WO2000017346A2 - Molecules hybridantes en epingle a cheveux servant a moduler l'expression genique - Google Patents

Molecules hybridantes en epingle a cheveux servant a moduler l'expression genique Download PDF

Info

Publication number
WO2000017346A2
WO2000017346A2 PCT/US1999/021865 US9921865W WO0017346A2 WO 2000017346 A2 WO2000017346 A2 WO 2000017346A2 US 9921865 W US9921865 W US 9921865W WO 0017346 A2 WO0017346 A2 WO 0017346A2
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
acid molecule
independently
hybridizer
haiφin
Prior art date
Application number
PCT/US1999/021865
Other languages
English (en)
Other versions
WO2000017346A3 (fr
Inventor
Thomas Hartmann
Michael Zwick
James Thompson
Thale Jarvis
Original Assignee
Ribozyme Pharmaceuticals, Inc.
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 Ribozyme Pharmaceuticals, Inc. filed Critical Ribozyme Pharmaceuticals, Inc.
Priority to CA002343067A priority Critical patent/CA2343067A1/fr
Priority to JP2000574246A priority patent/JP2002526072A/ja
Priority to AU60552/99A priority patent/AU6055299A/en
Priority to EP99969438A priority patent/EP1115859A2/fr
Publication of WO2000017346A2 publication Critical patent/WO2000017346A2/fr
Publication of WO2000017346A3 publication Critical patent/WO2000017346A3/fr

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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12N2310/315—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
    • C12N2310/31—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
    • C12N2310/32—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
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
    • 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
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • HPH hairpin hybridizer
  • nucleic acid molecules have been designed which are capable of binding to specific mRNA sequences by Watson-Crick base-pairing interaction and blocking translation (Crooke, 1996, Medicinal Res. Rev. 16, 319-344).
  • Another approach involves complexation of DNA with triplex-forming oligonucleotides to prevent transcription of bound DNA sequences thereby inhibiting gene expression (Kim et al., 1998, Biochemistry. 37, 2299-2304).
  • the interaction of antisense oligonucleotides, 2-5A antisense chimera, or ribozymes with target RNAs have been used to modulate gene expression. All of these nucleic acid molecules are 5 highly specific to their matching target sequences and therefore may offer lower toxicity compared to traditional approaches such as chemotherapy.
  • E. coli OmpF and OmpC genes are regulated by an antisense RNA mechanism (Mukopadhyay & Roth, 1996, Critical Rev. In Oncogenesis 7, 151-190).
  • Antisense oligonucleotides can be used to down-regulate target mRNA by a
  • Pseudo-targets are nucleic acid sequences, which may have sequence identity or homology to a target sequence.
  • the number of pseudo- o targets for a given sequence, especially human genes, is largely unknown at this point, since only a minor fraction of the human genome is currently sequenced.
  • the molecular switch consists of a probe sequence of 20 to 60 nudeotides
  • RNA cassette system for silencing the lad gene.
  • the molecules used consisted of a hairpin structure, which was used for target sequence recognition and an inhibitor region which was either an antisense or ribozyme sequence.
  • RNA molecule with an intramolecular stem-loop structure of greater than or equal to eight base-pairs is a RNA molecule with an intramolecular stem-loop structure of greater than or equal to eight base-pairs.
  • This invention relates to nucleic acid molecules capable of binding and blocking i o the function of target nucleic acid molecules, thereby modulating cellular or viral mechanisms including splicing, editing, replication or gene expression, and translation.
  • the invention concerns novel nucleic acid molecules with a hairpin- secondary structure capable of down regulating protein expression by binding (steric blocker) and optionally facilitating the cleavage of target RNA through an RNase H or 1 5 other mechanism.
  • the nucleic acid molecules of the instant invention shall be referred to as hairpin hybridizer (HPH) molecules.
  • HPH molecules to down-regulate gene expression in bacterial, microbial, fungal, eukaryotic systems including plant, or mammalian cells.
  • Down-regulation of specific target sequences may ⁇ either have a therapeutic effect in many diseases or disease states or aid in the identification of gene function and/or new therapeutic gene targets.
  • the HPH molecules of the present invention can be used for in vitro or in vivo applications well known in the art.
  • the present invention features a method of modulating the function of a target
  • HPH molecules include target-binding region and a hairpin region, where the target-binding region is capable of binding to the target sequence in a sequence specific manner in vitro or in vivo to modulate the function of the target sequence.
  • the hairpin region of the HPH molecule provides an improved specificity characteristic to the HPH molecule.
  • the hairpin region is expected ⁇ provide improved resistance to nuclease degradation, is expected to help the HPH 5 molecule with localization inside a cell, and is expected to help in improved uptake of the HPH molecule by the cells compared to a molecule lacking such a hairpin structure.
  • the target-binding region of the HPH molecule may also include an RNase H- activating region where such a region includes a greater than or equal to 4 deoxyribonucleotide nucleotide sequence with phosphorothioate, phosphodiester,
  • the RNase H-activating region interacts with the target RNA to form a DNA:RNA complex which is recognized by the cellular RNase H enzyme, which binds the DNA:RNA complex and cleaves the RNA portion of the DNA:RNA complex.
  • RNase H cleavage of the target RNA by RNase H causes the target RNA to lose its normal i s function by causing inhibition of its translation into proteins, its replication, its packaging into viral particles, or other functions.
  • a method of modulating the function, such as expression, of a target sequence comprising the step of contacting said target sequence with a HPH nucleic acid molecule under conditions suitable for the
  • HPH nucleic acid molecule includes the following formulae:
  • each P, Y, N and M represents independently a nucleotide which may be
  • N' is a nucleotide complementary to N
  • o is an integer greater than or equal to 3, more specifically 4, 5, 6, 7, 8 or 9
  • k is zero or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20
  • t is zero or an integer greater than or equal to 3 and preferably less than about 100
  • (N»N') 0 represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate or others known in the art).
  • a chemical linkage e.g. a phosphate ester linkage, amide linkage, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate or others known in the art.
  • N' is a nucleotide complementary to N; o is an integer greater than or equal to 3, more specifically 4, 5, 6, 7, 8 or 9; k is zero or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; t is zero or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; kl is zero or an integer greater
  • N' is a nucleotide complementary to N; o is an integer greater than or equal to 3, more specifically 4, 5, 6, 7, 8 or 9; k is zero or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; t is zero or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; w is an integer greater than or
  • I O equal to 4 and preferably less than 100, more specifically 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20;
  • P) t and (P) k are oligonucleotides preferably including at least one position that is not deoxynucleotide (e.g.
  • a chemical linkage e.g. a phosphate ester linkage, amide linkage, i o phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate or others known in the art).
  • N represents a ribonucleotide which may be the same or different; N' is a nucleotide complementary to N; • indicates hydrogen bond formation between two adjacent ribonucleotides; o is an integer greater than or equal to 3 and less than or equal to 9, more specifically 4, 5, 6, 7, 8 or 9; S, A, and B are oligoribonucleotides which are independently equal to 5 and preferably less than 100 nudeotides in length, more
  • S is an oligonucleotide of sufficient length to stably interact independently with a target sequence (the target can be an
  • RNA, DNA or RNA/DNA mixed polymers RNA, DNA or RNA/DNA mixed polymers); and represents a phosphodiester linkage.
  • each P, N, F, V, Z, and M represents independently a nucleotide which may be same or different; • indicates hydrogen bond formation between two adjacent i o nudeotides, N' is a nucleotide complementary to N; F' is a nucleotide complementary to F; o is an integer greater than or equal to 3, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, or 15; k is zero or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; t is zero or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, i s 7, 8, 9, 10, 11, 12, 15, or 20; w is an integer greater than or equal to 4 and preferably less than 100, more specifically 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; d is an integer greater than or equal to 3 and preferably less than about 20, more specifically 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, or 18; h is an integer greater than or equal
  • N and/or N' in (N»N') 0 , F and/or F' in (F»F') h and/or (Z) c may optionally be able to independently interact with a target sequence.
  • each P, N, F, Z, and M represents independently a nucleotide which may be same or different; • indicates hydrogen bond formation between two adjacent nudeotides, N' is a nucleotide complementary to N; F' is a nucleotide complementary to F; o is an integer greater than or equal to 3, more specifically 4, 5, 6, 7, 9, 10, 11, 12, 5 or 15; k is zero or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; kl is zero or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; t is zero or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; tl i o is zero or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; w is an integer greater than or equal to
  • (P) t ,(P) k , and (Z) c is an oligonucleotide preferably including at least one position that is not deoxynucleotide (e.g.
  • each said (P) connector (P) k , (P) tl , (P) kl ,and (M) w is an oligonucleotide of sufficient length to stably interact independently with a target sequence (the target can be an RNA, DNA or RNA/DNA mixed polymers); each B and B' independently represents a cap structure which may independently be present or absent; and represents a chemical linkage (e.g.
  • N and/or N' in (N»N') 0 , F and/or F' in (F»F') h and/or (Z) c may optionally be able to independently interact with a target sequence.
  • each P, N, F, V, Z, and M represents independently a nucleotide which i o may be same or different; • indicates hydrogen bond formation between two adjacent nudeotides, N' is a nucleotide complementary to N; F' is a nucleotide complementary to F; o is an integer greater than or equal to 3, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, or 15; k is zero or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; t is zero or an integer 15 greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; w is an integer greater than or equal to 4 and preferably less than 100, more specifically 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; d is an integer greater than or equal to 3 and preferably less than about 20, more specifically 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, or 18; h is an integer greater than or equal to 2 and
  • N and/or N' in (N » N') 0 , F and/or F' in (F*F') h and/or (Z) c may optionally be able to independently interact with a target ⁇ sequence.
  • each F, D, O, K, W and T represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and may include phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate linkers and the like or a combination thereof; F 5 and D independently or in combination form RNaseH-activating domain, wherein F and D are of length greater than or equal to 4 nudeotides if they form independent RNaseH domains and are of length greater than or equal to 2 nudeotides if they form
  • RNaseH domain in combination • indicates hydrogen bond formation between two adjacent nudeotides within an oligonucleotide; D comprises nucleotide sequence that is i o complementary to the nucleotide sequence of O; K comprises nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs; K and T form greater than or equal to two base pairs with each other that are contiguous or non-contiguous,
  • K and T form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs; the K»T and O*D base-paired regions may be contiguous or non-contiguous to each other; K, T, O and W are of length greater than or equal to 3 nudeotides and preferably less than about 100 nudeotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; F, D, K, T, W and O together are of sufficient length to stably interact with a target nucleic acid ⁇ sequence (the target can be an RNA, DNA or RNA/DNA mixed polymers); each B and B' independently represents a cap structure which may independently be present or absent; and represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate or others known in the art).
  • B B' independently represents a cap structure which may independently be present or absent; and represents a chemical link
  • each F, D, O, K, W and T represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and may include phosphodiester, phosphorothioate, 5'-thiophosphate,
  • F and D independently form RNaseH-activating domain, wherein F and D are of length greater than or equal to 4 nudeotides; • indicates hydrogen bond formation between two adjacent nudeotides within an oligonucleotide; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises nucleotide i o sequence that is complementary to the nucleotide sequence of T; D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs; K and T form greater than or equal to two base pairs with each other that are contiguous or noncontiguous, more specifically K and T form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs; the
  • K, T, O, and W are of length greater than or equal to 3 nudeotides and preferably less than about 100 nudeotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20;
  • F, D, K, T, W and O together are of sufficient length to stably interact with a target nucleic acid sequence (the target can be an RNA, DNA or RNA/DNA mixed 0 polymers);
  • each B and B' independently represents a cap structure which may independently be present or absent; and represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage, 5'-thiophosphate, phosphorothioate, phosphorodithioate, methylphosphonate or others known in the art).
  • each F, D, O, K, W and T represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and may include phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate linkers and the like or a combination thereof; F and D independently form RNaseH-activating domain, wherein F and D are of length i o greater than or equal to 4 nudeotides; • indicates hydrogen bond formation between two adjacent nudeotides within an oligonucleotide; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous,
  • K and T form greater than or equal to two base pairs with each other that are contiguous or noncontiguous, more specifically K and T form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs; the K»T and O»D base-paired regions may be contiguous or non-contiguous to each other; K, T, O, and W are of length greater than or equal to 3 nudeotides and preferably less
  • each B and B' independently represents a cap structure which may independently be present or absent; and represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphorothioate, phosphorodithioate, 5 ⁇ thiophosphate, methylphosphonate or others known in the art).
  • a target nucleic acid sequence the target can be an RNA, DNA or RNA/DNA mixed polymers
  • each B and B' independently represents a cap structure which may independently be present or absent; and represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphorothioate, phosphorodithioate, 5 ⁇ thiophosphate, methylphosphonate or others known in the art).
  • each F, D, O, K, W and T represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different I O length and may include phosphodiester, phosphorothioate, 5 '-thiophosphate, phosphorodithioate, methylphosphonate linkers and the like or a combination thereof; F and D independently or in combination form RNaseH-activating domain, wherein F and D are of length greater than or equal to 4 nudeotides if they form independent RNaseH domains and are of length greater than or equal to 2 nudeotides if they form
  • K comprises nucleotide sequence that is complementary to the nucleotide sequence of T
  • D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more ⁇ specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs
  • K and T form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically K and T form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs
  • the K»T and O «D base-paired regions may be contiguous or non-contiguous to each other
  • K, T, O and W are of length greater than or equal to 3 nudeotides and preferably less than about 100 nudeotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20
  • each B and B' independently represents a cap structure which may independently be present or absent; and represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphorothioate, 5 '-thiophosphate, phosphorodithioate, methylphosphonate or others known in the art).
  • a chemical linkage e.g. a phosphate ester linkage, amide linkage, phosphorothioate, 5 '-thiophosphate, phosphorodithioate, methylphosphonate or others known in the art.
  • each D, O and W represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and
  • 1 5 may include phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate linkers and the like or a combination thereof; D independently forms an RNaseH- activating domain of length greater than or equal to 4 nudeotides; • indicates hydrogen bond formation between two adjacent nudeotides within an oligonucleotide; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; 2o D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs; O and W are of length greater than or equal to 3 nudeotides and preferably less than about 100 nudeotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; D, W and O together are of sufficient length to stably interact with a target nucleic acid sequence (the target can be an RNA, DNA or RNA DNA mixed polymers); each B and B
  • each D, O and W represents independently an oligonucleotide whose i o nucleotide sequence may be same or different, may be of same or different length and may include phosphodiester, phosphorothioate, phosphorodithioate, 5 '-thiophosphate, methylphosphonate linkers and the like or a combination thereof; D independently forms an RNaseH-activating domain of length greater than or equal to 4 nudeotides; • indicates hydrogen bond formation between two adjacent nudeotides within an i s oligonucleotide; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs; O and W are of length greater than or equal to 3 nudeotides and preferably less than about 100 nudeotides, more specifically 4, 5, 6, 7, 20 8, 9, 10,
  • each A, D, O, K, W and T represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and may include phosphodiester, phosphorothioate, 5 '-thiophosphate, phosphorodithioate, methylphosphonate linkers and the like or a combination thereof;
  • i o D independently forms an RNaseH-activating domain of length greater than or equal to
  • D comprises nucleotide sequence that is complementary to the nucleotide sequence of O
  • K comprises nucleotide sequence that is complementary to the nucleotide sequence of T
  • D and O form greater than or equal to two base pairs
  • K and T form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically K and T form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs; the K*T and O «D base-paired regions may be contiguous or non-contiguous to each other;
  • A, K, T, O and W are of length o greater than or equal to 3 nudeotides and preferably less than about 100 nudeotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20;
  • A, D, K, T, W and O together are of sufficient length to stably interact with a target nucleic acid sequence (the target can be an RNA, DNA or RNA/DNA mixed polymers); each B and B' independently represents a cap structure which may independently be present or absent; and represents a chemical linkage (e.g. a phosphate ester linkage,
  • each A, D, O, K, W and T represents independently an oligonucleotide i o whose nucleotide sequence may be same or different, may be of same or different length and may include phosphodiester, phosphorothioate, 5 '-thiophosphate, phosphorodithioate, methylphosphonate linkers and the like or a combination thereof;
  • D independently forms an RNaseH-activating domain of length greater than or equal to
  • D comprises nucleotide sequence that is complementary to the nucleotide sequence of O
  • K comprises nucleotide sequence that is complementary to the nucleotide sequence of T
  • D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs
  • K and T form greater than or equal to two base
  • each B and B' independently represents a cap structure which may independently be present or absent; and represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphorothioate, 5 '-thiophosphate, phosphorodithioate, methylphosphonate or others known in the art).
  • a target nucleic acid sequence the target can be an RNA, DNA or RNA/DNA mixed polymers
  • each B and B' independently represents a cap structure which may independently be present or absent; and represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphorothioate, 5 '-thiophosphate, phosphorodithioate, methylphosphonate or others known in the art).
  • each A, D, O, K, W and T represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and may include phosphodiester, phosphorothioate, 5 '-thiophosphate,
  • D independently forms an RNaseH-activating domain of length greater than or equal to
  • K comprises nucleotide sequence that is complementary 2o to the nucleotide sequence of T; D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs; K and T form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically K and T form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs; the K*T and O*D base-paired regions may be contiguous or non-contiguous to each other; A, K, T, O and W are of length greater than or equal to 3 nudeotides and preferably less than about 100 nudeotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; A, D,
  • each A, D, O, K, W and T represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and may include phosphodiester, phosphorothioate, 5 '-thiophosphate, phosphorodithioate, methylphosphonate linkers and the like or a combination thereof; D independently forms an RNaseH-activating domain of length greater than or equal to
  • K comprises nucleotide sequence that is complementary to the nucleotide sequence of T
  • D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs
  • K and T form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically K and T 5 form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs
  • the K*T and O»D base-paired regions may be contiguous or non-contiguous to each other
  • A, K, T, O and W are of length greater than or equal to 3 nudeotides and preferably less than about 100 nudeotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20
  • A, D comprises nucleotide sequence that is complementary to the nucleotide sequence of T
  • D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous,
  • each A, D, O, and W represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and
  • D 20 may include phosphodiester, phosphorothioate, phosphorodithioate, 5 '-thiophosphate, methylphosphonate linkers and the like or a combination thereof;
  • D independently forms an RNaseH-activating domain of length greater than or equal to 4 nudeotides; • indicates hydrogen bond formation between two adjacent nudeotides within an oligonucleotide;
  • D comprises nucleotide sequence that is complementary to the nucleotide sequence of O;
  • D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically D and O form 2, 3 4, 5, 6, 7, 8, 9, 10 or 11 base pairs;
  • A, O and W are of length greater than or equal to 3 5 nudeotides and preferably less than about 100 nudeotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20;
  • A, D, W and O together are of sufficient length to stably interact with a target nucleic acid sequence
  • I O linkage e.g. a phosphate ester linkage, amide linkage, phosphorothioate, phosphorodithioate, 5 '-thiophosphate, methylphosphonate or others known in the art.
  • a phosphate ester linkage e.g. a phosphate ester linkage, amide linkage, phosphorothioate, phosphorodithioate, 5 '-thiophosphate, methylphosphonate or others known in the art.
  • each A, D, O, and W represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and may include phosphodiester, phosphorothioate, phosphorodithioate, 5 '-thiophosphate, methylphosphonate linkers and the like or a combination thereof; D independently forms an RNaseH-activating domain of length greater than or equal to 4 nudeotides; • ⁇ indicates hydrogen bond formation between two adjacent nudeotides within an oligonucleotide; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs; A, O and W are of length greater than or equal to 3 nudeotides and preferably less than about 100 nudeotides, more specifically 4, 5, 6, 7, 8, 9, 10,
  • each A, D, O, and W represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and may include phosphodiester, phosphorothioate, phosphorodithioate, 5 '-thiophosphate, i s and/or methylphosphonate linkers and the like or a combination thereof;
  • F and D independently form RNaseH-activating domains of length greater than or equal to 4 nudeotides; • indicates hydrogen bond formation between two adjacent nudeotides within an oligonucleotide;
  • D comprises nucleotide sequence that is complementary to the nucleotide sequence of O;
  • D and O form greater than or equal to two base pairs with 2o each other that are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs;
  • O and W are of length greater than or equal to 3 nudeotides and preferably less than about 100 nudeo
  • each F, D, O, and W represents independently an oligonucleotide whose i o nucleotide sequence may be same or different, may be of same or different length and may include phosphodiester, phosphorothioate, phosphorodithioate, 5 '-thiophosphate, methylphosphonate linkers and the like or a combination thereof; F and D independently or in combination form RNaseH-activating domain, wherein F and D are of length greater than or equal to 4 nudeotides if they form independent RNaseH i 5 domains and are of length greater than or equal to 2 nudeotides if they form RNaseH domain in combination; • indicates hydrogen bond formation between two adjacent nudeotides within an oligonucleotide; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form greater than or equal o to two base pairs with each other
  • each B and B' independently represents a cap structure which may independently be present or absent; and represents a chemical linkage (e.g. a phosphate ester linkage, amide linkage, phosphorothioate, phosphorodithioate, 5 '-thiophosphate, methylphosphonate or others known in the art).
  • a chemical linkage e.g. a phosphate ester linkage, amide linkage, phosphorothioate, phosphorodithioate, 5 '-thiophosphate, methylphosphonate or others known in the art.
  • each A, D, O, and W represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and
  • 1 5 may include phosphodiester, phosphorothioate, phosphorodithioate, 5 '-thiophosphate, methylphosphonate linkers and the like or a combination thereof;
  • D independently forms an RNaseH-activating domain of length greater than or equal to 4 nudeotides; • indicates hydrogen bond formation between two adjacent nudeotides within an oligonucleotide;
  • D comprises nucleotide sequence that is complementary to the 2 ⁇ nucleotide sequence of O;
  • D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs;
  • A, O and W are of length greater than or equal to 3 nudeotides and preferably less than about 100 nudeotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20;
  • A, D, W and O together are of sufficient length to stably interact with a target nucleic acid
  • each A, D, O, and W represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and may include phosphodiester, phosphorothioate, phosphorodithioate, 5 '-thiophosphate, methylphosphonate linkers and the like or a combination thereof; D independently forms an RNaseH-activating domain of length greater than or equal to 4 nudeotides; • i s indicates hydrogen bond formation between two adjacent nudeotides within an oligonucleotide; D comprises nucleotide sequence that is complementary to the nucleotide sequence of O; D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs; A, O and W are of length greater than or equal to 3 0 nudeotides and preferably less than about 100 nudeo
  • each F, D, O, and W represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and may include phosphodiester, phosphorothioate, phosphorodithioate, 5 '-thiophosphate, I O methylphosphonate linkers and the like or a combination thereof; F and D independently or in combination form RNaseH-activating domain, wherein F and D are of length greater than or equal to 4 nudeotides if they form independent RNaseH domains and are of length greater than or equal to 2 nudeotides if they form RNaseH domain in combination; • indicates hydrogen bond formation between two adjacent
  • D comprises nucleotide sequence that is complementary to the nucleotide sequence of O
  • K comprises nucleotide sequence that is complementary to the nucleotide sequence of T
  • D and O form greater than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs
  • K and T form greater 0 than or equal to two base pairs with each other that are contiguous or non-contiguous, more specifically K and T form 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 base pairs
  • the K»T and O «D base-paired regions may be contiguous or non-contiguous to each other
  • O and W are of length greater than or equal to 3 nudeotides and preferably less than about 100 nudeotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20
  • F, D, W and O together are of sufficient length to stably interact with a target nucle
  • each A, D, O, and W represents independently an oligonucleotide whose nucleotide sequence may be same or different, may be of same or different length and may include phosphodiester, phosphorothioate, phosphorodithioate, 5 '-thiophosphate, methylphosphonate linkers and the like or a combination thereof; F and D
  • each B and B' independently represents a cap structure which may independently be present or absent; and represents a chemical linkage (e.g.
  • the invention features an HPH molecule of any of formulae I-III, and V-VII, where the (M) w optionally includes an RNase H-activating region.
  • RNase H-activating region or "RNase H-activating Region” is meant, a region (generally greater than or equal to 4 nudeotides long, preferably 5, 6, 7, 8, 9, 10 i o or 11 nudeotides) of a nucleic acid molecule capable of binding to a target RNA to form, for example, a (M) w »target RNA complex that is recognized by cellular RNase H enzyme, where the RNase H enzyme will then bind to the (M) w » target RNA complex and cleave the target sequence.
  • the RNase H-activating region comprises, phosphodiester, phosphorothioate (preferably four of the nudeotides are
  • the RNase H-activating region comprises deoxyribose, arabino, fluoroarabino or a combination thereof, nucleotide
  • nucleotide as used herein is as recognized in the art to include natural bases
  • Nudeotides generally comprise a base, sugar and a phosphate group.
  • the nudeotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nudeotides, non-natural nudeotides, non-standard nudeotides and other; see for example, Usman and McSwiggen, supra; Eckstein et al, International PCT Publication No. WO 92/07065; Usman et al, International PCT Publication No.
  • nucleic acid bases examples include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3 -methyl uracil, dihydrouridine, naphthyl,
  • 5-alkylcytidines e.g., 5-methylcytidine
  • 5-alkyluridines e.g., ribothymidine
  • 5-halouridine e.g., 5-bromouridine
  • 6-azapyrimidines or 6- alkylpyrimidines e.g. 6-methyluridine
  • propyne and others (Burgin et al, 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).
  • modified bases in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1'
  • bases may be used at any position, for example, within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate- binding regions of the nucleic acid molecule.
  • ribonucleotide is meant a nucleotide with one of the bases adenine, cytosine, guanine, or uracil joined to the 1' carbon of ⁇ -D-ribo-furanose.
  • unmodified nucleotide is meant a nucleotide with one of the bases adenine, cytosine, guanine, thymine, uracil joined to the 1' carbon of ⁇ -D-ribo-furanose.
  • modified nucleotide is meant a nucleotide that contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate.
  • abasic nucleic acid sugar moieties lacking a base or having other 25 chemical groups in place of base at the 1' position.
  • sufficient length is generally meant an oligonucleotide of greater than or equal to 4 nudeotides, or an equivalent chemical moiety able to bind and interact with a target nucleic acid molecule in solution and/or in a cell under physiological condition ' s " .
  • nucleic acid can form hydrogen bond(s) 5 with another RNA sequence by either traditional Watson-Crick or other non-traditional types.
  • binding free energy for a nucleic acid molecule with its target or complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., ribozyme cleavage, antisense or triple helix inhibition. Determination of binding free energies for
  • I O nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785.
  • a percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a i 5 second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).
  • Perfectly complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • stably interact is meant an interaction of the oligonucleotides with target 20 nucleic acid (e.g., by forming hydrogen bonds with complementary nudeotides in the target under physiological conditions).
  • the term shall also mean the interaction of HPH molecules with the target molecule for a duration, under physiological conditions, in solution or in a cell, sufficient for the HPH molecule to interfere with the function of the target nucleic acid molecule.
  • antisense molecules will be complementary to a target sequence along a single contiguous sequence of the antisense molecule.
  • an antisense molecule may bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule may bind such that the antisense molecule forms a loop.
  • the antisense molecule may be complementary to two ( ⁇ even more) non-contiguous substrate sequences or two (or even more) non-contiguous 5 sequence portions of an antisense molecule may be complementary to a target sequence or both.
  • nucleic acid molecule as used herein is meant a molecule comprising nudeotides.
  • the nucleic acid can be composed of modified or unmodified nudeotides or non-nucleotides or various mixtures and combinations thereof.
  • inhibitor it is meant that the activity of target genes or level of mRNAs or equivalent RNAs encoding target genes is reduced below that observed in the absence of the nucleic acid molecule of the invention. In one embodiment, inhibition with HPH molecules preferably is below that level observed in the presence of an mismatched nucleic acid molecule that is not able to stably bind to the same site on the mRNA.
  • inhibition with HPH nucleic acid molecules is preferably greater than that observed in the presence of for example, an oligonucleotide with scrambled sequence or with mismatches.
  • inhibition of target genes with the nucleic acid molecule of the instant invention is greater than in the presence of the nucleic acid molecule than in its absence.
  • inhibitor is also meant, an impediment to ⁇ normal function of a macromolecule caused by the introduction a foreign substance, such as the HPH molecule.
  • target sequence or “target nucleic acid molecule” is meant, a gene or partial sequence thereof, and those elements necessary for its expression, regulation, or its transcription or replication product or intermediates or portions thereof, including
  • Non-limiting examples of target sequence include c-raf mRNA, hepatitis C RNA, vascular endothelial growth factor receptor (e.g., fit- and KDR), ras RNA, and the like.
  • RNA By “gene” it is meant a nucleic acid that encodes an RNA.
  • antisense is meant a nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al, 5 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review see Stein and Cheng, 1993 Science 261, 1004).
  • RNA-RNA or RNA-DNA or RNA-PNA protein nucleic acid
  • RNA to target genes is meant to include those naturally occurring RNA molecules having homology (partial or complete) to genes or encoding for proteins with similar function as genes in various animals, including human, rodent, i o primate, rabbit and pig.
  • the equivalent RNA sequence also includes in addition to the coding region, regions such as 5 '-untranslated region, 3 '-untranslated region, introns, intron-exon junction and the like.
  • cap structure is meant chemical modifications which have been incorporated at the terminus of the oligonucleotide (e.g., B and B' in formulae above). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and may help in delivery and/or localization within a cell.
  • (M) w , (Y) r , (Y) f , D, K, T, W and/or E independently include modifications selected from a group comprising 2'-Oalkyl (e.g. 2'-O-allyl; Sproat et al, supra) sometimes referred to as RNA modifications; 2'-O-alkylthioalkyl (e.g. 2'-0-methylthiomethyl; Karpeisky et al, 1998, Nucleosides & Nudeotides 16, 955-958); L-nucleotides
  • B' is selected from a group comprising inverted abasic residue,. 4',5'-methylene nucleotide; l-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L- nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; t ⁇ reo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4-
  • the terminal nudeotides are phosphorothiote substitutions); phosphorodithioate; or bridging or non-bridging methylphosphonate moiety (for more details see Beigelman et al, International PCT publication No. WO 97/26270, incorporated by reference herein).
  • B is selected from a group comprising,
  • alkyl refers to a saturated aliphatic hydrocarbon, including straight- chain, branched-chain, and cyclic alkyl groups.
  • the alkyl group has 1 to 12 carbons. More preferably it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons.
  • the alkyl group may be substituted or unsubstituted. When substituted
  • the term also includes alkenyl groups which are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups.
  • the alkenyl group has 1 to 12 carbons. More preferably it is a lower alkenyl of from 1 to 7 carbons, more
  • alkenyl group may be substituted or unsubstituted.
  • alkyl also includes alkynyl groups which have an unsaturated hydrocarbon group containing at least one carbon- carbon triple bond, including straight-chain, branched-chain, and cyclic groups.
  • the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons.
  • alkyl groups may also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups.
  • An "aryl” group refers to an aromatic group which has at least one ring having a conjugated p electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted.
  • the preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups.
  • alkylaryl refers to an alkyl group (as described above) covalently joined to an aryl group (as described above.
  • Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted.
  • Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms.
  • Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted.
  • An "amide” refers to an -C(O)-NH-R, where R is either alkyl, aryl, alkylaryl or hydrogen.
  • An “ester” refers to an -C(O)-OR', where R is either alkyl, aryl, alkylaryl or hydrogen.
  • the HPH molecules including the molecules described in formulae I-XXV are capable of binding to the target nucleic acid molecules in a sequence-specific manner.
  • the stable interaction between the HPH molecule and the target molecules interferes with the normal function of the target molecule.
  • Such interaction may cause inhibition of the function of the target molecule, such as transcription, translation, and replication.
  • the HPH molecules of the invention interact and interfere with the target molecule in vitro or in vivo in a bacterial cell, microbial system, plant system, or mammalian system to modulate the function of the target molecule in such biological systems.
  • the HPH molecules of the instant invention are used to inhibit target-gene expression in a biological system, more specifically in a cell, tissue, organ, and animal.
  • the HPH nucleic acid molecules including the molecules of formulae I-III and V-XXV comprise at least one phosphate backbone modification, where such a modification is phosphorothioate (preferably four of the 5 nudeotides have phosphorothiote substitutions; more specifically, either 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 19, 21, 23 or 25 of the nudeotides have phosphorothiote substitutions), phosphorodithioate, 5 '-thiophosphate, or methylphosphonate backbone chemistry or a combination thereof.
  • phosphorothioate preferably four of the 5 nudeotides have phosphorothiote substitutions; more specifically, either 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 19, 21, 23 or 25 of the nudeotides have phosphorothiote substitutions
  • phosphorodithioate phosphorodithioate
  • 5 '-thiophosphate or methylphosphonate backbone chemistry or a combination thereof.
  • the HPH nucleic acid molecules including the i o molecules of formulae I-XXV are added directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells.
  • the nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, infusion pump or stent, with or without their inco ⁇ oration in biopolymers.
  • the HPH nucleic acid molecules described in formulae IV are expressed from transcription units inserted into DNA or RNA vectors.
  • the recombinant vectors are preferably DNA plasmids or viral vectors.
  • HPH molecule expressing viral vectors could be constructed based on, but not limited to, adeno- associated virus, retrovirus, adenovirus, or alphavirus.
  • the recombinant ⁇ vectors capable of expressing the HPH molecules are delivered as described above, and persist in target cells.
  • viral vectors may be used that provide for transient expression of HPH nucleic acid molecules. Such vectors might be repeatedly administered as necessary. Once expressed, the nucleic acid molecules bind to target mRNA.
  • nucleic acid molecules expressing vectors could be systemic, such 5 as by intravenous or intramuscular administration, by administration to target cells ex- planted from the patient followed by reintroduction into the patient, or by any other means that would allow for introduction into the desired target cell (for a review see Couture and Stinchcomb, 1996, TIG., 12, 510).
  • nucleic acid molecules that bind target molecules and inhibit cell proliferation are expressed from transcription units inserted into DNA, RNA, or viral vectors.
  • the recombinant vectors capable of expressing the HPH molecules are locally delivered as described above, and transiently persist in smooth muscle cells.
  • other mammalian cell vectors that direct the expression of RNA may be used for this purpose.
  • phenotype is meant, the entire physical, biochemical, and physiological i o makeup of an organism as determined both genetically or environmentally and any one or any group of such traits.
  • the 5' and/or 3' portions of the hairpin region of the HPH molecule is independently complementary to the target sequence.
  • N and/or N' portion of the (N»N') 0 in formulae I- VII is independently complementary to 1 5 the target sequence.
  • the 5' and/or 3' portions of the hairpin region of the HPH molecule is independently complementary to the target sequence.
  • N and/or N' portion or the (F»F') h in formulae I- VII is independently complementary to the target sequence.
  • the invention features a method of modulating the function of a target sequence including the steps of contacting the target sequence with the HPH molecules, including the molecules of formulae I-XXV, under conditions suitable for the modulation of the function of the target sequence.
  • modulation can take place in vitro or in vivo, in microbial, plant, or mammalian systems where the 5 modulation of function may include inhibition of gene expression, modification of cellular function, change in the organism's phenotype, inhibition of replication of a virus and/or viral RNA, inhibition of motility, migration of a cell and others.
  • patient is meant an organism that is a donor or recipient of explanted cells or the cells themselves.
  • Patient also refers to an organism to which enzymatic nucleic 5 acid molecules can be administered.
  • a patient is a mammal or mammalian cells. More preferably, a patient is a human or human cells.
  • vectors any nucleic acid- and/or viral-based technique used to deliver a desired nucleic acid.
  • the nucleic acid molecule of the present invention is i o administered individually or in combination or in conjunction with other drugs, and can be used to treat diseases or conditions.
  • the patient may be treated, or other appropriate cells may be treated, as is evident to those skilled in the art.
  • Figure 1 is a schematic representation of the binding of the hairpin hybridizer (HPH) molecule to a target RNA.
  • HPH hairpin hybridizer
  • both the 5' and 3' sequences of the hairpin region may be non-complementary to the target sequence.
  • either the 5' or 3' sequence may be complementary to the target RNA molecule i o independently.
  • Figure 2A displays the hairpin structure of the unbound HPH nucleic acid molecule including a 4 base pair stem and an internal 9-nucleotide DNA sequence. The figure further displays the structure of the nucleic acid molecule before and after binding to RNA. This molecule's 5' and 3' sequences form the hairpin structure but do
  • Figure 2B displays the hairpin structure of the unbound nucleic acid molecule also including a 4-base-pair stem and an internal 9- nucleotide DNA sequence. This molecule's 5' and 3' sequence forms the hairpin structure. In certain embodiments, the 5' and/or 3' sequence is capable of binding to the target RNA molecule independently.
  • Figure 3 displays non-limiting structures of the HPH molecules that are within the scope of the present invention.
  • (1) represents a circular nucleic acid molecule with an internal base-paired hairpin stem structure, each loop within the molecule comprises an RNase H-activating Region and a Non-RNase H-activating Region and is capable of binding to a Target Sequence; (2) represents a molecule
  • (6) represents a discontinuous circular nucleic acid molecule comprising an RNase H- activating Region and a Non-RNase H-activating Region capable of interacting with the Target Sequence and an internal hai ⁇ in stem structure, each loop at the 3' and the 5' ends of the hai ⁇ in region is independently capable of binding to a target sequence.
  • (1) represents a HPH nucleic acid molecule structure, where the RNase H- activating Region is at the 5' end of the molecule and a portion of the RNase H- activating Region forms a hai ⁇ in stem structure with a portion of the 3' region of the HPH molecule. Both the RNase H-activating Region and the Non-RNase H-activating Regions are independently or in combination are capable of interacting with the Target
  • Both the RNase H-activating Region and the Non-RNase H-activating Regions are independently or in combination are capable of interacting with the Target sequence in a sequence-specific manner; (3) and (6) represent a HPH nucleic acid molecule structure, where the a portion of the RNase H-activating Region and a portion of the Non-RNase H-activating region forms a hai ⁇ in stem structure with a portion of 5 the Non-RNase H-activating region located in a different part of the HPH molecule.
  • Both the RNase H-activating Region and the Non-RNase H-activating Regions are independently or in combination are capable of interacting with the Target sequence in a sequence-specific manner;
  • (4) represents a HPH nucleic acid molecule structure, where the RNase H-activating Region is at the 3' end of the molecule and a portion of i o the RNase H-activating Region forms a hai ⁇ in stem structure with a portion of the 5' region of the HPH molecule.
  • Both the RNase H-activating Region and the Non-RNase H-activating Regions are independently or in combination are capable of interacting with the Target sequence in a sequence-specific manner.
  • (1) and (3) represents a HPH nucleic acid molecule structure, where a portion of the RNase H-
  • 1 5 activating Region forms a hai ⁇ in stem structure with a portion of the non-RNaseH- activating region of the HPH molecule.
  • Both the RNase H-activating Region and the Non-RNase H-activating Regions are independently or in combination are capable of interacting with the Target sequence in a sequence-specific manner;
  • (2) and (4) represents a HPH nucleic acid molecule structure, where the a portion of the RNase H- ⁇ activating Region and a portion of the Non-RNase H-activating region form a hai ⁇ in stem structure with a portion of the Non-RNase H-activating region located in a different part of the HPH molecule.
  • Both the RNase H-activating Region and the Non- RNase H-activating Regions are independently or in combination are capable of interacting with the Target sequence in a sequence-specific manner.
  • Figure 4 displays a graph demonstrating the effect of a 31 mer HPH nucleic acid molecule of the present invention on reducing c-raf mRNA levels in PC-3 cells compared to untreated and mismatch controls. The cells were treated with nucleic acid molecules for 1, 3, or 5 days and then harvested to quantify the c-raf RNA.
  • Figure 5 displays a graph demonstrating the effect of a 33mer nucleic acid molecule of the present invention on reducing c-raf mRNA levels in PC-3 cells compared to untreated and mismatch controls.
  • the cells were treated with the HPH nucleic acid molecules for 1, 3, or 5 days and then harvested to quantify the c-raf RNA.
  • 5 Figure 6 displays a graph demonstrating the effect of a 35mer HPH nucleic acid molecule of the present invention on reducing c-raf mRNA levels in PC-3 cells compared to untreated and mismatch controls.
  • the cells were treated with nucleic acid molecules for 1, 3, or 5 days and then harvested to quantify the c-raf RNA.
  • Figure 7 displays a graph demonstrating the effect of a 31 mer HPH nucleic acid
  • I O molecule of the present invention on reducing c-raf mRNA levels in PC-3 cells compared to a mismatch control.
  • Figure 8 displays a graph demonstrating the effect of a 31 mer HPH linear antisense molecule on reducing c-raf mRNA levels in PC-3 cells compared to a mismatch control.
  • Figure 9 displays the HPH nucleic acid molecule-based specific inhibition of c-raf
  • RNA levels in PC-3 cells and the effect of 1, 2 and 4 base mismatches on this inhibition.
  • Figure 10 displays several non-limiting examples of psuedoknot hai ⁇ in hybridizer molecules.
  • Figure 10A is a psuedoknot hai ⁇ in hybridizer molecule ⁇ comprised of 2 hai ⁇ in structures, and a target binding sequence located in closer proximity to the 5' end of the nucleic acid molecule compared to the 3' end.
  • Figure 10B is a psuedoknot hai ⁇ in hybridizer molecule comprised of 2 hai ⁇ in structures, and a target binding sequence located in closer proximity to the 3' end of the nucleic acid molecule compared to the 5' end.
  • Figure IOC is a psuedoknot hai ⁇ in hybridizer
  • Figure 10D is a psuedoknot hai ⁇ in hybridizer molecule comprised of 2 hai ⁇ in structures, a target binding sequence located in closer proximity to the 3' end of the nucleic acid molecule compared to the 5' end, and additional nucleotide sequences attached at the 5' and 3' ends of the hai ⁇ in hybridizer molecule. These additional sequences may be of equal or unequal length.
  • Figure 10E is a psuedoknot hai ⁇ in hybridizer molecule comprised of 2 hai ⁇ in structures, 2 target binding sequences, and additional nucleotide sequences attached at the 5' and 3' ends of the hai ⁇ in hybridizer molecule.
  • Figure 1 OF is a psuedoknot hai ⁇ in hybridizer molecule comprised of 2 hai ⁇ in structures, a target binding sequence located in closer proximity to the 5' end of the nucleic acid molecule compared to the 3' end, and an additional nucleotide sequence attached at the 5' of the hai ⁇ in hybridizer molecule.
  • Figure 10G is a psuedoknot hai ⁇ in hybridizer molecule i o comprised of 2 hai ⁇ in structures, a target binding sequence located in closer proximity to the 5' end of the nucleic acid molecule compared to the 3' end, and additional nucleotide sequences attached at the 5' and 3' ends of the hai ⁇ in hybridizer molecule. These additional sequences may be of equal or unequal length.
  • Figure 11 displays a graph demonstrating the effect of HPH nucleic acid molecule
  • Figure 12 displays a graph demonstrating the effect of a HPH nucleic acid molecule of the varying sizes and configurations of the present invention on reducing IMPDH II mRNA levels in PC-3 cells compared to untreated and mismatch controls.
  • Figure 13 displays a graph demonstrating the effect of a HPH nucleic acid molecule of the varying sizes and configurations of the present invention on reducing IMPDH II mRNA levels in PC-3 at an oligonucleotide concentration of 100 nM.
  • Antisense molecules known in the art are usually RNA or DNA oligonucleotides and primarily function by specifically binding to complementary (matching) sequences resulting in inhibition of peptide synthesis (Wu-Pong, Nov 1994, BioPharm, 20-33).
  • the oligonucleotide binds to target RNA by Watson Crick base-pairing and blocks gene expression by preventing ribosomal translation of the bound sequences by either steric blocking or RNase H-mediated degradation of target RNA.
  • Antisense molecules may also alter protein synthesis by interfering with RNA processing or transport from 5 the nucleus into the cytoplasm (Mukhopadhyay & Roth, 1996, Crit. Rev. in Oncogenesis 7, 151-190).
  • antisense molecules have been described that utilize novel configurations of chemically modified nudeotides, secondary structure, and/or RNase H substrate domains (Woolf et al, International PCT Publication No. WO 98/13526;
  • the antisense molecules described in the art are essentially single-stranded linear oligonucleotides which are known to tolerate a number of mismatches and still form stable hybrids with a target sequence raising the concern of safety and toxicity in ⁇ organisms. While these molecules are functional, for certain applications, including pharmaceutical compositions, greater specificity, lower toxicity and higher stability is desirable.
  • oligonucleotides described above may be increased by using the HPH nucleic acid molecule of the present invention which form internal hai ⁇ in
  • oligonucleotides such as molecular beacons which have a 10 base pair or more internal hai ⁇ in stems are capable of binding to a target sequence in a highly sequence specific manner in solution.
  • the specific interaction of a hai ⁇ in D ⁇ A with target R ⁇ A was also demonstrated in cells (Kostrikis. et al, 1998 , Science 279, 1228-1229) where the hai ⁇ in D ⁇ A was used to i o detect the presence of bFGF R ⁇ A, these oligonucleotides however were not used to inhibit gene expression.
  • the intramolecular bonding of the hai ⁇ in hybridizer molecules can result in increased stability.
  • Hai ⁇ in sequences located at the respective ends of the oligonucleotide may increase the stability of these
  • HPH molecules may be enhanced by the addition of nudeotides which act as substrates for R ⁇ ase H within the molecule.
  • binding of D ⁇ A to R ⁇ A is not as thermodynamically favorable as an R ⁇ A to R ⁇ A interaction (Altmann et al., 1996, Chimia 50, 168-176). Therefore a molecule with both
  • R ⁇ A and D ⁇ A nudeotides may be able to bind efficiently as well as promote degradation of the R ⁇ A molecule by R ⁇ ase H.
  • the region of oligodeoxynucleotides in such a chimeric molecule is recognized by RNase H when bound to target RNA; and facilitates cleavage of target RNA by RNase H. (Inoe & Ohtsuka, 1987, EERS Lett.
  • nucleic Acids Res. 15, 4403 were proposed to interact with target 5 RNA more stably than an all DNA oligonucleotide.
  • the nucleic acid molecule may function by binding to the target molecule that results in steric hindrance for ribosomal translation.
  • a number of chemical modifications may be utilized with this strategy including insertion of 2'-0-methyl modification at every nucleotide in the molecule.
  • i o One of the most studied and utilized chemical alterations in oligonucleotides has been backbone modifications such as phosphorothioates, phosphorodithioates, and 5'thiophosphates.
  • Phosphorothioate oligonucleotides are nucleic acid molecules whose phosphodiester linkage has been modified by substituting a sulfur atom in place of an oxygen atom.
  • phosphorothioate, i s phosphorodithioate, and 5'thiophosphates oligonucleotides are substrates for ribonuclease H (RNase H) (Monia, supra; Crooke et al., 1995, Biochem. J. 3112, 599- 608).
  • RNase H is an endonuclease which catalyzes the degradation of RNA in an RNA-DNA heteroduplex (Hostomsky et al., 1993 in Nucleases, Linn et al., eds., Cold Spring Harbor Laboratory Press, NY, 341-376).
  • RNA/DNA heteroduplexes called 0 Okazaki fragments, are formed naturally during DNA replication. Therefore, the normal function of RNase H is to degrade the RNA portion of the heteroduplex to complete DNA replication.
  • the phosphorothioate oligonucleotide activated the enzyme more efficiently (2-5 fold) compared to a standard phosphodiester containing oligonucleotide (Crooke, 1995, supra).
  • nucleic acids greater than 100 nudeotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive.
  • small nucleic acid motifs (“small refers to nucleic acid motifs no more than 5 100 nudeotides in length, preferably no more than 80 nudeotides in length, and most preferably no more than 40 nudeotides in length; e.g., HPH nucleic acid molecules) are used for exogenous delivery.
  • the simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of RNA structure.
  • oligodeoxyribonucleotides molecules of the instant invention were chemically i o synthesized using standard protocols as described in Caruthers et al., 1992, Methods in Enzymology 211,3-19, which is inco ⁇ orated herein by reference.
  • synthesizer included detritylation solution with 3% TCA n methylene chloride (ABI); capping was performed with 16% N-methyl imidazole in 5 THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution was 16.9 mM I 2 , 49 mM pyridine, 9% water in THF (PERSEPTIVETM). Burdick & Jackson Synthesis Grade acetonitrile was used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) was made up from the solid obtained from American International Chemical, Inc.
  • i o Deprotection of the the oligonucleotides of the instant invention was performed using either a two-pot or one-pot protocol.
  • the polymer- bound trityl-on oligoribonucleotide was transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aq. methylamine (1 mL) at 65 °C for 10 min. After cooling to -20 °C, the supernatant was removed from the polymer support. The i 5 support was washed three times with 1.0 mL of EtOH:MeC ⁇ :H2O/3 : 1 : 1 , vortexed and the supernatant was then added to the first supernatant.
  • the polymer-bound trityl-on oligoribonucleotide was transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO:l/l (0.8 mL) at 65 °C for 15 min.
  • the 5 vial was brought to room temperature.
  • TEA 3HF (0.1 mL) was added and the vial was heated at 65 °C for 15 min.
  • the sample was cooled at -20 °C and then quenched with 1.5 M NH 4 HCO 3 .
  • oligonucleotides of the instant inventions can be purified by other methods known in the art, for example, see Sproat et al, 1999, Nucleic Acids Res., 27, 1950).
  • nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example by ligation (Moore et al, 1992, Science 256, 9923; Draper et al, International PCT publication No. WO 93/23569; Shabarova et al, 1991, Nucleic Acids Research 19, 4247; Bellon et al, 1997, Nucleosides & Nudeotides, 16, 951; Bellon et al, 1997 Bioconjugate Chem. 8, 204).
  • RNA molecules may be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by inco ⁇ oration into other vehicles, such as hydrogeis, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.
  • nucleic acid molecules may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles.
  • the nucleic acid/vehicle combination is locally delivered by direct injection or by use of a catheter, infusion pump or stent.
  • routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, i o intraperitoneal and/or intrathecal delivery. More detailed descriptions of nucleic-acid delivery and administration are provided in Sullivan et al, supra and Draper et al, PCT WO93/23569 which are inco ⁇ orated by reference herein.
  • the molecules of the instant invention can be used as pharmaceutical agents.
  • Pharmaceutical agents prevent, inhibit the occurrence, or treat (alleviate a symptom to 15 some extent, preferably all of the symptoms) of a disease state in a patient.
  • the negatively charged polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced into a patient by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition.
  • RNA, DNA or protein e.g., RNA, DNA or protein
  • standard protocols for ⁇ formation of liposomes can be followed.
  • the compositions of the present invention may also be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions; suspensions for injectable administration; and the like.
  • the present invention also includes pharmaceutically acceptable formulations of 25 the compounds described.
  • formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.
  • a pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a 5 cell or patient, preferably a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation to reach a target cell (i.e., a cell to which the negatively charged polymer is desired to be delivered to). For example, pharmacological compositions injected into the blood stream should be soluble. Other i o factors are known in the art, and include considerations such as toxicity and forms which prevent the composition or formulation from exerting its effect.
  • systemic administration in vivo systemic abso ⁇ tion or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Administration routes which lead to systemic abso ⁇ tion include, without
  • 2 ⁇ instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES).
  • tissue types such as the tissues of the reticular endothelial system (RES).
  • RES reticular endothelial system
  • a liposome formulation which can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach may provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage
  • the invention also features the use of the composition comprising surface- modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long- circulating liposomes or stealth liposomes).
  • PEG-modified, or long- circulating liposomes or stealth liposomes These formulations offer an method for- increasing the accumulation of drugs in target tissues.
  • This class of drug carriers resists 5 opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011).
  • Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized-
  • the long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al, J. Biol Chem. 1995, 42, 24864-24870; Choi et al, International PCT i s Publication No. WO 96/10391; Ansell et al, International PCT Publication No. WO 96/10390; Holland et al, International PCT Publication No.
  • Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive o MPS tissues such as the liver and spleen.
  • compositions prepared for storage or administration which include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, 25 for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985) hereby inco ⁇ orated by reference herein.
  • preservatives, stabilizers, dyes and flavoring agents may be provided. These include sodium benzoate, sorbic acid and esters of /j-hydroxybenzoic acid.
  • antioxidants and suspending agents may be used.
  • a pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the 5 symptoms) of a disease state.
  • the pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors which those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day i o of active ingredients is administered dependent upon potency of the negatively charged polymer.
  • the nucleic acid molecules of the present invention may also be administered to a patient in combination with other therapeutic compounds to increase the overall therapeutic effect.
  • the use of multiple compounds to treat an indication may increase 1 5 the beneficial effects while reducing the presence of side effects.
  • nucleic acid molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and
  • nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector.
  • the activity of such nucleic acids can be augmented by their release from the primary transcript by a ribozyme (Draper et al, PCT WO 93/23569, and Sullivan et al, PCT WO 94/02595; Ohkawa et al, 1992 Nucleic Acids Symp.
  • RNA molecules of the present invention are preferably expressed from transcription units (see for example Couture et al, 1996, TIG., 12, 510) inserted into DNA or RNA vectors.
  • the recombinant vectors are i o preferably DNA plasmids or viral vectors. Ribozyme expressing viral vectors could be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus.
  • the recombinant vectors capable of expressing the nucleic acid molecules are delivered as described above, and persist in target cells.
  • viral vectors may be used that provide for transient expression of nucleic
  • nucleic acid molecule expressing vectors could be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that would allow for
  • an expression vector comprising nucleic acid sequence encoding at least one of the nucleic acid molecules of the instant invention is disclosed.
  • the nucleic acid sequence encoding the nucleic acid molecule is disclosed.
  • an expression vector comprising: a transcription-initiation region (e.g., eukaryotic pol I, II or III initiation region); b) a transcription-termination region (e.g., eukaryotic pol I, II or III termination region); c) a ⁇ gene encoding at least one of the nucleic-acid catalysts of the instant invention; and 5 wherein said gene is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
  • the vector may optionally include an open reading frame (ORF) for a protein operably linked on the 5' side or the 3'-side of the gene encoding the nucleic- acid catalyst of the invention; and/or an intron (intervening sequences).
  • RNA polymerase I RNA polymerase I
  • polymerase II RNA polymerase II
  • poly III RNA polymerase III
  • Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) i 5 present nearby.
  • Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy- Stein and Moss, 1990 Proc. Natl. Acad. Sci.
  • nucleic acid molecules such as ribozymes expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992 Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992 Proc. Natl. Acad. Sci. U S A, 89, 10802-6; Chen et al, 1992 Nucleic Acids Res., 20, 4581-9; Yu et al, 1993 Proc. Natl. Acad. Sci. US A, 90, 6340-4; L'Huillier et al, 1992 EMBO J. 11, 4411-8; Lisziewicz et al,
  • transcription units such as the ones derived from genes encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful in generating high concentrations of desired RNA molecules such as ribozymes in cells (Thompson et al, supra; Couture and Stinchcomb, 1996, supra; Noonberg et al, 1994, Nucleic Acid Res ⁇ 22, 2830; Noonberg et al, US Patent No.
  • ribozyme-transcription units can be inco ⁇ orated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or viral RNA vectors (such as retroviral i o or alphavirus vectors) (for a review see Couture and Stinchcomb, 1996, supra).
  • plasmid DNA vectors such as adenovirus or adeno-associated virus vectors
  • viral RNA vectors such as retroviral i o or alphavirus vectors
  • the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the nucleic acid molecules of the invention, in a manner which allows expression of that nucleic acid molecule.
  • the expression vector comprises in one embodiment; a) a transcription-initiation region; b)
  • the expression vector comprises: a) a transcription initiation region; b) a transcription-termination region; c) an open reading o frame; d) a gene encoding at least one said nucleic acid molecule, wherein said gene is operably linked to the 3'-end of said open reading frame; and wherein said gene is operably linked to said initiation region, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
  • the expression vector comprises: a) a transcription- initiation region; b) a transcription-termination region; c) an intron; d) a gene encoding at least one said nucleic acid molecule; and wherein said gene is operably linked to said initiation region, said intron and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
  • the expression vector comprises: a) a transcription- initiation region; b) a transcription-termination region; c) an intron; d) an open reading 5 frame; e) a gene encoding at least one said nucleic acid molecule, wherein said gene is operably linked to the 3'-end of said open reading frame; and wherein said gene is operably linked to said initiation region, said intron, said open reading frame and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
  • oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O-methyl, 2'- H, nucleotide base modifications (for a review see Usman and Cedergren, 1992 TIBS 17, 34; Usman et al, 1994 Nucleic Acids Symp. Ser. 31, 163; Burgin et al, 1996 Biochemistry 35, 14090). Sugar modifications of nucleic acid molecules have been 5 extensively described in the art (see Eckstein et al., International Publication PCT No.
  • Nucleic acid molecules having chemical modifications which maintain or enhance activity are disclosed herein. Such nucleic acid is also generally more resistant to nucleases than unmodified nucleic acid. Thus, in a cell and/or in vivo the activity may not be significantly lowered. Therapeutic HPH molecules delivered exogenously must optimally be stable within cells until translation of the target RNA has beerr inhibited long enough to reduce the levels of the undesirable protein. This period of 5 time varies between hours to days depending upon the disease state. Clearly, nucleic acid molecules must be resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of RNA and DNA (Wincott et al, 1995 Nucleic Acids Res.
  • HPH molecules will lead to better treatment of disease progression by affording the possibility of combination therapies (e.g., multiple HPH molecules targeted to different genes, HPH molecules coupled with known small-molecule 1 5 inhibitors, or intermittent treatment with combinations of HPH molecules (including different HPH motifs) and/or other chemical or biological molecules)).
  • combination therapies e.g., multiple HPH molecules targeted to different genes, HPH molecules coupled with known small-molecule 1 5 inhibitors, or intermittent treatment with combinations of HPH molecules (including different HPH motifs) and/or other chemical or biological molecules).
  • the treatment of patients with nucleic acid molecules may also include combinations of different types of nucleic acid molecules.
  • the nucleic acid molecules of the present invention can inhibit gene expression i o in a highly specific manner by binding to and causing the cleavage of the mRNA corresponding to the gene of interest, and thereby prevent production of the gene product (Christoffersen, Nature Biotech, 1997, 2, 483-484).
  • Appropriate delivery vehicles can be combined with these nucleic acid molecules (including polymers, cationic lipids, liposomes and the like) and delivered to appropriate cell culture or in i 5 vivo animal disease models as described above.
  • the process may be both fast and highly selective, and allow for the process to be used at any point in the development of the organism.
  • Example 1 Identification of Potential Binding Sites for the HPH Molecule in the Target Sequence
  • RNAs were screened for accessible sites using a computer-folding algorithm. Regions of the mRNA that did not form secondary
  • HPH oligonucleotides targeting exon 11 of the human c-raf gene with 4-6 i o complementary nudeotides at the 5' and the 3' end were synthesized using standard protocols (Wincott et al, supra). These 5' and 3' sequences were not complementary to the c-raf target. Of the 23 nudeotides complementary to the target sequence, 11 nudeotides in the DNA core and RNA arms were exchanged to generate a control molecule which lacks the capability to down-regulate c-raf mRNA in a sequence-
  • Prostate cancer cells were grown in a growth media consisting of Kaighn's F-12K media, 10% FBS, 1% glutamine, 20 mM HEPES, and 1% pen/strep to sub-confluent densities.
  • a 4X ⁇ concentration (10 ⁇ g/mL) of GSV was prepared from a 2 mg/mL stock solution as well as a lO ⁇ M solution of the nucleic acid molecule of the present invention and its antisense control.
  • Complexes of antisense and GSV were formed in a 96-well plate by channel pipetting in antisense and GSV to form complex solutions which are twice the final concentrations.
  • RNA 15 purified using Qiagen' s instructions and RNA was quantified using TaqManTM reagents and the 7700 Prism (Perkin Elmer) using the manufacturer's protocol. The ratio of c-raf mRNA over ⁇ -actin mRNA was determined by real-time PCR after reverse transcription.
  • Example 3 Comparison of c-raf inhibition between the Hai ⁇ in Hybridizer Molecule and a Linear Antisense Molecule.
  • hai ⁇ in and linear antisense molecules were synthesized (Wincott et al., supra).
  • PC-3 cells were treated for 1,3 or 5 days with lipid-complexed hai ⁇ in oligonucleotides or a lipid complexed linear antisense molecule.
  • the hai ⁇ in molecule (Seq. I.D. No. 5) was 31 nudeotides in length and the results of c-raf inhibition were compared to a mismatch control (Seq. I.D. No. 6).
  • the potency of the antisense molecule was also compared to its mismatch control. After each designated time period, PC-3 cells were harvested with 150 ⁇ L of RLT lysis buffer (Qiagen). RNA was purified using Qiagen's instructions and RNA ⁇ was quantified using TaqManTM reagents and the 7700 Prism (Perkin Elmer) using the 5 manufacturer's protocol. The ratio of c-raf mRNA over ⁇ -actin mRNA was determined by real-time PCR after reverse transcription. The data is given in figures 7 and 8. The HPH molecules significantly reduce the c-raf RNA level while the mismatch molecules did not cause any significant reduction (figure 6, 7).
  • nucleic acid molecules of the present invention were designed to bind to c-
  • Example 5 Inhibition of IMDPH II RNA Expression With Hai ⁇ in Hybridizing Molecules of Varying Lengths.
  • PC3 Prostate cancer cells
  • Nucleic acids were complexed and applied to cells as described, with the exception that a cationic lipid was 5 used.
  • the final oligonucleotide concentration was 100 nM.
  • RNA levels were measured by TaqManTM analysis as described above.
  • oligonucleotides targeted to IMPDH II had, in one case, a 23mer target-hybridizing i o region plus a 6 base hai ⁇ in hybridizing region at the 3' end that annealed to the 5' end (Seq. ID No.l l, and 2 base mismatch control Seq. ID No. 12).
  • the oligonucleotide had a 19mer target-hybridizing region plus a 4 base hai ⁇ in-hybridizing region at the 3' end that annealed to the 5' end (Seq. ID No. 13, and 2 base mismatch control Seq. ID No. 14).
  • the hai ⁇ in hybridizer molecule As shown in Fig. 12, the hai ⁇ in hybridizer molecule
  • Example 6 Alternative Hai ⁇ in Annealing Domains Confer Comparable Efficacy in Cell Culture.
  • oligonucleotide delivery method Using the cell culture and oligonucleotide delivery method described above, PC3 cells were treated for 24 hours with lipid-complexed oligonucleotides.
  • the oligonucleotides targeted c-Raf exon 11, and consisted of DNA core regions (an example of RNase H-activating Region) at or near the 5' end of the oligonucleotide, and a 3' hai ⁇ in hybridizing regions that could anneal to different regions of the target- 5 complementary region, including the DNA core and/or the RNA arms. As shown in Fig.
  • a 21mer target-hybridizing region with a 6 base hai ⁇ in that anneals to the 5' end of the oligo overlapping part of the DNA core shows greater than 80% inhibition of target RNA expression. Scrambling the 3' end to prevent formation of an intramolecular hai ⁇ in (Seq. ID No. 16) neither enhances nor interferes with the ⁇ cell efficacy in this assay, indicating that the oligonucleotide may be able to basepair to the target RNA equivalently with or without the hai ⁇ in structure.
  • An 18mer target- hybridizing (complementary) region with varying 6 nucleotide self-complementary structures (Seq. ID Nos.
  • PC3 cells were treated for 24 hours with lipid-complexed oligonucleotides.
  • the i o oligonucleotides targeted IMPDH II, and consisted of DNA core regions in the center of the oligonucleotide, and a 3' hai ⁇ in hybridizing regions that could anneal to the DNA core.
  • a linear antisense oligonucleotide with a 23mer target- hybridizing region (Seq. ID No. 22) gave 70% inhibition, while random sequence and scrambled sequence negative controls (Seq. ID No. 21, 23) gave virtually no inhibition.
  • the instant invention is not limited to the HPH molecules used in the foregoing examples.
  • the instant invention broadly features HPH oligonucleotides of varying structures, including those hybridizing to an internal RNase H-activating regions, hybridizing to both the RNase H-activating region and the Non-RNase H-activating region, and those hybridizing to a RNase H-activating
  • the hai ⁇ in structure could provide protection against exonucleolytic and/or endonucleolytic degradation, thus increasing stability both in vivo and in vitro.
  • the hai ⁇ in creates a duplex region that juxtaposes various chemical end-modifications that may confer altered in vivo pharmacokinetics or tissue distribution.
  • these ⁇ molecules may have advantages compared to traditional linear antisense molecules for use as therapeutics or as tools for in vivo target validation. Diagnostic uses
  • Nucleic acid molecules of this invention may be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of 5 specific RNAs in a cell.
  • the close relationship between antisense activity and the structure of the target RNA allows the detection of mutations in any region of the molecule which alters the base-pairing and three-dimensional structure of the target RNA.
  • nucleic acid molecules described in this invention one may map nucleotide changes which are important to RNA structure and function in vitro, as i o well as in cells and tissues.
  • Inhibition of target RNAs with nucleic acid molecules may be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of disease. In this manner, other genetic targets may be defined as important mediators of the disease.
  • Wait time does not include contact time during delivery.
  • Table IV HPH Molecules targeting IMPDH and c-Raf RNA

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne de nouvelles molécules hybridantes en épingle à cheveux d'acides nucléiques capables de moduler l'expression génique; elles peuvent servir dans la validation de cibles, l'identification des fonctions de gènes et la thérapie de l'humain.
PCT/US1999/021865 1998-09-21 1999-09-20 Molecules hybridantes en epingle a cheveux servant a moduler l'expression genique WO2000017346A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CA002343067A CA2343067A1 (fr) 1998-09-21 1999-09-20 Molecules hybridantes en epingle a cheveux servant a moduler l'expression genique
JP2000574246A JP2002526072A (ja) 1998-09-21 1999-09-20 遺伝子発現の調節のためのヘアピンハイブリダイザー分子
AU60552/99A AU6055299A (en) 1998-09-21 1999-09-20 Hairpin hybridizer molecules for modulation of gene expression
EP99969438A EP1115859A2 (fr) 1998-09-21 1999-09-20 Molecules hybridantes en epingle a cheveux servant a moduler l'expression genique

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US10117498P 1998-09-21 1998-09-21
US60/101,174 1998-09-21

Publications (2)

Publication Number Publication Date
WO2000017346A2 true WO2000017346A2 (fr) 2000-03-30
WO2000017346A3 WO2000017346A3 (fr) 2000-08-03

Family

ID=22283374

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/021865 WO2000017346A2 (fr) 1998-09-21 1999-09-20 Molecules hybridantes en epingle a cheveux servant a moduler l'expression genique

Country Status (5)

Country Link
EP (1) EP1115859A2 (fr)
JP (1) JP2002526072A (fr)
AU (1) AU6055299A (fr)
CA (1) CA2343067A1 (fr)
WO (1) WO2000017346A2 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002006531A2 (fr) * 2000-07-14 2002-01-24 Applied Gene Technologies, Inc. Sondes d'acides nucleiques a structure en epingle a cheveux et utilisations associees
EP1838875A2 (fr) * 2004-12-30 2007-10-03 Todd M. Hauser Compositions et procedes pour la modulation de l'expression genique par oligonucleotides a autoprotection
EP2363467A1 (fr) * 2008-10-23 2011-09-07 The University of Tokyo Procédé d'inhibition de la fonction des micro-arn
US20130045520A1 (en) * 2002-02-01 2013-02-21 Life Technologies Corporation Double-stranded oligonucleotides
US9777275B2 (en) 2002-02-01 2017-10-03 Life Technologies Corporation Oligonucleotide compositions with enhanced efficiency
US11555188B2 (en) 2017-07-26 2023-01-17 Nissan Chemical Corporation Single-stranded oligonucleotide

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116064544A (zh) 2016-01-26 2023-05-05 日产化学株式会社 单链寡核苷酸
MX2019009305A (es) 2017-02-06 2019-09-19 Nissan Chemical Corp Oligonucleotido de cadena simple.

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992019732A1 (fr) * 1991-04-25 1992-11-12 Genset Oligonucleotides fermes, antisens et sens, et leurs applications
WO1999054459A2 (fr) * 1998-04-20 1999-10-28 Ribozyme Pharmaceuticals, Inc. Molecules d'acides nucleiques presentant de nouvelles compositions chimiques capables de moduler l'expression genique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992019732A1 (fr) * 1991-04-25 1992-11-12 Genset Oligonucleotides fermes, antisens et sens, et leurs applications
WO1999054459A2 (fr) * 1998-04-20 1999-10-28 Ribozyme Pharmaceuticals, Inc. Molecules d'acides nucleiques presentant de nouvelles compositions chimiques capables de moduler l'expression genique

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HOSONO K ET AL: "Properties and anti-HIV activating of hairpin antisense oligonucleotides containing 2'-methoxynucleosides with base-pairing in the stem region at the 3'-end." ANTIVIRAL CHEMISTRY & CHEMOTHERAPY, vol. 7, no. 2, 1996, pages 86-93, XP000877270 ISSN: 0956-3202 *
LI JING ET AL: "The base-paired strand of a stable hairpin DNA can template RNase H cleavage of a RNA target site. A potential stringency clamp approach to antisense strategy." FASEB JOURNAL, vol. 12, no. 5, 20 March 1998 (1998-03-20), page A775 XP002131931 ISSN: 0892-6638 & ANNUAL MEETING OF THE PROFESSIONAL RESEARCH SCIENTISTS ON EXPERIMENTAL BIOLOGY 98, PART II;SAN FRANCISCO, CALIFORNIA, USA; APRIL 18-22, 1998, *
MONIA B P ET AL: "ANTITUMOR ACTIVITY OF A PHOSPHOROTHIOATE ANTISENSE OLIGODEOXYNUCLEOTIDE TARGETED AGAINST C-RAF KINASE" NATURE MEDICINE, vol. 2, no. 6, 6 June 1996 (1996-06-06), pages 668-675, XP002073792 ISSN: 1078-8956 *
TANG J Y ET AL: "SELF-STABILIZED ANTISENSE OLIGODEOXYNUCLEOTIDE PHOSPHOROTHIOATES: PROPERTIES AND ANTI-HIV ACTIVITY" NUCLEIC ACIDS RESEARCH, vol. 21, no. 11, 1993, pages 2729-2735, XP002030620 ISSN: 0305-1048 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002006531A3 (fr) * 2000-07-14 2003-07-10 Applied Gene Technologies Inc Sondes d'acides nucleiques a structure en epingle a cheveux et utilisations associees
US6596490B2 (en) 2000-07-14 2003-07-22 Applied Gene Technologies, Inc. Nucleic acid hairpin probes and uses thereof
WO2002006531A2 (fr) * 2000-07-14 2002-01-24 Applied Gene Technologies, Inc. Sondes d'acides nucleiques a structure en epingle a cheveux et utilisations associees
US9796978B1 (en) 2002-02-01 2017-10-24 Life Technologies Corporation Oligonucleotide compositions with enhanced efficiency
US10626398B2 (en) 2002-02-01 2020-04-21 Life Technologies Corporation Oligonucleotide compositions with enhanced efficiency
US10196640B1 (en) 2002-02-01 2019-02-05 Life Technologies Corporation Oligonucleotide compositions with enhanced efficiency
US10106793B2 (en) 2002-02-01 2018-10-23 Life Technologies Corporation Double-stranded oligonucleotides
US20130045520A1 (en) * 2002-02-01 2013-02-21 Life Technologies Corporation Double-stranded oligonucleotides
US10036025B2 (en) 2002-02-01 2018-07-31 Life Technologies Corporation Oligonucleotide compositions with enhanced efficiency
US8815821B2 (en) * 2002-02-01 2014-08-26 Life Technologies Corporation Double-stranded oligonucleotides
US9777275B2 (en) 2002-02-01 2017-10-03 Life Technologies Corporation Oligonucleotide compositions with enhanced efficiency
EP1838875A4 (fr) * 2004-12-30 2010-08-25 Todd M Hauser Compositions et procedes pour la modulation de l'expression genique par oligonucleotides a autoprotection
EP1838875A2 (fr) * 2004-12-30 2007-10-03 Todd M. Hauser Compositions et procedes pour la modulation de l'expression genique par oligonucleotides a autoprotection
US8563709B2 (en) 2008-10-23 2013-10-22 The University Of Tokyo Method for inhibiting function of micro-RNA
EP2363467A4 (fr) * 2008-10-23 2013-02-20 Univ Tokyo Procédé d'inhibition de la fonction des micro-arn
EP2363467A1 (fr) * 2008-10-23 2011-09-07 The University of Tokyo Procédé d'inhibition de la fonction des micro-arn
US11555188B2 (en) 2017-07-26 2023-01-17 Nissan Chemical Corporation Single-stranded oligonucleotide

Also Published As

Publication number Publication date
AU6055299A (en) 2000-04-10
CA2343067A1 (fr) 2000-03-30
JP2002526072A (ja) 2002-08-20
WO2000017346A3 (fr) 2000-08-03
EP1115859A2 (fr) 2001-07-18

Similar Documents

Publication Publication Date Title
US6673611B2 (en) Nucleic acid molecules with novel chemical compositions capable of modulating gene expression
AU750947C (en) Nucleic acid catalysts with endonuclease activity
US6831171B2 (en) Nucleic acid catalysts with endonuclease activity
US20030064945A1 (en) Enzymatic nucleic acid treatment of diseases or conditions related to levels of epidermal growth factor receptors
US20030105051A1 (en) Nucleic acid treatment of diseases or conditions related to levels of HER2
US6797815B2 (en) Xylofuranosly-containing nucleoside phosphoramidites and polynucleotides
WO2001059103A9 (fr) Methode et reactif destines a la modulation et au diagnostic de l'expression genetique de cd20 et de nogo
WO2001057206A2 (fr) Procede et reactif destines a inhiber la checkpoint kinase-1 (chk 1)
WO1998032846A2 (fr) Traitement par acides nucleiques enzymatiques des maladies ou des etats lies aux niveaux de c-fos
WO1998032846A9 (fr) Traitement par acides nucleiques enzymatiques des maladies ou des etats lies aux niveaux de c-fos
CA2288640A1 (fr) Traitement a l'acide nucleique enzymatique de maladies et etats pathologiques associes aux niveaux d'expression de c-raf
EP1115859A2 (fr) Molecules hybridantes en epingle a cheveux servant a moduler l'expression genique
EP1257639A2 (fr) Nucleozymes a activite d'endonuclease
EP1165758A2 (fr) Regulation de genes represseur avec des molecules d'acide nucleique
EP1086212A2 (fr) Procede et reactifs pour le traitement de maladies ou d'affections associees a des molecules impliquees dans les reactions angiogeniques
US20030050259A1 (en) Method and reagent for the treatment of cardiac disease
US20030087847A1 (en) Method and reagent for the inhibition of checkpoint kinase-1 (Chk1) enzyme
US20030073207A1 (en) Enzymatic nucleic acid treatment of diseases or conditions related to levels of epidermal growth factor receptors
EP1626086A2 (fr) Molécules d'acides nucléiques double-brin présentant de nouvelles compositions chimiques capables de moduler l'expression de gènes
US20030186909A1 (en) Nucleic acid treatment of diseases or conditions related to levels of epidermal growth factor receptors
MXPA01002953A (en) Hairpin hybridizer molecules for modulation of gene expression
WO2002011674A2 (fr) Procede et reactif permettant d'inhiber le canal chlorure active par le calcium 1 (clca-1)
WO2001062911A2 (fr) Methode et reactif d'inhibition de grid
US20030143708A1 (en) Method and reagent for the treatment of alzheimer's disease
US20030092646A1 (en) Method and reagent for the inhibition of CD20

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN 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 ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW SD SL SZ TZ 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
AK Designated states

Kind code of ref document: A3

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN 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 ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW SD SL SZ TZ 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

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 60552/99

Country of ref document: AU

ENP Entry into the national phase in:

Ref country code: CA

Ref document number: 2343067

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: PA/a/2001/002953

Country of ref document: MX

ENP Entry into the national phase in:

Ref country code: JP

Ref document number: 2000 574246

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 1999969438

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1999969438

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

ENP Entry into the national phase in:

Ref document number: 2343067

Country of ref document: CA

WWW Wipo information: withdrawn in national office

Ref document number: 1999969438

Country of ref document: EP