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WO1996031600A1 - Procede de modulation de l'expression genique sans depletion de complement - Google Patents

Procede de modulation de l'expression genique sans depletion de complement Download PDF

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Publication number
WO1996031600A1
WO1996031600A1 PCT/US1996/004327 US9604327W WO9631600A1 WO 1996031600 A1 WO1996031600 A1 WO 1996031600A1 US 9604327 W US9604327 W US 9604327W WO 9631600 A1 WO9631600 A1 WO 9631600A1
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oligonucleotide
oligonucleotides
oligo
hybrid
chimeric
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PCT/US1996/004327
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English (en)
Inventor
Sudhir Agrawal
Denise R. Shaw
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Hybridon, Inc.
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Priority to AU53256/96A priority Critical patent/AU5325696A/en
Publication of WO1996031600A1 publication Critical patent/WO1996031600A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base

Definitions

  • the present invention relates to the control of gene expression. More particularly, this invention relates to the use of synthetic oligonucleotides to modulate the expression of a gene in an animal .
  • Paterson et al (Proc. Natl. Acad. Sci. (USA) (1977) 74:4370-4374) discloses that cell-free translation of mRNA can be inhibited by the binding of an oligonucleotide complementary to the mRNA. Stephenson et al. (Proc. Natl. Acad. Sci. (USA)
  • vesicular stomatitis virus see, e.g., Agris et al. (1986) Biochem. 25:6268-6275
  • herpes simplex see, e.g., Gao et al. (1990) Antimicrob. Agents Chem. 34:808-812)
  • • SV40 see, e.g., Birg et al. (1990) (Nucleic Acids Res. 18:2901-2908)
  • human papilloma virus see, e.g., Storey et al. (1991) (Nucleic Acids Res. 19:4109-4114).
  • the use of synthetic oligonucleotides and their analogs as antiviral agents has recently been extensively reviewed by Agrawal (Trends in Biotech. (1992) 10:152- 158) .
  • synthetic oligonucleotides have been used to inhibit a variety of non-viral pathogens, as well as to selectively inhibit the expression of certain cellular genes.
  • synthetic oligonucleotides as agents to inhibit virus propagation, propagation of non- viral, pathogens and selective expression of cellular genes has been well established.
  • oligonucleotides have more recently been developed that have greater efficacy in inhibiting such viruses, pathogens and selective gene expression. Some of these oligonucleotides having modifications in their internucleotide linkages have been shown to be more effective than their unmodified counterparts. For example, Agrawal et al. (Proc. Natl. Acad. Sci. (USA) (1988) 85:7079-7083) teaches that oligonucleotide phosphorothioates and certain oligonucleotide phosphoramidates are more effective at inhibiting HIV-l than conventional phosphodiester-linked oligodeoxynucleotides . Agrawal et al . (Proc. Natl. Acad. Sci. (USA) (1989) 86:7790-7794) discloses the advantage of oligonucleotide phosphorothioates in inhibiting HIV-l in early and chronically infected cells .
  • chimeric oligonucleotides having more than one type of internucleotide linkage within the oligonucleotide have been developed.
  • Pederson et al . U.S. Patent Nos. 5,149,797 and 5,220,007 discloses chimeric oligonucleotides having an oligonucleotide phosphodiester or oligonucleotide phosphorothioate core sequence flanked by nucleotide methylphosphonates or phosphoramidates.
  • Furdon et al . Nucleic Acids Res.
  • Hybrid oligonucleotides having both deoxyribonucleotides and ribonucleotides have been developed.
  • Metelev et al . International Publication No. WO 94/024978 disclose hybrid oligonucleotides having both deoxyribonucleotides and ribonucleotides or 21- substituted ribonucleotides which have superior properties of duplex formation with RNA, nuclease resistance, and the ability to activate RNase H.
  • oligonucleotides that are chimeric and/or hybrid can modulate the expression of a gene to which the nucleic acid sequence oligonucleotide is complementary without depleting complement, and therefore without causing any complications which lack of complement causes in an animal.
  • oligonucleotide sequence that is complementary to a nucleic acid sequence is intended to mean an oligonucleotide sequence (6 to about 50 nucleotides) that binds to the nucleic acid sequence under physiological conditions, e.g., by Watson-Crick base pairing (interaction between oligonucleotide and single-stranded nucleic acid) or by Hoogsteen base pairing (interaction between oligonucleotide and double-stranded nucleic acid) or by any other means including in the case of a oligonucleotide binding to RNA, pseudoknot formation.
  • Watson-Crick base pairing interaction between oligonucleotide and single-stranded nucleic acid
  • Hoogsteen base pairing interaction between oligonucleotide and double-stranded nucleic acid
  • any other means including in the case of a oligonucleotide binding to RNA, pseudoknot formation.
  • a method of modulating gene expression in an animal without depleting complement is provided.
  • a therapeutic formulation is administered which comprises an oligonucleotide complementary to the gene in a pharmaceutically acceptable carrier, the oligonucleotide being hybrid and/or chimeric.
  • animal is meant to encompass humans as well as - 7 - other mammals, as well as reptiles, amphibians, and insects.
  • the oligonucleotide is a hybrid oligonucleotide, while in other preferred embodiments the oligonucleotide is chimeric. In some embodiments, the oligonucleotide is chimeric and hybrid.
  • the oligonucleotide has at least one 2' -substituted ribonucleotide such as a 2' -O-alkyl-substituted ribonucleotide. In one particular embodiment, the oligonucleotide comprises at least one 2' -O-methyl-substituted ribonucleotide.
  • the term "2'- substituted oligonucleotide” refers to an oligonucleotide having a sugar attached to a chemical group other that a hydroxyl group at its 2' position.
  • the 2' -OH of the ribose molecule can be substituted with -O-lower alkyl containing 1-6 carbon atoms, aryl or substituted aryl or allyl having 2-6 carbon atoms, e.g., 2'-0-allyl, 2'-0- aryl, 2'-0-alkyl (such as a 2' -O-methyl) , 2'- halo, or 2' -amino, but not with 2'-H, wherein allyl, aryl, or alkyl groups may be unsubstituted or substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl
  • the hybrid and/or chimeric oligonucleotide is modified, and in some particular embodiments, the oligonucleotide comprises at least one phosphorothioate internucleotide linkage.
  • a method of modulating gene expression in an animal without inhibiting normal blood clotting in the animal is provided.
  • a therapeutic formulation is administered which comprises an oligonucleotide complementary to the gene in a pharmaceutically acceptable carrier, the oligonucleotide being hybrid and/or chimeric.
  • the oligonucleotide is a hybrid oligonucleotide, while in other preferred embodiments the oligonucleotide is chimeric. In some embodiments, the oligonucleotide is chimeric and hybrid.
  • the oligonucleotide has at least one 2' -substituted ribonucleotide such as a 2' -O-alkyl-substituted ribonucleotide. In one particular embodiment, the oligonucleotide comprises at least one 2' -O-methyl-substituted ribonucleotide.
  • the hybrid and/or chimeric oligonucleotide is modified, and in some particular embodiments, the oligonucleotide comprises at least one phosphorothioate internucleotide linkage.
  • FIG. 1A is a graphic representation showing hemolysis in serum in the presence of different concentrations of oligonucleotide 1 (SEQ ID N0:1) in the presence or absence of protamine;
  • FIG. IB is a graphic representation showing hemolysis in serum in the presence of different concentrations of oligonucleotide 1 (SEQ ID N0:1) in the presence or absence of C4d fragment generation protamine;
  • FIG. 1C is a graphic representation showing hemolysis in serum in the presence of different concentrations of oligonucleotide 1 (SEQ ID N0:1) in the presence or absence of Bb fragment generation protamine;
  • FIG. 2 is a graphic representation of the anticoagulation activity of various oligonucleotides used in the method of the invention
  • FIG. 3 is a graphic representation of the CH50 percent of baseline at varying concentrations of oligonucleotides used in the method of the invention
  • FIG. 4 is a graphic representation of the effect of different concentrations of oligonucleotides used in the method of the invention on the proliferation of murine splenic lymphocytes in vitro .
  • FIG. 5 is a graphic representation showing the mutual neutralization of anti-coagulation activities by different concentrations of oligonucleotide 1 and different concentrations of protamine.
  • the present invention provides methods of modulating or regulating gene expression in an animal without depleting complement or without inhibiting normal blood clotting in the animal.
  • This method is also a means of examining the function of various genes in an animal, including those essential to animal development.
  • gene function can only be examined by the arduous task of making a "knock out” animal such as a mouse. This task is difficult, time-consuming and cannot be accomplished for genes essential to animal development since the "knock out” would produce a lethal phenotype.
  • the present invention overcomes the shortcomings of this model.
  • the oligonucleotide administered is complementary to a gene of a virus, pathogenic organism, or a cellular gene in some embodiments of the invention.
  • the oligonucleotide is complementary to a gene of a virus involved in AIDS, oral or genital herpes, papilloma warts, influenza, foot and mouth disease, yellow fever, chicken pox, shingles, adult T-cell leukemia, Burkitt's lymphoma, nasopharyngeal carcinoma, or hepatitis.
  • the oligonucleotide is complementary to an HIV gene and includes about 15 to 26 nucleotides linked by phosphorothioate internucleotide linkages, at least one of the nucleotides at the 3' terminus being a 2'- substituted ribonucleotide, and at least four contiguous deoxyribonucleotides.
  • the oligonucleotide is complementary to a gene encoding a protein in associated with Alzheimer's disease.
  • the oligonucleotide is complementary to a gene encoding a protein expressed in a parasite that causes a parasitic disease such as amebiasis, Chagas' disease, toxoplasmosis, pneumocytosis, giardiasis, cryptoporidiosis, trichomoniasis, malaria, ascariasis, filariasis, trichinosis, or schistosomiasis infections.
  • a parasitic disease such as amebiasis, Chagas' disease, toxoplasmosis, pneumocytosis, giardiasis, cryptoporidiosis, trichomoniasis, malaria, ascariasis, filariasis, trichinosis, or schistosomiasis infections.
  • hybrid oligonucleotides is used herein to describe a molecule comprising at least six 5' to 3' -linked nucleotides wherein both deoxyribonucleotides and ribonucleotides are present. At least one deoxyribonucleotide or at least one ribonucleotide is present in a hybrid oligonucleotide.
  • Hybrid oligonucleotides are described in International Publication No. WO 94/02498, herein incorporated by reference.
  • Oligonucleotides useful in the method of the invention may also be chimeric oligonucleotides.
  • chimeric oligonucleotides refers to oligonucleotides having more than one type of internucleotide linkage.
  • chimeric oligonucleotides of the invention may contain a phosphodiester internucleotide linkage and at least one nonphosphodiester linkage. Chimeric oligonucleotides are described in U.S. Patent No. 5,149,797 herein incorporated by reference.
  • non-phosphodiester-linked oligonucleotide is an oligonucleotide in which all of its nucleotides are covalently linked via a synthetic linkage, i.e., a linkage other than a phosphodiester between the 5' end of one nucleotide and the 3' end of another nucleotide in which the 5' nucleotide phosphate has been replaced with any number of chemical groups.
  • Preferable synthetic linkages include alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, phosphora idates, phosphoramidites, phosphate esters, carbamates, carbonates, phosphate triesters, acetamidate, and carboxymethyl esters.
  • the all of the nucleotides of the oligonucleotide comprises are linked via phosphorothioate and/or phosphorodithioate linkages.
  • the oligonucleotides administered are modified.
  • the term "modified oligonucleotide” encompasses oligonucleotides with modified nucleic acid(s), base(s), and/or sugar(s) other than those found in nature.
  • a 3', 5'- substituted oligonucleotide is an oligonucleotide having a sugar which, at both its 3' and 5' - I m ⁇ positions is attached to a chemical group other than a hydroxyl group (at its 3' position) and other than a phosphate group (at its 5' position) .
  • a modified oligonucleotide may also be one with added substituents such as diamines, cholestryl, or other lipophilic groups, or a capped species.
  • unoxidized or partially oxidized oligonucleotides having a substitution in one nonbridging oxygen per nucleotide in the molecule are also considered to be modified oligonucleotides.
  • modified oligonucleotides are oligonucleotides having nuclease resistance-conferring bulky substituents at their 3' and/or 5' end(s) and/or various other structural modifications not found in vivo without human intervention are also considered herein as modified.
  • an oligonucleotide can bind to a target single-stranded nucleic acid molecule according to the Watson-Crick or the Hoogsteen rule of base pairing, and in doing so, disrupt the function of the target by one of several mechanisms: by preventing the binding of factors required for normal transcription, splicing, or translation; by triggering the enzymatic destruction of mRNA by RNase H if a contiguous region of deoxyribonucleotides exists in the oligonucleotide, and/or by destroying the target via reactive groups attached directly to the antisense oligonucleotide.
  • antisense oligonucleotide can bind to a target single-stranded nucleic acid molecule according to the Watson-Crick or the Hoogsteen rule of base pairing, and in doing so, disrupt the function of the target by one of several mechanisms: by preventing the binding of factors required for normal transcription, splicing, or translation; by triggering the enzymatic destruction of
  • the oligonucleotides useful in the method of the invention are at least 6 nucleotides in length, but are preferably 6 to 50 nucleotides long, with 15 to 30mers being the most common. They are composed of deoxyribonucleotides, ribonucleotides, or a combination of both, with the 5' end of one nucleotide and the 3' end of another nucleotide being covalently linked by non- phosphodiester internucleotide linkages.
  • Such linkages include alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters.
  • Oligonucleotides with these linkages can be prepared according to known methods such as phosphoramidate or H-phosphonate chemistry which can be carried out manually or by an automated synthesizer as described by Brown (A Brief History of Oligonucleotide Synthesis. Protocols for Oligonucleotides and Analogs, Methods in Molecular Biology (1994) 20:1-8) .
  • oligonucleotides of the composition may also be modified in a number of ways without compromising their ability to hybridize to the target nucleic acid.
  • Such modifications include, for example, those which are internal or at the end(s) of the oligonucleotide molecule and include additions to the molecule of the internucleoside phosphate linkages, such as cholesteryl or diamine compounds with varying numbers of carbon residues between the amino groups and terminal ribose, deoxyribose and phosphate modifications which cleave, or crosslink to the opposite chains or to associated enzymes or other proteins which bind to the viral genome.
  • cholesteryl or diamine compounds with varying numbers of carbon residues between the amino groups and terminal ribose, deoxyribose and phosphate modifications which cleave, or crosslink to the opposite chains or to associated enzymes or other proteins which bind to the viral genome.
  • modified oligonucleotides include oligonucleotides with a modified base and/or sugar such as arabinose instead of ribose, or a 3' , 5' -substituted oligonucleotide having a sugar which, at both its 3' and 5' positions is attached to a chemical group other than a hydroxyl group (at its 3' position) and other than a phosphate group (at its 5' position) .
  • Other modified oligonucleotides are capped with a nuclease resistance-conferring bulky substituent at their 3' and/or 5' end(s) , or have a substitution in one nonbridging oxygen per nucleotide.
  • Such modifications can be at some or all of the internucleoside linkages, as well as at either or both ends of the oligonucleotide and/or in the interior of the molecule.
  • Agrawal (1994) Methods in Molecular Biology 26; Uhlmann et al . (1990) Chem. Rev. 90:543-583) The preparation of these unmodified and modified oligonucleotides is well known in the art (reviewed in Agrawal et al. (1992) Trends Biotechnol. 10:152-158) (see, e.g., Uhlmann et al . (1990) Chem. Rev. 90:543-584; and (1987) Tetrahedron. Lett. 28: (31) :3539- 3542) ; Agrawal (1994) Methods in Molecular Biology 20:63- 80) .
  • oligonucleotides are provided with additional stability by having non-phosphodiester internucleotide linkages such as alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, phosphoramidates, phosphoramidites, phosphate esters, carbamates, carbonates, phosphate triesters, acetamidate, and carboxymethyl esters.
  • Particularly useful oligonucleotides are linked with phosphorothioate and/or phosphorodithioate internucleoside linkages.
  • the oligonucleotides administered to the animal may be hybrid oligonucleotides in that they contain both deoxyribonucleotides and at least one 2' substituted ribonucleotide.
  • the term "2' -substituted" means substitution of the 2' -OH of the ribose molecule with, e.g., 2'-0-allyl, 2'-0-alkyl, 2' -halo, or 2'-amino, but not with 2'-H, wherein allyl, aryl, or alkyl groups may be unsubstituted or substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl or amino groups.
  • the hybrid DNA/RNA oligonucleotides useful in the method of the invention resist nucleolytic degradation, form stable duplexes with RNA or DNA, and preferably activate RNase H when hybridized with RNA. They may additionally include at least one unsubstituted ribonucleotide.
  • an oligonucleotide useful in the method of the invention may contain all deoxyribonucleotides with the exception of one 2' substituted ribonucleotide at the 3' terminus of the oligonucleotide.
  • the oligonucleotide may have at least one substituted ribonucleotide at both its 3' and 5' termini.
  • the 2' substituted ribonucleotide (s) in the oligonucleotide may contain at the 2' position of the ribose, a -0-lower alkyl containing 1-6 carbon atoms, aryl or substituted aryl or allyl having 2- 6 carbon atoms e.g., 2'-0-allyl, 2'-0-aryl, 2'-0- alkyl, 2' -halo, or 2' -amino, but not with 2'-H, wherein allyl, aryl, or alkyl groups may be unsubstituted or substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl or amino groups.
  • Useful substituted ribonucleotides are 2'-0-alkyls such as 2' -0-methyl.
  • oligonucleotides useful in the method of the invention will range from about 2 to about 50 nucleotides in length, and most preferably from about 15 to about 25 nucleotides in length.
  • oligonucleotides according to the invention will have from 14 to 24 non-phosphodiester internucleotide linkages.
  • oligonucleotides according to the invention are effective in inhibiting the expression of various genes in viruses, pathogenic organisms, or in inhibiting the expression of cellular genes.
  • the ability to inhibit such agents is clearly important to the treatment of a variety of disease states.
  • oligonucleotides according to the method of the invention have a nucleotide sequence which is complementary to a nucleic acid sequence that is from a virus, a pathogenic organism or a cellular gene.
  • oligonucleotides are from about 6 to about 50 nucleotides in length.
  • nucleic acid sequence to which an oligonucleotide according to the invention is complementary will vary, depending upon the gene to be down-regulated.
  • the target gene or nucleic acid sequence will be a virus nucleic acid sequence.
  • antisense oligonucleotides to inhibit various viruses is well known (reviewed in Agrawal (1992) Trends in
  • Viral nucleic acid sequences that are complementary to effective antisense oligonucleotides have been described for many viruses, including human immunodeficiency virus type 1 (HIV-l) (U.S. Patent No. 4,806,463) , herpes simplex virus (U.S. Patent No. 4,689,320) , influenza virus (U.S. Patent No. 5,194,428) , and human papilloma virus (Storey et al . (1991) Nucleic Acids Res. 19:4109-4114 ) .
  • HMV-l human immunodeficiency virus type 1
  • herpes simplex virus U.S. Patent No. 4,689,320
  • influenza virus U.S. Patent No. 5,194,428
  • human papilloma virus Storey et al . (1991) Nucleic Acids Res. 19:4109-4114 ) .
  • oligonucleotides can be used for oligonucleotides according to the invention, as can be oligonucleotide sequences complementary to nucleic acid sequences from any other virus .
  • Additional viruses that have known nucleic acid sequences against which antisense oligonucleotides can be prepared include, but are not limited to, foot and mouth disease virus (see, Robertson et al. (1985) J. Virol. 54:651; Harris et al . (1980) Virol. 36:659) , yellow fever virus (see Rice et al . (1985) Science 229:726) , varicella-zoster virus
  • an oligonucleotide has been designed which is complementary to a portion of the HIV-l gag gene (oligonucleotide having SEQ ID N0:1) , and as such, has significant anti-HIV effects (Agrawal (1992) Antisense Res. Development
  • This oligonucleotide has been found to be conserved among various HIV-l isolates. It is 56% G + C rich, water soluble, and relatively stable under physiological conditions. This oligonucleotide binds to a complementary RNA target under physiological conditions, with the Tm of the duplex approximately being 56°C. The antiviral activity of this oligonucleotide has been tested in several models, including acutely and chronically infected CEM cells, long-term cultures mimicking in vivo conditions, human peripheral blood lymphocytes and macrophages, and isolates from HIV-l infected patients (Lisziewicz et al . (Proc. Natl. Acad. Sci.
  • the oligonucleotides according to the invention alternatively can have an oligonucleotide sequence complementary to a nucleic acid sequence of a pathogenic organism.
  • the nucleic acid sequences of many pathogenic organisms have been described, including the malaria organism, Plasmodium falciparum, and many pathogenic bacteria.
  • Oligonucleotide sequences complementary to nucleic acid sequences from any such pathogenic organism can be used in oligonucleotides according to the invention.
  • Examples of pathogenic eucaryotes having known nucleic acid sequences against which antisense oligonucleotides can be prepared include Trypanosom abrucei gambiense and Leishmania (See Campbell et al . ,
  • Fasciola hepatica See Zurita et al., Proc. Natl. Acad. Sci. USA 84:2340 (1987) .
  • Antifungal oligonucleotides can be prepared using a target hybridizing region having an oligonucleotide sequence that is complementary to a nucleic acid sequence from, e.g., the chitin synthetase gene, and antibacterial oligonucleotides can be prepared using, e.g., the alanine racemase gene.
  • fungal diseases that may be treatable by the method of treatment according to the invention are candidiasis, histoplasmosis, cryptococcocis, blastomycosis, aspergillosis, sporotrichosis, chromomycosis, dermatophytosis, and coccidioidomycosis.
  • the method might also be used to treat rickettsial diseases (e.g., typhus, Rocky Mountain spotted fever) , as well as sexually transmitted diseases caused by Chlamydia trachomatis or Lymphogranuloma venereum .
  • rickettsial diseases e.g., typhus, Rocky Mountain spotted fever
  • sexually transmitted diseases caused by Chlamydia trachomatis or Lymphogranuloma venereum .
  • a variety of parasitic diseases may be treated by the method according to the invention, including amebiasis, Chagas' disease, toxoplasmosis, pneumocystosis, giardiasis, cryptosporidiosis, trichomoniasis, and Pneumocystis carini pneumonia; also worm (helminthic) diseases such as ascariasis, filariasis, trichinosis, schistosomiasis and nematode or cestode infections. Malaria may be treated by the method of treatment of the invention regardless of whether it is caused by P. falcip arum, P. vivas, P. orale, or P. mala ⁇ ae .
  • infectious diseases identified above may all be treated by the method of treatment according to the invention because the infectious agents for these diseases are known and thus oligonucleotides according to the invention can be prepared, having oligonucleotide sequence that is complementary to a nucleic acid sequence that is an essential nucleic acid sequence for the propagation of the infectious agent, such as an essential gene.
  • Other disease states or conditions that may be treatable by the method according to the invention are those which result from an abnormal expression or product of a cellular gene. These conditions may be treated by administration of oligonucleotides according to the invention, and have been discussed earlier in this disclosure.
  • oligonucleotides according to the invention can have a nucleotide sequence complementary to a cellular gene or gene transcript, the abnormal expression or product of which results in a disease state.
  • the nucleic acid sequences of several such cellular genes have been described, including prion protein (Stahl et al. (1991) FASEB J. 5:2799-2807), the amyloid-like protein associated with Alzheimer's disease (U.S. Patent No. 5,015,570), and various well-known oncogenes and proto-oncogenes, such as c-myb, c- myc, c-abl, and n-ras .
  • oligonucleotides that inhibit the synthesis of structural proteins or enzymes involved largely or exclusively in spermatogenesis, sperm motility, the binding of the sperm to the egg or any other step affecting sperm viability may be used as contraceptives.
  • contraceptives for women may be oligonucleotides that inhibit proteins or enzymes involved in ovulation, fertilization, implantation or in the biosynthesis of hormones involved in those processes.
  • Hypertension may be controlled by oligonucleotides that down-regulate the synthesis of angiotensin converting enzyme or related enzymes in the renin/angiotensin system.
  • Platelet aggregation may be controlled by suppression of the synthesis of enzymes necessary for the synthesis of thromboxane A2 for use in myocardial and cerebral circulatory disorders, infarcts, arteriosclerosis, embolism and thrombosis.
  • Deposition of cholesterol in arterial wall may be inhibited by suppression of the synthesis of fatty acid co-enzyme A: cholesterol acyl transferase in arteriosclerosis. Inhibition of the synthesis of cholinephosphotransferase may be useful in hypolipidemia.
  • hybridization arrest may be used to reduce or eliminate adverse effects of the disorder.
  • suppression of the synthesis of monoamine oxidase may be used in Parkinson's disease.
  • Suppression of catechol o-methyl transferase may be used to treat depression; and suppression of indole N-methyl transferase may be used in treating schizophrenia.
  • Suppression of selected enzymes in the arachidonic acid cascade which leads to prostaglandins and leukotrienes may be useful in the control of platelet aggregation, allergy, inflammation, pain and asthma.
  • mdr- 1 multidrug resistance
  • Oligonucleotide sequences complementary to nucleic acid sequences from any of these genes can be used for oligonucleotides according to the invention, as can be oligonucleotide sequences complementary to any other cellular gene transcript, the abnormal expression or product of which results in a disease state.
  • oligonucleotides described herein are administered orally or enterally to the animal subject in the form of therapeutic pharmaceutical formulations that are effective for treating virus infection, infections by pathogenic organisms, or disease resulting from abnormal gene expression or from the expression of an abnormal gene product.
  • the oligonucleotides are administered in conjunction with other therapeutic agents, e.g., AZT in the case of AIDS.
  • the therapeutic pharmaceutical formulation containing the oligonucleotide includes a physiologically acceptable carrier, such as an inert diluent or an assimilable edible carrier with which the oligonucleotide is administered.
  • a physiologically acceptable carrier such as an inert diluent or an assimilable edible carrier with which the oligonucleotide is administered.
  • suitable formulations that include pharmaceutically acceptable excipients for introducing compounds to the bloodstream by other than injection routes can be found in Remington 's
  • the oligonucleotide and other ingredients may be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the individual's diet.
  • the oligonucleotide may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • the oligonucleotide When the oligonucleotide is administered orally, it may be mixed with other food forms and pharmaceutically acceptable flavor enhancers.
  • oligonucleotide When the oligonucleotide is administered enterally, they may be introduced in a solid, semi-solid, suspension, or emulsion form and may be compounded with any number of well- known, pharmaceutically acceptable additives. Sustained release oral delivery systems and/or enteric coatings for orally administered dosage forms are also contemplated such as those described in U.S. Patent Nos. 4,704,295, 4,556,552, 4,309,404, and 4,309,406.
  • compositions or preparations according to the present invention are prepared so that an oral dosage unit contains between from about 50 micrograms to about 200 mg per kg body weight of the animal, with 10 mg to 100 mg per kg being most preferable.
  • unit content of active ingredient or ingredients contained in an individual dose of each dosage form need not in itself constitute an effective amount since the necessary effective amount can be reached by administration of a plurality of dosage units (such as capsules or tablets or combinations thereof) .
  • Oligo 1 inhibits hemolytic complement activity when normal serum samples from either humans, Rhesus monkeys or guinea pigs are spiked in vitro with Oligo 1, in a dose-dependent manner (Tables 9 and 10) .
  • Oligo 1 177 (0.50) 51 (0.43) 130 (0.75) 73 (0.56) 500 ⁇ g/ml
  • Hemolytic complement is also inhibited when oligo 1 is present in blood during clotting to prepare serum (Table 12) .
  • Control 354 (1.00) 111 (1.00) >60.0 305 (1.00) 98 (1.00) >60.0 Oligo 1 184 (0.52) 81 (0.73) 14.5 128 (0.42) 69 (0.70) 10.3
  • Oligo 1 inhibits hemolytic complement assays with both classical and alternative pathway targets (Tables 9 and 11) .
  • oligo 1 By ELISA quantitation of complement pathway by-products in treated serum, oligo 1 appears to act primarily by activation of the classical pathway (e.g., C4d generation) as compared to the alternative pathway (e.g., Bb generation) (Table 13 and Fig. 4) . However, it is possible that oligo 1 activates both pathways of complement.
  • C4d generation e.g., C4d generation
  • Bb generation alternative pathway
  • oligo 1 By visual inspection, the presence of relatively high doses of oligo 1 in vacutainers used to collect peripheral blood for preparation of serum from normal human volunteers (e.g., final concentrations of 250 ⁇ g/ml or greater) results in gross inhibition of blood clotting after 1 - 24 hours at room temperature. An apparent total inhibition of blood clotting has been observed with oligo 1 concentrations of 400-500 ⁇ g/ml.
  • Hemolytic complement was analyzed in the serum of guinea pigs administered oligo 1 intravenously. The results are shown in Table 15 below. Within 15 minutes of oligo 1 infusion, serum hemolytic complement was significantly reduced and remained so at 60 minutes post infusion, as directly compared to control guinea pigs receiving infusions of saline alone. Thus, results in guinea pigs were similar to those obtained by others with oligo 1 in monkeys. Guinea pigs may provide a more economical preclinical model for analysis of oligo 1 effects on complement and coagulation in vivo .
  • Oligo 1 shares several noteworthy properties with the pharmaceutical anti-coagulant heparin: (i) both have a high density of negatively-charged residues due to sulfuration, and (ii) both act as anti-coagulants in whole blood. This led to the hypothesis that the pharmaceutical anti-heparin compound, protamine sulfate, might also exhibit neutralizing activity for the anti-coagulant and anti-complementary effects of oligo 1.
  • heparin and oligo 1 demonstrate similar anti-coagulant activities in vitro, the anti- complement activity of oligo 1 was not mimicked by heparin (Table 16) . Additionally, genomic DNA did not demonstrate significant anti-complement activity when tested in parallel with oligo 1 (Table 9 above) .
  • protamine effectively antagonized the anti-complement activity of oligo 1 treatment of human serum in vitro (Table 16, and Fig. 4 above) .
  • Protamine was also effective in neutralizing the anti-coagulant properties of oligo 1 in plasma treated in vitro, as measured by either clotting times or PFl.2 generation (FIG. 5 and Table 16) .
  • the dose ratio for complete neutralization of oligo 1 by protamine in these experiments was estimated to be 0.5 ⁇ g protamine per 1.0 ⁇ g oligo 1.
  • protamine at doses of ⁇ 50 ⁇ g/ml alone will prolong PTT measurements (FIG. 5) .
  • the initial oligo 1 derived modified oligonucleotides demonstrated inhibition of coagulation with potency similar to that observed for inhibition of complement hemolysis.
  • Residual aliquots of serum treated with test compounds were stored at -70°C until used in quantitative ELISA for determination of C4d, C3a and Bb using kits from Quidel (San Diego, CA) .
  • Plasma samples Normal donor blood anti-coagulated with sodium citrate were used to prepare platelet- depleted plasma. Aliquots of plasma were mixed with test compounds and then quickly frozen on dry ice and stored at -70°C. The activated partial thromboplastin time (PTT) , prothrombin time (PT) and thrombin clotting time (TCT) were determined by standard clinical coagulation tests.
  • PTT partial thromboplastin time
  • PT prothrombin time
  • TCT thrombin clotting time

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Abstract

L'invention se rapporte à un procédé de modulation de l'expression génique chez un animal sans déplétion du complément. Le procédé consiste à administrer une formulation thérapeutique comprenant un oligonucléotide complémentaire au gène dans un excipient pharmaceutiquement acceptable, l'oligonucléotide étant hybride et/ou chimère.
PCT/US1996/004327 1995-04-03 1996-03-29 Procede de modulation de l'expression genique sans depletion de complement WO1996031600A1 (fr)

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WO1997011171A1 (fr) * 1995-09-22 1997-03-27 Hybridon, Inc. Oligonucleotides modifiees specifiques de la proteine kinase a et leurs modes d'utilisation
EP0856579A1 (fr) * 1997-01-31 1998-08-05 BIOGNOSTIK GESELLSCHAFT FÜR BIOMOLEKULARE DIAGNOSTIK mbH Procédé pour la préparation d'oligonucléotides antisens
WO1998040479A1 (fr) * 1997-03-12 1998-09-17 Hybridon, Inc. Oligonucleotides modifies specifiques de la proteine kinase a et methodes d'utilisation
WO2000071703A3 (fr) * 1999-05-03 2001-07-19 Methylgene Inc Inhibition d'histone deacetylase
US6624293B1 (en) 1995-08-17 2003-09-23 Hybridon, Inc. Modified protein kinase A-specific oligonucleotides and methods of their use
US7074768B2 (en) 1995-08-17 2006-07-11 Idera Pharmaceuticals, Inc. Modified protein kinase A-specific oligonucleotides and methods of their use
US9758786B2 (en) 2016-02-09 2017-09-12 Autotelic, Llc Compositions and methods for treating pancreatic cancer

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6624293B1 (en) 1995-08-17 2003-09-23 Hybridon, Inc. Modified protein kinase A-specific oligonucleotides and methods of their use
US7074768B2 (en) 1995-08-17 2006-07-11 Idera Pharmaceuticals, Inc. Modified protein kinase A-specific oligonucleotides and methods of their use
WO1997011171A1 (fr) * 1995-09-22 1997-03-27 Hybridon, Inc. Oligonucleotides modifiees specifiques de la proteine kinase a et leurs modes d'utilisation
EP0856579A1 (fr) * 1997-01-31 1998-08-05 BIOGNOSTIK GESELLSCHAFT FÜR BIOMOLEKULARE DIAGNOSTIK mbH Procédé pour la préparation d'oligonucléotides antisens
WO1998033904A3 (fr) * 1997-01-31 1999-05-14 Biognostik Ges Procede de preparation d'un oligonucleotide antisens
US6972171B1 (en) 1997-01-31 2005-12-06 Biognostik Ges. Fur Biomolekulare Diagnostik Mbh Antisense oligonucleotide preparation method
US7563778B2 (en) 1997-01-31 2009-07-21 Biognostik Ges. Fur Biomolekulare Diagnostik Mbh Antisense oligonucleotide preparation method
EP2028274A3 (fr) * 1997-01-31 2012-06-13 Biognostik Gesellschaft für biomolekulare Diagnostik mbH Un oligonucléotide antisens dirigé contre le gène de TGF-beta2
WO1998040479A1 (fr) * 1997-03-12 1998-09-17 Hybridon, Inc. Oligonucleotides modifies specifiques de la proteine kinase a et methodes d'utilisation
WO2000071703A3 (fr) * 1999-05-03 2001-07-19 Methylgene Inc Inhibition d'histone deacetylase
US9758786B2 (en) 2016-02-09 2017-09-12 Autotelic, Llc Compositions and methods for treating pancreatic cancer
US9963703B2 (en) 2016-02-09 2018-05-08 Autotelic Llc Compositions and methods for treating pancreatic cancer

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