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  Antisense antiviral compounds and methods for treating a filovirus infectionUSPTO Application #: 20060205693Title: Antisense antiviral compounds and methods for treating a filovirus infection Abstract: The invention provides antisense antiviral compounds and methods of their use and production in inhibition of growth of viruses of the Filoviridae family, and in the treatment of a viral infection. The compounds and methods relate to the treatment of viral infections in mammals including primates by Ebola and Marburg viruses. The antisense antiviral compounds are substantially uncharged morpholino oligonucleotides having: a) a nuclease resistant backbone, b) 15-40 nucleotide bases, and c) a targeting sequence of at least 15 bases in length that hybridizes to a target region selected from the following: i) the AUG start site region of VP35, as exemplified by antisense compounds SEQ ID NO:21-26, ii) the AUG start site region of VP24, as exemplified by antisense compound SEQ ID NO:34, iii) the region 85 to 65 base pairs upstream of the AUG start site of VP24, as exemplified by SEQ ID NO:39, iv) the AUG start site region of polymerase L, as exemplified by antisense compound SEQ ID NO: 17, and v) combinations of (i), (ii), (iii), and/or (iv). (end of abstract)
Agent: Perkins Coie LLP - Menlo Park, CA, US Inventors: David A. Stein, Patrick L. Iversen, Sina Bavari USPTO Applicaton #: 20060205693 - Class: 514081000 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), Phosphorus Containing Other Than Solely As Part Of An Inorganic Ion In An Addition Salt Doai, Nitrogen Containing Hetero Ring, Polycylo Ring System Having A Ring Nitrogen In The System, Nonshared Hetero Atoms In At Least Two Rings Of The Polycyclo Ring System The Patent Description & Claims data below is from USPTO Patent Application 20060205693. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application claims priority to U.S. provisional Patent Application No. 60/671,694 filed Apr. 14, 2005, and U.S. provisional Patent Application No. 60/624,277 filed Nov. 1, 2004, which are both incorporated herein in their entirety by reference. FIELD OF THE INVENTION [0002] This invention relates to antisense oligonucleotide compounds for use in treating an infection by a virus of the Filoviridae family and antiviral treatment methods employing the compounds. More specifically, it relates to treatment methods and compounds for treating viral infections in mammals including primates by Ebola and Marburg viruses. [0003] Agrawal, S., S. H. Mayrand, et al. (1990). "Site-specific excision from RNA by RNase H and mixed-phosphate-backbone oligodeoxynucleotides." Proc Natl Acad Sci USA 87(4): 1401-5. [0004] Arora, V. and P. L. Iversen (2001). "Redirection of drug metabolism using antisense technology." Curr Opin Mol Ther 3(3): 249-57. [0005] Blommers, M. J., U. Pieles, et al. (1994). "An approach to the structure determination of nucleic acid analogues hybridized to RNA. NMR studies of a duplex between 2'-OMe RNA and an oligonucleotide containing a single amide backbone modification." Nucleic Acids Res 22(20): 4187-94. [0006] Bonham, M. A., S. Brown, et al. (1995). "An assessment of the antisense properties of RNase H-competent and steric-blocking oligomers." Nucleic Acids Res 23(7): 1197-203. [0007] Borio, L., T. Inglesby, et al. (2002). "Hemorrhagic fever viruses as biological weapons: medical and public health management." Jama 287(18): 2391-405. [0008] Boudvillain, M., M. Guerin, et al. (1997). "Transplatin-modified oligo(2'-O-methyl ribonucleotide)s: a new tool for selective modulation of gene expression." Biochemistry 36(10): 2925-31. [0009] Bray, M., K. Davis, et al. (1998). "A mouse model for evaluation of prophylaxis and therapy of Ebola hemorrhagic fever." J Infect Dis 178(3): 651-61. [0010] Burnett, J., E. A. Henchal, et al. (2005). "The evolving field of biodefence: Therapeutic developments and diagnostics." Nat Rev Drug Disc 4: 281-297. [0011] Connolly, B. M., K. E. Steele, et al. (1999). "Pathogenesis of experimental Ebola virus infection in guinea pigs." J Infect Dis 179 Suppl 1: S203-17. [0012] Cross, C. W., J. S. Rice, et al. (1997). "Solution structure of an RNA.times.DNA hybrid duplex containing a 3'-thioformacetal linker and an RNA A-tract." Biochemistry 36(14): 4096-107. [0013] Dagle, J. M., J. L. Littig, et al. (2000). "Targeted elimination of zygotic messages in Xenopus laevis embryos by modified oligonucleotides possessing terminal cationic linkages." Nucleic Acids Res 28(10): 2153-7. [0014] Ding, D., S. M. Grayaznov, et al. (1996). "An oligodeoxyribonucleotide N3'- ->P5'phosphoramidate duplex forms an A-type helix in solution." Nucleic Acids Res 24(2): 354-60. [0015] Egholm, M., O. Buchardt, et al. (1993). "PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules." Nature 365(6446): 566-8. [0016] Feldmann, H., S. Jones, et al. (2003). "Ebola virus: from discovery to vaccine." Nat Rev Immunol 3(8): 677-85. [0017] Feldmann, H. and M. P. Kiley (1999). "Classification, structure, and replication of filoviruses. " Curr Top Microbiol Immunol 235: 1-21. [0018] Feldmann, H., H. D. Klenk, et al. (1993). "Molecular biology and evolution of filoviruses." Arch Virol Suppl 7: 81-100. [0019] Felgner, P. L., T. R. Gadek, et al. (1987). "Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure." Proc Natl Acad Sci USA 84(21): 7413-7. [0020] Gait, M. J., A. S. Jones, et al. (1974). "Synthetic-analogues of polynucleotides XII. Synthesis of thymidine derivatives containing an oxyacetamido- or an oxyformamido-linkage instead of a phosphodiester group." J Chem Soc [Perkin 1]0(14): 1684-6. [0021] Gee, J. E., I. Robbins, et al. (1998). "Assessment of high-affinity hybridization, RNase H cleavage, and covalent linkage in translation arrest by antisense oligonucleotides." Antisense Nucleic Acid Drug Dev 8(2): 103-11. [0022] Geisbert, T. W. and L. E. Hensley (2004). "Ebola virus: new insights into disease aetiopathology and possible therapeutic interventions." Expert Rev Mol Med 6(20): 1-24. [0023] Geisbert, T. W., L. E. Hensley, et al. (2003). "Treatment of Ebola virus infection with a recombinant inhibitor of factor VIIa/tissue factor: a study in rhesus monkeys." Lancet 362(9400): 1953-8. [0024] Jahrling, P. B., T. W. Geisbert, et al. (1999). "Evaluation of immune globulin and recombinant interferon-alpha2b for treatment of experimental Ebola virus infections." J Infect Dis 179 Suppl 1: S224-34. [0025] Lesnikowski, Z. J., M. Jaworska, et al. (1990). "Octa(thymidine methanephosphonates) of partially defined stereochemistry: synthesis and effect of chirality at phosphorus on binding to pentadecadeoxyriboadenylic acid." Nucleic Acids Res 18(8): 2109-15. [0026] Mertes, M. P. and E. A. Coats (1969). "Synthesis of carbonate analogs of dinucleosides. 3'-Thymidinyl 5'-thymidinyl carbonate, 3'-thymidinyl 5'-(5-fluoro-2'-deoxyuridinyl) carbonate, and 3'-(5-fluoro-2'-deoxyuridinyl) 5'-thymidinyl carbonate." J Med Chem 12(1): 154-7. [0027] Moulton, H. M., M. H. Nelson, et al. (2004). "Cellular uptake of antisense morpholino oligomers conjugated to arginine-rich peptides." Bioconjug Chem 15(2): 290-9. [0028] Nelson, M. H., D. A. Stein, et al. (2005). "Arginine-rich peptide conjugation to morpholino oligomers: effects on antisense activity and specificity." Bioconjug Chem 16(4): 959-66. [0029] Peters, C. J. and J. W. LeDuc (1999). "An introduction to Ebola: the virus and the disease." J Infect Dis 179 Suppl 1: ix-xvi. [0030] Sanchez, A., M. P. Kiley, et al. (1993). "Sequence analysis of the Ebolavirus genome: organization, genetic elements, and comparison with the genome of Marburg virus." Virus Res 29(3): 215-40. [0031] Strauss, J. H. and E. G. Strauss (2002). Viruses and Human Disease. San Diego, Academic Press. [0032] Summerton, J. and D. Weller (1997). "Morpholino antisense oligomers: design, preparation, and properties." Antisense Nucleic Acid Drug Dev 7(3): 187-95. [0033] Toulme, J. J., R. L. Tinevez, et al. (1996). "Targeting RNA structures by antisense oligonucleotides." Biochimie 78(7): 663-73. [0034] Warfield, K. L., J. G. Perkins, et al. (2004). "Role of natural killer cells in innate protection against lethal ebola virus infection." J Exp Med 200(2): 169-79. BACKGROUND OF THE INVENTION [0035] Minus-strand (-) RNA viruses are major causes of human suffering that cause epidemics of serious human illness. In humans the diseases caused by these viruses include influenza (Orthomyxoviridae), mumps, measles, upper and lower respiratory tract disease (Paramyxoviridae), rabies (Rhabdoviridae), hemorrhagic fever (Filoviridae, Bunyaviridae and Arenaviridae), encephalitis (Bunyaviridae) and neurological illness (Bomaviridae). Virtually the entire human population is thought to be infected by many of these viruses (e.g. respiratory syncytial virus) (Strauss and Strauss 2002). [0036] The order Mononegavirales is composed of four minus strand RNA virus families, the Rhabdoviridae, the Paramyxoviridae, the Filoviridae and the Bornaviridae. The viruses in these families contain a single strand of non-segmented negative-sense RNA and are responsible for a wide range of significant diseases in fish, plants, and animals. Viruses with segmented (-) RNA genomes belong to the Arenaviridae, Bunyaviridae and Orthomyxoviridae families and possess genomes with two, three and seven or eight segments, respectively. [0037] The expression of the five to ten genes encoded by the members of the Mononegavirales is controlled at the level of transcription by the order of the genes on the genome relative to the single 3' promoter. Gene order throughout the Mononegavirales is highly conserved. Genes encoding products required in stoichiometric amounts for replication are always at or near the 3' end of the genome while those whose products are needed in catalytic amounts are more promoter distal (Strauss and Strauss 2002). The segmented (-) RNA viruses encode genes with similar functions to those encoded by the Mononegavirales. Other features of virion structure and replication pathways are also shared among the (-) RNA viruses. [0038] For some (-) RNA viruses, effective vaccines are available (e.g. influenza, mumps and measles virus) whereas for others there are no effective vaccines (e.g. Ebola virus and Marburg virus). In general, no effective antiviral therapies are available to treat an infection by any of these viruses. As with many other human viral pathogens, available treatment involves supportive measures such as anti-pyretics to control fever, fluids, antibiotics for secondary bacterial infections and respiratory support as necessary. [0039] The development of a successful therapeutic for filoviruses Ebola and Marburg virus is a long-sought and seemingly difficult endeavor (Geisbert and Hensley 2004). Although they cause only a few hundred deaths worldwide each year, filoviruses are considered a significant world health threat and have many of the characteristics commonly associated with biological weapons since they can be grown in large quantities, can be fairly stable, are highly infectious as an aerosol, and are exceptionally deadly (Borio, Inglesby et al. 2002). Filoviruses are relatively simple viruses of 19 Kb genomes and consist of seven genes which encode nucleoprotein (NP), glycoprotein (GP), four smaller viral proteins (VP24, VP30, VP35 and VP40), and the RNA-dependent RNA polymerase (L protein) all in a single strand of negative-sensed RNA (Feldmann and Kiley 1999). The development of an effective therapeutic for Ebola virus has been hindered by a lack of reagents and a clear understanding of filovirus pathogenesis, disparity between animal models, and both the difficulty and danger of working with Ebola virus in biosafety level (BSL)-4 conditions (Geisbert and Hensley 2004; Burnett, Henchal et al. 2005). Administration of type I interferons, therapeutic vaccines, immune globulins, ribavirin, and other nucleoside analogues have been somewhat successful in rodent Ebola virus models, but not in infected nonhuman primates (Jahrling, Geisbert et al. 1999; Geisbert and Hensley 2004; Warfield, Perkins et al. 2004). Ebola virus frequently causes severe disseminated intravascular coagulation and administration of a recombinant clotting inhibitor has recently shown to protect 33% of rhesus monkeys (Geisbert, Hensley et al. 2003; Geisbert and Hensley 2004). Host-directed therapeutics alone have not proven to be a sufficiently efficacious therapeutic approach. A well-orchestrated sequence-specific attack on viral gene expression is required for a highly successful anti-filovirus therapeutic and treatment regimen. [0040] In view of the severity of the diseases caused by (-) RNA viruses, in particular members of the Filoviridae family of viruses, and the lack of effective prevention or therapies, it is therefore an object of the present invention to provide therapeutic compounds and methods for treating a host infected with a (-) RNA virus. SUMMARY OF THE INVENTION [0041] The invention includes, in one aspect, an anti-viral antisense composition effective in inhibiting replication within a host cell of an Ebola virus or Marburg virus. The composition contains one or more antisense compounds that target viral RNA sequences within a region of the positive-strand mRNA that includes the region 5' and/or the 25-basepair region just downstream of the AUG start site of the (i) VP35 polymerase, (ii) the L polymerase, (iii) the VP24 membrane associated protein, (iv) the VP40 membrane-associated protein, and (v) the VP30 nucleoprotein. The antiviral compound(s) in the composition include an oligonucleotide analog having: [0042] a) a nuclease-resistant backbone, [0043] b) 15-40 nucleotide bases, and [0044] c) a targeting sequence of at least 15 bases in length that hybridizes to a target region selected from the following: [0045] i) the AUG start site region of VP35, as exemplified by antisense compounds SEQ ID NOs:21-26, [0046] ii) the AUG start site region of VP24, as exemplified by antisense compound SEQ ID NO:34, [0047] iii) the region 85 to 65 base pairs upstream of the AUG start site of VP24, as exemplified by SEQ ID NO:39, [0048] iv) the AUG start site region of polymerase L, as exemplified by antisense compound SEQ ID NO:17, and [0049] v) combinations of (i), (ii), (iii) and/or (iv). The oligonucleotide analog also has: [0050] a) the capability of being actively taken up by mammalian host cells, and [0051] b) the ability to form a heteroduplex structure with the viral target region, wherein said heteroduplex structure is: [0052] i) composed of the positive or negative sense strand of the virus and the oligonucleotide compound, and [0053] ii) characterized by a Tm of dissociation of at least 45.degree. C. [0054] The compound may be composed of morpholino subunits linked by uncharged, phosphorus-containing intersubunit linkages, joining a morpholino nitrogen of one subunit to a 5' exocyclic carbon of an adjacent subunit. In one embodiment, the intersubunit linkages are phosphorodiamidate linkages, such as those having the structure: where Y.sub.1.dbd.O, Z.dbd.O, Pj is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide, and X is alkyl, alkoxy, thioalkoxy, or alkyl amino e.g., wherein X.dbd.NR.sub.2, where each R is independently hydrogen or methyl. The compound may contain one or more cationic linkages wherein X.dbd.(1-piperazino). [0055] The compound may also be a covalent conjugate of an oligonucleotide analog moiety capable of forming such a heteroduplex structure with the positive or negative sense strand of the virus, and an arginine-rich polypeptide effective to enhance the uptake of the compound into host cells. Exemplary polypeptides have one of the sequences identified as SEQ ID NOs:61-66. [0056] Exemplary compositions include one or more antisense compounds that target a positive strand RNA region that includes: [0057] (i) the AUG start site region of VP35, as exemplified by antisense compounds SEQ ID NOs:21-26, [0058] (ii) the AUG start site region of VP24, as exemplified by antisense compound SEQ ID NO:34, [0059] (iii) the region 85 to 65 base pairs upstream of the AUG start site of VP24, as exemplified by SEQ ID NO:39, [0060] (iv) the AUG start site region of polymerase L, as exemplified by antisense compound SEQ ID NO:17, and [0061] (v) combinations of (i) , (ii), (iii) and/or (iv). [0062] The antisense compound(s) in the composition preferably target(s) at least 18, more preferably, at least 20 target base pairs. [0063] In another aspect, the invention includes a method of treating an Ebola or Marburg virus infection in a mammalian host, by administering to the host, a therapeutically effective amount of a composition of the type described above. The method includes, in exemplary embodiments, administering a composition having a combination of antisense compounds targeted against different viral proteins, such as the VP35, VP24, and L polymerase proteins. [0064] In a related, more general aspect, the invention includes a method of vaccinating a mammalian subject against Ebola or Marburg virus by pretreating the subject with the composition of the invention, and exposing the subject to the Ebola virus, preferably in an attenuated form. [0065] These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE FIGURES [0066] FIGS. 1A-1D show the repeating subunit segment of several preferred morpholino oligonucleotides, designated A through D, constructed using subunits having 5-atom (A), six-atom (B) and seven-atom (C-D) linking groups suitable for forming polymers. [0067] FIGS. 2A-2G show the backbone structures of various oligonucleotide analogs with uncharged backbones and FIG. 2H shows a cationic linkage structure. [0068] FIGS. 3A-3C illustrate the components and morphology of a filovirus (3A), and show the arrangement of viral genes in the Ebola virus (Zaire) (3B), and the Marburg virus (3C). [0069] FIGS. 4A-4B show the target regions of 6 antisense compounds targeted against the VP35 gene in Ebola virus. Continue reading... Full patent description for Antisense antiviral compounds and methods for treating a filovirus infection Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Antisense antiviral compounds and methods for treating a filovirus infection patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. 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