WO2000014219A2 - Molecules chimeres oligoadelynates-acides nucleiques peptidiques - Google Patents

Molecules chimeres oligoadelynates-acides nucleiques peptidiques Download PDF

Info

Publication number
WO2000014219A2
WO2000014219A2 PCT/US1999/020159 US9920159W WO0014219A2 WO 2000014219 A2 WO2000014219 A2 WO 2000014219A2 US 9920159 W US9920159 W US 9920159W WO 0014219 A2 WO0014219 A2 WO 0014219A2
Authority
WO
WIPO (PCT)
Prior art keywords
antisense
moiety
rna
chimeric molecule
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1999/020159
Other languages
English (en)
Other versions
WO2000014219A9 (fr
WO2000014219A3 (fr
Inventor
Paul F. Torrence
Jacques H. Van Boom
Jeroen C. Verheijen
Gijsbert A. Van Der Marel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universiteit Leiden
US Department of Health and Human Services
Original Assignee
Universiteit Leiden
US Department of Health and Human Services
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universiteit Leiden, US Department of Health and Human Services filed Critical Universiteit Leiden
Priority to AU57034/99A priority Critical patent/AU5703499A/en
Publication of WO2000014219A2 publication Critical patent/WO2000014219A2/fr
Publication of WO2000014219A3 publication Critical patent/WO2000014219A3/fr
Publication of WO2000014219A9 publication Critical patent/WO2000014219A9/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • C12N15/1131—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 against viruses
    • 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
    • C12N15/1137—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 against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2310/317—Chemical structure of the backbone with an inverted bond, e.g. a cap structure
    • 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
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C12N2310/3181—Peptide nucleic acid, PNA
    • 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
    • C12N2310/319—Chemical structure of the backbone linked by 2'-5' linkages, i.e. having a free 3'-position

Definitions

  • the present invention relates to chimeric molecules comprising activators of RNase L covalently linked to peptide nucleic acid oligomers, and to compositions thereof.
  • the invention also relates to methods for sequence-specific cleavage of RNA using these chimeric molecules in vitro and in vivo.
  • Antisense oligonucleotides hold considerable promise both as research tools for inhibiting gene expression and as agents for the treatment of a myriad of human diseases (Stein and Cheng, Science, 261:1004 (1993); Milligan et al, J. Med. Chem., 36:1923-1937 (1993)).
  • the targeted destruction of RNA using antisense oligonucleotides has been difficult to achieve in a versatile, efficient, and reliable manner.
  • Miller et al. were the first to attempt to capitalize on nucleic acid hybridization through the preparation of a series of trinucleotides modified through phosphotriester, 2'-O-methyl or methylphosphonate substitution (Miller, et al, Biochemistry, 13:4887 (1974)). These short, modified DNA sequences, complementary to t-RNA anticodon regions, were found to be able to inhibit protein translation. Zamecnik and Stephenson (Proc. Natl. Acad. Sci. U.S.A., 74:280 (1978)) used a similar strategy to synthesize a 21-deoxyribonucleotide sequence which inhibited the replication of Rous sarcoma virus. Enthusiasm for this approach was generated by the demonstration that human immunodeficiency virus
  • HIV HIV
  • antisense oligonucleotides Zamecnik, et al, Proc. Natl. Acad. Sci. U.S.A., 83:7706 (1986)
  • RNase H the enzyme that is present in both eukaryotes and prokaryotes.
  • oligonucleotides which are able to induce chemical alteration or strand scission of a target RNA molecule.
  • photoreactive agents such as psoralen or porphyrin (Lee, et al, Nucleic Acids Res., 16:10681 (1988)); oxidative nuclease metal ion complexes such as po hyrin-iron (Doan, et al, Biochemistry, 25:6736 (1986)); phenanthroline-copper (Chen, et al,
  • ACCA sequence were annealed. Oligonucleotides with an ACCA sequence at one end, referred to as "external guide sequences" (EGS's), were hybridized to a specific sequence on an RNA molecule. The RNA molecule with the bound EGS thereby became a substrate for RNase P and was specifically cleaved by RNase P.
  • EGS's extra guide sequences
  • Minshull et al Another method of digesting RNA at a specific location with an antisense oligonucleotide and an RNase was demonstrated by Minshull et al (Nucleic Acids Research, 14:6433-6451 (1986)). Minshull cleaved a specific RNA molecule by first hybridizing an antisense DNA oligonucleotide to the RNA molecule and then treating the hybridized molecule with RNase H. Since RNase H ° specifically digests DNA/RNA hybrids, the RNA strand of the hybridized molecule was digested by RNase H.
  • Corey et al J. Am. Chem. Soc, 111 :8523-8525 (1989) also discussed a method of targeting a polynucleotide for destruction by a nuclease.
  • Corey fused an antisense oligonucleotide to a nonspecific nuclease.
  • the oligonucleotide was then hybridized to a polynucleotide with which it could anneal, the nuclease specifically cleaved the targeted polynucleotide strand.
  • This approach has not been applied in vivo, however, due to the difficulties involved in passing a molecule as large as a nuclease into an intact living cell.
  • the 1,10-phenanthro line-copper ion cleaved the target polynucleotide.
  • the latent endonuclease RNase L (E.C. 3.1.26.-), formerly known as "2-5A-dependent RNase” cleaves RNA in the presence of the unusual 2',5'- phosphodiester-linked trimeric oligoadenylate ppp5A2'p5A2'p5 (2-5A) (Kerr, et
  • RNase L is part of what has been termed the 2-5 A system, which is believed to be involved in the regulation of cell growth (Etienne-Smekins, et al, Proc. Natl. Acad. Sci. USA, 80:4609 (1983))
  • RNase L is potently induced by interferon, and appears to mediate certain actions of interferon such as the inhibition of picornavirus replication (Hassel et al, EMBO J., 12:3297-3304 (1993)). Cells and tissues from reptilian, avian, and mammalian species have been found to contain basal levels RNase L. For reviews, see Williams 5 et al , 'The 2-5-A System: Molecular and Clinical Aspects of the Interferon-Related Pathway" (Alan R. Liss, Inc., New York, 1985) and Player and Torrence, Pharmacol. Ther., 78:55-1 13 (1998).
  • PKR mRNA resulted in the ablation of PKR mRNA and enzyme activity such that the double-stranded RNA-mediated activation of transcription factor NF- ⁇ B was ablated.
  • RNase L the activation of RNase L by an activator- antisense DNA chimera results in the catalytic degradation of PKR mRNA (K cat of about 7 sec "1 ) (Maitra et al, J. Biol. Chem., 270:15071-15075 (1995)).
  • Antisense oligonucleotides have great potential as therapeutic agents, due to their ability to inhibit gene expression in a sequence-specific manner.
  • PNAs peptide nucleic acids
  • PNA oligomers which lack phosphodiester bonds, are entirely resistant to nucleases. See, for example, Nielsen et al, patent application PCT/US97/12811, publication No. WO 98/03542.; Hyrup and Nielsen, Bioorg. Med. Chem. 4:5-23 (1996); and Nielsen et al, Science 254:1497-1500 (1991).
  • a PNA antisense agent will hybridize with high affinity and selectivity to the complementary mRNA strand (Demidov et al;., Biochem.
  • RNA-PNA duplex is resistant to RNase H, the enzyme specific for DNA/RNA hybrids that plays a major role in the standard mechanism of action of antisense oligonucleotides.
  • RNase H the enzyme specific for DNA/RNA hybrids that plays a major role in the standard mechanism of action of antisense oligonucleotides.
  • the effect of an antisense PNA has therefore been limited to steric inhibition of translation, because of the absence of catalytic RNase H degradation of PNA-RNA hybrids (Hanvey et al, Science 258:1481-1485 (1992)). Because hybridization of the sense and antisense strands is reversible, a relatively high concentration of antisense PNA must be attained in order to bind a high proportion of the complementary mRNA at equilibrium, if significant inhibition of translation is to be observed.
  • oligonucleotide refers to any number of nucleotides linked by phosphodiester, phosphorothiodiester, methylphosphonodiester, or methylphosphonothiodiester moieties.
  • activator of RNase L refers to any compound or chemical moiety that is capable of activating RNase L.
  • polyamide nucleic acid and "PNA” refer to an oligomer of amino acid monomers, wherein each monomer comprises a terminal amino group and a terminal carboxyl group, and the monomers are linked through amide bonds between the terminal amino group of one monomer and the terminal carboxyl group of the next monomer.
  • Each monomer further comprises a covalently attached purine or pyrimidine base, and the bases are oriented along the oligomer chain so as to be capable of forming Watson-Crick base pairs with a single-stranded oligonucleic acid.
  • peptide nucleic acid refers to a PNA which is an oligomer of N-(2-aminoethyl)glycine, wherein purine and pyrimidine bases are covalently attached through an acetyl linker to the glycine alpha nitrogens.
  • nucleobase refers to a purine or pyrimidine base that is capable of forming Watson-Crick base pairs to one or more of the bases adenine, thymine, guanosine, cytosine, and uracil.
  • the term is intended to include analogues of purines and pyrimidines as well, such as deazapurines and deazapyimidines, that are likewise capable of forming Watson-Crick base pairs with to one or more of the bases adenine, thymine, guanosine, cytosine, and uracil.
  • Nucleobase sequences preceded by "(pna)" refer to polyamide nucleic acids having that sequence of nucleobases, and do not refer to any particular polarity of the polyamide chain, i.e. the amino and carboxy termini of the polyamide backbone could be at either end of the given nucleobase sequence.
  • Nucleobase sequences preceded by "pna” refer to oligo[N-(2- aminoethyl)-N-(l-(purinyl or pyrimidinyl)acetyl)glycine] nucleic acids having that sequence of nucleobases. Sequences not preceded by "(pna)” or “pna” refer to ordinary oligonucleotides having the given sequence. Description of the invention One object of the invention is to provide 2-5A-polyamide nucleic acid chimeric molecules, which comprise a polyamide nucleic acid (PNA) moiety covalently attached to an RNase L activating moiety.
  • PNA polyamide nucleic acid
  • chimeric molecules are capable of binding to and activating RNase L, and can direct the nuclease activity of RNase L to a target RNA molecule having a sequence complementary to that of the polyamide nucleic acid moiety of the chimeric molecule.
  • the peptide nucleic acid moiety is preferably an oligo[N-(2-aminoethyl)-N-(l-(purinyl or pyrimidinyl)acetyl)glycine], and the activator moiety preferably comprises a 2', 5'- oligoadenylate oligonucleotide.
  • the activator moiety is preferably attached through its 2' end to the peptide nucleic acid, and preferably to the amino terminus of the peptide nucleic acid.
  • the activator moiety may optionally comprise one or more 5' thiophosphate groups.
  • Another object of the invention is to provide compositions comprising the chimeric molecules of this invention.
  • Another object of the invention is to provide methods for cleaving a specifically selected strand of RNA (the "target RNA”), using the chimeric molecules of this invention.
  • Yet another object of the invention is to provide methods of degrading and/or reducing the concentration of a target RNA molecule, by contacting the target RNA with RNase L in the presence of a 2,5-A-peptide nucleic acid chimeric molecule of this invention having a polyamide nucleic acid sequence complementary to that of the target RNA.
  • Another object of this invention is to provide pharmaceutical compositions comprising the chimeric molecules of the invention, and methods of treating mammals with these compositions.
  • FIG. 1 Structure of 5'-O-phosphoryladenylyl(2'->5')adenylyl- (2'->5')adenylyl(2'- ⁇ 5')adenosine, a 2',5'-oligoadenylate example of a 2-5A activator of RNase L.
  • Figure 2. Retrosynthetic analysis of target 2-5A-PNAs 2-4. (DMTr, dimethoxytrityl; MMTr, monomethoxytrityl; TBDMS, t-butyldimethylsilyl; CNE, cyanoethyl; HMBA, p-hydroxymethylbenzoate.)
  • Figure 4 The effect of 2-5 A and 2-5A-PNA chimeras on the ability of RNase L to cleave a radiolabeled poly(U) substrate, plotted as a function of concentration.
  • Lane 1 no 2-5A; lanes 2-6, 10 "7 -10 "n M 2-5A; lanes 7-11, 10 "5 -10 "9 M 2-5A-pnaA 4 ; lanes 12-16, 10 "5 -10 “9 M 2-5A-pnaA 8 ; lanes 17-21, 10 "5 -10 "9 M 2-5A-pnaA 12 .
  • Figure 6 The displacement of (2-5 A-[ 32 P]pCp) from binding sites in
  • the invention provides chimeric molecules which comprise an antisense polyamide nucleic acid moiety that binds or anneals to the target strand of RNA, and an activator of RNase L attached to the antisense polyamide nucleic acid moiety.
  • the antisense peptide nucleic acid moiety may comprise any polyamide nucleic acid oligomer that is capable of forming a double helix with single-stranded nucleic acids having a complementary sequence of base pairs.
  • the polyamide nucleic acid backbone may be based on any amino acids known to the art, such as for example N-(2-aminoethyl)glycine (Nielsen et al. , patent application PCT/US97/12811, publication No. WO 98/03542) or N-(l-aminoacyl)proline (Lowe, patent application PCT/GB97/02820, publication No. WO 98/16550).
  • PNA backbone polymers are also known to the art: Dueholm et al, Bioorg. Med. Chem. Lett., 4:1077 (1994); Hyrup et al, J. Chem. Soc. Chem. Commun., 518 (1993); Hyrup et al, J. Am. Chem. Soc, 116:7964 (1994), Krotz et al, Tetrahedron
  • the polyamide nucleic acid backbone is preferably based on N-(2-aminoethyl)glycine, and is more preferably an oligo[N-(2-aminoethyl)-N-(l- (purinyl or pyrimidinyl)acetyl)glycine].
  • the antisense polyamide nucleic acid moiety has a nucleobase sequence complementary to the sequence of the selected target RNA.
  • the activator of RNase L may be any RNase L activator.
  • activators are known in the art.
  • a 3'-deoxyadenosine may be substituted for an adenosine within the 2-5 A moiety (Torrence et al, J. Biol. Chem., 263:131-1139 (1988)).
  • 3'-O-methylation at the 2' terminal of the 2-5A moiety also generates an activator (Baglioni et al, J. Biol. Chem., 256:3253-3257 (1981)).
  • the RNase L activator moiety preferably comprises a 2',5'- oligoadenylate oligonucleotide having three or more adenosine residues, more preferably three or four adenosine residues.
  • the 2',5'-oligoadenylate oligonucleotide is p5A2'p5A2'p5'A2'p5A.
  • a mono-, di-, or tri-phosphate group, or one of the thio analogues thereof, is present at the 5' end of the 2-5 A moiety.
  • the RNase activator moiety comprises one or more 5' thiophosphate groups.
  • the RNase L activator moiety is coupled at its 2' end, preferably through the 2' position, to the PNA moiety, and is preferably coupled through a flexible linker.
  • the linker may be any bifunctional linker that provides sufficient flexibility and spacing between the activator moiety and the PNA moiety to allow RNase L-mediated cleavage of the target RNA strand.
  • Linkers suitable for attaching oligonucleotides to peptide nucleic acids are known to the art, such as, for example, N-(2-hydroxyethyl)-N-[l-(purinyl or pyrimidinyl)acetyl]glycine (Petersen et al, Bioorg. Med. Chem.
  • linker is an oligo[butanediol monophosphate] linker as exemplified herein.
  • the polarity of the antisense nucleobase sequence with respect to the RNase L activator moiety is not critical to antisense activity (Xiao et al. , J. Med. Chem. 41 : 1531 - 1539 ( 1998)) .
  • the RNase L activator therefore may be linked to either end of the antisense PNA moiety.
  • the RNase L activator moiety is preferably linked to the amino terminus of the PNA moiety as exemplified herein.
  • the polarity of the polyamide backbone with respect to the antisense nucleobase sequence is also not critical, (Lagriffoul et al, Bioorg. Med.
  • the antisense polyamide nucleic acid moiety is preferably between about 6 and about 50 nucleobases in length, more preferably between about 10 and about 30 nucleobases in length, and most preferably between about 12 and about 25 nucleobases in length.
  • the antisense moiety hybridizes, binds, anneals to, or otherwise forms a complex with, a single stranded region of the target strand of RNA contained in the cell.
  • the target RNA is the RNA component of telomerase, the mRNA encoded by a mutant gene associated with a dominant autosomal disease, or a transcript of an oncogene or a proto- oncogene, or is the transcript of a viral gene or the genome or antigenome of an RNA virus.
  • the present invention relates to methods of inhibiting the action of teleomerase with chimeric molecules comprising an activator of RNase L covalently attached to an antisense PNA that is capable of binding to the RNA component of telomerase.
  • a polyamide nucleic acid having a nucleobase sequence having a nucleobase sequence
  • (pna)GCGCGGGGAGCAAAAGCAC covalently linked to an activator of RNase L, will be useful for inhibiting telomerase.
  • the activator of RNase L is preferably a 2',5'-oligoadenylate oligonucleotide having three or more adenosine residues, more preferably three or four adenosine residues. Such chimeras, and pharmaceutical compositions comprising them, would be useful for inhibiting the growth of tumors in mammals.
  • An embodiment of this invention suitable for ablating the bcr/abl mRNA, the chimeras and methods of which may also be useful for treating chronic myelogenous leukemias includes a polyamide nucleic acid having the following nucleobase sequence:
  • An embodiment of this invention suitable for ablating the ICAM-1 mRNA, the chimeras and methods of which may also be useful for treating Crohns Disease and other inflammatory conditions includes a polyamide nucleic acid having the following nucleobase sequence:
  • An embodiment of this invention invention suitable for ablating the ha-ras mRNA, the chimeras and methods of which may also be useful for treating cancer includes a polyamide nucleic acid having the following nucleobase sequence:
  • This embodiment of the invention is directed to the mRNA transcribed from the ha-ras gene. (Monia et al., J. Biol. Chem., 271 :14533-14540 (1996)).
  • An embodiment of this invention suitable for ablating the HIV gag gene or gag mRNA, the chimeras and methods of which may also be useful for treating HIV infection includes a polyamide nucleic acid having the following nucleobase sequence:
  • An embodiment of this invention suitable for ablating the CMV immediate-early RNA, the chimeras and methods of which may also be useful for treating CMV retinitis includes a polyamide nucleic acid having the following nucleobase sequence:
  • This embodiment of the invention is directed to the mRNA of cytomegalovirus.
  • a phosphorothioate antisense oligonucleotide having this sequence (VitraveneTM, fomivirsen) has been approved for treatment of CMV retinitis. (Anderson et al.,. Antimicrob. Agents Chemother.,
  • the present invention also relates to covalently-linked chimeras of an activator of RNase L and a polyamide nucleic acid (PNA) that is capable of binding to the genomic RNA strand of an RNA virus and/or binding to the antigenomic or mRNA of a negative strand RNA virus.
  • PNA polyamide nucleic acid
  • the methods and chimeras of the invention may be applied to target any negative strand RNA virus, including, but not limited to, parainfluenza virus, mumps virus, rabies, influenza virus, and arenaviruses such as LCMV (lymphocytic chorio- 0 meningitis virus), Lassa virus, Machupo virus, Sabia virus and Junin virus.
  • LCMV lymphocytic chorio- 0 meningitis virus
  • Lassa virus Machupo virus
  • Sabia virus Junin virus.
  • the invention in one embodiment relates to a covalently linked chimera of a PNA that is capable of binding to the genomic or antigenomic template RNA strand of a negative strand RNA virus and/or binding to an mRNA of a viral protein (an i c "antisense PNA") coupled to an activator of RNase L
  • Negative strand RNA viruses have multiple genes, i.e., the virion contains the complement of the coding strand.
  • the genome On entry into a host cell the genome is transcribed to produce the various mRNA encoding the viral proteins and also to produce an entire complementary RNA, i.e., the antigenome, from which the 0 genomic strands of the progeny virus are transcribed.
  • the sequence of the antisense PNA is selected so that the activator-antisense chimera binds to and thereby causes the catalytic destruction of the RNA virus genomic or antigenomic strand.
  • the present invention also relates to methods of inhibiting infection by RNA viruses with chimeric molecules comprising an activator of RNase L covalently attached to an antisense PNA that is capable of binding to the genome, antigenome, or mRNA of an RNA virus.
  • the chimeric molecules specifically lead to cleavage of the viral genomic or antigenomic RNA or 0 mRNA.
  • the antisense PNA moiety of the chimera is complementary to a region of the virus RNA antigenome which is characterized by an absence of self-hybridizing secondary structure. 5
  • the non-self-hybridizing portion of the antigenome to be targeted by the antisense PNA moiety can be determined from the sequence of the
  • RNA antigenome by using a secondary-structure-determining algorithm such as MFOLDTM.
  • a suitable portion of the antigenome is one that is normally in a single stranded conformation, i.e., forms a loop in the stem and loop secondary structure of the RNA.
  • a similar selection of non-self-hybridizing portions of the viral mRNA may be carried out.
  • the antisense activator chimeras are designed to target antigenomic RNA, they are also complementary to the mRNA that directs translation of the viral proteins.
  • the antisense PNA moiety is complementary to a portion of the RSV genome or antigenome that is normally single stranded.
  • the activator is attached through a linker to either the amino or the carboxy terminus of the antisense PNA moiety by a linker.
  • the RNase L activator-antisense PNA chimeras of the present invention can be designed to be complementary to either the genomic or antigenome of any negative strand RNA virus.
  • This embodiment of the present invention is illustrated by the following description of chimeras directed to RSV strain A2, but the invention can be practiced with any other RNA virus having a known genomic sequence.
  • the antigenomic sequence of negative strand RNA viruses can be derived therefrom by routine techniques.
  • the present invention also relates to a chimera of an activator of RNase L, coupled to a PNA which is complementary to a region of the virus RNA genomic strand characterized by repeated, conserved, or consensus sequences.
  • the antisense is complementary to a region of the virus RNA genomic strand characterized by repeated, conserved, or consensus sequences.
  • the PNA moiety of the chimera is complementary to a region of the viral genomic RNA strand characterized by repeated or consensus sequences.
  • the antisense PNA moiety has a sequence of approximately 17 nucleobases that is complementary to a number of repeated or consensus sequences that occur within the critical gene-end, intragenic, and gene-start signals of the RSV virus RNA genome.
  • Each gene of the RSV genomic RNA begins with a conserved nine-nucleotide gene-start signal, 3'CCCCGUUUA, with the exception of the L gene, which has the signal 3'CCCUGUUUUA. Transcription begins at the first nucleotide of the gene start signal.
  • Each RSV gene terminates with a semi- conserved 12- to 13- nucleotide gene-end signal, 3' UCAAUUNAUAUAUUUU, which directs transcriptional termination and polyadenylation.
  • the following sequence is used as the antisense PNA moiety of the chimera:
  • the genomic strand of RSV may be targeted with a chimera having an antisense PNA moiety which comprises any of the following sequences:
  • this consensus PNA antisense sequence may additionally target other critical regions with lowered but significant efficiency.
  • the nucleotide sequence signal at the F/intragenic M2 gene start signal has only two mismatches to the consensus antisense sequence. Moreover, one of these is a terminal mismatch which would have a relatively small effect on hybrid duplex stability.
  • the signal at the NS2-intragenic-NS2 gene-start has three mismatches, but only one is of the more significant internal variety. Following this logic, the expected order of hybridization efficiency of the consensus antisense PNA
  • the activator-antisense chimeras can also be designed to target repeated, conserved, or consensus sequences of the genomic strand of other negative strand RNA viruses.
  • Sendai, vesicular stomatitis and influenza viral genes are transcribed from 3' to 5' from a single promoter at the 3' terminus.
  • the 3' and 5' termini also contain sequences required for viral replication and viral packaging.
  • these sequences are targeted by the antisense PNA chimeras of the present invention to specifically cleave the genomic strand of these negative strand RNA genomes.
  • the PNA chimeras of this invention possess an RNase L activation potency similar to that of 2-5A-antisense chimeras with an all-nucleic- acid backbone, they represent an important advance in the application of both the 2- 5 A-antisense and PNA strategies to the control of gene expression.
  • the 2-5A-dependent RNase L augments the PNA-antisense agents with a catalytic mode of targeted RNA destruction.
  • the in vivo lifetime of these chimeras, relative to the corresponding 2-5A-oligonucleotide antisense conjugates, will be improved since the PNA backbone is highly resistant to attack by the nucleases present in cells and sera (Demidov et al;., Biochem. Pharmacol. 48:1310- 1313 (1994)); and this longevity is gained without compromise to the affinity or selectivity of target hybridization (Knudsen and Nielsen, Nucleic Acids Res. 24:494-500 (1996)).
  • PNA antisense oligomer Another advantage of the PNA antisense oligomer is the synthetic flexibility of the pseudopeptide linkage which facilitates further modifications, for example those directed toward improved cellular uptake of the chimera [de
  • chimeras of the present invention are their ability to broaden the substrate specificity of RNase L, as illustrated by the examples herein.
  • This enzyme is normally specific for single-stranded RNA, but surprisingly, cleavage induced by the chimeras of this invention may occur at sites where the target RNA is presumably hybridized to the antisense PNA moiety. Also surprising is that the chimeras induce RNase L to cleave the targe RNA at sites other than at the uridine residues normally preferred by RNase L.
  • the invention also provides a method of cleaving a specifically selected (target) strand of RNA, which comprises the steps of: (a) hybridizing the strand of RNA with a chimeric molecule of this invention to form a complex of the RNA strand and the chimeric molecule, and (b) allowing the resulting complex to react with RNase L; thereby specifically cleaving the target strand of RNA.
  • the chimeric molecules of the invention comprise (a) an antisense peptide nucleic acid moiety that binds or anneals to the target strand of RNA and (b) an activator of RNase L attached to the antisense peptide nucleic acid moiety.
  • Another aspect of this invention is a method of cleaving a specifically selected target strand of RNA contained in a cell, wherein the cell contains RNase L, comprising the steps of: (a)contacting the cell with a chimeric molecule of the invention, (b) entry of the chimeric molecule into the cell; (c) allowing the chimeric molecule to form a complex with the target strand of RNA; and (d) allowing the complex to react with RNase L; whereby the target strand of RNA is cleaved.
  • the cell may be an isolated cell in culture, or may be a cell in its natural environment as part of a multicellular animal. The animal is preferably a mammal, and most preferably a human.
  • the chimeric molecule is contacted with said cell at a concentration of between 0.1 ⁇ M and 100 ⁇ M, more preferably at a concentration of between about 1.0 ⁇ M and 5.0 ⁇ M.
  • PNA-DNA chimeras are known to be capable of passing into cells at concentrations within this range (Uhlmann et al, Angew. Chem. Int. ed. Engl. 35:2632-2635 (1996)).
  • methods of enhancing the passing of oligonucleotides into cells may be employed, for example by electroporation and by complexation with ionic surfactants, as is known to those skilled in the art. It is within the ability of those skilled of the art to vary the dosage, if necessary or desired, and depending upon the mode of delivery and the cellular milieu, so as to obtain the desired concentration of chimeric molecules in a multicellular animal.
  • compositions comprising the chimeric molecules of this invention.
  • Pharmaceutical compositions comprising the chimeric molecules of this invention will comprise an effective amount of the chimera, and may additionally comprise pharmaceutically acceptable carriers, solvents, diluents, and additives known to the art.
  • additional pharmaceutically acceptable components of these pharmaceutical compositions include water, saline, buffers, surfactants, dispersants, and preservatives.
  • the pharmaceutical compositions are preferably sterilized by ultrafiltration or other methods known to the art.
  • the compositions may take the form of sterile solutions or lyophilized powders, or may be in solid dosage forms for implantation or oral or rectal administration.
  • the chimeric molecules and compositions of this invention are expected to be useful for reducing the concentration of RNA species in vitro and in vivo, in any application where antisense molecules are of use.
  • the chimeric molecules of the present invention are also expected to be useful wherever ribozymes are of use.
  • the sequence-specific inhibition of a targeted mRNA in order to effectively "knock out" the associated gene, as an aid to elucidating the function of the encoded protein.
  • the therapeutic uses for the chimeras and compositions of this invention are the treatment of diseases or infections, particularly viral diseases and infections, by ablation of viral mRNA, genomic, or antigenomic RNA, by the reduction of expression of proteins associated with pathophysiological effects (such as TNF and auto-antibodies), and by the reduction of transcription of oncogenes or tumor-promoting gene products (such as angiogenic growth factors).
  • Another therapeutic use for the chimeras of the invention is the inhibition of tumor growth in mammals by chimeras targeted to the RNA component of telomerase.
  • chimeric molecules targeted to the mRNA encoded by mutated genes responsible for autosomal dominant inherited diseases are expected to be useful for ameliorating the symptoms of such diseases, whenever the DNA sequence of the mutant gene is known.
  • the invention provides a method for treating such diseases by administration of a chimeric molecule of the invention, wherein the antisense polyamide nucleic acid moiety of the chimera has a nucleobase sequence complementary to the mRNA encoded by said gene, in an amount sufficient to reduce the transcription rate of the mutant gene.
  • the activator-antisense chimeras of the invention may also be used to inhibit infection by a negative strand RNA virus to which the activator-antisense chimera is targeted, for example RSV infection.
  • the activator-antisense chimeras of the invention can be administered to a subject having an RSV or other respiratory viral infection by any route effective to deliver the activator-antisense chimeras to the epithelium of the bronchi, bronchioles and alveoli of the subject.
  • the activator-antisense chimeras are delivered by use of an inhaled aerosol, according to the techniques well known in the art for the delivery of ribavirin.
  • a mixture of ribavirin and a chimera of the invention can be administered in a common pharmaceutical carrier.
  • the chimera can be administered parentally, e.g. , by intravenous infusion.
  • the dose of the chimera can be determined by routine methods well known to pharmacologists so that the serum concentration approximates the concentration at which RNA cleavage activity is seen in the in vitro examples described herein.
  • the dose should be selected so that the tissue concentration in the lung approximates the concentration at which RNA cleavage activity is seen in the in vitro examples.
  • Another aspect of the invention relates to methods of preparing the chimeras of the invention.
  • the synthesis process comprises the steps of
  • step (b) attaching to the N-terminal of said polyamide nucleic acid moiety an N-(2-hydroxyethyl)-N-[l-(purinly or pyrimidinyl)acetyl]glycyl moiety, (c) attaching to the terminal hydroxyl group introduced in step (b) an O-(4-hydroxybutyl)phosphoryl moiety,
  • step (d) attaching to the terminal hydroxyl group introduced in step (c) an additional O-(4-hydroxybutyl)phosphoryl moiety,
  • step (e) attaching to the terminal hydroxyl group introduced in step (d) an O-(2'-adenosyl)phosphoryl moiety,
  • step (g) repeating step (f) between one and three times,
  • highly cross-linked polystyrene beads were functionalized with a glycine unit via a/ hydroxymethylbenzoic acid linker, resulting in immobilized 10 ( Figure 2).
  • the PNA part of the chimera was constructed by sequential elongation of the amino-terminus using monomethoxytrityl protected PNA adeninyl monomer 5 (Will et al, Tetrahedron 51 :12069-12082 (1995)) and HATU as the coupling reagent.
  • the synthesis of the PNA sequence was completed by introduction of the 3'-linker by reaction of 6 (Petersen et al, Bioorg. Med. Chem. Lett., 5:1119-1124 (1995)) with the amino group of immobilized PNA 11 to give 12.
  • elongation of the solid-phase bound oligomer was feasible using standard phosphoramidite chemistry.
  • the butyl spacers were introduced by the o- nitrophenyltetrazole mediated reaction (D. Filipov, Ph.D. Thesis, Leiden University (1988)) of(2-cyanoethyl)-N,N-diisopropyl-4-O-(4,4'- dimethoxytrityl)butylphosphoramidite 7 (Lesiak et al, Bioconjugate Chem., 4:467- 472 (1993)) with the free hydroxyl group in 12.
  • the 2',5'-oligoadenylate part of the chimera was appended to the growing oligomer by elongation of 13 with the protected adenosine 2'-phosphoramidite 8 in four consecutive coupling cycles.
  • the 5'-terminal phosphate triester was introduced by reaction of the free hydroxyl in resulting 14 with 2-[[2-(4,4'-dimethoxytrityloxy)ethyl]sulfonyl]ethyl-(2- cyanoethyl)-N,N-diisopropylphosphoramidite 9, to afford fully protected 2-5A-PNA adduct 15.
  • the chimeric oligonucleotide 15 was released from the resin with concomitant deprotection using methanolic ammonia and desilylation with tetraethylammonium fluoride.
  • the crude mixture was desalted by gel filtration and purified by reverse-phase HPLC. This procedure furnished 2-
  • n 4, 8, or 12.
  • the monomers Prior to coupling, the monomers were pre-activated for 1 min by mixing equal amounts of the PNA monomer (15 equiv per ⁇ mol support), HATU and DiPEA solutions.
  • the protocol for one PNA chain extension cycle consisted of (1) wash: acetonitrile/dimethylformamide (1/1, v/v), 2.5 ml; (2) coupling: PNA + HATU + DiPEA in acetonitrile/dimethylformamide (1/1, v/v), 15 min; (3) wash: acetonitrile/dimethylformamide (1/1, v/v), 2.5 ml, acetonitrile, 2.5 ml; (4) capping: Ac 2 ⁇ /lutidine/N-methylimidazole/tetrahydrofuran (1/1/1/7, v/v/v/v), 2.0 ml; (5) wash: acetonitrile, 2.5 ml, dichloromethane, 3.5 ml; (6) detritylation:
  • the oligomers were cleaved from the support with concomitant deprotection of the phosphate groups and exocyclic amino groups by treatment with methanolic ammonia (1.5 ml) at 50°C for 16 h.
  • the samples were filtered and the silyl protective groups were removed by treatment with tetraethylammonium fluoride (0.5 M in dry acetonitrile) at ambient temperature for 16 h. Desalting was established using a G-25 column with a 0.15 M solution of ammonium bicarbonate as the eluting agent.
  • RP-HPLC purification and analysis were carried out with a LiChrospherTM 100 RP-18 endcapped column (10.0 x 250 mm and 4.0 x 250 mm, respectively).
  • Gradient elution was performed at 40°C by building up a gradient starting with buffer A (50 mM triethylammonium acetate in water) and applying buffer B (50 mM triethylammonium acetate in acetonitrile/water, 1/1 , v/v) with a flow rate of 1.0 ml/min or 5.0 ml/min for analysis and purification, respectively.
  • the identity of the oligomers was confirmed using electrospray mass spectrometry.
  • Chimeric molecules having other nucleobase sequences may be prepared by the method described above, by altering the order in which the PNA monomers are introduced to the reaction vessel and coupled to the growing oligomer. RNase L cleavage
  • the first assay employed radiolabeled poly(U) as the substrate.
  • the 2-5A tetramer 1 activated pure recombinant human RNase L to degrade poly(U)-[ PJpCp, with 50% cleavage occurring at a concentration (EC 50 ) of 0.4 ⁇ 0.04 nM ( Figure 4).
  • Each of the three 2-5A-PNA analogues showed a concentration-dependent ability to cause cleavage of the poly(U). The most effective was the tetraadenylate derivative 2, which possessed an EC 50 of 4 ⁇ 0.5 nM.
  • C re ⁇ was used which defines the relative activity of the 2-5 A tetramer as unity, and compares (ratio of EC 5 o's) the relative concentration of other activators needed to effect 50% cleavage. The greater the C re ⁇ , the less effective the activator.
  • the 2-5A-PNA chimera tetramer 2 showed a mean EC 50 of 3 ⁇ 1 nM , a 10-fold reduction in RNase L activation ability.
  • the octaadenylate congener 3 gave an average EC 50 of 5 ⁇ 1 nM
  • compound 4 (the dodecaadenylate chimera) had a mean EC 50 of 32 ⁇ 1 nM, a 100-fold reduction in activation compared to the tetrameric 2-5A standard.
  • a C rel is the EC 50 (or IC 50 ) of the 2-5A-PNA chimera divided by the EC 50 (or IC 50 ) of the 2-5 A standard, with 2-5 A arbitrarily assigned a C re ⁇ value of 1. These values are given as the mean ⁇ standard deviation, with the number of experiments (n) given in brackets.
  • the experimental IC 50 for probe displacement by 2-5 A in the radiobinding assay was 3.3 ⁇
  • a lOx cleavage buffer 100 mM HEPES, pH 7.5, 1.0 M KC1, 50 mM Mg(OAc) 2 , 10 mM ATP, and 143 mM 2-mercaptoethanol
  • 12-16 ⁇ L of RNase-free water were used in each cleavage reaction.
  • 2 ⁇ L of a 1 Ox solution of 2-5 A analogue final concentrations 10-5 to 10-10 M
  • recombinant RNase L enzyme final concentration of 130 nM
  • poly(U)-[ 32 P]pCp substrate final concentration lOuM in UMP equivalents
  • RNA was added, and then 10 M ammonium acetate to a final concentration of 2-2.5 M. After mixing with 2 volumes of cold ethanol, the reaction mixtures were left on ice for 30 min, and the precipitated RNA pelleted with a brief spin at 4 °C (12 000 x g for 2 min.). The presence of cleaved fragments of poly(U)- [ 32 P]pCp was assessed by counting aliquots of the supernatant in scintillation fluid. RNase L activation and cleavage of a synthetic oligonucleotide substrate.
  • Oligoribonucleotide rC ⁇ U 2 C 7 was prepared by Midland Certified Reagent Co. (Midland, TX) and 5 '-labeled with polynucleotide kinase and ⁇ - 32 P-ATP (DuPont- NEN, Wilmington, DE).
  • the cleavage buffer was 25 mM Tris HC1, pH 7.4, 100 mM KC1, 10 mM MgCl 2 , 100 ⁇ M ATP, and 10 mM DTT.
  • cleavage buffer 14 - 18 ⁇ L cleavage buffer, 2 ⁇ L 2-5A-antisense chimera at lOx the desired final concentration, and 2 ⁇ L of an RNase L solution to give a final concentration of 130 nM RNase L.
  • Cleavage reaction mixtures were held for 10 min on ice after addition of RNase L.
  • the substrate [ 32 P]pCnU 2 C 7 (2 ⁇ L of a solution to give a final substrate concentration of 10 nM) was added last and the mixture incubated at 37 °C for 15 min.
  • the EC 50 was defined (after background subtraction) as the effective concentration that brought about 50% degradation of RNA substrate.
  • the binding data were in accord with the data from the two activation assays since there was a correspondence between a diminished ability to activate RNase L and the ability to bind to RNase L Radiobinding assays.
  • Assay mixtures were incubated at 4°C for 2 h, after which they were applied to nitrocellulose filters that were subsequently washed (3x) with water. The filters were placed in scintillant and counted in a liquid scintillation counter.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Virology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne la conjugaison covalente d'une fraction 2',5'-oligoadélynate lié 5'- phosphorylé (2-5A) à un oligomère antisens d'acide nucléique peptidique (ANP), qui constitue un réactif chimère nouveau. Ce dernier procède au clivage sélectif et spécifique d'un ARN cible choisi. Les chimères ANP antisens-2-5A lient l'ARN cible avec une spécificité et une affinité élevées et sont résistantes aux nucléases. La partie antisens de la chimère recrute un ARN choisi comme substrat pour le clivage, la partie 2-5-A de la chimère liant et activant la RNase L, ce qui constitue une nouvelle approche de l'ablation ciblée d'un ARNm cible et diminue dans l'expression de sa protéine spécifique. On estime que les molécules chimères seront utiles en tant qu'outils de recherche et agents thérapeutiques.
PCT/US1999/020159 1998-09-04 1999-09-02 Molecules chimeres oligoadelynates-acides nucleiques peptidiques Ceased WO2000014219A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU57034/99A AU5703499A (en) 1998-09-04 1999-09-02 Peptide nucleic acid-oligoadenylate chimeric molecules

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US9917398P 1998-09-04 1998-09-04
US60/099,173 1998-09-04

Publications (3)

Publication Number Publication Date
WO2000014219A2 true WO2000014219A2 (fr) 2000-03-16
WO2000014219A3 WO2000014219A3 (fr) 2000-07-06
WO2000014219A9 WO2000014219A9 (fr) 2000-08-10

Family

ID=22273307

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/020159 Ceased WO2000014219A2 (fr) 1998-09-04 1999-09-02 Molecules chimeres oligoadelynates-acides nucleiques peptidiques

Country Status (2)

Country Link
AU (1) AU5703499A (fr)
WO (1) WO2000014219A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3784222A4 (fr) * 2018-04-24 2022-03-30 The Scripps Research Institute Recrutement ciblé de petites molécules d'une nucléase à l'arn

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5583032A (en) * 1992-10-21 1996-12-10 The Cleveland Clinic Foundation And National Institutes Of Health Method of cleaving specific strands of RNA
US6214805B1 (en) * 1996-02-15 2001-04-10 The United States Of America As Represented By The Department Of Health And Human Services RNase L activators and antisense oligonucleotides effective to treat RSV infections
US6015710A (en) * 1996-04-09 2000-01-18 The University Of Texas System Modulation of mammalian telomerase by peptide nucleic acids

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3784222A4 (fr) * 2018-04-24 2022-03-30 The Scripps Research Institute Recrutement ciblé de petites molécules d'une nucléase à l'arn

Also Published As

Publication number Publication date
AU5703499A (en) 2000-03-27
WO2000014219A9 (fr) 2000-08-10
WO2000014219A3 (fr) 2000-07-06

Similar Documents

Publication Publication Date Title
US5532130A (en) Methods and compositions for sequence-specific hybridization of RNA by 2'-5' oligonucleotides
Uhlmann et al. PNA: synthetic polyamide nucleic acids with unusual binding properties
JP3529135B2 (ja) 特定のｒｎａ鎖の切断方法
EP0866865B1 (fr) Oligomeres chimeriques possedant une activite de clivage de l'arn
US6380169B1 (en) Metal complex containing oligonucleoside cleavage compounds and therapies
HUT76094A (en) Oligonucleotide n3'-p5' phosphoramidates: synthesis and compounds; hybridization and nuclease resistance properties
AU7976198A (en) Hammerhead ribozymes with extended cleavage rule
US5849900A (en) Inhibition of viruses by antisense oligomers capable of binding to polypurine rich tract of single-stranded RNA or RNA-DNA hybrids
WO1999022742A1 (fr) Activateurs de rnase l et oligonucleotides antisens efficaces pour traiter des infections a vrs (virus respiratoire syncytial)
AU2003202376A1 (en) Oligonucleotides comprising alternating segments and uses thereof
WO2003064441A2 (fr) Oligonucleotides comportant des segments alternatifs et utilisations associees
AU6527094A (en) Modified oligonucleotides having improved anti-influenza activity
JPH11236396A (ja) ペプチド核酸の高次構造および結合
WO1997029757A1 (fr) Activateurs de rnase l et oligonucleotides antisens efficaces dans le traitement des infections a rsv
WO1994007367A9 (fr) Oligomeres antiviraux liant des voies de polypurine d'arn monocatenaire ou d'hybrides d'arn-adn
JPH11513881A (ja) テンプレートおよびプライマーに基づく酵素的に切断可能なオリゴヌクレオチドの合成
Verheijen et al. 2′, 5′-Oligoadenylate-peptide nucleic acids (2–5A-PNAs) activate RNase L
US20030203356A1 (en) RNase L activator-antisense complexes
WO2000014219A2 (fr) Molecules chimeres oligoadelynates-acides nucleiques peptidiques
Lowe et al. Hybridization studies with chiral peptide nucleic acids
EP1574572B1 (fr) Composés pour l'hydrolyse d'acides ribonucléiques (RNAs)
AU736470C (en) RNase L activators and antisense oligonucleotides effective to treat RSV infections
Kumar Dna Applications In Biology
WO1999000401A1 (fr) Oligonucleotides 3'-monophosphorylisants
HK1013103B (en) Method of cleaving specific strands of rna

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

AK Designated states

Kind code of ref document: C2

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: C2

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

COP Corrected version of pamphlet

Free format text: PAGES 1/6-6/6, DRAWINGS, REPLACED BY NEW PAGES 1/6-6/6; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase