) are available and no effective antiviral therapies are scriptional regulatory sequence contained within one of the available to treat an infection. As with many other human 50 sequences SEQ ID NOS: 1-9. Exemplary compound viral pathogens, available treatment involves supportive mea sequences in this embodiment include SEQ ID NOS: 20-35. sures such as anti-pyretics to keep fever down, ?uids, antibi The compound may be composed of morpholino subunits otics for secondary bacterial infections and respiratory sup linked with the uncharged linkages described above inter port as necessary. spersed with linkages that are positively charged at physi In view of the severity of the diseases caused by Nidovi 55 ological pH. The total number of positively charged linkages ruses and the lack of effective prevention or therapies, it is is between 2 and no more than half of the total number of therefore an object of the present invention to provide thera linkages. The positively charged linkages may have the struc peutic compounds and methods for treating a host infected ture above, where X is 1-piperazine. with a coronavirus, torovirus or arterivirus. For use in inhibiting replication of human SARS virus, the 60 compound may contain one of sequences SEQ ID NOS: 26 SUMMARY OF THE INVENTION and 27. For use in inhibiting replication of human coronavius 229E or human coronavirus-OC43, the compound may con In one aspect, the invention includes an oligonucleotide tain one of the sequences SEQ ID NOS: 22 or 23, for the compound for use in inhibiting replication of a nidovirus in coronavirus-229E, and the sequence SEQ ID NOS: 24 or 25, human cells. The compound is characterized by: (i) a 65 for the coronavirus-OC43. For use in inhibiting replication of nuclease-resistant backbone, (ii) capable of uptake by virus feline coronavirus, the compound may contain SEQ ID NOS: infected human cells, (iii) containing between 8-25 nucle 20 or 21. US 8,759,307 B2 5 6 In another general embodiment, the compound has a FIG. 3A-3G show the backbone structures of various oli sequence complementary to at least 8 bases contained in the gonucleotide analogs with uncharged backbones and FIG. 3H negative-strand 3' sub genomic region of the virus exempli?ed shows a preferred cationic linkage. by the group SEQ ID NOS: 10-19. The compound preferably FIGS. 4A-4D show the repeating subunit segment of exem has a sequence complementary to at least a portion of the plary morpholino oligonucleotides, designated 3A-3D. minus-strand body transcriptional regulatory sequence con FIG. 5 shows graphically the reduction of SARS viral titer tained within one of the sequences SEQ ID NOS: 10-19. when SARS-infected cells are cultured in the presence of Exemplary compound sequences in this embodiment contain either of two antisense PMOs targeted at the leader transcrip a sequence from the group SEQ ID NOS: 36-45. tional regulatory sequence. For use in inhibiting replication of human SARS virus, the FIG. 6 shows graphically the reduction in SARS plaque compound may contain one of the sequences SEQ ID NOS: size when infected cells are cultured in the presence of either 3 6-43. For use in inhibiting replication of simian hemorrhagic of two antisense PMOs targeted at the leader transcriptional regulatory sequence. fever virus, the compound may contain the SEQ ID NOS: 44 FIGS. 7A and 7B show photomicrographs of SARS-in and 45. fected cells, control cells and the corresponding SARS-in In the method of the invention for inhibiting nidovirus duced cytopathic effects in the presence of a variety of anti replication in virus-infected cells, the cells are exposed to the sense PMO compounds including two antisense PMOs oligonucleotide compound, in an amount suf?cient to inhibit targeted at the leader transcriptional regulatory sequence. nidovirus replication in the virus-infected cells. The inhibi FIGS. 8A-8C show photomicrographs of EAV-infected tion is due to base-pair binding of the compound to (l) a 20 cells, control cells and the corresponding immuno?uores sequence in a 5' leader sequence of the nidovirus’ positive cence using labeled antibodies to EAV-speci?c in the strand genomic RNA that includes an internal leader tran presence of an antisense PMO that targets the leader TRS of scriptional regulatory sequence, or (2) a sequence in a nega EAV. tive-strand 3' subgenomic region of the virus that includes an FIG. 9 shows graphically the reduction of MHV viral titer internal body transcriptional regulatory sequence that is sub 25 when MHV-infected cells are cultured in the presence of a stantially complementary to the corresponding transcrip PMO targeted to the leader TRS of MHV. tional regulatory sequence contained the 5' leader sequence. FIG. 10 shows the synthetic steps to produce subunits used This base-pair binding forms a viral-RNA/compound hetero to produce +PMO containing the (l -piperazino) phosphi duplex characterized by (l) a Tm of dissociation of at least 45° nylideneoxy cationic linkage as shown in FIG. 3H. C., and (2) a disrupted base pairing between the transcrip 30 tional regulatory sequences in the 5' leader region of the DETAILED DESCRIPTION OF THE INVENTION positive-strand viral genome and negative-strand 3' subge I. De?nitions nomic region. Various embodiments of the compound, noted above, are incorporated into the method. 35 The terms below, as used herein, have the following mean For use in treating a nidovirus infection in a human or ings, unless indicated otherwise: veterinary-animal subject, the compound may be adminis The terms “oligonucleotide analog” refers to oligonucle tered orally to the subject, to contact the compound with the otide having (i) a modi?ed backbone structure, e.g., a back virus-infected cells. The method may further include moni bone other than the standard pho sphodiester linkage found in toring a subject body ?uid for the appearance of a heterodu 40 natural oligo- and polynucleotides, and (ii) optionally, modi plex composed of the oligonucleotide compound and a ?ed sugar moieties, e.g., morpholino moieties rather than complementary portion of the viral genome in positive- or ribose or deoxyribose moieties. The analog supports bases negative-strand form. capable of hydrogen bonding by Watson-Crick base pairing These and other objects and features of the invention will to standard polynucleotide bases, where the analog backbone become more fully apparent when the following detailed 45 presents the bases in a manner to permit such hydrogen bond description of the invention is read in conjunction with the ing in a sequence-speci?c fashion between the oligonucle accompanying drawings. otide analog molecule and bases in a standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA). Pre BRIEF DESCRIPTION OF THE DRAWINGS ferred analogs are those having a substantially uncharged, 50 phosphorus containing backbone. FIG. 1 shows a genetic map and predicted viral proteins of A substantially uncharged, phosphorus containing back the SARS coronavirus which is representative of the genome bone in an oligonucleotide analog is one in which a majority organization of all Nidoviruses. Also shown are the eight of the subunit linkages, e.g., between 50-100%, are subgenomic mRNAs (mRNA 2-8) that are produced. The uncharged at physiological pH, and contain a single phospho small black boxes at the 5' end of the genomic and subge 55 rous atom. The analog contains between 8 and 40 subunits, nomic RNAs represent the common 72 nucleotide leader typically about 8-25 subunits, and preferably about 12 to 25 RNA sequence derived from the 5' terminal 72 of subunits. The analog may have exact sequence complemen the genomic RNA during discontinuous transcription. tarity to the target sequence or near complementarity, as FIG. 2A-2B is a schematic representation of Nidovirus de?ned below. RNA replication and the process of discontinuous transcrip 60 A “subunit” of an oligonucleotide analog refers to one tion that results in a nested set of 3'co-terminal subgenomic nucleotide (or nucleotide analog) unit of the analog. The term mRNAs with a common 5' leader sequence. FIG. 2B indicates may refer to the nucleotide unit with or without the attached the point at which antisense oligomers targeted to the 5' intersubunit linkage, although, when referring to a “charged positive-strand leader transcriptional regulatory sequence subunit”, the charge typically resides within the intersubunit interfere with this process. Also shown in FIG. 2B is an 65 linkage (e.g. a phosphate or phosphorothioate linkage). antisense oligomer targeted to the 3' minus-strand body tran A “morpholino oligonucleotide analog” is an oligonucle scriptional regulatory sequence. otide analog composed of morpholino subunit structures of US 8,759,307 B2 7 8 the form shown in FIGS. 3A-3D, where (i) the structures are that is, the sequence to which the oligonucleotide analog will linked together by phosphorus-containing linkages, one to hybridize. The target sequence includes at least a portion of three atoms long, joining the morpholino nitrogen of one one of the sequences identi?ed as SEQ ID NOS: 1-9, repre subunit to the 5' exocyclic carbon of an adjacent subunit, and senting the genome’s leader transcriptional regulatory (ii) PI. and P]. are purine or pyrimidine base-pairing moieties sequence in a member of the Nidovirales, as discussed further effective to bind, by base-speci?c hydrogen bonding, to a below. Another target sequence includes at least a portion of base in a polynucleotide. The purine or pyrimidine base the 3' minus-strand body transcriptional regulatory sequence pairing moiety is typically adenine, cytosine, guanine, uracil exempli?ed by SEQ ID NOS: 10-19. or thymine. The synthesis, structures, and binding character The term “targeting sequence” is the sequence in the oli istics of morpholino oligomers are detailed in US. Pat. Nos. gonucleotide analog that is complementary or substantially 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, complementary to the target sequence in the RNA genome. 5,521,063, and 5,506,337, all of which are incorporated The entire sequence, or only a portion of, the analog may be herein by reference. complementary to the target sequence, or only a portion of the The subunit and linkage shown in FIG. 3B are used for total analog sequence. For example, in an analog having 20 six-atom repeating-unit backbones, as shown in FIG. 3B bases, only 8-12 may be targeting sequences. Typically, the (where the six atoms include: a morpholino nitrogen, the targeting sequence is formed of contiguous bases in the ana connected phosphorus atom, the atom (usually oxygen) link log, but may alternatively be formed of non-contiguous ing the phosphorus atom to the 5' exocyclic carbon, the 5' sequences that whenplaced together, e.g., from opposite ends exocyclic carbon, and two carbon atoms of the next mor 20 of the analog, constitute sequence that spans the target pholino ring). In these structures, the atom Yl linking the 5' sequence. As will be seen, the target and targeting sequences exocyclic morpholino carbon to the phosphorus group may be are selected such that binding of the analog to part of the viral sulfur, nitrogen, carbon or, preferably, oxygen. The X moiety genome acts to disrupt or prevent formation of subgenomic pendant from the phosphorus is any stable group which does RNA formed by the interaction between leader and body not interfere with base-speci?c hydrogen bonding. Preferred 25 transcriptional regulatory sequences. X groups include ?uoro, alkyl, alkoxy, thioalkoxy, and alkyl Target and targeting sequences are described as “comple amino, including cyclic amines, all of which can be variously mentary” to one another when hybridization occurs in an substituted, as long as base-speci?c bonding is not disrupted. antiparallel con?guration. A double-stranded polynucleotide Alkyl, alkoxy and thioalkoxy preferably include 1-6 carbon can be “complementary” to another polynucleotide. A target atoms. Alkyl amino preferably refers to lower alkyl (C 1 to C6) 30 ing sequence may have “near” or “substantial” complemen substitution, and cyclic amines are preferably 5- to 7-mem tarity to the target sequence and still function for the purpose bered nitrogen heterocycles optionally containing 1-2 addi of the present invention. Preferably, the oligonucleotide ana tional heteroatoms selected from oxygen, nitrogen, and sul logs employed in the present invention have at most one fur. Z is sulfur or oxygen, and is preferably oxygen. 35 mismatch with the target sequence out of 10 nucleotides, and A preferred morpholino oligomer is a phosphorodiami preferably at mo st one mismatch out of 20. Alternatively, the date-linked morpholino oligomer, referred to herein as a antisense oligomers employed have at least 90% sequence PMO. Such oligomers are composed of morpholino subunit homology, and preferably at least 95% sequence homology, structures such as shown in FIG. 2B, where X:NH2, NHR, with the exemplary targeting sequences as designated herein. or NR2 (where R is lower alkyl, preferably methyl), Y:O, 40 An oligonucleotide analog “speci?cally hybridizes” to a and Z:O, and Pi and Pj are purine or pyrimidine base target polynucleotide if the oligomer hybridizes to the target pairing moieties effective to bind, by base-speci?c hydrogen under physiological conditions, with a Tm substantially bonding, to a base in a polynucleotide, as seen in FIG. 3G. greater than 45° C., preferably at least 50° C., and typically Also preferred are morpholino oligomers where the pho spho 60° C.-80° C. or higher. Such hybridization preferably corre rdiamidate linkages are uncharged linkages as shown in FIG. 45 sponds to stringent hybridization conditions. At a given ionic 3G interspersed with cationic linkages as shown in FIG. 3H strength and pH, the Tm is the temperature at which 50% of a where, in FIG. 2B, X:1-piperazino. In another FIG. 2B target sequence hybridizes to a complementary polynucle embodiment, XIlower alkoxy, such as methoxy or ethoxy, otide. Again, such hybridization may occur with “near” or Y:NH or NR, where R is lower alkyl, and Z:O. “substantial” complementary of the antisense oligomer to the The term “substituted”, particularly with respect to an 50 target sequence, as well as with exact complementarity. alkyl, alkoxy, thioalkoxy, or alkylamino group, refers to A “nuclease-resistant” oligomeric molecule (oligomer) replacement of a hydrogen atom on carbon with a heteroa refers to one whose backbone is substantially resistant to tom-containing substituent, such as, for example, halogen, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino, imino, nuclease cleavage, in non-hybridized or hybridized form; by common extracellular and intracellular nucleases in the body; oxo (keto), nitro, cyano, or various acids or esters such as 55 carboxylic, sulfonic, or phosphonic. It may also refer to that is, the oligomer shows little or no nuclease cleavage replacement of a hydrogen atom on a heteroatom (such as an under normal nuclease conditions in the body to which the amine hydrogen) with an alkyl, carbonyl or other carbon oligomer is exposed. containing group. A “heteroduplex” refers to a duplex between an oligoncu As used herein, the term “target”, relative to the viral 60 leotide analog and the complementary portion of a target genomic RNA, refers to a viral genomic RNA, and may RNA. A “nuclease-resistant heteroduplex” refers to a hetero include either the positive strand RNA which is the replicative duplex formed by the binding of an antisense oligomer to its strand of the virus, or the negative or antisense strand which complementary target, such that the heteroduplex is substan is formed in producing multiple new copies of the positive tially resistant to in vivo degradation by intracellular and strand RNA. 65 extracellular nucleases, such as RNAseH, which are capable The term “target sequence” refers to a portion of the target of cutting double-stranded RNA/RNA or RNA/DNA com RNA against which the oligonucleotide analog is directed, plexes. US 8,759,307 B2 10 A “base-speci?c intracellular binding event involving a TABLE 1 target RN ” refers to the speci?c binding of an oligonucle otide analog to a target RNA sequence inside a cell. The base Representative Nidoviruses speci?city of such binding is sequence speci?c. For example, Virus Name Abbreviation a single-stranded polynucleotide can speci?cally bind to a single-stranded polynucleotide that is complementary in Canine coronavirus CCoV Feline coronavirus FCoV sequence. Human coronavirus 229E HCoV—229E An “effective amount” of an antisense oligomer, targeted Porcine epidemic diarrhea virus PEDV against an infecting ssRNA virus, is an amount effective to Transmissible gastroenteritis virus TGEV Porcine Respiratory Coronavirus PRCV reduce the rate of replication of the infecting virus, and/or Bovine coronavirus BCoV viral load, and/or symptoms associated with the viral infec Human coronavirus OC43 HCoV—OC43 tion. Murine hepatitis virus MHV Rat coronavirus RCV As used herein, the term “body ?uid” encompasses a vari Infectious bronchitis virus IBV ety of sample types obtained from a subject including, urine, Turkey coronavirus TCoV saliva, plasma, blood, spinal ?uid, or other sample of biologi Rabbit coronavirus RbCoV SARS coronavirus SARS—CoV cal origin, such as skin cells or dermal debris, and may refer Human torovirus HuTV to cells or cell fragments suspended therein, or the liquid Equine arteritis virus EAV medium and its solutes. Porcine reproductive and PRRSV respiratory syndrome virus The term “relative amount” is used where a comparison is 20 Porcine hemagglutinating PHEV made between a test measurement and a control measure encephalomyelitis virus ment. The relative amount of a reagent forming a complex in Simian hemorrhagic fever virus SHFV a reaction is the amount reacting with a test specimen, com pared with the amount reacting with a control specimen. The control specimen may be run separately in the same assay, or 25 II. Target Coronaviruses and Arteriviruses it may be part of the same sample (for example, normal tissue surrounding a malignant area in a tissue section). The present invention is based on the discovery that effec “Treatment” of an individual or a cell is any type of inter tive inhibition of coronavirus replication can be achieved by vention provided as a means to alter the natural course of the exposing coronavirus-infected cells to oligomeric analogs (i) individual or cell. Treatment includes, but is not limited to, 30 targeted to the transcriptional regulatory sequence (TRS) administration of e. g., a pharmaceutical composition, and region of coronavirus RNA and (ii) having physical and phar may be performed either prophylactically, or subsequent to macokinetic features which allow effective interaction the initiation of a pathologic event or contact with an etiologic between the analog and the viral RNA within host cells. In agent. The related term “improved therapeutic outcome” rela 35 one aspect, the analogs can be used in treating a mammalian tive to a patient diagnosed as infected with a particular virus, subject infected with the virus. refers to a slowing or diminution in the growth of virus, or The invention targets members of the Coronaviridae and viral load, or detectable symptoms associated with infection Arteriviridae families of the Nidovirales order including, but by that particular virus. not limited to, the viruses described below. Various physical, An agent is “actively taken up by mammalian cells” when 40 morphological, and biological characteristics of the genes, the agent can enter the cell by a mechanism other than passive and members therein, can be found, for example, in (Strauss diffusion across the cell membrane. The agent may be trans and Strauss 2002), and in one or more of the references cited ported, for example, by “active transport”, referring to trans herein. Some of the key biological, pathological and epide port of agents across a mammalian cell membrane by e.g. an miological characteristics of representative nidoviruses are ATP-dependent transport mechanism, or by “facilitated 45 summarized below. Recent reviews on coronaviruses are transport”, referring to transport of antisense agents across available (e. g. see (Siddell 1995; Lai and Cavanagh 1997) and the cell membrane by a transport mechanism that requires are incorporated herein in their entirety. binding of the agent to a transport protein, which then facili A. Human Coronaviruses tates passage of the bound agent across the membrane. For Coronaviruses are known to cause approximately 30% of both active and facilitated transport, the oligonucleotide ana 50 common colds in humans. The most studied of these are log preferably has a substantially uncharged backbone, as human coronaviruses HCoV-229E and HCoV-OC43. These de?ned below. Alternatively, the antisense compound may be viruses are spread by the respiratory route and, unlike rhi noviruses, cause both upper and lower respiratory tract infec formulated in a complexed form, such as an agent having an tions. Lower respiratory tract infections are considered more anionic backbone complexed with cationic lipids or lipo 55 serious clinically. In addition to these viruses, human torovi somes, which can be taken into cells by an endocytotic rus (HuTV) causes gastroenteritis and diarrhea in infected mechanism. The analog may be conjugated, e.g., at its 5' or 3' individuals. end, to an arginine rich peptide, e.g., the HIV TAT protein, or The SARS-CoV causes a life-threatening form of pneumo polyarginine, to facilitate transport into the target host cell. nia called severe acute respiratory syndrome (SARS). From The term “coronavirus” is used herein to include all mem 60 its likely zoonotic origin in Guangdong Province, China in bers of the Coronaviridae family including viruses of the November 2002, the SARS-CoV spread rapidly to 29 coun Coronavirus and Torovirus genera. The term “arterivirus” tries, infected over 8400 individuals and caused more than includes members of the Arteriviridae family which includes 750 deaths (Peiris, Yuen et al 2003). The rapid transmission the Arterivirus genera. The term “Nidovirus” refers to viruses by aerosols and probably the fecal-oral route coupled with the of the Nidovirales order which includes the families Coro 65 high mortality rate make SARS a global threat for which no naviridae and Arteriviridae. Representative Nidoviruses are e?icacious therapy is available. There is evidence that natural listed in Table 1. below. infection with SARS-CoV occurs in a number of animal US 8,759,307 B2 11 12 species indigenous to China and parts of southeast Asia 3' end of the viral genome, each one provided at its 5' end with (Guan, Zheng et al 2003). Genome sequences of SARS-CoV a sequence identical to the genomic 5' “leader” sequence. The isolates obtained from a number of index patients have been mRNAs are each functionally monocistronic (i.e. the proteins published recently (Marra, Jones et al 2003; Rota, Oberste et are translated only from the 5'-most ORF). A schematic that al 2003). A virus closely related genetically to SARS-CoV illustrates the gene organization and sg mRNAs of SARS was isolated from several animals obtained from a market in CoV is shown in FIG. 1 (Thiel, Ivanov et al 2003). Quangdong Province. Sequencing of the viruses obtained The control of gene expression by discontinuous transcrip from these animals demonstrated that the most signi?cant tion is novel and unique to a small number of single stranded difference between them and SARS-CoV was an additional RNA viruses, most notably the Nidovirales order (de Vries, 29 base-pair sequence in the animal viruses. The role of Horzinek et al. 1997). FIG. 2A provides a schematic of the animals in the transmission of SARS-CoV to humans and replication and transcriptional mechanism employed by whether an animal reservoir for the virus exists is the subject members of the Nidovirales (Pasternak, van den Born et al. of active, ongoing research. B. Animal Coronaviruses and Arteriviruses 2001). As with most single stranded, positive-sense RNA Coronaviruses and Arteriviruses for many other animals viruses, the infecting RNA is translated to yield the viral are known, including mice chickens, pigs, and cats. Associ encoded RNA polymerase or replicase. The RNA polymerase ated diseases include respiratory disease, gastroenteritis, then produces either full length negative-sense RNA, used as hepatitis, and a syndrome similar to multiple sclerosis of a template for additional full-length, positive-sense genomic humans, among many other illnesses. Mouse hepatitis virus RNA synthesis, or a series of negative-sense subgenomic (sg) (MHV) has been particularly well studied and, along with the 20 RNAs which serve as templates for synthesis of the comple Arterivirus equine arteritis virus (EAV) has been the proto mentary, positive-sense sg mRNAs. This process is termed typic molecular model system for the Nidovirales order. discontinuous transcription (Sawicki and Sawicki 1998). One animal coronavirus that causes signi?cant animal Negative-sense sg RNAs are produced by a mechanism in mortality is feline infectious peritonitis virus, the leading which the viral RNA polymerase copies the 3' end of the cause of death in young domestic cats. The feline coronavi 25 genomic RNA template and completes synthesis by “jump ruses (FCOV) generally do not cause infections with high ing” to ?nish copying the immediate 5' end. The negative morbidity but in a small percentage of cases, the virus mutates sense RNAs are then used to synthesize individual sg to become more virulent. This virus, feline infectious perito mRNAs. Each sg mRNA contains the same 5' end sequence nitis virus (FIPV), causes severe disease in young cats. This approximately 70 nucleotides long (approximately 200 disease is in large part immunopathological and understand 30 nucleotides in the Arteriviridae) called the leader sequence. ing it is a major goal of coronavirus research. The infection Coronavirus gene expression is regulated at the level of tran causes lesions in many organs, most prominently in the liver scription primarily through the mechanism of discontinuous and spleen. The disease is further characterized by dissemi transcription of the negative-sense templates. nated in?ammation and serositis in the abdominal and tho The fusion of the noncontiguous sequences during discon racic cavities. In addition to this “wet” or effusive form, a 35 “dry” or noneffusive form of feline infectious peritonitis tinuous transcription is believed to be achieved through the (FIP) also occurs. Both forms are different manifestations of involvement of transcriptional regulatory sequences (TRSs). the same infection. Despite many studies, the pathogenesis of The TRS sequences are short, 6-12 nucleotide regions that are FIP is still not well understood. found at the 3' end of the leader sequence (leader TRS), and Another serious non-human Nidovirus is porcine repro 40 upstream of the genes in the 3'-proximal part of the genome ductive and respiratory syndrome virus (PRRSV), an arterivi (body TRSs). The TRSs are homologous and are believed to rus similar to EAV and SHFV. The disease caused by PRRSV function by a mechanism where the minus-strand body TRS causes reproductive failure in sows, pre-weaning mortality “jumps” to the complementary leader TRS on the positive and respiratory tract illness that can have severe conse sense strand where it “lands” (i.e., the two TRSs hybridize) quences, especially in piglets (Allende, Lewis et al. 1999). 45 allowing completion of the synthesis of the negative-sense This virus causes signi?cant losses in the pig industry with leader sequence (i.e., see FIG. 2A) (Pasternak, van den Born associated economic rami?cations. et al. 2001). III. Coronavirus and Arterivirus Replication and IV. Antisense Targeting of Nidovirus Transcriptional Gene Expression 50 Regulatory Sequences

Coronaviruses encode an RNA genome that is capped, The preferred target sequences are those nucleotide polyadenylated, nonsegmented, infectious, positive-strand, sequences adjacent and including at least a portion, e.g., at approximately 30 kb and considered the largest of all known least 2-8 bases, of the leader TRS of the positiveiRNA or the viral RNA genomes. Arteriviruses have a 13 kb genome that 55 minus-strand body TRS of Nidovirus RNA. As discussed is very similar in organization and expression strategy to that above, the TRSs appear to play a critical role in viral tran of coronaviruses. The 5' two-thirds of the coronavirus scription by bringing into close proximity the minus-strand genome is occupied by the open reading frame (ORF) 1a and body TRS and positive-strand leader TRS in order to com ORF lb which encode the replicase proteins of the virus. plete synthesis of the negative-sense sg RNA templates. FIG. These genes are translated from infecting genomic RNA into 60 2B provides a schematic representation of how and where two polyprotein precursors which produce the viral replica antisense oligomers interfere with the process of discontinu tion and transcription functions. Downstream of ORF lb a ous transcription at these two target sites. number of genes occur that encode structural and several A variety of Nidovirus genome sequences are available nonstructural, accessory proteins. These genes are expressed from well known sources, such as the NCBI Genbank data through a 3'-coterminal nested set of sub genomic mRNAs (s g 65 bases. Alternatively, a person skilled in the art can ?nd mRNAs) that are synthesized by a process of discontinuous sequences for many of the subject viruses in the open litera transcription. The sg mRNAs represent variable lengths of the ture, e.g., by searching for references that disclose sequence US 8,759,307 B2 13 14 information on designated viruses. Once a complete or partial TABLE 2 — cont inued viral sequence is obtained, the leader TRS sequence of the virus is identi?ed. SHFV AF18039114699— 18 ucagcacgucgucguggu GenBank references for exemplary viral nucleic acid 14719 uga sequences containing the leader TRS or body TRS in the 5 corresponding viral genomes are listed in Table 2 below. It SHFV AF18039115280— 19 agccauacuuccucaggu 15300 uaa will be appreciated that these sequences are only illustrative of other Nidovirus sequences, as may be available from avail able gene-sequence databases or literature or patent The degree of complementarity between the target and resources. The sequences below, identi?ed as SEQ ID NOS: targeting sequence is suf?cient to form a stable duplex. The 1-19, are also listed in Table 4 at the end of the speci?cation. region of complementarity of the antisense oligomers with The bold nucleotides in Table 2 identify the core leader TRS the target RNA sequence may be as short as 8-1 1 bases, but is or the body minus-strand TRS. preferably 12-15 bases or more, e.g. 12-20 bases, or 12-25 bases. An antisense oligomer of about 15 bases is generally TABLE 2 long enough to have a unique complementary sequence in the viral genome. In addition, a minimum length of complemen Exemplary TRS Target Sequences tary bases may be required to achieve the requisite binding Leader SEQ Tm, as discussed below. GenBank TRS ID Target Sequence 20 Oligomers as long as 40 bases may be suitable, where at Virus Acc. No.ths. NO.(5‘ to 3W least a minimum number ofbases, e.g., 12 bases, are comple

FCoV AY204705 109—135 1 cggacaccaacucgaacu mentary to the target sequence. In general, however, facili aaacgaaau tated or active uptake in cells is optimized at oligomer lengths less than about 30, preferably less than 25. For PMO oligo TGEV AJ271965 78—104 2 cggacaccaacucgaacu aaacgaaau 25 mers, described further below, an optimum balance of bind ing stability and uptake generally occurs at lengths of 15-22 HCoV—229E AF304460 55—78 3 cuacuuuucucaacuaaa bases. cgaaau The oligomer may be 100% complementary to the viral HCoV—OC43 AY391777 51—74 4 gaucuuuuuguaaucuaa nucleic acid target sequence, or it may include mismatches, acuuua 30 e.g., to accommodate variants, as long as a heteroduplex formed between the oligomer and viral nucleic acid target SARS—CoV AY274119 53—76 5 gaucuguucucuaaacga acuuua sequence is suf?ciently stable to withstand the action of cel lular nucleases and other modes of degradation which may MHV AF029248 50—75 6 guaguuuaaaucuaaucu occur in vivo. Oligomer backbones which are less susceptible aaacuuua 35 to cleavage by nucleases are discussed below. Mismatches, if SHFV AF180391 188—215 7 gauuugcagacccuccuu present, are less destabilizing toward the end regions of the aaccauguuc hybrid duplex than in the middle. The number of mismatches

PRRSV AF046869 168—194 8 cggucucuccaccccuuu allowed will depend on the length of the oligomer, the per aaccauguc 40 centage of G:C base pairs in the duplex, and the position of the mismatch(es) in the duplex, according to well understood EAV X53459 189—228 9 caucgucgucgaucucua ucaacuacccuugcgacu principles of duplex stability. Although such an antisense augg oligomer is not necessarily 100% complementary to the viral nucleic acid target sequence, it is effective to stably and Body Minus— SEQTarget Sequence 45 speci?cally bind to the target sequence, such that a biological GenBank Strand ID (5‘ to 3‘— activity of the nucleic acid target, e. g. discontinous transcrip Virus Acc. No.TRS ths. NO.minus strand) tion, is modulated. The stability of the duplex formed between the oligomer SARS—CoV AY29131521482— 10 aaaauaaacauguucguu 21502 uag and the target sequence is a function of the binding Tm and the 50 susceptibility of the duplex to cellular enzymatic cleavage. SARS—CoV AY29131525257— 11 acaaauccauaaguucgu The Tm of an antisense compound with respect to comple 25277 uua mentary-sequence RNA may be measured by conventional SARS—CoV AY29131526109— 12 cgaaugaguacauaaguu methods, such as those described by Hames et al., Nucleic 26129 cgu Acid Hybridization, IRL Press, 1985, pp. 107-108. Each anti 55 sense oligomer should have a binding Tm, with respect to a SARS—CoV AY29131526343— 13 auaauaguuaguucguuu 26363 aga complementary-sequence RNA, of greater than body tem perature and preferably greater than 45° C. Tm’s in the range SARS—CoV AY29131526913— 14 uuguaauaagaaagcguu 60-80° C. or greater are preferred. According to well known 26933 cgu principles, the Tm of an oligomer compound, with respect to 60 a complementary-based RNA hybrid, can be increased by SARS—CoV AY29131527265— 15 gaauaauuuucauguucg 27285 uuu increasing the ratio of CG paired bases in the duplex, and/or by increasing the length (in base pairs) of the heteroduplex. At SARS—CoV AY29131527768— 16 gaaguuucauguucguuu the same time, for purposes of optimizing cellular uptake, it 27788 aga may be advantageous to limit the size of the oligomer. For this SARS—CoV AY29131528103— 17 acauuuuaauuuguucgu 65 reason, compounds that show high Tm (50° C. or greater) at a 28123 uua length of 15 bases or less are generally preferred over those requiring 20+ bases for high Tm values.