US20100004320A1 - Pharmaceutical Composition - Google Patents

Pharmaceutical Composition Download PDF

Info

Publication number
US20100004320A1
US20100004320A1 US12/296,084 US29608407A US2010004320A1 US 20100004320 A1 US20100004320 A1 US 20100004320A1 US 29608407 A US29608407 A US 29608407A US 2010004320 A1 US2010004320 A1 US 2010004320A1
Authority
US
United States
Prior art keywords
mir
hsa
xxxxxx
lna
oligonucleotide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US12/296,084
Other languages
English (en)
Inventor
Joacim Elmen
Phil Kearney
Sakari Kauppinen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Roche Innovation Center Copenhagen AS
Original Assignee
Santaris Pharma AS
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 Santaris Pharma AS filed Critical Santaris Pharma AS
Priority to US12/296,084 priority Critical patent/US20100004320A1/en
Assigned to SANTARIS PHARMA A/S reassignment SANTARIS PHARMA A/S ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ELMEN, JOACIM, KAUPPINEN, SAKARI, KEARNEY, PHIL
Publication of US20100004320A1 publication Critical patent/US20100004320A1/en
Abandoned legal-status Critical Current

Links

Images

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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • 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
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • 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
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-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
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • 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
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol

Definitions

  • the present invention concerns pharmaceutical compositions comprising LNA-containing single stranded oligonucleotides capable of inhibiting disease-inducing microRNAs.
  • miRNAs are an abundant class of short endogenous RNAs that act as post-transcriptional regulators of gene expression by base-pairing with their target mRNAs.
  • the mature miRNAs are processed sequentially from longer hairpin transcripts by the RNAse III ribonucleases Drosha (Lee et al. 2003) and Dicer (Hutvagner et al. 2001, Ketting et al. 2001).
  • Drosha Lee et al. 2003
  • Dicer Dicer
  • miRNAs are involved in a wide variety of human diseases.
  • SMA spinal muscular atrophy
  • SMI motor neurodegenerative disease caused by reduced protein levels or loss-of-function mutations of the survival of motor neurons (SMN) gene
  • SLITRK1 motor neurons
  • HCV hepatitis C virus
  • FXMR frag-ile X mental retardation
  • FMRP fragile X mental retardation protein
  • miRNAs have also been shown to be deregulated in breast cancer (Iorio et al. 2005), lung cancer (Johnson et al. 2005) and colon cancer (Michael et al. 2004), while the miR-17-92 cluster, which is amplified in human B-cell lymphomas and miR-155 which is upregulated in Burkitt's lymphoma have been reported as the first human miRNA oncogenes (Eis et al. 2005, He et al. 2005). Thus, human miRNAs would not only be highly useful as biomarkers for future cancer diagnostics, but are rapidly emerging as attractive targets for disease intervention by oligonucleotide technologies.
  • WO03/029459 (Tuschl) claims oligonucleotides which encode microRNAs and their complements of between 18-25 nucleotides in length which may comprise nucleotide analogues.
  • LNA is suggested as a possible nucleotide analogue, although no LNA containing oligonucleotides are disclosed.
  • US2005/0182005 discloses a 24mer 2′OMe RNA oligoribonucleotide complementary to the longest form of miR 21 which was found to reduce miR 21 induced repression, whereas an equivalent DNA containing oligonucleotide did not.
  • 2′OMe-RNA refers to an RNA analogue where there is a substitution to methyl at the 2′ position (2′OMethyl).
  • US20050261218 claims an oligomeric compound comprising a first region and a second region, wherein at least one region comprises a modification and a portion of the oligomeric compound is targeted to a small non-coding RNA target nucleic acid, wherein the small non-coding RNA target nucleic acid is a miRNA.
  • Oligomeric compounds of between 17 and 25 nucleotides in length are claimed. The examples refer to entirely 2′ OMe PS compounds, 21mers and 20mer and 2′OMe gapmer oligonucleotides targeted against a range of pre-miRNA and mature miRNA targets.
  • Naguibneva (Naguibneva et al. Nature Cell Biology 2006 8 describes the use of mixmer DNA-LNA-DNA antisense oligonucleotide anti-mir to inhibit microRNA miR-181 function in vitro, in which a block of 8 LNA nucleotides is located at the center of the molecule flanked by 6 DNA nucleotides at the 5′ end, and 9 DNA nucleotides at the 3′ end, respectively.
  • a major drawback of this antisense design is low in vivo stability due to low nuclease resistance of the flanking DNA ends.
  • the present invention is based upon the discovery that the use of short oligonucleotides designed to bind with high affinity to miRNA targets are highly effective in alleviating the repression of mRNA by microRNAs in vivo.
  • the evidence disclosed herein indicates that the highly efficient targeting of miRNAs in vivo is achieved by designing oligonucleotides with the aim of forming a highly stable duplex with the miRNA target in vivo.
  • This is achieved by the use of high affinity nucleotide analogues such as at least one LNA units and suitably further high affinity nucleotide analogues, such as LNA, 2′-MOE RNA of 2′-Fluoro nucleotide analogues, in a short, such as 10-17 or 10-16 nucleobase oligonucleotides.
  • the aim is to generate an oligonucleotide of a length which is unlikely to form a siRNA complex (i.e. a short oligonucleotide), and with sufficient loading of high affinity nucleotide analogues that the oligonucleotide sticks almost permanently to its miRNA target, effectively forming a stable and non-functional duplex with the miRNA target.
  • oligonucleotide of a length which is unlikely to form a siRNA complex (i.e. a short oligonucleotide)
  • 2′fluor-DNA refers to an DNA analogue where the is a substitution to fluor at the 2′ position (2′F).
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a single stranded oligonucleotide having a length of between 8 and 17, such as 10 and 17, such as 8-16 or 10-16 nucleobase units, a pharmaceutically acceptable diluent, carrier, or adjuvant, wherein at least one of the nucleobase units of the single stranded oligonucleotide is a high affinity nucleotide analogue, such as a Locked Nucleic Acid (LNA) nucleobase unit, and wherein the single stranded oligonucleotide is complementary to a human microRNA sequence.
  • LNA Locked Nucleic Acid
  • the high affinity nucleotide analogues are nucleotide analogues which result in oligonucleotide which has a higher thermal duplex stability with a complementary RNA nucleotide than the binding affinity of an equivalent DNA nucleotide. This is typically determined by measuring the T m .
  • the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a single stranded oligonucleotide having a length of between 8 and 17 nucleobase units, such as between 10 and 17 nucleobase units, such as between 10 and 16 nucleobase units, and a pharmaceutically acceptable diluent, carrier, or adjuvant, wherein at least one of the nucleobase units of the single stranded oligonucleotide is a Locked Nucleic Acid (LNA) nucleobase unit, and wherein the single stranded oligonucleotide is complementary to a human microRNA sequence.
  • LNA Locked Nucleic Acid
  • the invention further provides for the use of an oligonucleotide according to the invention, such as those which may form part of the pharmaceutical composition, for the manufacture of a medicament for the treatment of a disease or medical disorder associated with the presence or over-expression (upregulation) of the microRNA.
  • the invention further provides for a method for the treatment of a disease or medical disorder associated with the presence or over-expression of the microRNA, comprising the step of administering a composition (such as the pharmaceutical composition) according to the invention to a person in need of treatment.
  • a composition such as the pharmaceutical composition
  • the invention further provides for a method for reducing the effective amount of a miRNA in a cell or an organism, comprising administering a composition (such as the pharmaceutical composition) according to the invention or a single stranded oligonucleotide according to the invention to the cell or the organism.
  • a composition such as the pharmaceutical composition
  • a single stranded oligonucleotide according to the invention to the cell or the organism.
  • Reducing the effective amount in this context refers to the reduction of functional miRNA present in the cell or organism.
  • the preferred oligonucleotides according to the invention may not always significantly reduce the actual amount of miRNA in the cell or organism as they typically form very stable duplexes with their miRNA targets.
  • the invention further provides for a method for de-repression of a target mRNA of a miRNA in a cell or an organism, comprising administering a composition (such as the pharmaceutical composition) or a single stranded oligonucleotide according to the invention to the cell or the organism.
  • a composition such as the pharmaceutical composition
  • a single stranded oligonucleotide according to the invention
  • the invention further provides for the use of a single stranded oligonucleotide of between 8-16 such as 10-16 nucleobases in length, for the manufacture of a medicament for the treatment of a disease or medical disorder associated with the presence or over-expression of the microRNA.
  • the invention further provides for a method for the treatment of a disease or medical disorder associated with the presence or over-expression of the microRNA, comprising the step of administering a composition (such as the pharmaceutical composition) comprising a single stranded oligonucleotide of between between 8-16 such as between 10-16 nucleobases in length to a person in need of treatment.
  • a composition such as the pharmaceutical composition
  • the invention further provides for a method for reducing the effective amount of a miRNA target (i.e. ‘available’ miRNA) in a cell or an organism, comprising administering a composition (such as the pharmaceutical composition) comprising a single stranded oligonucleotide of between 8-16 such as between 10-16 nucleobases to the cell or the organism.
  • a miRNA target i.e. ‘available’ miRNA
  • a composition such as the pharmaceutical composition
  • a single stranded oligonucleotide of between 8-16 such as between 10-16 nucleobases
  • the invention further provides for a method for de-repression of a target mRNA of a miRNA in a cell or an organism, comprising a single stranded oligonucleotide of between 8-16 such as between 10-16 nucleobases or (or a composition comprising said oligonucleotide) to the cell or the organism.
  • the invention further provides for a method for the synthesis of a single stranded oligonucleotide targeted against a human microRNA, such as a single stranded oligonucleotide described herein, said method comprising the steps of:
  • the synthesis is performed by sequential synthesis of the regions defined in steps a-f, wherein said synthesis may be performed in either the 3′-5′ (a to f) or 5′-3′ (f to a) direction, and wherein said single stranded oligonucleotide is complementary to a sequence of the miRNA target.
  • the oligonucleotide of the invention is designed not to be recruited by RISC or to mediate RISC directed cleavage of the miRNA target. It has been considered that by using long oligonucleotides, e.g. 21 or 22mers, particularly RNA oligonucleotides, or RNA ‘analogue’ oligonucleotide which are complementary to the miRNA target, the oligonucleotide can compete against the target mRNA in terms of RISC complex association, and thereby alleviate miRNA repression of miRNA target mRNAs via the introduction of an oligonucleotide which competes as a substrate for the miRNA.
  • long oligonucleotides e.g. 21 or 22mers, particularly RNA oligonucleotides, or RNA ‘analogue’ oligonucleotide which are complementary to the miRNA target.
  • the present invention seeks to prevent such undesirable target mRNA cleavage or translational inhibition by providing oligonucleotides capable of complementary, and apparently in some cases almost irreversible binding to the mature microRNA. This appears to result in a form of protection against degradation or cleavage (e.g. by RISC or RNAseH or other endo or exo-nucleases), which may not result in substantial or even significant reduction of the miRNA (e.g. as detected by northern blot using LNA probes) within a cell, but ensures that the effective amount of the miRNA, as measured by de-repression analysis is reduced considerably.
  • the invention provides oligonucleotides which are purposefully designed not to be compatible with the RISC complex, but to remove miRNA by titration by the oligonucleotide.
  • the oligonucleotides of the present invention work through non-competitive inhibition of miRNA function as they effectively remove the available miRNA from the cytoplasm, where as the prior art oligonucleotides provide an alternative miRNA substrate, which may act as a competitor inhibitor, the effectiveness of which would be far more dependant upon the concentration of the oligonucleotide in the cytoplasm, as well as the concentration of the target mRNA and miRNA.
  • oligonucleotides of approximately similar length to the miRNA targets, is that the oligonucleotides could form a siRNA like duplex with the miRNA target, a situation which would reduce the effectiveness of the oligonucleotide. It is also possible that the oligonucleotides themselves could be used as the guiding strand within the RISC complex, thereby generating the possibility of RISC directed degradation of non-specific targets which just happen to have sufficient complementarity to the oligonucleotide guide.
  • Short oligonucleotides which incorporate LNA are known from the reagents area, such as the LNA (see for example WO2005/098029 and WO 2006/069584).
  • the molecules designed for diagnostic or reagent use are very different in design than those for pharmaceutical use.
  • the terminal nucleobases of the reagent oligos are typically not LNA, but DNA, and the internucleoside linkages are typically other than phosphorothioate, the preferred linkage for use in the oligonucleotides of the present invention.
  • the invention therefore provides for a novel class of oligonucleotide per se.
  • the invention further provides for a (single stranded) oligonucleotide as described in the context of the pharmaceutical composition of the invention, wherein said oligonucleotide comprises either
  • the oligonucleotide is fully phosphorothiolated—the exception being for therapeutic oligonucleotides for use in the CNS, such as in the brain or spine where phosphorothioation can be toxic, and due to the absence of nucleases, phosphodieater bonds may be used, even between consecutive DNA units.
  • the second 3′ nucleobase, and/or the 9 th and 10 th (from the 3′ end), may also be LNA.
  • RNA cleavage such as exo-nuclease degradation in blood serum, or RISC associated cleavage of the oligonucleotide according to the invention are possible, and as such the invention also provides for a single stranded oligonucleotide which comprises of either:
  • oligonucleotides Whilst the benefits of these other aspects may be seen with longer oligonucleotides, such as nucleotide of up to 26 nucleobase units in length, it is considered these features may also be used with the shorter oligonucleotides referred to herein, such as the oligonucleotides of between 10-17 or 10-16 nucleobases described herein. It is highly preferably that the oligonucleotides comprise high affinity nucleotide analogues, such as those referred to herein, most preferably LNA units.
  • oligonucleotides comprising locked nucleic acid (LNA) units in a particular order show significant silencing of microRNAs, resulting in reduced microRNA levels. It was found that tight binding of said oligonucleotides to the so-called seed sequence, nucleotides 2 to 8 or 2-7, counting from the 5′ end, of the target microRNAs was important. Nucleotide 1 of the target microRNAs is a non-pairing base and is most likely hidden in a binding pocket in the Ago 2 protein.
  • LNA locked nucleic acid
  • the present inventors consider that by selecting the seed region sequences, particularly with oligonucleotides that comprise LNA, preferably LNA units in the region which is complementary to the seed region, the duplex between miRNA and oligonucleotide is particularly effective in targeting miRNAs, avoiding off target effects, and possibly providing a further feature which prevents RISC directed miRNA function.
  • the inventors have surprisingly found that microRNA silencing is even more enhanced when LNA-modified single stranded oligonucleotides do not contain a nucleotide at the 3′ end corresponding to this non-paired nucleotide 1. It was further found that two LNA units in the 3′ end of the oligonucleotides according to the present invention made said oligonucleotides highly nuclease resistant.
  • the oligonucleotides of the invention which have at least one nucleotide analogue, such as an LNA nucleotide in the positions corresponding to positions 10 and 11, counting from the 5′ end, of the target microRNA may prevent cleavage of the oligonucleotides of the invention
  • oligonucleotide having a length of from 12 to 26 nucleotides, wherein
  • the invention further provides for the oligonucleotides as defined herein for use as a medicament.
  • the invention further relates to compositions comprising the oligonucleotides defined herein and a pharmaceutically acceptable carrier.
  • a fourth aspect of the invention relates to the use of an oligonucleotide as defined herein for the manufacture of a medicament for the treatment of a disease associated with the expression of microRNAs selected from the group consisting of spinal muscular atrophy, Tourette's syndrome, hepatitis C virus, fragile X mental retardation, DiGeorge syndrome and cancer, such as chronic lymphocytic leukemia, breast cancer, lung cancer and colon cancer, in particular cancer.
  • a further aspect of the invention is a method to reduce the levels of target microRNA by contacting the target microRNA to an oligonucleotide as defined herein, wherein the oligonucleotide
  • the invention further provides for an oligonucleotide comprising a nucleobase sequence selected from the group consisting of SEQ IDs NO 1-534, SEQ ID NOs 539-544, SEQ ID NOs 549-554, SEQ ID NOs 559-564, SEQ ID NOs 569-574 and SEQ ID NOs 594-598, and SEQ ID NOs 579-584, or a pharmaceutical composition comprising said oligonucleotide.
  • the oligonucleotide may have a nucleobase sequence of between 1-17 nucleobases, such as 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 nucleobases, and as such the oligonucleobase in such an embodiment may be a contiguous subsequence within the oligonucleotides disclosed herein.
  • FIG. 1 The effect of treatment with different LNA anti-miR oligonucleotides on target nucleic acid expression in the miR-122a expressing cell line Huh-7. Shown are amounts of miR-122a (arbitrary units) derived from miR-122a specific qRT-PCR as compared to untreated cells (mock). The LNA anti-miR oligonucleotides were used at two concentrations, 1 and 100 nM, respectively. Included is also a mismatch control (SPC3350) to SPC3349 (also referred to herein as SPC3549).
  • SPC3350 mismatch control
  • SPC3549 also referred to herein as SPC3549.
  • FIG. 2 Assessment of LNA anti-miR-122a knock-down dose-response for SPC3548 and SPC3549 in comparison with SPC3372 in vivo in mice livers using miR-122a real-time RT-PCR.
  • FIG. 2 b miR-122 levels in the mouse liver after treatment with different LNA-antimiRs.
  • the LNA-antimiR molecules SPC3372 and SPC3649 were administered into normal mice by three i.p. injections on every second day over a six-day-period at indicated doses and sacrificed 48 hours after last dose.
  • Total RNA was extracted from the mice livers and miR-122 was measured by miR-122 specific qPCR.
  • FIG. 3 Assessment of plasma cholesterol levels in LNA-antimiR-122a treated mice compared to the control mice that received saline.
  • FIG. 4 a Assessment of relative Bckdk mRNA levels in LNA antimiR-122a treated mice in comparison with saline control mice using real-time quantitative RT-PCR.
  • FIG. 4 b Assessment of relative aldolase A mRNA levels in LNA antimiR-122a treated mice in comparison with saline control mice using real-time quantitative RT-PCR.
  • FIG. 4 c Assessment of GAPDH mRNA levels in LNA antimiR-122a treated mice (animals 4-30) in comparison with saline control mice (animals 1-3) using real-time quantitative RT-PCR.
  • FIG. 5 Assessment of LNA-antimiRTM-122a knock-down dose-response in vivo in mice livers using miR-122a real-time RT-PCR.
  • FIG. 6 Northern blot comparing SPC3649 with SPC3372. Total RNA from one mouse in each group were subjected to miR-122 specific northern blot. Mature miR-122 and the duplex (blocked microRNA) formed between the LNA-antimiR and miR-122 is indicated.
  • FIG. 9 Dose dependent miR-122a target mRNA induction by SPC3372 inhibition of miR-122a.
  • Mice were treated with different SPC3372 doses for three consecutive days, as described above and sacrificed 24 hours after last dose.
  • Total RNA extracted from liver was subjected to qPCR.
  • Genes with predicted miR-122 target site and observed to be upregulated by microarray analysis were investigated for dose-dependent induction by increasing SPC3372 doses using qPCR.
  • FIG. 10 Transient induction of miR-122a target mRNAs following SPC3372 treatment.
  • NMRI female mice were treated with 25 mg/kg/day SPC3372 along with saline control for three consecutive days and sacrificed 1, 2 or 3 weeks after last dose, respectively.
  • RNA was extracted from livers and mRNA levels of predicted miR-122a target mRNAs, selected by microarray data were investigated by qPCR. Three animals from each group were analysed.
  • FIG. 11 Induction of Vldlr in liver by SPC3372 treatment.
  • FIG. 12 Stability of miR-122a/SPC3372 duplex in mouse plasma. Stability of SPC3372 and SPC3372/miR-122a duplex were tested in mouse plasma at 37° C. over 96 hours. Shown in FIG. 12 is a SYBR-Gold stained PAGE.
  • FIG. 13 Sequestering of mature miR-122a by SPC3372 leads to duplex formation. Shown in FIG. 13 is a membrane probed with a miR-122a specific probe (upper panel) and re-probed with a Let-7 specific probe (lower panel). With the miR-122 probe, two bands could be detected, one corresponding to mature miR-122 and one corresponding to a duplex between SPC3372 and miR-122.
  • FIG. 14 miR-122a sequestering by SPC3372 along with SPC3372 distribution assessed by in situ hybridization of liver sections. Liver cryo-sections from treated animals were
  • FIG. 15 Liver gene expression in miR-122 LNA-antimiR treated mice.
  • Temporal liver gene expression profiles in LNA-antimiR treated mice Mice were treated with 25 mg/kg/day LNA-antimiR or saline for three consecutive days and sacrificed 1, 2 or 3 weeks after last dose. Included are also the values from the animals sacrificed 24 hours after last dose. (c,3) RNA samples from different time points were also subjected to expression profiling. Hierarchical cluster analysis of expression profiles of genes identified as differentially expressed between LNA-antimiR and saline treated mice 24 hours, one week or three weeks post treatment. (d,4) Expression profiles of genes identified as differentially expressed between LNA-antimiR and saline treated mice 24 hours post treatment were followed over time. The expression ratios of up- and down-regulated genes in LNA-antimiR treated mice approach 1 over the time-course, indicating a reversible effect of the LNA-antimiR treatment.
  • FIG. 16 The effect of treatment with SPC3372 and 3595 on miR-122 levels in mice livers.
  • FIG. 17 The effect of treatment with SPC3372 and 3595 on Aldolase A levels in mice livers.
  • FIG. 18 The effect of treatment with SPC3372 and 3595 on Bckdk levels in mice livers.
  • FIG. 19 The effect of treatment with SPC3372 and 3595 on CD320 levels in mice livers.
  • FIG. 20 The effect of treatment with SPC3372 and 3595 on Ndrg3 levels in mice livers.
  • FIG. 21 The effect of long-term treatment with SPC3649 on total plasma cholesterol in hypercholesterolemic and normal mice. Weekly samples of blood plasma were obtained from the SPC3649 treated and saline control mice once weekly followed by assessment of total plasma cholesterol. The mice were treated with 5 mg/kg SPC3649, SPC3744 or saline twice weekly. Normal mice given were treated in parallel.
  • FIG. 22 The effect of long-term treatment with SPC3649 on miR-122 levels in hypercholesterolemic and normal mice.
  • FIG. 23 The effect of long-term treatment with SPC3649 on Aldolase A levels in hypercholesterolemic and normal mice.
  • FIG. 24 The effect of long-term treatment with SPC3649 on Bckdk levels in hypercholesterolemic and normal mice.
  • FIG. 25 The effect of long-term treatment with SPC3649 on AST levels in hypercholesterolemic and normal mice.
  • FIG. 26 The effect of long-term treatment with SPC3649 on ALT levels in hypercholesterolemic and normal mice.
  • FIG. 27 Functional de-repression of renilla luciferase with miR-155 target by miR-155 blocking oligonucleotides in an endogenously miR-155 expressing cell line, 518A2.
  • psiCheck2 is the plasmid without miR-155 target, i.e. full expression
  • miR-155 target is the corresponding plasmid with miR-155 target but not co-transfected with oligo blocking miR-155 and hence represent fully miR-155 repressed renilla luciferace expression.
  • FIG. 28 Functional de-repression of renilla luciferase with miR-19b target by miR-19b blocking oligonucleotides in an endogenously miR-19b expressing cell line, HeLa.
  • miR-19b target is the plasmid with miR-19b target but not co-transfected with oligo blocking miR-19b and hence represent fully miR-19b repressed renilla luciferace expression.
  • FIG. 29 Functional de-repression of renilla luciferase with miR-122 target by miR-122 blocking oligonucleotides in an endogenously miR-122 expressing cell line, Huh-7.
  • miR-122 target is the corresponding plasmid with miR-122 target but not co-transfected with oligo blocking miR-122 and hence represent fully miR-122 repressed renilla luciferace expression.
  • FIG. 30 Diagram illustrating the alignment of an oligonucleotide according to the invention and a microRNA target.
  • the invention provides pharmaceutical compositions comprising short single stranded oligonucleotides, of length of between 8 and 17 such as between 10 and 17 nucleobases which are complementary to human microRNAs.
  • the short oligonucleotides are particularly effective at alleviating miRNA repression in vivo. It is found that the incorporation of high affinity nucleotide analogues into the oligonucleotides results in highly effective anti-microRNA molecules which appear to function via the formation of almost irreversible duplexes with the miRNA target, rather than RNA cleavage based mechanisms, such as mechanisms associated with RNaseH or RISC.
  • the single stranded oligonucleotide according to the invention comprises a region of contiguous nucleobase sequence which is 100% complementary to the human microRNA seed region.
  • single stranded oligonucleotide according to the invention is complementary to the mature human microRNA sequence.
  • the single stranded oligonucleotide according to the invention is complementary to a microRNA sequence, such as a microRNA sequence selected from the group consisting of: hsa-let-7a, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f, hsa-miR-15a, hsa-miR-16, hsa-miR-17-5p, hsa-miR-17-3p, hsa-miR-18a, hsa-miR-19a, hsa-miR-19b, hsa-miR-20a, hsa-miR-21, hsa-miR-22, hsa-miR-23a, hsa-miR-189, hsa-miR-24, hsa-miR-25, hsa microRNA
  • the single stranded oligonucleotide according to the invention is complementary to a microRNA sequence, such as a microRNA sequence selected from the group consisting of: hsa-let-7a, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f, hsa-miR-15a, hsa-miR-16, hsa-miR-17-5p, hsa-miR-17-3p, hsa-miR-18a, hsa-miR-19a, hsa-miR-20a, hsa-miR-22, hsa-miR-23a, hsa-miR-189, hsa-miR-24, hsa-miR-25, hsa-miR-26a, hsa-miR-26b,
  • Preferred single stranded oligonucleotide according to the invention are complementary to a microRNA sequence selected from the group consisting of has-miR19b, hsa-miR21, hsa-miR 122, hsa-miR 142 a7b, hsa-miR 155, hsa-miR 375.
  • Preferred single stranded oligonucleotide according to the invention are complementary to a microRNA sequence selected from the group consisting of hsa-miR196b and has-181a.
  • the oligonucleotide according to the invention does not comprise a nucleobase at the 3′ end that corresponds to the first 5′ end nucleotide of the target microRNA.
  • the first nucleobase of the single stranded oligonucleotide according to the invention, counting from the 3′ end is a nucleotide analogue, such as an LNA unit.
  • the second nucleobase of the single stranded oligonucleotide according to the invention, counting from the 3′ end is a nucleotide analogue, such as an LNA unit.
  • the ninth and/or the tenth nucleotide of the single stranded oligonucleotide according to the invention, counting from the 3′ end is a nucleotide analogue, such as an LNA unit.
  • the ninth nucleobase of the single stranded oligonucleotide according to the invention, counting from the 3′ end is a nucleotide analogue, such as an LNA unit.
  • the tenth nucleobase of the single stranded oligonucleotide according to the invention, counting from the 3′ end is a nucleotide analogue, such as an LNA unit.
  • both the ninth and the tenth nucleobase of the single stranded oligonucleotide according to the invention, calculated from the 3′ end is a nucleotide analogue, such as an LNA unit.
  • the single stranded oligonucleotide according to the invention does not comprise a region of more than 5 consecutive DNA nucleotide units. In one embodiment, the single stranded oligonucleotide according to the invention does not comprise a region of more than 6 consecutive DNA nucleotide units. In one embodiment, the single stranded oligonucleotide according to the invention does not comprise a region of more than 7 consecutive DNA nucleotide units. In one embodiment, the single stranded oligonucleotide according to the invention does not comprise a region of more than 8 consecutive DNA nucleotide units.
  • the single stranded oligonucleotide according to the invention does not comprise a region of more than 3 consecutive DNA nucleotide units. In one embodiment, the single stranded oligonucleotide according to the invention does not comprise a region of more than 2 consecutive DNA nucleotide units.
  • the single stranded oligonucleotide comprises at least region consisting of at least two consecutive nucleotide analogue units, such as at least two consecutive LNA units.
  • the single stranded oligonucleotide comprises at least region consisting of at least three consecutive nucleotide analogue units, such as at least three consecutive LNA units.
  • the single stranded oligonucleotide of the invention does not comprise a region of more than 7 consecutive nucleotide analogue units, such as LNA units. In one embodiment, the single stranded oligonucleotide of the invention does not comprise a region of more than 6 consecutive nucleotide analogue units, such as LNA units. In one embodiment, the single stranded oligonucleotide of the invention does not comprise a region of more than 5 consecutive nucleotide analogue units, such as LNA units. In one embodiment, the single stranded oligonucleotide of the invention does not comprise a region of more than 4 consecutive nucleotide analogue units, such as LNA units.
  • the single stranded oligonucleotide of the invention does not comprise a region of more than 3 consecutive nucleotide analogue units, such as LNA units. In one embodiment, the single stranded oligonucleotide of the invention does not comprise a region of more than 2 consecutive nucleotide analogue units, such as LNA units.
  • the first or second 3′ nucleobase of the single stranded oligonucleotide corresponds to the second 5′ nucleotide of the microRNA sequence.
  • nucleobase units 1 to 6 (inclusive) of the single stranded oligonucleotide as measured from the 3′ end the region of the single stranded oligonucleotide are complementary to the microRNA seed region sequence.
  • nucleobase units 1 to 7 (inclusive) of the single stranded oligonucleotide as measured from the 3′ end the region of the single stranded oligonucleotide are complementary to the microRNA seed region sequence.
  • nucleobase units 2 to 7 (inclusive) of the single stranded oligonucleotide as measured from the 3′ end the region of the single stranded oligonucleotide are complementary to the microRNA seed region sequence.
  • the single stranded oligonucleotide comprises at least one nucleotide analogue unit, such as at least one LNA unit, in a position which is within the region complementary to the miRNA seed region.
  • the single stranded oligonucleotide may, in one embodiment comprise at between one and 6 or between 1 and 7 nucleotide analogue units, such as between 1 and 6 and 1 and 7 LNA units, in a position which is within the region complementary to the miRNA seed region.
  • the nucleobase sequence of the single stranded oligonucleotide which is complementary to the sequence of the microRNA seed region is selected from the group consisting of (X)Xxxxxx, (X)xXxxxx, (X)xxXxxx, (X)xxxXxx, (X)xxxxXx and (X)xxxxxX, as read in a 3′-5′direction, wherein “X” denotes a nucleotide analogue, (X) denotes an optional nucleotide analogue, such as an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.
  • the single stranded oligonucleotide comprises at least two nucleotide analogue units, such as at least two LNA units, in positions which are complementary to the miRNA seed region.
  • the nucleobase sequence of the single stranded oligonucleotide which is complementary to the sequence of the microRNA seed region is selected from the group consisting of (X)XXxxxx, (X)XxXxxx, (X)XxxXxx, (X)XxxxxxX, (X)xXxXxx, (X)xXxxXx, (X)xXxxxX, (X)xxXXxx, (X)xxXxXx, (X)xxXxXx, (X)xxXxxX, (X)xxXX, (X)xxxXXx, (X)xxxXXx, (X)xxxXxX and (X)xxxxXX, wherein “X” denotes a nucleotide analogue, such as an LNA unit, (X) denotes an optional nucleotide analogue, such as an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.
  • the single stranded oligonucleotide comprises at least three nucleotide analogue units, such as at least three LNA units, in positions which are complementary to the miRNA seed region.
  • the nucleobase sequence of the single stranded oligonucleotide which is complementary to the sequence of the microRNA seed region is selected from the group consisting of (X)XXXxxx, (X)xXXXxx, (X)xxXXXx, (X)xxxXXX, (X)XXxxxX, (X)xXXxXx, (X)xXXxxX, (X)xxXXxX, (X)XxXxx, (X)XxxXXX, (X)X)XxxxXX, (X)xXxXXx, (X)xXxxXX, (X)xXxXXx, (X)xXxxXX, (X)xxXXX, (X)xxXXX, (X)xXxXxX and (X)XxXxXx, wherein “X” denotes a nucleotide analogue, such as an LNA unit, (X) denotes an optional nucleot
  • the single stranded oligonucleotide comprises at least four nucleotide analogue units, such as at least four LNA units, in positions which are complementary to the miRNA seed region.
  • nucleobase sequence of the single stranded oligonucleotide which is complementary to the sequence of the microRNA seed region is selected from the group consisting of (X)xxXXX, (X)xXxXXX, (X)xXXxX, (X))xXXXx, (X))xXXXx, (X)XxxXXX, (X)XxXxX, (X)XxXXxX, (X)XxXx, (X)XXxxXX, (X)XXxXxX, (X)XXxXXx, (X)XXXxxX, (X)XXXxXx, (X)XXXxxX, (X)XXXxXx, and (X)XXXXxx, wherein “X” denotes a nucleotide analogue, such as an LNA unit, (X) denotes an optional nucleotide analogue, such as an LNA unit, and “x”
  • the single stranded oligonucleotide comprises at least five nucleotide analogue units, such as at least five LNA units, in positions which are complementary to the miRNA seed region.
  • the nucleobase sequence of the single stranded oligonucleotide which is complementary to the sequence of the microRNA seed region is selected from the group consisting of (X)xXXXXX, (X)XxXXXX, (X)XXxXXX, (X)XXXxXX, (X)XXXxX and (X)XXXXXx, wherein “X” denotes a nucleotide analogue, such as an LNA unit, (X) denotes an optional nucleotide analogue, such as an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.
  • the single stranded oligonucleotide comprises six or seven nucleotide analogue units, such as six or seven LNA units, in positions which are complementary to the miRNA seed region.
  • the nucleobase sequence of the single stranded oligonucleotide which is complementary to the sequence of the microRNA seed region is selected from the group consisting of XXXXXX, XxXXXXX, XXxXXXX, XXXxXXX, XXXXxX and XXXXXx, wherein “X” denotes a nucleotide analogue, such as an LNA unit, such as an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.
  • the two nucleobase motif at position 7 to 8, counting from the 3′ end of the single stranded oligonucleotide is selected from the group consisting of xx, XX, xX and Xx, wherein “X” denotes a nucleotide analogue, such as an LNA unit, such as an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.
  • the two nucleobase motif at position 7 to 8, counting from the 3′ end of the single stranded oligonucleotide is selected from the group consisting of XX, xX and Xx, wherein “X” denotes a nucleotide analogue, such as an LNA unit, such as an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.
  • the single stranded oligonucleotide comprises at least 12 nucleobases and wherein the two nucleobase motif at position 11 to 12, counting from the 3′ end of the single stranded oligonucleotide is selected from the group consisting of xx, XX, xX and Xx, wherein “X” denotes a nucleotide analogue, such as an LNA unit, such as an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.
  • the single stranded oligonucleotide comprises at least 12 nucleobases and wherein the two nucleobase motif at position 11 to 12, counting from the 3′ end of the single stranded oligonucleotide is selected from the group consisting of XX, xX and Xx, wherein “X” denotes a nucleotide analogue, such as an LNA unit, such as an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.
  • the single stranded oligonucleotide comprises at least 13 nucleobases and wherein the three nucleobase motif at position 11 to 13, counting from the 3′ end, is selected from the group consisting of xxx, Xxx, xXx, xxX, XXx, XxX, xXX and XXX, wherein “X” denotes a nucleotide analogue, such as an LNA unit, such as an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.
  • the three nucleobase motif at position 11 to 13, counting from the 3′ end of the single stranded oligonucleotide is selected from the group consisting of Xxx, xXx, xxX, XXx, XxX, xXX and XXX, wherein “X” denotes a nucleotide analogue, such as an LNA unit, such as an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.
  • the single stranded oligonucleotide comprises at least 14 nucleobases and wherein the four nucleobase motif at positions 11 to 14, counting from the 3′ end, is selected from the group consisting of xxxx, Xxxx, xXxx, xxxX, XXxx, XxXx, XxxX, xXXx, xXxX, xxXX, XXXx, XxXX, xXXX, xXXX, XXxX and XXXX wherein “X” denotes a nucleotide analogue, such as an LNA unit, such as an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.
  • the four nucleobase motif at position 11 to 14 of the single stranded oligonucleotide, counting from the 3′ end is selected from the group consisting of Xxxx, xXxx, xxXx, xxxX, XxXx, XxxX, xXXx, xXxX, xxXX, XXXx, XxXX, xXXX, XXxX and XXXX, wherein “X” denotes a nucleotide analogue, such as an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.
  • the single stranded oligonucleotide comprises 15 nucleobases and the five nucleobase motif at position 11 to 15, counting from the 3′ end, is selected from the group consisting of Xxxxx, xXxxx, xxXxx, xxxX, XXxxx, XxXxx, XxXxx, xXxXx, xXxxX, xxXXx, xxXxX, xxxXX, XXxxX, XXX, XxxXX, XXXX, XxxXX, XXXX, xXXXx, xxXXXX, XXxXX, XxXX, XXXxX, XXXxX, XXXxX, XXXxX, XXXxX, XXxX, XXxX, XXxX, XXxX, XXxX, XXxX, X
  • the single stranded oligonucleotide comprises 16 nucleobases and the six nucleobase motif at positions 11 to 16, counting from the 3′ end, is selected from the group consisting of Xxxxxx, xXxxxx, xxXxxx, xxxxXx, xxxxxX, XXxxxx, XxXxxx, XxxxxX, xXXxxx, xXxXxx, xXxxxX, xXxxxX, xxXxxX, xxXxXx, xxxXxX, xxxxXX, xxxXX, xxxXxX, xxxxXX, XXxxx, XXXxx, XXXxx, XXXxx, XXXxx,
  • the six nucleobase motif at positions 11 to 16 of the single stranded oligonucleotide, counting from the 3′ end is xxXxxX, wherein “X” denotes a nucleotide analogue, such as an LNA unit, such as an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.
  • the three 5′ most nucleobases is selected from the group consisting of Xxx, xXx, xxX, XXx, XxX, xXX and XXX, wherein “X” denotes a nucleotide analogue, such as an LNA unit, such as an LNA unit, and “x” denotes a DNA or RNA nucleotide unit. In one embodiment, x” denotes a DNA unit.
  • the single stranded oligonucleotide comprises a nucleotide analogue unit, such as an LNA unit, at the 5′ end.
  • a nucleotide analogue unit such as an LNA unit
  • the nucleotide analogue units are independently selected form the group consisting of: 2′-O-alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA unit, PNA unit, HNA unit, INA unit.
  • all the nucleobases of the single stranded oligonucleotide of the invention are nucleotide analogue units.
  • nucleotide analogue units such as X
  • the single stranded oligonucleotide comprises said at least one LNA analogue unit and at least one further nucleotide analogue unit other than LNA.
  • the non-LNA nucleotide analogue unit or units are independently selected from 2′-OMe RNA units and 2′-fluoro DNA units.
  • the single stranded oligonucleotide consists of at least one sequence XYX or YXY, wherein X is LNA and Y is either a 2′-OMe RNA unit and 2′-fluoro DNA unit.
  • sequence of nucleobases of the single stranded oligonucleotide consists of alternative X and Y units.
  • the single stranded oligonucleotide comprises alternating LNA and DNA units (Xx) or (xX).
  • the single stranded oligonucleotide comprises a motif of alternating LNA followed by 2 DNA units (Xxx), xXx or xxX.
  • At least one of the DNA or non-LNA nucleotide analogue units are replaced with a LNA nucleobase in a position selected from the positions identified as LNA nucleobase units in any one of the embodiments referred to above.
  • “X” donates an LNA unit.
  • the single stranded oligonucleotide comprises at least 2 nucleotide analogue units, such as at least 3 nucleotide analogue units, such as at least 4 nucleotide analogue units, such as at least 5 nucleotide analogue units, such as at least 6 nucleotide analogue units, such as at least 7 nucleotide analogue units, such as at least 8 nucleotide analogue units, such as at least 9 nucleotide analogue units, such as at least 10 nucleotide analogue units.
  • the single stranded oligonucleotide comprises at least 2 LNA units, such as at least 3 LNA units, such as at least 4 LNA units, such as at least 5 LNA units, such as at least 6 LNA units, such as at least 7 LNA units, such as at least 8 LNA units, such as at least 9 LNA units, such as at least 10 LNA units.
  • nucleotide analogues such as LNA units
  • cytosine or guanine such as between 1-10 of the of the nucleotide analogues, such as LNA units
  • cytosine or guanine such as 2, 3, 4, 5, 6, 7, 8, or 9 of the of the nucleotide analogues, such as LNA units, is either cytosine or guanine.
  • At least two of the nucleotide analogues such as LNA units is either cytosine or guanine. In one embodiment at least three of the nucleotide analogues such as LNA units is either cytosine or guanine. In one embodiment at least four of the nucleotide analogues such as LNA units is either cytosine or guanine. In one embodiment at least five of the nucleotide analogues such as LNA units is either cytosine or guanine. In one embodiment at least six of the nucleotide analogues such as LNA units is either cytosine or guanine.
  • At least seven of the nucleotide analogues such as LNA units is either cytosine or guanine. In one embodiment at least eight of the nucleotide analogues such as LNA units is either cytosine or guanine.
  • the nucleotide analogues have a higher thermal duplex stability a complementary RNA nucleotide than the binding affinity of an equivalent DNA nucleotide to said complementary RNA nucleotide.
  • the nucleotide analogues confer enhanced serum stability to the single stranded oligonucleotide.
  • the single stranded oligonucleotide forms an A-helix conformation with a complementary single stranded RNA molecule.
  • a duplex between two RNA molecules typically exists in an A-form conformation, where as a duplex between two DNA molecules typically exits in a B-form conformation.
  • a duplex between a DNA and RNA molecule typically exists in a intermediate conformation (A/B form).
  • nucleotide analogues such as beta-D-oxy LNA can be used to promote a more A form like conformation.
  • Standard circular dichromisms (CD) or NMR analysis is used to determine the form of duplexes between the oligonucleotides of the invention and complementary RNA molecules.
  • the oligonucleotides according to the present invention may, in one embodiment form a A/B-form duplex with a complementary RNA molecule.
  • nucleotide analogues which promote the A-form structure can also be effective, such as the alpha-L isomer of LNA.
  • the single stranded oligonucleotide forms an A/B-form conformation with a complementary single stranded RNA molecule.
  • the single stranded oligonucleotide forms an A-form conformation with a complementary single stranded RNA molecule.
  • the single stranded oligonucleotide according to the invention does not mediate RNAseH based cleavage of a complementary single stranded RNA molecule.
  • a stretch of at least 5 typically not effective for RNAse H recruitment
  • more preferably at least 6, more preferably at least 7 or 8 consecutive DNA nucleobases or alternative nucleobases which can recruit RNAseH, such as alpha L-amino LNA
  • EP 1 222 309 provides in vitro methods for determining RNaseH activity, which may be used to determine the ability to recruit RNaseH.
  • a compound is deemed capable of recruiting RNase H if, when provided with the complementary RNA target, it has an initial rate, as measured in pmol/l/min, of at least 1%, such as at least 5%, such as at least 10% or less than 20% of the equivalent DNA only oligonucleotide, with no 2′ substitutions, with phosphorothiote linkage groups between all nucleotides in the oligonucleotide, using the methodology provided by Example 91-95 of EP 1 222 309.
  • a compound is deemed essentially incapable of recruiting RNaseH if, when provided with the complementary RNA target, and RNaseH, the RNaseH initial rate, as measured in pmol/l/min, is less than 1%, such as less than 5%, such as less than 10% or less than 20% of the initial rate determined using the equivalent DNA only oligonucleotide, with no 2′ substitutions, with phosphiothiote linkage groups between all nucleotides in the oligonucleotide, using the methodology provided by Example 91-95 of EP 1 222 309.
  • the single stranded oligonucleotide of the invention is capable of forming a duplex with a complementary single stranded RNA nucleic acid molecule (typically of about the same length of said single stranded oligonucleotide) with phosphodiester internucleoside linkages, wherein the duplex has a T m of at least about 60° C.
  • the single stranded oligonucleotide is capable of forming a duplex with a complementary single stranded RNA nucleic acid molecule with phosphodiester internucleoside linkages, wherein the duplex has a T m of between about 70° C. to about 95° C., such as a T m of between about 70° C. to about 90° C., such as between about 70° C. and about 85° C.
  • the single stranded oligonucleotide is capable of forming a duplex with a complementary single stranded DNA nucleic acid molecule with phosphodiester internucleoside linkages, wherein the duplex has a T m of between about 50° C. to about 95° C., such as between about 50° C. to about 90° C., such as at least about 55° C., such as at least about 60° C., or no more than about 95° C.
  • the single stranded oligonucleotide may, in one embodiment have a length of between 14-16 nucleobases, including 15 nucleobases.
  • the LNA unit or units are independently selected from the group consisting of oxy-LNA, thio-LNA, and amino-LNA, in either of the D- ⁇ and L- ⁇ configurations or combinations thereof.
  • the LNA units may be an ENA nucleobase.
  • the LNA units are beta D oxy-LNA.
  • the LNA units are in alpha-L amino LNA.
  • the single stranded oligonucleotide comprises between 3 and 17 LNA units.
  • the single stranded oligonucleotide comprises at least one internucleoside linkage group which differs from phosphate.
  • the single stranded oligonucleotide comprises at least one phosphorothioate internucleoside linkage.
  • the single stranded oligonucleotide comprises phosphodiester and phosphorothioate linkages.
  • the all the internucleoside linkages are phosphorothioate linkages.
  • the single stranded oligonucleotide comprises at least one phosphodiester internucleoside linkage.
  • all the internucleoside linkages of the single stranded oligonucleotide of the invention are phosphodiester linkages.
  • composition according to the invention comprises a carrier such as saline or buffered saline.
  • the method for the synthesis of a single stranded oligonucleotide targeted against a human microRNA is performed in the 3′ to 5′ direction a-f.
  • the method for the synthesis of the single stranded oligonucleotide according to the invention may be performed using standard solid phase oligonucleotide synthesis.
  • nucleobase refers to nucleotides, such as DNA and RNA, and nucleotide analogues.
  • oligonucleotide refers, in the context of the present invention, to a molecule formed by covalent linkage of two or more nucleobases.
  • oligonucleotide may have, in one embodiment, for example between 8-26 nucleobases, such as between 10 to 26 nucleobases such between 12 to 26 nucleobases.
  • the oligonucleotide of the invention has a length of between 8-17 nucleobases, such as between 20-27 nucleobases such as between 8-16 nucleobases, such as between 12-15 nucleobases,
  • the oligonucleotide of the invention may have a length of 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleobases.
  • nucleobases are nucleotide analogues, such as at least about 33%, such as at least about 40%, or at least about 50% or at least about 60%, such as at least about 66%, such as at least about 70%, such as at least about 80%, or at least about 90%.
  • the oligonucleotide may comprise of a nucleobase sequence which consists of only nucleotide analogue sequences.
  • nucleobases A, C, T and G such as the DNA nucleobases A, C, T and G, the RNA nucleobases A, C, U and G, as well as non-DNA/RNA nucleobases, such as 5-methylcytosine ( Me C), isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 5-propyny-6-fluoroluracil, 5-methylthiazoleuracil, 6-aminopurine, 2-aminopurine, inosine, 2,6-diaminopurine, 7-propyne-7-deazaadenine, 7-propyne-7-deazaguanine and 2-chloro-6-aminopurine, in particular Me C.
  • purines and pyrimidines such as the DNA nucleobases A, C, T and G, the RNA nucleobases A, C, U and G, as well as non-DNA/RNA nucleobases
  • non-DNA/RNA nucleobase will depend on the corresponding (or matching) nucleotide present in the microRNA strand which the oligonucleotide is intended to target.
  • the corresponding nucleotide is G it will normally be necessary to select a non-DNA/RNA nucleobase which is capable of establishing hydrogen bonds to G.
  • a typical example of a preferred non-DNA/RNA nucleobase is Me C.
  • nucleoside linkage group is intended to mean a group capable of covalently coupling together two nucleobases, such as between DNA units, between DNA units and nucleotide analogues, between two non-LNA units, between a non-LNA unit and an LNA unit, and between two LNA units, etc.
  • Preferred examples include phosphate, phosphodiester groups and phosphorothioate groups.
  • the internucleoside linkage may be selected form the group consisting of: —O—P(O) 2 —O—, —O—P(O,S)—O—, —O—P(S) 2 —O—, —S—P(O) 2 —O—, —S—P(O,S)—O—, —S—P(O) 2 —O—, —O—P(O) 2 —S—, —O—P(O,S)—S—, —O—PO(R H )—O—, O—PO(OCH 3 )—O—, —O—PO(NR H )—O—, —O—PO(OCH 2 CH 2 S—R)—O—, —O—PO(BH 3 )—O—, —O—PO(NHR H )—O—, —O—P(O) 2 —NR H —, —NR H —, —NR H —
  • corresponding to and “corresponds to” as used in the context of oligonucleotides refers to the comparison between either a nucleobase sequence of the compound of the invention, and the reverse complement thereof, or in one embodiment between a nucleobase sequence and an equivalent (identical) nucleobase sequence which may for example comprise other nucleobases but retains the same base sequence, or complement thereof.
  • Nucleotide analogues are compared directly to their equivalent or corresponding natural nucleotides. Sequences which form the reverse complement of a sequence are referred to as the complement sequence of the sequence.
  • the length of a nucleotide molecule corresponds to the number of monomer units, i.e. nucleobases, irrespective as to whether those monomer units are nucleotides or nucleotide analogues.
  • nucleobases the terms monomer and unit are used interchangeably herein.
  • Preferred DNA analogues includes DNA analogues where the 2′-H group is substituted with a substitution other than —OH (RNA) e.g. by substitution with —O—CH 3 , —O—CH 2 —CH 2 —O—CH 3 , —O—CH 2 —CH 2 —CH 2 —NH 2 , —O—CH 2 —CH 2 —CH 2 —OH or —F.
  • RNA DNA analogues where the 2′-H group is substituted with a substitution other than —OH (RNA) e.g. by substitution with —O—CH 3 , —O—CH 2 —CH 2 —O—CH 3 , —O—CH 2 —CH 2 —CH 2 —NH 2 , —O—CH 2 —CH 2 —CH 2 —OH or —F.
  • RNA analogues includes RNA analogues which have been modified in its 2′-OH group, e.g. by substitution with a group other than —H (DNA), for example —O—CH 3 , —O—CH 2 —CH 2 —O—CH 3 , —O—CH 2 —CH 2 —CH 2 —NH 2 , —O—CH 2 —CH 2 —CH 2 —OH or —F.
  • nucleotide analogue is “ENA”.
  • LNA unit LNA monomer
  • LNA residue locked nucleic acid unit
  • locked nucleic acid monomer or “locked nucleic acid residue”
  • LNA units are described in inter alia WO 99/14226, WO 00/56746, WO 00/56748, WO 01/25248, WO 02/28875, WO 03/006475 and WO 03/095467.
  • the LNA unit may also be defined with respect to its chemical formula.
  • an “LNA unit”, as used herein, has the chemical structure shown in Scheme 1 below:
  • corresponding LNA unit is intended to mean that the DNA unit has been replaced by an LNA unit containing the same nitrogenous base as the DNA unit that it has replaced, e.g. the corresponding LNA unit of a DNA unit containing the nitrogenous base A also contains the nitrogenous base A.
  • the corresponding LNA unit may contain the base C or the base Me C, preferably Me C.
  • non-LNA unit refers to a nucleoside different from an LNA-unit, i.e. the term “non-LNA unit” includes a DNA unit as well as an RNA unit.
  • a preferred non-LNA unit is a DNA unit.
  • At least one encompasses an integer larger than or equal to 1, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and so forth.
  • a and “an” as used about a nucleotide, an agent, an LNA unit, etc. is intended to mean one or more.
  • the expression “a component (such as a nucleotide, an agent, an LNA unit, or the like) selected from the group consisting of . . . ” is intended to mean that one or more of the cited components may be selected.
  • expressions like “a component selected from the group consisting of A, B and C” is intended to include all combinations of A, B and C, i.e. A, B, C, A+B, A+C, B+C and A+B+C.
  • thio-LNA unit refers to an LNA unit in which X in Scheme 1 is S.
  • a thio-LNA unit can be in both the beta-D form and in the alpha-L form.
  • beta-D form of the thio-LNA unit is preferred.
  • the beta-D-form and alpha-L-form of a thio-LNA unit are shown in Scheme 3 as compounds 3A and 3B, respectively.
  • amino-LNA unit refers to an LNA unit in which X in Scheme 1 is NH or NR H , where R H is hydrogen or C 1-4 -alkyl.
  • An amino-LNA unit can be in both the beta-D form and in the alpha-L form. Generally, the beta-D form of the amino-LNA unit is preferred.
  • the beta-D-form and alpha-L-form of an amino-LNA unit are shown in Scheme 4 as compounds 4A and 4B, respectively.
  • oxy-LNA unit refers to an LNA unit in which X in Scheme 1 is O.
  • An Oxy-LNA unit can be in both the beta-D form and in the alpha-L form.
  • the beta-D form of the oxy-LNA unit is preferred.
  • the beta-D form and the alpha-L form of an oxy-LNA unit are shown in Scheme 5 as compounds 5A and 5B, respectively.
  • C 1-6 -alkyl is intended to mean a linear or branched saturated hydrocarbon chain wherein the longest chains has from one to six carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl and hexyl.
  • a branched hydrocarbon chain is intended to mean a C 1-6 -alkyl substituted at any carbon with a hydrocarbon chain.
  • C 1-4 -alkyl is intended to mean a linear or branched saturated hydrocarbon chain wherein the longest chains has from one to four carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.
  • a branched hydrocarbon chain is intended to mean a C 1-4 -alkyl substituted at any carbon with a hydrocarbon chain.
  • C 1-6 -alkoxy is intended to mean C 1-6 -alkyl-oxy, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, isopentoxy, neopentoxy and hexoxy.
  • C 2-6 -alkenyl is intended to mean a linear or branched hydrocarbon group having from two to six carbon atoms and containing one or more double bonds.
  • Illustrative examples of C 2-6 -alkenyl groups include allyl, homo-allyl, vinyl, crotyl, butenyl, butadienyl, pentenyl, pentadienyl, hexenyl and hexadienyl.
  • the position of the unsaturation may be at any position along the carbon chain.
  • C 2-6 -alkynyl is intended to mean linear or branched hydrocarbon groups containing from two to six carbon atoms and containing one or more triple bonds.
  • Illustrative examples of C 2-6 -alkynyl groups include acetylene, propynyl, butynyl, pentynyl and hexynyl.
  • the position of unsaturation may be at any position along the carbon chain. More than one bond may be unsaturated such that the “C 2-6 -alkynyl” is a di-yne or enedi-yne as is known to the person skilled in the art.
  • hybridisation means hydrogen bonding, which may be Watson-Crick, Hoogsteen, reversed Hoogsteen hydrogen bonding, etc., between complementary nucleoside or nucleotide bases.
  • the four nucleobases commonly found in DNA are G, A, T and C of which G pairs with C, and A pairs with T.
  • RNA T is replaced with uracil (U), which then pairs with A.
  • the chemical groups in the nucleobases that participate in standard duplex formation constitute the Watson-Crick face.
  • Hoogsteen showed a couple of years later that the purine nucleobases (G and A) in addition to their Watson-Crick face have a Hoogsteen face that can be recognised from the outside of a duplex, and used to bind pyrimidine oligonucleotides via hydrogen bonding, thereby forming a triple helix structure.
  • complementary refers to the capacity for precise pairing between two nucleotides sequences with one another. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the corresponding position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position.
  • the DNA or RNA strand are considered complementary to each other when a sufficient number of nucleotides in the oligonucleotide can form hydrogen bonds with corresponding nucleotides in the target DNA or RNA to enable the formation of a stable complex.
  • an oligonucleotide need not be 100% complementary to its target microRNA.
  • complementary and specifically hybridisable thus imply that the oligonucleotide binds sufficiently strong and specific to the target molecule to provide the desired interference with the normal function of the target whilst leaving the function of non-target RNAs unaffected.
  • the oligonucleotide of the invention is 100% complementary to a human microRNA sequence, such as one of the microRNA sequences referred to herein.
  • the oligonucleotide of the invention comprises a contiguous sequence which is 100% complementary to the seed region of the human microRNA sequence.
  • MicroRNAs are short, non-coding RNAs derived from endogenous genes that act as post-transcriptional regulators of gene expression. They are processed from longer (ca 70-80 nt) hairpin-like precursors termed pre-miRNAs by the RNAse III enzyme Dicer. MicroRNAs assemble in ribonucleoprotein complexes termed miRNPs and recognize their target sites by antisense complementarity thereby mediating down-regulation of their target genes. Near-perfect or perfect complementarity between the miRNA and its target site results in target mRNA cleavage, whereas limited complementarity between the microRNA and the target site results in translational inhibition of the target gene.
  • microRNA or “miRNA”, in the context of the present invention, means an RNA oligonucleotide consisting of between 18 to 25 nucleotides in length. In functional terms miRNAs are typically regulatory endogenous RNA molecules.
  • target microRNA or “target miRNA” refer to a microRNA with a biological role in human disease, e.g. an upregulated, oncogenic miRNA or a tumor suppressor miRNA in cancer, thereby being a target for therapeutic intervention of the disease in question.
  • target gene refers to regulatory mRNA targets of microRNAs, in which said “target gene” or “target mRNA” is regulated post-transcriptionally by the microRNA based on near-perfect or perfect complementarity between the miRNA and its target site resulting in target mRNA cleavage; or limited complementarity, often conferred to complementarity between the so-called seed sequence (nucleotides 2-7 of the miRNA) and the target site resulting in translational inhibition of the target mRNA.
  • the oligonucleotide is single stranded, this refers to the situation where the oligonucleotide is in the absence of a complementary oligonucleotide—i.e. it is not a double stranded oligonucleotide complex, such as an siRNA.
  • the composition according to the invention does not comprise a further oligonucleotide which has a region of complementarity with the single stranded oligonucleotide of five or more consecutive nucleobases, such as eight or more, or 12 or more of more consecutive nucleobases. It is considered that the further oligonucleotide is not covalently linked to the single stranded oligonucleotide.
  • the LNA units may be replaced with other nucleotide analogues, such as those referred to herein.
  • X may, therefore be selected from the group consisting of 2′-O-alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA unit, PNA unit, HNA unit, INA unit.
  • x is preferably DNA or RNA, most preferably DNA.
  • the oligonucleotides of the invention are modified in positions 3 to 8, counting from the 3′ end.
  • the design of this sequence may be defined by the number of non-LNA units present or by the number of LNA units present.
  • at least one, such as one, of the nucleotides in positions three to eight, counting from the 3′ end is a non-LNA unit.
  • at least two, such as two, of the nucleotides in positions three to eight, counting from the 3′ end are non-LNA units.
  • at least three, such as three, of the nucleotides in positions three to eight, counting from the 3′ end are non-LNA units.
  • At least four, such as four, of the nucleotides in positions three to eight, counting from the 3′ end, are non-LNA units.
  • at least five, such as five, of the nucleotides in positions three to eight, counting from the 3′ end are non-LNA units.
  • all six nucleotides in positions three to eight, counting from the 3′ end are non-LNA units.
  • said non-LNA unit is a DNA unit.
  • the oligonucleotide according to the invention comprises at least one LNA unit in positions three to eight, counting from the 3′ end.
  • the oligonucleotide according to the present invention comprises one LNA unit in positions three to eight, counting from the 3′ end.
  • the substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end may be selected from the group consisting of Xxxxxx, xXxxxx, xxXxxx, xxxXxx, xxxxXx and xxxxxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the oligonucleotide according to the present invention comprises at least two LNA units in positions three to eight, counting from the 3′ end. In an embodiment thereof, the oligonucleotide according to the present invention comprises two LNA units in positions three to eight, counting from the 3′ end.
  • substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end may be selected from the group consisting of XXxxxx, XxXxxx, XxxXxx, XxxxXx, XxxxxX, xXxXxx, xXxxxX, xxXXxx, xxXxXx, xxXxxX, xxxXXx, xxxXxX and xxxxXX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end is selected from the group consisting of XxXxxx, XxxXxx, XxxxXx, XxxxxX, xXxxXx, xXxxxX, xxXxXx, xxXxxX and xxxXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end is selected from the group consisting of xXxXxx, xXxxXx, xXxxxX, xxXxXx, xxXxxX and xxxXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end is selected from the group consisting of xXxXxx, xXxxXx and xxXxXx, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end is xXxXxx, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the oligonucleotide according to the present invention comprises at least three LNA units in positions three to eight, counting from the 3′ end. In an embodiment thereof, the oligonucleotide according to the present invention comprises three LNA units in positions three to eight, counting from the 3′ end.
  • substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end may be selected from the group consisting of XXXxxx, xXXXxx, xxXXXx, xxxXXX, XXxXxx, XXxxxX, xXXxXx, xXXxxX, xxXXxX, XxXXxx, XxxXXX, XxxxXX, XxxxXX, xXxXXx, xXxxXXX, xxXXX, xXxXxX and XxXxXx, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end is selected from the group consisting of XXxXxx, XXxxXx, XXxxxX, xXXxXx, xXXxxX, xxXXxX, XxxXXx, XxxxXX, xXxXXx, xXxxXX, xxXxXX, xXxXxX and XxXxXx, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end is selected from the group consisting of xXXxXx, xXXxxX, xxXXxX, xXxXXx, xXxxXX, xxXxXX and xXxXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end is xXxXxX or XxXxXx, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end is xXxXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the oligonucleotide according to the present invention comprises at least four LNA units in positions three to eight, counting from the 3′ end. In an embodiment thereof, the oligonucleotide according to the present invention comprises four LNA units in positions three to eight, counting from the 3′ end.
  • substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end may be selected from the group consisting of xxXXXX, xXxXXX, xXXxXX, xXXXxX, xXXXx, XxxXXX, XxXxX, XxXXxX, XxXXx, XXxxXX, XXxXxX, XXxXx, XXxxX, XXXxXxX, XXxXx, XXxxX, XXXxXx and XXXXxx, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the oligonucleotide according to the present invention comprises at least five LNA units in positions three to eight, counting from the 3′ end. In an embodiment thereof, the oligonucleotide according to the present invention comprises five LNA units in positions three to eight, counting from the 3′ end.
  • the substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end may be selected from the group consisting of xXXXXX, XxXXXX, XXxXXX, XXXxXX, XXXxX and XXXXx, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the oligonucleotide according to the present invention comprises one or two LNA units in positions three to eight, counting from the 3′ end. This is considered advantageous for the stability of the A-helix formed by the oligo:microRNA duplex, a duplex resembling an RNA:RNA duplex in structure.
  • said non-LNA unit is a DNA unit.
  • the length of the oligonucleotides of the invention need not match the length of the target microRNAs exactly. Accordingly, the length of the oligonucleotides of the invention may vary. Indeed it is considered advantageous to have short oligonucleotides, such as between 10-17 or 10-16 nucleobases.
  • the oligonucleotide according to the present has a length of from 8 to 24 nucleotides, such as 10 to 24, between 12 to 24 nucleotides, such as a length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, preferably a length of from 10-22, such as between 12 to 22 nucleotides, such as a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 nucleotides, more preferably a length of from 10-20, such as between 12 to 20 nucleotides, such as a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides, even more preferably a length of from 10 to 19, such as between 12 to 19 nucleotides, such as a length of 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides, e.g.
  • the substitution pattern for the nucleotides from position 11, counting from the 3′ end, to the 5′ end may include nucleotide analogue units (such as LNA) or it may not.
  • the oligonucleotide according to the present invention comprises at least one nucleotide analogue unit (such as LNA), such as one nucleotide analogue unit, from position 11, counting from the 3′ end, to the 5′ end.
  • the oligonucleotide according to the present invention comprises at least two nucleotide analogue units, such as LNA units, such as two nucleotide analogue units, from position 11, counting from the 3′ end, to the 5′ end.
  • the LNA units may be replaced with other nucleotide analogues, such as those referred to herein.
  • X may, therefore be selected from the group consisting of 2′-O-alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA unit, PNA unit, HNA unit, INA unit.
  • x is preferably DNA or RNA, most preferably DNA.
  • the oligonucleotide according to the present invention has the following substitution pattern, which is repeated from nucleotide eleven, counting from the 3′ end, to the 5′ end: xXxX or XxXx, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the oligonucleotide according to the present invention has the following substitution pattern, which is repeated from nucleotide eleven, counting from the 3′ end, to the 5′ end: XxxXxx, xXxxXx or xxXxxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the oligonucleotide according to the present invention has the following substitution pattern, which is repeated from nucleotide eleven, counting from the 3′ end, to the 5′ end: XxxxXxxx, xXxxxXxx, xxXxxxXx or xxxXxxxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the specific substitution pattern for the nucleotides from position 11, counting from the 3′ end, to the 5′ end depends on the number of nucleotides in the oligonucleotides according to the present invention.
  • the oligonucleotide according to the present invention contains 12 nucleotides and the substitution pattern for positions 11 to 12, counting from the 3′ end, is selected from the group consisting of xX and Xx, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern for positions 11 to 12, counting from the 3′ end is xX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern is xx.
  • the oligonucleotide according to the present invention contains 13 nucleotides and the substitution pattern for positions 11 to 13, counting from the 3′ end, is selected from the group consisting of Xxx, xXx, xxX, XXx, XxX, xXX and XXX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern for positions 11 to 13, counting from the 3′ end is selected from the group consisting of xXx, xxX and xXX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern for positions 11 to 13, counting from the 3′ end is xxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern is xxx.
  • the oligonucleotide according to the present invention contains 14 nucleotides and the substitution pattern for positions 11 to 14, counting from the 3′ end, is selected from the group consisting of Xxxx, xXxx, xxXx, xxxX, XxXx, XxxX, xXXx, xXxX and xxXX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern for positions 11 to 14, counting from the 3′ end is selected from the group consisting of xXxx, xxXx, xxxX, xXxX and xxXX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern for positions 11 to 14, counting from the 3′ end is xXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • no LNA units are present in positions 11 to 14, counting from the 3′ end, i.e. the substitution pattern is xxxx
  • the oligonucleotide according to the present invention contains 15 nucleotides and the substitution pattern for positions 11 to 15, counting from the 3′ end, is selected from the group consisting of Xxxxx, xXxxx, xxXxx, xxxXx, XxXxx, XxxxX, xXXxx, xXxXx, xXxxX, xxXXx, xxXxX, xxxXX and XxXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern for positions 11 to 15, counting from the 3′ end is selected from the group consisting of xxXxx, XxXxx, XxxXx, xXxXx, xXxxX and xxXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern for positions 11 to 15, counting from the 3′ end is selected from the group consisting of xxXxx, xXxXx, xXxxX and xxXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern for positions 11 to 15, counting from the 3′ end is selected from the group consisting of xXxxX and xxXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern for positions 11 to 15, counting from the 3′ end is xxXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern is xxxxx
  • the oligonucleotide according to the present invention contains 16 nucleotides and the substitution pattern for positions 11 to 16, counting from the 3′ end, is selected from the group consisting of Xxxxxx, xXxxxx, xxXxxx, xxxxXx, xxxxxX, XxXxxx, XxxxxxXx, XxxxxX, xXXxxx, xXxXxx, xXxxxX, xXxxxX, xxXxxX, xxXXx, xxxXxX, xxxxXX, xxxXXx, xxxXxX, xxxxXX, XXxxx, XXXxx, XXXxx, XXXxx, XXXxx,
  • the substitution pattern for positions 11 to 16, counting from the 3′ end is selected from the group consisting of XxxXxx, xXxXxx, xXxxXx, xxXxXx, xxXxxX, XxXxXx, XxXxxX, XxxXxX, xXxXxX, xXxxXX and xxXxXX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern for positions 11 to 16, counting from the 3′ end is selected from the group consisting of xXxXxx, xXxxXx, xxXxXx, xxXxxX, xXxXxX, xXxxXX and xxXxXX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern for positions 11 to 16, counting from the 3′ end is selected from the group consisting of xxXxxX, xXxXxX, xXxxXX and xxXxXX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern for positions 11 to 16, counting from the 3′ end is selected from the group consisting of xxXxxX and xXxXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern for positions 11 to 16, counting from the 3′ end is xxXxxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the substitution pattern is xxxxxx
  • the oligonucleotide according to the present invention contains an LNA unit at the 5′ end. In another preferred embodiment, the oligonucleotide according to the present invention contains an LNA unit at the first two positions, counting from the 5′ end.
  • the oligonucleotide according to the present invention contains 13 nucleotides and the substitution pattern, starting from the 3′ end, is XXxXxXxxXXxxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the preferred sequence for this embodiment, starting from the 3′ end, is CCtCaCacTGttA, wherein a capital letter denotes a nitrogenous base in an LNA-unit and a small letter denotes a nitrogenous base in a non-LNA unit.
  • the oligonucleotide according to the present invention contains 15 nucleotides and the substitution pattern, starting from the 3′ end, is XXxXxXxxXXxxXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • the preferred sequence for this embodiment, starting from the 3′ end, is CCtCaCacTGttAcC, wherein a capital letter denotes a nitrogenous base in an LNA-unit and a small letter denotes a nitrogenous base in a non-LNA unit.
  • Typical internucleoside linkage groups in oligonucleotides are phosphate groups, but these may be replaced by internucleoside linkage groups differing from phosphate.
  • the oligonucleotide of the invention is modified in its internucleoside linkage group structure, i.e. the modified oligonucleotide comprises an internucleoside linkage group which differs from phosphate. Accordingly, in a preferred embodiment, the oligonucleotide according to the present invention comprises at least one internucleoside linkage group which differs from phosphate.
  • internucleoside linkage groups which differ from phosphate (—O—P(O) 2 —O—) include —O—P(O,S)—O—, —O—P(S) 2 —O—, —S—P(O) 2 —O—, —S—P(O,S)—O—, —S—P(S) 2 —O—, —O—P(O) 2 —S—, —O—P(O,S)—S—, —O—PO(R H )—O—, O—PO(OCH 3 )—O—, —O—PO(NR H )—O—, —O—PO(OCH 2 CH 2 S—R)—O—, —O—PO(BH 3 )—O—, —O—PO(NHR H )—O—, —O—P(O) 2 —NR H —, —NR H —, —NR H —
  • the internucleoside linkage group is preferably a phosphorothioate group (—O—P(O,S)—O—).
  • all internucleoside linkage groups of the oligonucleotides according to the present invention are phosphorothioate.
  • the LNA Unit The LNA Unit
  • the LNA unit has the general chemical structure shown in Scheme 1 below:
  • r is 1 or 2
  • a preferred LNA unit has the chemical structure shown in Scheme 2 below:
  • the LNA units incorporated in the oligonucleotides of the invention are independently selected from the group consisting of thio-LNA units, amino-LNA units and oxy-LNA units.
  • the thio-LNA unit may have the chemical structure shown in Scheme 3 below:
  • the thio-LNA unit is in its beta-D-form, i.e. having the structure shown in 3A above.
  • amino-LNA unit may have the chemical structure shown in Scheme 4 below:
  • the amino-LNA unit is in its beta-D-form, i.e. having the structure shown in 4A above.
  • the oxy-LNA unit may have the chemical structure shown in Scheme 5 below:
  • the oxy-LNA unit is in its beta-D-form, i.e. having the structure shown in 5A above.
  • B is a nitrogenous base which may be of natural or non-natural origin.
  • nitrogenous bases include adenine (A), cytosine (C), 5-methylcytosine ( Me C), isocytosine, pseudoisocytosine, guanine (G), thymine (T), uracil (U), 5-bromouracil, 5-propynyluracil, 5-propyny-6, 5-methylthiazoleuracil, 6-aminopurine, 2-aminopurine, inosine, 2,6-diaminopurine, 7-propyne-7-deazaadenine, 7-propyne-7-deazaguanine and 2-chloro-6-aminopurine.
  • terminal groups include terminal groups selected from the group consisting of hydrogen, azido, halogen, cyano, nitro, hydroxy, Prot-O—, mercapto, Prot-S—, C 1-6 -alkylthio, amino, Prot-N(R H )—, mono- or di(C 1-6 -alkyl)amino, optionally substituted C 1-6 -alkoxy, optionally substituted C 1-6 -alkyl, optionally substituted C 2-6 -alkenyl, optionally substituted C 2-6 -alkenyloxy, optionally substituted C 2-6 -alkynyl, optionally substituted C 2-6 -alkynyloxy, monophosphate including protected monophosphate, monothiophosphate including protected monothiophosphate, diphosphate including protected diphosphate, dithiophosphate including protected dithiophosphate, triphosphate including protected triphosphate, trithiophosphate including protected trithiophosphate, where Prot is a protection group for
  • phosphate protection groups include S-acetylthioethyl (SATE) and S-pivaloylthioethyl (t-butyl-SATE).
  • terminal groups include DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, ligands, carboxy, sulphono, hydroxymethyl, Prot-O—CH 2 —, Act-O—CH 2 —, aminomethyl, Prot-N(R H )—CH 2 —, Act-N(R H )—CH 2 —, carboxymethyl, sulphonomethyl, where Prot is a protection group for —OH, —SH and —NH(R H ), and Act is an activation group for —OH, —SH, and —NH(R H ), and R H is hydrogen or C 1-6 -alkyl.
  • protection groups for —OH and —SH groups include substituted trityl, such as 4,4′-dimethoxytrityloxy (DMT), 4-monomethoxytrityloxy (MMT); trityloxy, optionally substituted 9-(9-phenyl)xanthenyloxy (pixyl), optionally substituted methoxytetrahydro-pyranyloxy (mthp); silyloxy, such as trimethylsilyloxy (TMS), triisopropylsilyloxy (TIPS), tert-butyldimethylsilyloxy (TBDMS), triethylsilyloxy, phenyldimethylsilyloxy; tert-butylethers; acetals (including two hydroxy groups); acyloxy, such as acetyl or halogen-substituted acetyls, e.g.
  • DMT 4,4′-dimethoxytrityloxy
  • amine protection groups include fluorenylmethoxycarbonylamino (Fmoc), tert-butyloxycarbonylamino (BOC), trifluoroacetylamino, allyloxycarbonylamino (alloc, AOC), Z-benzyloxycarbonylamino (Cbz), substituted benzyloxycarbonylamino, such as 2-chloro benzyloxycarbonylamino (2-ClZ), monomethoxytritylamino (MMT), dimethoxytritylamino (DMT), phthaloylamino, and 9-(9-phenyl)xanthenylamino (pixyl).
  • Fmoc fluorenylmethoxycarbonylamino
  • BOC tert-butyloxycarbonylamino
  • trifluoroacetylamino allyloxycarbonylamino (alloc, AOC)
  • the term “phosphoramidite” means a group of the formula —P(OR x )—N(R y ) 2 , wherein R x designates an optionally substituted alkyl group, e.g. methyl, 2-cyanoethyl, or benzyl, and each of R y designates optionally substituted alkyl groups, e.g. 5 ethyl or isopropyl, or the group —N(R y ) 2 forms a morpholino group (—N(CH 2 CH 2 ) 2 O).
  • R x preferably designates 2-cyanoethyl and the two R y are preferably identical and designates isopropyl. Accordingly, a particularly preferred phosphoramidite is N,N-diisopropyl-O-(2-cyanoethyl)phosphoramidite.
  • the most preferred terminal groups are hydroxy, mercapto and amino, in particular hydroxy.
  • oligonucleotide such as those used in pharmaceutical compositions, as compared to prior art type of molecules.
  • Oligo #, target microRNA, oligo sequence Design SEQ ID target: hsa-miR-122a MIMAT0000421 uggagugugacaaugguguuugu SEQ ID NO 535 screened in HUH-7 cell line expressing miR-122 3962: miR-122 5′-ACAAacaccattgtcacacTCCA-3′ Full complement, gap SEQ ID NO 536 3965: miR-122 5′-acaaacACCATTGTcacactcca-3′ Full complement, block SEQ ID NO 537 3972: miR-122 5′-acAaaCacCatTgtCacActCca-3′ Full complement, LNA_3 SEQ ID NO 538 3549 (3649): miR-122 5′-CcAttGTcaCaCtCC-3′ New design SEQ ID NO 539 3975: miR-122 5′-CcAtTGTcaCACtCC-3′ Enhanced new design SEQ ID NO 540 3975′:
  • the LNA cytosines may optionally be methylated).
  • Capital letters followed by a superscript M refer to 2′OME RNA units,
  • Capital letters followed by a superscript F refer to 2′fluoro DNA units, lowercase letter refer to DNA.
  • the above oligos may in one embodiment be entirely phosphorothioate, but other nucleobase linkages as herein described bay be used. In one embodiment the nucleobase linkages are all phosphodiester. It is considered that for use within the brain/spinal cord it is preferable to use phosphodiester linkages, for example for the use of antimiRs targeting miR21.
  • Table 2 below provides non-limiting examples of oligonucleotide designs against known human microRNA sequences in miRBase microRNA database version 8.1.
  • oligonucleotides according to the invention may, in one embodiment, have a sequence of nucleobases 5′-3′ selected form the group consisting of:
  • L LNA unit
  • d DNA units
  • M 2′MOE RNA
  • F 2′Fluoro and residues in brackets are optional
  • the invention also provides for conjugates comprising the oligonucleotide according to the invention.
  • the oligomeric compound is linked to ligands/conjugates, which may be used, e.g. to increase the cellular uptake of antisense oligonucleotides.
  • This conjugation can take place at the terminal positions 5′/3′-OH but the ligands may also take place at the sugars and/or the bases.
  • the growth factor to which the antisense oligonucleotide may be conjugated may comprise transferrin or folate. Transferrin-polylysine-oligonucleotide complexes or folate-polylysine-oligonucleotide complexes may be prepared for uptake by cells expressing high levels of transferrin or folate receptor.
  • conjugates/ligands are cholesterol moieties, duplex intercalators such as acridine, poly-L-lysine, “end-capping” with one or more nuclease-resistant linkage groups such as phosphoromonothioate, and the like.
  • the invention also provides for a conjugate comprising the compound according to the invention as herein described, and at least one non-nucleotide or non-polynucleotide moiety covalently attached to said compound. Therefore, in one embodiment where the compound of the invention consists of s specified nucleic acid, as herein disclosed, the compound may also comprise at least one non-nucleotide or non-polynucleotide moiety (e.g. not comprising one or more nucleotides or nucleotide analogues) covalently attached to said compound.
  • the non-nucleobase moiety may for instance be or comprise a sterol such as cholesterol.
  • the oligonucleotide of the invention such as the oligonucleotide used in pharmaceutical (therapeutic) formulations may comprise further non-nucleobase components, such as the conjugates herein defined.
  • the oligonucleotides of the invention will constitute suitable drugs with improved properties.
  • the design of a potent and safe drug requires the fine-tuning of various parameters such as affinity/specificity, stability in biological fluids, cellular uptake, mode of action, pharmacokinetic properties and toxicity.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an oligonucleotide according to the invention and a pharmaceutically acceptable diluent, carrier or adjuvant.
  • a pharmaceutically acceptable diluent, carrier or adjuvant is saline of buffered saline.
  • the present invention relates to an oligonucleotide according to the present invention for use as a medicament.
  • dosing is dependent on severity and responsiveness of the disease state to be treated, and the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved.
  • Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient.
  • Optimum dosages may vary depending on the relative potency of individual oligonucleotides. Generally it can be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ⁇ g to 1 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 10 years or by continuous infusion for hours up to several months. The repetition rates for dosing can be estimated based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state.
  • the invention also relates to a pharmaceutical composition, which comprises at least one oligonucleotide of the invention as an active ingredient.
  • the pharmaceutical composition according to the invention optionally comprises a pharmaceutical carrier, and that the pharmaceutical composition optionally comprises further compounds, such as chemotherapeutic compounds, anti-inflammatory compounds, antiviral compounds and/or immuno-modulating compounds.
  • oligonucleotides of the invention can be used “as is” or in form of a variety of pharmaceutically acceptable salts.
  • pharmaceutically acceptable salts refers to salts that retain the desired biological activity of the herein-identified oligonucleotides and exhibit minimal undesired toxicological effects.
  • Non-limiting examples of such salts can be formed with organic amino acid and base addition salts formed with metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, and the like, or with a cation formed from ammonia, N,N-dibenzylethylene-diamine, D-glucosamine, tetraethylammonium, or ethylenediamine.
  • metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, and the like, or with a cation formed from ammonia, N,N-dibenzylethylene-diamine, D-glucosamine, tetraethylammonium, or ethylenediamine.
  • the oligonucleotide may be in the form of a pro-drug. Oligonucleotides are by virtue negatively charged ions. Due to the lipophilic nature of cell membranes the cellular uptake of oligonucleotides are reduced compared to neutral or lipophilic equivalents. This polarity “hindrance” can be avoided by using the pro-drug approach (see e.g. Crooke, R. M. (1998) in Crooke, S. T. Antisense research and Application. Springer-Verlag, Berlin, Germany, vol. 131, pp. 103-140). Pharmaceutically acceptable binding agents and adjuvants may comprise part of the formulated drug.
  • compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Delivery of drug to tumour tissue may be enhanced by carrier-mediated delivery including, but not limited to, cationic liposomes, cyclodextrins, porphyrin derivatives, branched chain dendrimers, polyethylen-imine polymers, nanoparticles and microspheres (Dass C R. J Pharm Pharmacol 2002; 54(1):3-27).
  • compositions of the present invention may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • the compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels and suppositories.
  • the compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethyl-cellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the compounds of the invention may also be conjugated to active drug substances, for example, aspirin, ibuprofen, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
  • compositions of the invention may contain one or more oligonucleotide compounds, targeted to a first microRNA and one or more additional oligonucleotide compounds targeted to a second microRNA target. Two or more combined compounds may be used together or sequentially.
  • therapeutic methods of the invention include administration of a therapeutically effective amount of an oligonucleotide to a mammal, particularly a human.
  • the present invention provides pharmaceutical compositions containing (a) one or more compounds of the invention, and (b) one or more chemotherapeutic agents.
  • chemotherapeutic agents When used with the compounds of the invention, such chemotherapeutic agents may be used individually, sequentially, or in combination with one or more other such chemotherapeutic agents or in combination with radiotherapy. All chemotherapeutic agents known to a person skilled in the art are here incorporated as combination treatments with compound according to the invention.
  • anti-inflammatory drugs including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, antiviral drugs, and immuno-modulating drugs may also be combined in compositions of the invention. Two or more combined compounds may be used together or sequentially.
  • microRNA Possible medical indications miR-21 Glioblastoma, breast cancer miR-122 hypercholesterolemia, hepatitis C, hemochromatosis miR-19b lymphoma and other tumour types miR-155 lymphoma, breast and lung cancer miR-375 diabetes, metabolic disorders miR-181 myoblast differentiation, auto immune disorders
  • TPM1 Tumor suppressor gene tropomysin 1
  • mtpn Myotrophin
  • the present invention relates to the use of an oligonucleotide according to the invention for the manufacture of a medicament for the treatment of a disease selected from the group consisting of: atherosclerosis, hypercholesterolemia and hyperlipidemia; cancer, glioblastoma, breast cancer, lymphoma, lung cancer; diabetes, metabolic disorders; myoblast differentiation; immune disorders.
  • a disease selected from the group consisting of: atherosclerosis, hypercholesterolemia and hyperlipidemia; cancer, glioblastoma, breast cancer, lymphoma, lung cancer; diabetes, metabolic disorders; myoblast differentiation; immune disorders.
  • the invention further refers to an oligonucleotides according to the invention for the use in the treatment of from a disease selected from the group consisting of: atherosclerosis, hypercholesterolemia and hyperlipidemia; cancer, glioblastoma, breast cancer, lymphoma, lung cancer; diabetes, metabolic disorders; myoblast differentiation; immune disorders.
  • a disease selected from the group consisting of: atherosclerosis, hypercholesterolemia and hyperlipidemia; cancer, glioblastoma, breast cancer, lymphoma, lung cancer; diabetes, metabolic disorders; myoblast differentiation; immune disorders.
  • the invention provides for a method of treating a subject suffering from a disease or condition selected from from the group consisting of: atherosclerosis, hypercholesterolemia and hyperlipidemia; cancer, glioblastoma, breast cancer, lymphoma, lung cancer; diabetes, metabolic disorders; myoblast differentiation; immune disorders, the method comprising the step of administering an oligonucleotide or pharmaceutical composition of the invention to the subject in need thereof.
  • a disease or condition selected from from the group consisting of: atherosclerosis, hypercholesterolemia and hyperlipidemia; cancer, glioblastoma, breast cancer, lymphoma, lung cancer; diabetes, metabolic disorders; myoblast differentiation; immune disorders
  • the present invention relates to the use of an oligonucleotide according to the invention for the manufacture of a medicament for the treatment of cancer.
  • the present invention concerns a method for treatment of, or prophylaxis against, cancer, said method comprising administering an oligonucleotide of the invention or a pharmaceutical composition of the invention to a patient in need thereof.
  • Such cancers may include lymphoreticular neoplasia, lymphoblastic leukemia, brain tumors, gastric tumors, plasmacytomas, multiple myeloma, leukemia, connective tissue tumors, lymphomas, and solid tumors.
  • said cancer may suitably be in the form of a solid tumor.
  • said cancer in the method for treating cancer disclosed herein said cancer may suitably be in the form of a solid tumor.
  • said cancer is also suitably a carcinoma.
  • the carcinoma is typically selected from the group consisting of malignant melanoma, basal cell carcinoma, ovarian carcinoma, breast carcinoma, non-small cell lung cancer, renal cell carcinoma, bladder carcinoma, recurrent superficial bladder cancer, stomach carcinoma, prostatic carcinoma, pancreatic carcinoma, lung carcinoma, cervical carcinoma, cervical dysplasia, laryngeal papillomatosis, colon carcinoma, colorectal carcinoma and carcinoid tumors. More typically, said carcinoma is selected from the group consisting of malignant melanoma, non-small cell lung cancer, breast carcinoma, colon carcinoma and renal cell carcinoma.
  • the malignant melanoma is typically selected from the group consisting of superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral melagnoma, amelanotic melanoma and desmoplastic melanoma.
  • the cancer may suitably be a sarcoma.
  • the sarcoma is typically in the form selected from the group consisting of osteosarcoma, Ewing's sarcoma, chondrosarcoma, malignant fibrous histiocytoma, fibrosarcoma and Kaposi's sarcoma.
  • the cancer may suitably be a glioma.
  • a further embodiment is directed to the use of an oligonucleotide according to the invention for the manufacture of a medicament for the treatment of cancer, wherein said medicament further comprises a chemotherapeutic agent selected from the group consisting of adrenocorticosteroids, such as prednisone, dexamethasone or decadron; altretamine (hexalen, hexamethylmelamine (HMM)); amifostine (ethyol); aminoglutethimide (cytadren); amsacrine (M-AMSA); anastrozole (arimidex); androgens, such as testosterone; asparaginase (elspar); bacillus calmette-gurin; bicalutamide (casodex); bleomycin (blenoxane); busulfan (myleran); carboplatin (paraplatin); carmustine (BCNU, BiCNU); chlorambucil (leukeran); chlorodeoxyadenosine (2-C
  • the invention is further directed to the use of an oligonucleotide according to the invention for the manufacture of a medicament for the treatment of cancer, wherein said treatment further comprises the administration of a further chemotherapeutic agent selected from the group consisting of adrenocorticosteroids, such as prednisone, dexamethasone or decadron; altretamine (hexalen, hexamethylmelamine (HMM)); amifostine (ethyol); aminoglutethimide (cytadren); amsacrine (M-AMSA); anastrozole (arimidex); androgens, such as testosterone; asparaginase (elspar); bacillus calmette-gurin; bicalutamide (casodex); bleomycin (blenoxane); busulfan (myleran); carboplatin (paraplatin); carmustine (BCNU, BiCNU); chlorambucil (leukeran); chlorodeoxyaden
  • the invention is furthermore directed to a method for treating cancer, said method comprising administering an oligonucleotide of the invention or a pharmaceutical composition according to the invention to a patient in need thereof and further comprising the administration of a further chemotherapeutic agent.
  • Said further administration may be such that the further chemotherapeutic agent is conjugated to the compound of the invention, is present in the pharmaceutical composition, or is administered in a separate formulation.
  • the compounds of the invention may be broadly applicable to a broad range of infectious diseases, such as diphtheria, tetanus, pertussis, polio, hepatitis B, hepatitis C, hemophilus influenza, measles, mumps, and rubella.
  • infectious diseases such as diphtheria, tetanus, pertussis, polio, hepatitis B, hepatitis C, hemophilus influenza, measles, mumps, and rubella.
  • Hsa-miR122 is indicated in hepatitis C infection and as such oligonucleotides according to the invention which target miR-122 may be used to treat Hepatitis C infection.
  • the present invention relates the use of an oligonucleotide according to the invention for the manufacture of a medicament for the treatment of an infectious disease, as well as to a method for treating an infectious disease, said method comprising administering an oligonucleotide according to the invention or a pharmaceutical composition according to the invention to a patient in need thereof.
  • the inflammatory response is an essential mechanism of defense of the organism against the attack of infectious agents, and it is also implicated in the pathogenesis of many acute and chronic diseases, including autoimmune disorders.
  • Inflammation is a complex process normally triggered by tissue injury that includes activation of a large array of enzymes, the increase in vascular permeability and extravasation of blood fluids, cell migration and release of chemical mediators, all aimed to both destroy and repair the injured tissue.
  • the present invention relates to the use of an oligonucleotide according to the invention for the manufacture of a medicament for the treatment of an inflammatory disease, as well as to a method for treating an inflammatory disease, said method comprising administering an oligonucleotide according to the invention or a pharmaceutical composition according to the invention to a patient in need thereof.
  • the inflammatory disease is a rheumatic disease and/or a connective tissue diseases, such as rheumatoid arthritis, systemic lupus erythematous (SLE) or Lupus, scleroderma, polymyositis, inflammatory bowel disease, dermatomyositis, ulcerative colitis, Crohn's disease, vasculitis, psoriatic arthritis, exfoliative psoriatic dermatitis, pemphigus vulgaris and Sjorgren's syndrome, in particular inflammatory bowel disease and Crohn's disease.
  • SLE systemic lupus erythematous
  • Lupus scleroderma
  • polymyositis inflammatory bowel disease
  • dermatomyositis ulcerative colitis
  • Crohn's disease vasculitis
  • psoriatic arthritis exfoliative psoriatic dermatitis
  • pemphigus vulgaris and Sjorgren's syndrome
  • the inflammatory disease may be a non-rheumatic inflammation, like bursitis, synovitis, capsulitis, tendinitis and/or other inflammatory lesions of traumatic and/or university origin.
  • a metabolic disease is a disorder caused by the accumulation of chemicals produced naturally in the body. These diseases are usually serious, some even life threatening. Others may slow physical development or cause mental retardation. Most infants with these disorders, at first, show no obvious signs of disease. Proper screening at birth can often discover these problems. With early diagnosis and treatment, metabolic diseases can often be managed effectively.
  • the present invention relates to the use of an oligonucleotide according to the invention or a conjugate thereof for the manufacture of a medicament for the treatment of a metabolic disease, as well as to a method for treating a metabolic disease, said method comprising administering an oligonucleotide according to the invention or a conjugate thereof, or a pharmaceutical composition according to the invention to a patient in need thereof.
  • the metabolic disease is selected from the group consisting of Amyloidosis, Biotinidase, OMIM (Online Mendelian Inheritance in Man), Crigler Najjar Syndrome, Diabetes, Fabry Support & Information Group, Fatty acid Oxidation Disorders, Galactosemia, Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency, Glutaric aciduria, International Organization of Glutaric Acidemia, Glutaric Acidemia Type I, Glutaric Acidemia, Type II, Glutaric Acidemia Type I, Glutaric Acidemia Type-II, F-HYPDRR-Familial Hypophosphatemia, Vitamin D Resistant Rickets, Krabbe Disease, Long chain 3 hydroxyacyl CoA dehydrogenase deficiency (LCHAD), Mannosidosis Group, Maple Syrup Urine Disease, Mitochondrial disorders, Mucopolysaccharidosis Syndromes: Niemann Pick, Organic acidemias,
  • the present invention relates to the use of an oligonucleotide according to the invention or a conjugate thereof for the manufacture of a medicament for the treatment of a liver disorder, as well as to a method for treating a liver disorder, said method comprising administering an oligonucleotide according to the invention or a conjugate thereof, or a pharmaceutical composition according to the invention to a patient in need thereof.
  • the liver disorder is selected from the group consisting of Biliary Atresia, Alagille Syndrome, Alpha-1 Antitrypsin, Tyrosinemia, Neonatal Hepatitis, and Wilson Disease.
  • the oligonucleotides of the present invention can be utilized for as research reagents for diagnostics, therapeutics and prophylaxis.
  • the oligonucleotide may be used to specifically inhibit the synthesis of target genes in cells and experimental animals thereby facilitating functional analysis of the target or an appraisal of its usefulness as a target for therapeutic intervention.
  • the oligonucleotides may be used to detect and quantitate target expression in cell and tissues by Northern blotting, in-situ hybridisation or similar techniques.
  • an animal or a human, suspected of having a disease or disorder, which can be treated by modulating the expression of target is treated by administering the oligonucleotide compounds in accordance with this invention.
  • a LNA-antimiRTM such as SPC3372
  • targeting miR-122a reduces plasma cholesterol levels. Therefore, another aspect of the invention is use of the above described oligonucleotides targeting miR-122a as medicine. Still another aspect of the invention is use of the above described oligonucleotides targeting miR-122a for the preparation of a medicament for treatment of increased plasma cholesterol levels. The skilled man will appreciate that increased plasma cholesterol levels is undesirable as it increases the risk of various conditions, e.g. atherosclerosis. Still another aspect of the invention is use of the above described oligonucleotides targeting miR-122a for upregulating the mRNA levels of Nrdg3, Aldo A, Bckdk or CD320.
  • oligonucleotide having a length of from 12 to 26 nucleotides, wherein
  • oligonucleotide according to claim 1 wherein the ninth nucleotide, counting from the 3′ end, is an LNA unit.
  • oligonucleotide according to any of embodiments 1-4, wherein said oligonucleotide comprises at least one LNA unit in positions three to eight, counting from the 3′ end.
  • oligonucleotide according to embodiment 5 wherein said oligonucleotide comprises one LNA unit in positions three to eight, counting from the 3′ end.
  • oligonucleotide according to embodiment 5 wherein said oligonucleotide comprises at least two LNA units in positions three to eight, counting from the 3′ end.
  • oligonucleotide according to embodiment 8 wherein said oligonucleotide comprises two LNA units in positions three to eight, counting from the 3′ end.
  • substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end is selected from the group consisting of XXxxxx, XxXxxx, XxxXxx, XxxxXx, xXxXxx, xXxxxX, xxXXxx, xxXxXx, xxXxxX, xxxXXx, xxxXxX and xxxxXX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end is selected from the group consisting of XxXxxx, XxxXxx, XxxxXx, XxxxxXx, xXxxxX, xxXxXx, xxXxxX and xxxXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • oligonucleotide according to embodiment 11, wherein the substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end, is selected from the group consisting of xXxXxx, xXxxXx, xXxxxX, xxXxXx, xxXxxX and xxxXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • oligonucleotide according to embodiment 12, wherein the substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end, is selected from the group consisting of xXxXxx, xXxxXx and xxXxXx, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • oligonucleotide according to embodiment 5 wherein said oligonucleotide comprises at least three LNA units in positions three to eight, counting from the 3′ end.
  • substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end is selected from the group consisting of XXXxxx, xXXXxx, xxXXXx, xxxXXX, XXxXxx, XXxxxX, xXXxXx, xXXxxX, xxXXxX, XxXXxx, XxxXXx, XxxxXX, XxxxXX, XxxxXX, xXxXXx, xXxxXXX, xxXXX, xXxXxX and XxXxXx, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end is selected from the group consisting of XXxXxx, XXxxXx, XXxxxX, xXXxXx, xXXxxX, xxXXxX, XxxXXx, XxxxXX, xXxXXx, xXxxXX, xxXxXX, xXxXxX and XxXxXx, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end is selected from the group consisting of xXXxXx, xXXxxX, xxXXxX, xXxXXx, xXxxXX, xxXxXX and xXxXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • oligonucleotide according to embodiment 18, wherein the substitution pattern for the nucleotides in positions three to eight, counting from the 3′ end, is xXxXxX or XxXxXx, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • nucleotide has a length of from 12 to 24 nucleotides, such as a length of from 12 to 22 nucleotides, preferably a length of from 12 to 20 nucleotides, such as a length of from 12 to 19 nucleotides, more preferably a length of from 12 to 18 nucleotides, such as a length of from 12 to 17 nucleotides, even more preferably a length of from 12 to 16 nucleotides.
  • oligonucleotide according to any of the preceding embodiments, wherein said oligonucleotide comprises at least one LNA unit, such as one LNA unit, from position 11, counting from the 3′ end, to the 5′ end.
  • oligonucleotide according to any of the preceding embodiments, wherein said oligonucleotide comprises at least two LNA units, such as two LNA units, from position 11, counting from the 3′ end, to the 5′ end.
  • oligonucleotide according to embodiment 28 wherein the substitution pattern for positions 11 to 13, counting from the 3′ end, is xxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • oligonucleotide according to embodiment 32 wherein the substitution pattern for positions 11 to 15, counting from the 3′ end, is xxXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • oligonucleotide according to embodiment 34 wherein the substitution pattern for positions 11 to 16, counting from the 3′ end, is xxXxxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.
  • oligonucleotide according to any of the preceding embodiments, wherein the oligonucleotide comprises at least one internucleoside linkage group which differs from phosphate.
  • LNA units are independently selected from the group consisting of thio-LNA units, amino-LNA units and oxy-LNA units.
  • oligonucleotide for use as a medicament.
  • a pharmaceutical composition comprising an oligonucleotide according to any of embodiments 1-43 and a pharmaceutically acceptable carrier.
  • composition according to embodiment 45 wherein said carrier is saline or buffered saline.
  • a method for the treatment of cancer comprising the step of administering an oligonucleotide according to any of embodiments 1-43 or a composition according to embodiment 45.
  • LNA monomer building blocks and derivatives thereof were prepared following published procedures and references cited therein, see, e.g. WO 03/095467 A1 and D. S. Pedersen, C. Rosenbohm, T. Koch (2002) Preparation of LNA Phosphoramidites, Synthesis 6, 802-808.
  • Oligonucleotides were synthesized using the phosphoramidite approach on an Expedite 8900/MOSS synthesizer (Multiple Oligonucleotide Synthesis System) at 1 ⁇ mol or 15 ⁇ mol scale. For larger scale synthesis an ⁇ kta Oligo Pilot (GE Healthcare) was used. At the end of the synthesis (DMT-on), the oligonucleotides were cleaved from the solid support using aqueous ammonia for 1-2 hours at room temperature, and further deprotected for 4 hours at 65° C. The oligonucleotides were purified by reverse phase HPLC (RP-HPLC). After the removal of the DMT-group, the oligonucleotides were characterized by AE-HPLC, RP-HPLC, and CGE and the molecular mass was further confirmed by ESI-MS. See below for more details.
  • the coupling of phosphoramidites is performed by using a solution of 0.1 M of the 5′-O-DMT-protected amidite in acetonitrile and DCI (4,5-dicyanoimidazole) in acetonitrile (0.25 M) as activator.
  • the thiolation is carried out by using xanthane chloride (0.01 M in acetonitrile:pyridine 10%).
  • the rest of the reagents are the ones typically used for oligonucleotide synthesis.
  • Buffers 0.1 M ammonium acetate pH 8 and acetonitrile
  • Target microRNA miR-122a: 5′-uggagugugacaaugguguuugu-3′ SEQ ID NO: 535 miR-122a 3′ to 5′: 3′-uguuugugguaacagugugaggu-5′ (SEQ ID NO: 535 reverse orientation)
  • the melting temperatures were assessed towards the mature miR-122a sequence, using a synthetic miR-122a RNA oligonucleotide with phosphorothioate linkaged.
  • the LNA anti-miR/miR-122a oligo duplex was diluted to 3 ⁇ M in 500 ⁇ l RNase free H 2 0, which was then mixed with 500 ⁇ l 2 ⁇ dimerization buffer (final oligo/duplex conc. 1.5 ⁇ M, 2 ⁇ Tm buffer: 200 mM NaCl, 0.2 mM EDTA, 20 mM NaP, pH 7.0, DEPC treated to remove RNases). The mix was first heated to 95 degrees for 3 minutes, then allowed to cool at room temperature (RT) for 30 minutes.
  • RT room temperature
  • T m was measured on Lambda 40 UV/VIS Spectrophotometer with peltier temperature programmer PTP6 using PE Templab software (Perkin Elmer). The Temperature was ramped up from 20° C. to 95° C. and then down again to 20° C., continuously recording absorption at 260 nm. First derivative and local maximums of both the melting and annealing was used to assess melting/annealing point (T m ), both should give similar/same T m values. For the first derivative 91 points was used to calculate the slope.
  • the above assay can be used to determine the T m of other oligonucleotides such as the oligonucleotides according to the invention.
  • the T m may be made with a complementary DNA (phosphorothioate linkages) molecule.
  • the T m measured against a DNA complementary molecule is about 10° C. lower than the T m with an equivalent RNA complement.
  • the T m measured using the DNA complement may therefore be used in cases where the duplex has a very high T m .
  • T m assays may be insufficient to determine the T m .
  • the use of a phosphorothioated DNA complementary molecule may further lower the T m .
  • formamide is routine in the analysis of oligonucleotide hybridisation (see Hutton 1977, NAR 4 (10) 3537-3555).
  • the inclusion of 15% formamide typically lowers the T m by about 9° C.
  • the inclusion of 50% formamide typically lowers the T m by about 30° C.
  • an alternative method of determining the T m is to make titrations and run it out on a gel to see single strand versus duplex and by those concentrations and ratios determine Kd (the dissociation constant) which is related to deltaG and also T m .
  • LNA oligonucleotide stability was tested in plasma from human or rats (it could also be mouse, monkey or dog plasma). In 45 ⁇ l plasma, 5 ⁇ l LNA oligonucleotide is added (at a final concentration of 20 ⁇ M). The LNA oligonucleotides are incubated in plasma for times ranging from 0 to 96 hours at 37° C. (the plasma is tested for nuclease activity up to 96 hours and shows no difference in nuclease cleavage-pattern).
  • LNA oligonucleotides on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels.
  • Target can be expressed endogenously or by transient or stable transfection of a nucleic acid encoding said nucleic acid.
  • target nucleic acid can be routinely determined using, for example, Northern blot analysis (including microRNA northern), Quantitative PCR (including microRNA qPCR), Ribonuclease protection assays.
  • Northern blot analysis including microRNA northern
  • Quantitative PCR including microRNA qPCR
  • Ribonuclease protection assays The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen.
  • Cells were cultured in the appropriate medium as described below and maintained at 37° C. at 95-98% humidity and 5% CO 2 . Cells were routinely passaged 2-3 times weekly.
  • the human prostate cancer cell line 15PC3 was kindly donated by Dr. F. Baas, Neurozintuigen Laboratory, AMC, The Netherlands and was cultured in DMEM (Sigma)+10% fetal bovine serum (FBS)+Glutamax I+gentamicin.
  • PC3 The human prostate cancer cell line PC3 was purchased from ATCC and was cultured in F12 Coon's with glutamine (Gibco)+10% FBS+gentamicin.
  • the human melanoma cancer cell line 518A2 was kindly donated by Dr. B. Jansen, Section of experimental Oncology, Molecular Pharmacology, Department of Clinical Pharmacology, University of Vienna and was cultured in DMEM (Sigma)+10% fetal bovine serum (FBS)+Glutamax I+gentamicin.
  • HeLa The cervical carcinoma cell line HeLa was cultured in MEM (Sigma) containing 10% fetal bovine serum gentamicin at 37° C., 95% humidity and 5% CO 2 .
  • MPC-11 The murine multiple myeloma cell line MPC-11 was purchased from ATCC and maintained in DMEM with 4 mM Glutamax+10% Horse Serum.
  • the human prostate cancer cell line DU-145 was purchased from ATCC and maintained in RPMI with Glutamax+10% FBS.
  • RCC-4 ⁇ VHL The human renal cancer cell line RCC4 stably transfected with plasmid expressing VHL or empty plasmid was purchased from ECACC and maintained according to manufacturers instructions.
  • the human renal cell carcinoma cell line 786-0 was purchased from ATCC and maintained according to manufacturers instructions
  • HUVEC The human umbilical vein endothelial cell line HUVEC was purchased from Camcrex and maintained in EGM-2 medium.
  • K562 The human chronic myelogenous leukaemia cell line K562 was purchased from ECACC and maintained in RPMI with Glutamax+10% FBS.
  • U87MG The human glioblastoma cell line U87MG was purchased from ATCC and maintained according to the manufacturers instructions.
  • the murine melanoma cell line B16 was purchased from ATCC and maintained according to the manufacturers instructions.
  • LNCap The human prostate cancer cell line LNCap was purchased from ATCC and maintained in RPMI with Glutamax+10% FBS
  • Huh-7 Human liver, epithelial like cultivated in Eagles MEM with 10% FBS, 2 mM Glutamax I, 1 ⁇ non-essential amino acids, Gentamicin 25 ⁇ g/ml
  • L428 (Deutsche Sammlung für Mikroorganismen (DSM, Braunschwieg, Germany): Human B cell lymphoma maintained in RPMI 1640 supplemented with 10% FCS, L-glutamine and antibiotics.
  • L1236 (Deutsche Sammlung für Mikroorganismen (DSM, Braunschwieg, Germany): Human B cell lymphoma maintained in RPMI 1640 supplemented with 10% FCS, L-glutamine and antibiotics.
  • the miR-122a expressing cell line Huh-7 was transfected with LNA anti-miRs at 1 and 100 nM concentrations according to optimized lipofectamine 2000 (LF2000, Invitrogen) protocol (as follows).
  • Huh-7 cells were cultivated in Eagles MEM with 10% FBS, 2mM Glutamax I, 1 ⁇ non-essential amino acids, Gentamicin 25 ⁇ g/ml. The cells were seeded in 6-well plates (300000 cells per well), in a total vol. of 2.5 ml the day before transfection. At the day of transfection a solution containing LF2000 diluted in Optimem (Invitrogen) was prepared (1.2 ml optimem+3.75 ⁇ l LF2000 per well, final 2.5 pg LF2000/ml, final tot vol 1.5 ml).
  • LNA Oligonucleotides (LNA anti-miRs) were also diluted in optimem. 285 ⁇ l optimem+15 ⁇ l LNA oligonucleotide (10 ⁇ M oligonucleotide stock for final concentration 100 nM and 0.1 ⁇ M for final concentration 1 nM) Cells were washed once in optimem then the 1.2 ml optimem/LF2000 mix were added to each well. Cells were incubated 7 min at room temperature in the LF2000 mix where after the 300 ⁇ l oligonucleotide optimem solution was added.
  • miR-122a levels in the RNA samples were assessed on an ABI 7500 Fast real-time PCR instrument (Applied Biosystems, USA) using a miR-122a specific qRT-PCR kit, mirVana (Ambion, USA) and miR-122a primers (Ambion, USA). The procedure was conducted according to the manufacturers protocol.
  • the miR-122a -specific new LNA anti-miR oligonucleotide design (ie SPC3349 (also referred to as SPC 3549)), was more efficient in inhibiting miR-122a at 1 nM compared to previous design models, including “every-third” and “gap-mer” (SPC3370, SPC3372, SPC3375) motifs were at 100 nM.
  • the mismatch control was not found to inhibit miR-122a (SPC3350). Results are shown in FIG. 1 .
  • the labeling reactions contained 2-5 ⁇ g total RNA, 15 ⁇ M RNA linker, 50 mM Tris-HCl (pH 7.8), 10 mM MgCl2, 10 mM DTT, 1 mM ATP, 16% polyethylene glycol and 5 unit T4 RNA ligase (Ambion, USA) and were incubated at 30° C. for 2 hours followed by heat inactivation of the T4 RNA ligase at 80° C. for 5 minutes.
  • LNA-modified oligonucleotide capture probes comprising probes for all annotated miRNAs annotated from mouse ( Mus musculus ) and human ( Homo sapiens ) in the miRBase MicroRNA database Release 7.1 including a set of positive and negative control probes were purchased from Exiqon (Exiqon, Denmark) and used to print the microarrays for miRNA profiling.
  • the capture probes contain a 5′-terminal C6-amino modified linker and were designed to have a Tm of 72° C. against complementary target miRNAs by adjustment of the LNA content and length of the capture probes.
  • the capture probes were diluted to a final concentration of 10 ⁇ M in 150 mM sodium phosphate buffer (pH 8.5) and spotted in quadruplicate onto Codelink slides (Amersham Biosciences) using the MicroGrid II arrayer from BioRobotics at 45% humidity and at room temperature. Spotted slides were post-processed as recommended by the manufacturer.
  • RNA was hybridized to the LNA microarrays overnight at 65° C. in a hybridization mixture containing 4 ⁇ SSC, 0.1% SDS, 1 ⁇ g/ ⁇ l Herring Sperm DNA and 38% formamide.
  • the hybridized slides were washed three times in 2 ⁇ SSC, 0.025% SDS at 65° C., followed by three times in 0.08 ⁇ SSC and finally three times in 0.4 ⁇ SSC at room temperature.
  • the microarrays were scanned using the ArrayWorx scanner (Applied Precision, USA) according to the manufacturer's recommendations.
  • the scanned images were imported into TIGR Spotfinder version 3.1 (Saeed et al., 2003) for the extraction of mean spot intensities and median local background intensities, excluding spots with intensities below median local background+4 ⁇ standard deviations. Background-correlated intensities were normalized using variance stabilizing normalization package version 1.8.0 (Huber et al., 2002) for R (www.r-project.org). Intensities of replicate spots were averaged using Microsoft Excel. Probes displaying a coefficient of variance>100% were excluded from further data analysis.
  • Sections on slides are deparaffinized in xylene and then rehydrated through an ethanol dilution series (from 100% to 25%). Slides are submerged in DEPC-treated water and subject to HCl and 0.2% Glycine treatment, re-fixed in 4% paraformaldehyde and treated with acetic anhydride/triethanolamine; slides are rinsed in several washes of 1 ⁇ PBS in-between treatments. Slides are pre-hybridized in hyb solution (50% formamide, 5 ⁇ SSC, 500 mg/mL yeast tRNA, 1 ⁇ Denhardt) at 50° C. for 30 min.
  • hyb solution 50% formamide, 5 ⁇ SSC, 500 mg/mL yeast tRNA, 1 ⁇ Denhardt
  • a FITC-labeled LNA probe (Exiqon, Denmark) complementary to each selected miRNA is added to the hyb. solution and hybridized for one hour at a temperature 20-25° C. below the predicted Tm of the probe (typically between 45-55° C. depending on the miRNA sequence).
  • a tyramide signal amplification reaction was carried out using the Genpoint Fluorescein (FITC) kit (DakoCytomation, Denmark) following the vendor's recommendations.
  • slides are mounted with Prolong Gold solution. Fluorescence reaction is allowed to develop for 16-24 hr before documenting expression of the selected miRNA using an epifluorescence microscope.
  • hybridization buffer 50% Formamide, 5 ⁇ SSC, 0.1% Tween, 9.2 mM citric acid, 50 ug/ml heparin, 500 ug/ml yeast RNA
  • Hybridization is performed in fresh pre-heated hybridization buffer containing 10 nM of 3′ DIG-labeled LNA probe (Roche Diagnostics) complementary to each selected miRNA.
  • Post-hybridization washes are done at the hybridization temperature by successive incubations for 15 min in HM-(hybridization buffer without heparin and yeast RNA), 75% HM-/25% 2 ⁇ SSCT (SSC containing 0.1% Tween-20), 50% HM-/50% 2 ⁇ SSCT, 25% HM-/75% 2 ⁇ SSCT, 100% 2 ⁇ SSCT and 2 ⁇ 30 min in 0.2 ⁇ SSCT.
  • embryos are transferred to PBST through successive incubations for 10 min in 75% 0.2 ⁇ SSCT/25% PBST, 50% 0.2 ⁇ SSCT/50% PBST, 25% 0.2 ⁇ SSCT/75% PBST and 100% PBST.
  • blocking buffer 2% sheep serum/2 mg:ml BSA in PBST
  • the embryos are incubated overnight at 4° C. in blocking buffer containing anti-DIG-AP FAB fragments (Roche, 1/2000).
  • zebrafish embryos are washed 6 ⁇ 15 min in PBST, mouse and X. tropicalis embryos are washed 6 ⁇ 1 hour in TBST containing 2 mM levamisole and then for 2 days at 4° C. with regular refreshment of the wash buffer.
  • the embryos are washed 3 ⁇ 5 min in staining buffer (100 mM tris HCl pH9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% tween 20). Staining was done in buffer supplied with 4.5 ⁇ l/ml NBT (Roche, 50 mg/ml stock) and 3.5 ⁇ l/ml BCIP (Roche, 50 mg/ml stock). The reaction is stopped with 1 mM EDTA in PBST and the embryos are stored at 4° C.
  • staining buffer 100 mM tris HCl pH9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% tween 20. Staining was done in buffer supplied with 4.5 ⁇ l/ml NBT (Roche, 50 mg/ml stock) and 3.5 ⁇ l/ml BCIP (Roche, 50 mg/ml stock). The reaction is stopped with 1 mM EDTA in PBST and the embryos are stored at
  • the embryos are mounted in Murray's solution (2:1 benzylbenzoate:benzylalcohol) via an increasing methanol series (25% MeOH in PBST, 50% MeOH in PBST, 75% MeOH in PBST, 100% MeOH) prior to imaging.
  • RNA expression tissue samples were first homogenised using a Retsch 300MM homogeniser and total RNA was isolated using the Trizol reagent or the RNeasy mini kit as described by the manufacturer.
  • First strand synthesis (cDNA from mRNA) was performed using either OmniScript Reverse Transcriptase kit or M-MLV Reverse transcriptase (essentially described by manufacturer (Ambion)) according to the manufacturer's instructions (Qiagen).
  • OmniScript Reverse Transcriptase 0.5 ⁇ g total RNA each sample, was adjusted to 12 ⁇ l and mixed with 0.2 ⁇ l poly (dT) 12-18 (0.5 ⁇ g/ ⁇ l) (Life Technologies), 2 ⁇ l dNTP mix (5 mM each), 2 ⁇ l 10 ⁇ RT buffer, 0.5 ⁇ l RNAguardTM RNase Inhibitor (33 units/ml, Amersham) and 1 ⁇ l OmniScript Reverse Transcriptase followed by incubation at 37° C. for 60 min. and heat inactivation at 93° C. for 5 min.
  • RNA is synthesized at 42° C. for 60 min followed by heating inactivation step at 95° C. for 10 min and finally cooled to 4° C.
  • the cDNA can further be used for mRNA quantification by for example Real-time quantitative PCR.
  • mRNA expression can be assayed in a variety of ways known in the art. For example, mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), Ribonuclease protection assay (RPA) or real-time PCR. Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or mRNA.
  • PCR competitive polymerase chain reaction
  • RPA Ribonuclease protection assay
  • RNA analysis can be performed on total cellular RNA or mRNA.
  • RNA isolation and RNA analysis are routine in the art and is taught in, for example, Current Protocols in Molecular Biology, John Wiley and Sons.
  • Real-time quantitative can be conveniently accomplished using the commercially available iQ Multi-Color Real Time PCR Detection System available from BioRAD.
  • Real-time Quantitative PCR is a technique well-known in the art and is taught in for example Heid et al. Real time quantitative PCR, Genome Research (1996), 6: 986-994.
  • mice Six groups of animals (5 mice per group) were treated in the following manner. Group 1 animals were injected with 0.2 ml saline by i.v. on 3 successive days, Group 2 received 2.5 mg/kg SPC3372, Group 3 received 6.25 mg/kg, Group 4 received 12.5 mg/kg and Group 5 received 25 mg/kg, while Group 6 received 25 mg/kg SPC 3373 (mismatch LNA-antimiRTM oligonucleotide), all in the same manner. All doses were calculated from the Day 0 body weights of each animal.
  • retro-orbital blood was collected in tubes containing EDTA and the plasma fraction harvested and stored frozen ⁇ 80° C. for cholesterol analysis. At sacrifice livers were dissected and one portion was cut into 5 mm cubes and immersed in 5 volumes of ice-cold RNAlater. A second portion was snap frozen in liquid nitrogen and stored for cryo-sectioning.
  • FIG. 5 demonstrates a clear dose-response obtained with SPC3372 with an IC50 at ca 3-5 mg/kg, whereas no miR-122a inhibition was detected using the mismatch LNA antago-mir SPC 3373 for miR-122a.
  • the animals were sacrificed 48 hours after last dose (Day 6), retro-orbital blood was collected in tubes containing EDTA and the plasma fraction harvested and stored frozen ⁇ 80° C. for cholesterol analysis.
  • At sacrifice livers were dissected and one portion was cut into 5 mm cubes and immersed in 5 volumes of ice-cold RNAlater. A second portion was snap frozen in liquid nitrogen and stored for cryo-sectioning.
  • FIG. 2 demonstrates a clear dose-response obtained with all three LNA antimir-122a molecules (SPC3372, SPC3548, SPC3549). Both SPC3548 and SPC3549 show significantly improved efficacy in vivo in miR-122a silencing (as seen from the reduced miR-122a levels) compared to SPC3372, with SPC3549 being most potent (IC 50 ca 1 mg/kg).
  • Total cholesterol level was measured in plasma using a colometric assay Cholesterol CP from ABX Pentra. Cholesterol was measured following enzymatic hydrolysis and oxidation (2.3). 21.5 ⁇ l water was added to 1.5 ⁇ l plasma. 250 ⁇ l reagent was added and within 5 min the cholesterol content measured at a wavelength of 540 nM. Measurements on each animal were made in duplicate. The sensitivity and linearity was tested with 2-fold diluted control compound (ABX Pentra N control). The cholesterol level was determined by subtraction of the background and presented relative to the cholesterol levels in plasma of saline treated mice.
  • FIG. 3 demonstrates a markedly lowered level of plasma cholesterol in the mice that received SPC3548 and SPC3549 compared to the saline control at Day 6.
  • RNA levels were assessed by real-time quantitative RT-PCR for two miR-122a target genes, Bckdk (branched chain ketoacid dehydrogenase kinase, ENSMUSG0000030802) and aldolase A (aldoA, ENSMUSG00000030695), respectively, as well as for GAPDH as control, using Taqman assays according to the manufacturer's instructions (Applied biosystems, USA).
  • Bckdk branched chain ketoacid dehydrogenase kinase
  • aldolase A aldoA, ENSMUSG00000030695
  • FIGS. 4 a and 4 b demonstrate a clear dose-dependent upregulation of the two miR-122a target genes, Bckdk and AldoA, respectively, as a response to treatment with all three LNA antimiR-122a molecules (SPC3372, SPC3548, SPC3549).
  • the qPCR assays for GAPDH control did not reveal any differences in the GAPD mRNA levels in the LNA-antimiR-122a treated mice compared to the saline control animals ( FIG. 4 c ).
  • the Bckdk and AldoA mRNA levels were significantly higher in the SPC3548 and SPC3549 treated mice compared to the SPC3372 treated mice ( FIGS. 4 a and 4 b ), thereby demonstrating their improved in vivo efficacy.
  • mice Two groups of animals (21 mice per group) were treated in the following manner. Group 1 animals were injected with 0.2 ml saline by i.v. on 3 successive days, Group 2 received 25mg/kg SPC3372 in the same manner. All doses were calculated from the Day 0 body weights of each animal.
  • FIG. 7 (Sacrifice day 9, 16 or 23 correspond to sacrifice 1, 2 or 3 weeks after last dose) demonstrates a two-fold inhibition in the mice that received SPC3372 compared to the saline control, and this inhibition could still be detected at Day 16, while by Day 23 the mi122a levels approached those of the saline group.
  • mice Two groups of animals (21 mice per group) were treated in the following manner. Group 1 animals were injected with 0.2 ml saline by i.v. on 3 successive days, Group 2 received 25 mg/kg SPC3372 in the same manner. All doses were calculated from the Day 0 body weights of each animal.
  • FIG. 8 demonstrates a two-fold inhibition in the mice that received SPC3372 compared to the saline control, and this inhibition could still be detected at Day 16, while by Day23 the miR-122a levels approached those of the saline group.
  • NMRI mice were administered intravenously with SPC3372 using daily doses ranging from 2.5 to 25 mg/kg for three consecutive days. Animals were sacrificed 24 hours, 1, 2 or 3 weeks after last dose. Livers were harvested divided into pieces and submerged in RNAlater (Ambion) or snap-frozen. RNA was extracted with Trizol reagent according to the manufacturer's instructions (Invitrogen) from the RNAlater tissue, except that the precipitated RNA was washed in 80% ethanol and not vortexed. The RNA was used for mRNA TaqMan qPCR according to manufacturer (Applied biosystems) or northern blot (see below). The snap-frozen pieces were cryo-sectioned for in situ hybridizations.
  • SPC3372 is designated LNA-antimiR and SPC3373 (the mismatch control) is designated “mm” instead of using the SPC number.
  • mice were treated with different SPC3372 doses for three consecutive days, as described above and sacrificed 24 hours after last dose.
  • Total RNA extracted from liver was subjected to qPCR.
  • Genes with predicted miR-122 target site and observed to be upregulated by microarray analysis were investigated for dose-dependent induction by increasing SPC3372 doses using qPCR.
  • NMRI female mice were treated with 25 mg/kg/day SPC3372 along with saline control for three consecutive days and sacrificed 1, 2 or 3 weeks after last dose, respectively.
  • RNA was extracted from livers and mRNA levels of predicted miR-122a target mRNAs, selected by microarray data were investigated by qPCR. Three animals from each group were analysed.
  • liver RNA samples as in previous example were investigated for Vldlr induction.
  • Stability of SPC3372 and SPC3372/miR-122a duplex were tested in mouse plasma at 37° C. over 96 hours. Shown in FIG. 12 is a SYBR-Gold stained PAGE.
  • SPC3372 was completely stable over 96 hours.
  • the SPC3372/miR-122a duplex was immediately truncated (degradation of the single stranded miR-122a region not covered by SPC3372) but thereafter almost completely stable over 96 hours.
  • the liver RNA was also subjected to microRNA Northern blot. Shown in FIG. 13 is a membrane probed with a miR-122a specific probe (upper panel) and re-probed with a Let-7 specific probe (lower panel). With the miR-122 probe, two bands could be detected, one corresponding to mature miR-122 and one corresponding to a duplex between SPC3372 and miR-122.
  • liver RNA samples were subjected to small RNA northern blot analysis, which showed significantly reduced levels of detectable mature miR-122, in accordance with our real-time RT-PCR results.
  • the levels of the let-7a control were not altered.
  • we observed dose-dependent accumulation of a shifted miR-122/SPC3372 heteroduplex band suggesting that SPC3372 does not target miR-122 for degradation, but rather binds to the microRNA, thereby sterically hindering its function.
  • RNA was electrophoretically transferred to a GeneScreen plus Hybridization Transfer Membrane (PerkinElmer) at 200 mA for 35 min.
  • the LNA oligonucleotides were labelled and hybridized to the membrane as described in (Válóczi et al. 2004) except for the following changes:
  • the prehybridization and hybridization solutions contained 50% formamide, 0.5% SDS, 5 ⁇ SSC, 5 ⁇ Denhardt's solution and 20 ⁇ g/ml sheared denatured herring sperm DNA.
  • Hybridizations were performed at 45° C.
  • the blots were visualized by scanning in a Storm 860 scanner.
  • the signal of the background membrane was subtracted from the radioactive signals originating from the miRNA bands.
  • the values of the miR-122 signals were corrected for loading differences based on the let-7a signal.
  • the Decade Marker System was used according to the suppliers' recommendations.
  • Liver cryo-sections from treated animals were subjected to in situ hybridizations for detection and localization of miR-122 and SPC3372 ( FIG. 14 ).
  • a probe complementary to miR-122 could detect miR-122a.
  • a second probe was complementary to SPC3372.
  • Shown in FIG. 14 is an overlay, in green is distribution and apparent amounts of miR-122a and SPC3372 and blue is DAPI nuclear stain, at 10 ⁇ magnification. 100 ⁇ magnifications reveal the intracellular distribution of miR-122a and SPC3372 inside the mouse liver cells.
  • the liver sections from saline control animals showed a strong miR-122 staining pattern over the entire liver section, whereas the sections from SPC3372 treated mice showed a significantly reduced patchy staining pattern.
  • SPC3372 molecule was readily detected in SPC3372 treated liver, but not in the untreated saline control liver. Higher magnification localized miR-122a to the cytoplasm in the hepatocytes, where the miR-122 in situ pattern was clearly compartmentalized, while SPC3372 molecule was evenly distributed in the entire cytoplasm.
  • UTRs 3′ untranslated regions of the differentially expressed mRNAs for the presence of the 6 nt sequence CACTCC, corresponding to the reverse complement of the nucleotide 2-7 seed region in mature miR-122.
  • the number of transcripts having at least one miR-122 recognition sequence was 213 (51%) among the upregulated transcripts, and 10 (19%) within the downregulated transcripts, while the frequency in a random sequence population was 25%, implying that a significant pool of the upregulated mRNAs represent direct miR-122 targets in the liver ( FIG. 15 b ).
  • the LNA-antimiR treatment showed maximal reduction of miR-122 levels at 24 hours, 50% reduction at one week and matched saline controls at three weeks after last LNA dose (Example 12 “old design”). This coincided with a markedly reduced number of differentially expressed genes between the two mice groups at the later time points. Compared to the 509 mRNAs 24 hours after the last LNA dose we identified 251 differentially expressed genes after one week, but only 18 genes after three weeks post treatment ( FIGS. 15 c and 15 d ). In general genes upregulated 24 hours after LNA-antimiR treatment then reverted towards control levels over the next two weeks ( FIG. 15 d ).
  • livers of saline and LNA-antimiR treated mice were compared. NMRI female mice were treated with 25 mg/kg/day of LNA-antimiR along with saline control for three consecutive days and sacrificed 24 h, 1, 2 or 3 weeks after last dose. Additionally, expression profiles of livers of mice treated with the mismatch LNA control oligonucleotide 24 h after last dose were obtained. Three mice from each group were analyzed, yielding a total of 21 expression profiles. RNA quality and concentration was measured using an Agilent 2100 Bioanalyzer and Nanodrop ND-1000, respectively.
  • Transcripts with annotated 3′ UTRs were extracted from the Ensembl database (Release 41) using the EnsMart data mining tool30 and searched for the presence of the CACTCC sequence which is the reverse complement of the nucleotide 2-7 seed in the mature miR-122 sequence.
  • CACTCC sequence which is the reverse complement of the nucleotide 2-7 seed in the mature miR-122 sequence.
  • a set of 1000 sequences with a length of 1200 nt, corresponding to the mean 3′ UTR length of the up- and downregulated transcripts at 24 h after last LNA-antimiR dose were searched for the 6 nucleotide miR-122 seed matches. This was carried out 500 times and the mean count was used for comparison
  • miR-122 levels were analyzed by qPCR and normalized to the saline treated group.
  • Genes with predicted miR-122 target site and up regulated in the expression profiling AldoA, Nrdg3, Bckdk and CD320 showed dose-dependent de-repression by increasing LNA-antimiR doses measured by qPCR.
  • mice C57BL/6J female mice were fed on high fat diet for 13 weeks before the initiation of the SPC3649 treatment. This resulted in increased weight to 30-35 g compared to the weight of normal mice, which was just under 20 g, as weighed at the start of the LNA-antimiR treatment.
  • the high fat diet mice lead to significantly increased total plasma cholesterol level of about 130 mg/dl, thus rendering the mice hypercholesterolemic compared to the normal level of about 70 mg/dl.
  • Both hypercholesterolemic and normal mice were treated i.p. twice weekly with 5 mg/kg SPC3649 and the corresponding mismatch control SPC3744 for a study period of 51 ⁇ 2 weeks. Blood samples were collected weekly and total plasma cholesterol was measured during the entire course of the study. Upon sacrificing the mice, liver and blood samples were prepared for total RNA extraction, miRNA and mRNA quantification, assessment of the serum transaminase levels, and liver histology.
  • ALT and AST alanine and aspartate aminotransferase
  • mice C57BL/6J female mice (Taconic M&B Laboratory Animals, Ejby, Denmark) were used. All substances were formulated in physiological saline (0.9% NaCl) to final concentration allowing the mice to receive an intraperitoneal injection volume of 10 ml/kg.
  • mice received a high fat (60EN %) diet (D12492, Research Diets) for 13 weeks to increase their blood cholesterol level before the dosing started.
  • the dose regimen was stretched out to 51 ⁇ 2 weeks of 5 mg/kg LNA-antimiRTM twice weekly. Blood plasma was collected once a week during the entire dosing period. After completion of the experiment the mice were sacrificed and RNA extracted from the livers for further analysis. Serum was also collected for analysis of liver enzymes.
  • the miR-122 and let-7a microRNA levels were quantified with TaqMan microRNA Assay (Applied Biosystems) following the manufacturer's instructions.
  • the RT reaction was diluted ten times in water and subsequently used for real time PCR amplification according to the manufacturer's instructions.
  • a two-fold cDNA dilution series from liver total RNA of a saline-treated animal or mock transfected cells cDNA reaction (using 2.5 times more total RNA than in samples) served as standard to ensure a linear range (Ct versus relative copy number) of the amplification.
  • Applied Biosystems 7500 or 7900 real-time PCR instrument was used for amplification.
  • mRNA quantification of selected genes was done using standard TaqMan assays (Applied Biosystems). The reverse transcription reaction was carried out with random decamers, 0.5 ⁇ g total RNA, and the M-MLV RT enzyme from Ambion according to a standard protocol. First strand cDNA was subsequently diluted 10 times in nuclease-free water before addition to the RT-PCR reaction mixture. A two-fold cDNA dilution series from liver total RNA of a saline-treated animal or mock transfected cells cDNA reaction (using 2.5 times more total RNA than in samples) served as standard to ensure a linear range (Ct versus relative copy number) of the amplification. Applied Biosystems 7500 or 7900 real-time PCR instrument was used for amplification.
  • Serum from each individual mouse was prepared as follows: Blood samples were stored at room temperature for 2 h before centrifugation (10 min, 3000 rpm at room temperature). After centrifugation, serum was harvested and frozen at ⁇ 20° C.
  • ALT and AST measurement was performed in 96-well plates using ALT and AST reagents from ABX Pentra according to the manufacturer's instructions. In short, serum samples were diluted 2.5 fold with H 2 O and each sample was assayed in duplicate. After addition of 50 ⁇ l diluted sample or standard (multical from ABX Pentra) to each well, 200 ⁇ l of 37° C. AST or ALT reagent mix was added to each well. Kinetic measurements were performed for 5 min with an interval of 30 s at 340 nm and 37° C. using a spectrophotometer.
  • Oligos used in this example (uppercase: LNA, lowercase DNA, LNA Cs are methyl- m c, and LNAs are preferably B-D-oxy (o subscript after LNA residue e.g. C s o ):
  • SPC3649 (LNA-antimiR targeting miR-122, SEQ ID 558 was in the initial small scale synthesis designated SPC3549) 5′- m C s o c s A s o t s t s G s o T s o c s a s m C s o a s m C s o t s m C s om C o -3′
  • SPC3648 (LNA-antimiR targeting miR-122, was in the initial small scale synthesis designated SPC3548) 5′-A s o t s t s G s o T s o c s a s m C s o a s m C s o t s m C s o m C o -3′
  • SPC3550 (4 nt mismatch control to SPC3649) SEQ ID 592 5′- m C s
  • HCV Hepatitis C replication has been shown to be facilitated by miR-122 and consequently, antagonizing miR-122 has been demonstrated to affect HCV replication in a hepatoma cell model in vitro.
  • SPC3649 reducing HCV replication in the Huh-7 based cell model.
  • the different LNA-antimiR molecules along with a 2′ OMe antisense and scramble oligonucleotide are transfected into Huh-7 cells, HCV is allowed to replicate for 48 hours. Total RNA samples extracted from the Huh-7 cells are subjected to Northern blot analysis.
  • SPC3521 miR-21 5′-FAM TCAgtctgataaGCTa-3′ (SEQ ID NO 594) (gap-mer design) SPC3870 miR-21(mm) 5′-FAM TCCgtcttagaaGATa-3′ (SEQ ID NO 595) SPC3825 miR-21 5′-FAM TcTgtCAgaTaCgAT-3′ (SEQ ID NO 596) (new design) SPC3826 miR-21(mm) 5′-FAM TcAgtCTgaTaAgCT-3′ (SEQ ID NO 597) SPC3827 miR-21 5′-FAM TcAGtCTGaTaAgCT-3′ (SEQ ID NO 598) (new, enhanced design)
  • All compounds preferably have a fully or almost fully thiolated backbone (preferably fully) and have here also a FAM label in the 5′ end (optional).
  • miR-21 has been show to be up-regulated in both glioblastoma (Chan et al. Cancer Research 2005, 65 (14), p6029) and breast cancer (Iorio et al. Cancer Research 2005, 65 (16), p7065) and hence has been considered a potential ‘oncogenic’ microRNA. Chan et al. also show induction of apoptosis in glioblastoma cells by antagonising miR-21 with 2′OMe or LNA modified antisense oligonucleotides. Hence, agents antagonising miR-21 have the potential to become therapeutics for treatment of glioblastoma and other solid tumours, such as breast cancer.
  • Suitable therapeutic administration routes are, for example, intracranial injections in glioblastomas, intratumoural injections in glioblastoma and breast cancer, as well as systemic delivery in breast cancer
  • Efficacy of current LNA-antimiRTM is assessed by transfection at different concentrations, along with control oligonucleotides, into U373 and MCF-7 cell lines known to express miR-21 (or others miR-21 expressing cell lines as well). Transfection is performed using standard Lipofectamine2000 protocol (Invitrogen). 24 hours post transfection, the cells are harvested and total RNA extracted using the Trizol protocol (Invitrogen). Assessment of miR-21 levels, depending on treatment and concentration used is done by miR-21 specific, stem-loop real-time RT-PCR (Applied Biosystems), or alternatively by miR-21 specific non-radioactive northern blot analyses. The detected miR-21 levels compared to vehicle control reflects the inhibitory potential of the LNA-antimiRTM.
  • the effect of miR-21 antagonism is investigated through cloning of the perfect match miR-21 target sequence behind a standard Renilla luciferase reporter system (between coding sequence and 3′ UTR, psiCHECK-2, Promega)—see Example 29.
  • the reporter construct and LNA-antimiRTM will be co-transfected into miR-21 expressing cell lines (f. ex. U373, MCF-7).
  • the cells are harvested 24 hours post transfection in passive lysis buffer and the luciferase activity is measured according to a standard protocol (Promega, Dual Luciferase Reporter Assay System).
  • the induction of luciferase activity is used to demonstrate the functional effect of LNA-antimiRTM antagonising miR-21.
  • Oligos used in this example (uppercase: LNA, lowercase: DNA) to assess LNA-antimiR de-repressing effect on luciferase reporter with microRNA target sequence cloned by blocking respective microRNA:
  • a reporter plasmid (psiCheck-2 Promega) encoding both the Renilla and the Firefly variants of luciferase was engineered so that the 3′UTR of the Renilla luciferase includes a single copy of a sequence fully complementary to the miRNA under investigation.
  • LNA nucleotides are shown in uppercase letters, DNA nucleotides in lowercase letters, LNA C nucleotides denote LNA methyl-C (mC).
  • the LNA-antimiR oligonucleotides can be conjugated with a variety of haptens or fluorochromes for monitoring uptake into cells and tissues using standard methods.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Oncology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Virology (AREA)
  • Obesity (AREA)
  • Communicable Diseases (AREA)
  • Immunology (AREA)
  • Neurology (AREA)
  • Endocrinology (AREA)
  • Cardiology (AREA)
  • Vascular Medicine (AREA)
  • Pain & Pain Management (AREA)
  • Heart & Thoracic Surgery (AREA)
US12/296,084 2006-04-03 2007-03-30 Pharmaceutical Composition Abandoned US20100004320A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US12/296,084 US20100004320A1 (en) 2006-04-03 2007-03-30 Pharmaceutical Composition

Applications Claiming Priority (11)

Application Number Priority Date Filing Date Title
US78899506P 2006-04-03 2006-04-03
DKPA200600478 2006-04-03
DKPA200600478 2006-04-03
US79681306P 2006-05-01 2006-05-01
DKPA200600615 2006-05-01
DK200600615 2006-05-01
US83871006P 2006-08-18 2006-08-18
DK200601401 2006-10-30
DKPA200601401 2006-10-30
PCT/DK2007/000169 WO2007112754A2 (en) 2006-04-03 2007-03-30 Pharmaceutical compositions comprising anti-mirna antisense oligonucleotides
US12/296,084 US20100004320A1 (en) 2006-04-03 2007-03-30 Pharmaceutical Composition

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK2007/000169 A-371-Of-International WO2007112754A2 (en) 2006-04-03 2007-03-30 Pharmaceutical compositions comprising anti-mirna antisense oligonucleotides

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US13/006,099 Continuation US20120083596A1 (en) 2006-04-03 2011-01-13 Pharmaceutical Composition

Publications (1)

Publication Number Publication Date
US20100004320A1 true US20100004320A1 (en) 2010-01-07

Family

ID=38564007

Family Applications (8)

Application Number Title Priority Date Filing Date
US12/296,084 Abandoned US20100004320A1 (en) 2006-04-03 2007-03-30 Pharmaceutical Composition
US13/006,099 Abandoned US20120083596A1 (en) 2006-04-03 2011-01-13 Pharmaceutical Composition
US13/415,685 Active US8729250B2 (en) 2006-04-03 2012-03-08 Antisense oligonucleotides for inhibition of microRNA-21
US14/245,557 Active US9133455B2 (en) 2006-04-03 2014-04-04 Pharmaceutical composition comprising anti-miRNA antisense oligonucleotides
US14/844,088 Abandoned US20160060627A1 (en) 2006-04-03 2015-09-03 Pharmaceutical Composition for Inhibition of Disease-inducing microRNAs
US15/703,598 Abandoned US20180195062A1 (en) 2006-04-03 2017-09-13 Pharmaceutical Composition
US16/126,465 Abandoned US20190071672A1 (en) 2006-04-03 2018-09-10 Pharmaceutical Composition
US17/061,534 Abandoned US20210071181A1 (en) 2006-04-03 2020-10-01 Pharmaceutical composition

Family Applications After (7)

Application Number Title Priority Date Filing Date
US13/006,099 Abandoned US20120083596A1 (en) 2006-04-03 2011-01-13 Pharmaceutical Composition
US13/415,685 Active US8729250B2 (en) 2006-04-03 2012-03-08 Antisense oligonucleotides for inhibition of microRNA-21
US14/245,557 Active US9133455B2 (en) 2006-04-03 2014-04-04 Pharmaceutical composition comprising anti-miRNA antisense oligonucleotides
US14/844,088 Abandoned US20160060627A1 (en) 2006-04-03 2015-09-03 Pharmaceutical Composition for Inhibition of Disease-inducing microRNAs
US15/703,598 Abandoned US20180195062A1 (en) 2006-04-03 2017-09-13 Pharmaceutical Composition
US16/126,465 Abandoned US20190071672A1 (en) 2006-04-03 2018-09-10 Pharmaceutical Composition
US17/061,534 Abandoned US20210071181A1 (en) 2006-04-03 2020-10-01 Pharmaceutical composition

Country Status (12)

Country Link
US (8) US20100004320A1 (es)
EP (3) EP2007888A2 (es)
JP (5) JP5814505B2 (es)
KR (1) KR101407707B1 (es)
AU (1) AU2007234191B2 (es)
CA (4) CA2649045C (es)
DK (1) DK2666859T3 (es)
EA (1) EA015570B1 (es)
ES (1) ES2715625T3 (es)
IL (1) IL194007A0 (es)
MX (1) MX2008012219A (es)
WO (2) WO2007112753A2 (es)

Cited By (74)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090143326A1 (en) * 2007-10-04 2009-06-04 Santaris Pharma A/S MICROMIRs
US20090298916A1 (en) * 2008-03-07 2009-12-03 Santaris Pharma A/S Pharmaceutical compositions for treatment of microRNA related diseases
US20100144850A1 (en) * 2007-04-30 2010-06-10 The Ohio State University Research Foundation Methods for Differentiating Pancreatic Cancer from Normal Pancreatic Function and/or Chronic Pancreatitis
US20100285471A1 (en) * 2007-10-11 2010-11-11 The Ohio State University Research Foundation Methods and Compositions for the Diagnosis and Treatment of Esphageal Adenocarcinomas
US20100286234A1 (en) * 2006-04-03 2010-11-11 Joacim Elmen Pharmaceutical Composition Comprising Anti-Mirna Antisense Oligonucleotides
US20100330035A1 (en) * 2009-04-24 2010-12-30 Hildebrandt-Eriksen Elisabeth S Pharmaceutical Compositions for Treatment of HCV Patients that are Poor-Responders to Interferon
US20110034538A1 (en) * 2008-02-28 2011-02-10 The Ohio State University Research Foundation MicroRNA-Based Methods and Compositions for the Diagnosis, Prognosis and Treatment of Gastric Cancer
US20110190372A1 (en) * 2009-08-07 2011-08-04 New York University Compositions and methods for treating inflammatory disorders
US20110281933A1 (en) * 2010-05-13 2011-11-17 Saint Louis University Methods and compositions for the management of cardiovascular disease with oligonucleotides
WO2011144831A1 (fr) 2010-05-21 2011-11-24 Sine Sileo Agent édulcorant contenant un extrait de stévia rebaudiana bertoni
WO2012097261A2 (en) 2011-01-14 2012-07-19 The General Hospital Corporation Methods targeting mir-128 for regulating cholesterol/lipid metabolism
US20120184596A1 (en) * 2010-12-15 2012-07-19 Miragen Therapeutics Microrna inhibitors comprising locked nucleotides
US20120295962A1 (en) * 2007-10-29 2012-11-22 Rosetta Genomics Ltd. Targeting micrornas for the treatment of liver cancer
WO2013055865A1 (en) 2011-10-11 2013-04-18 The Brigham And Women's Hospital, Inc. Micrornas in neurodegenerative disorders
WO2013090556A1 (en) * 2011-12-13 2013-06-20 The Ohio State University Methods and compositions related to mir-21 and mir-29a, exosome inhibition, and cancer metastasis
US8492357B2 (en) 2008-08-01 2013-07-23 Santaris Pharma A/S Micro-RNA mediated modulation of colony stimulating factors
JP2013532141A (ja) * 2010-06-04 2013-08-15 ボード・オブ・リージエンツ，ザ・ユニバーシテイ・オブ・テキサス・システム ｍｉＲ−３７８による代謝調節
US20130345288A1 (en) * 2012-06-21 2013-12-26 Miragen Therapeutics Inhibitors of the mir-15 family of micro-rnas
US8729250B2 (en) 2006-04-03 2014-05-20 Joacim Elmén Antisense oligonucleotides for inhibition of microRNA-21
WO2014151835A1 (en) * 2013-03-15 2014-09-25 Miragen Therapeutics, Inc Locked nucleic acid inhibitor of mir-145 and uses thereof
US8859202B2 (en) 2012-01-20 2014-10-14 The Ohio State University Breast cancer biomarker signatures for invasiveness and prognosis
US8859519B2 (en) 2010-08-25 2014-10-14 The General Hospital Corporation Methods targeting miR-33 microRNAs for regulating lipid metabolism
US8865885B2 (en) 2006-03-20 2014-10-21 The Ohio State University Research Foundation MicroRNA fingerprints during human megakaryocytopoiesis
US20140323555A1 (en) * 2013-03-15 2014-10-30 The Board Of Trustees Of The Leland Stanford Junior University tRNA DERIVED SMALL RNAs (tsRNAs) INVOLVED IN CELL VIABILITY
US20140356459A1 (en) * 2011-12-15 2014-12-04 Oncostamen S.R.L. Micrornas and uses thereof
US8916533B2 (en) 2009-11-23 2014-12-23 The Ohio State University Materials and methods useful for affecting tumor cell growth, migration and invasion
US8946187B2 (en) 2010-11-12 2015-02-03 The Ohio State University Materials and methods related to microRNA-21, mismatch repair, and colorectal cancer
US9017939B2 (en) 2006-01-05 2015-04-28 The Ohio State University Methods for diagnosing breast, colon, lung, pancreatic and prostate cancer using miR-21 and miR-17-5p
US20150126579A1 (en) * 2011-04-12 2015-05-07 Beth Israel Deaconess Medical Center, Inc. Micro-rna inhibitors and their uses in disease
US20150133522A1 (en) * 2013-11-11 2015-05-14 Emory University Manipulating microrna for the management of neurological diseases or conditions and compositions related thereto
US9085804B2 (en) 2007-08-03 2015-07-21 The Ohio State University Research Foundation Ultraconserved regions encoding ncRNAs
US20150240232A1 (en) * 2009-10-19 2015-08-27 University Of Massachusetts Deducing Exon Connectivity by RNA-Templated DNA Ligation/Sequencing
US9150855B2 (en) 2010-05-21 2015-10-06 Universität Für Bodenkultur Wien Methods for diagnosing bone or cardiovascular disorders
US9206115B2 (en) 2010-05-21 2015-12-08 Technische Universität Graz ATGListatin and pharmaceutical composition comprising the same
WO2015200697A1 (en) 2014-06-25 2015-12-30 The General Hospital Corporation Targeting human satellite ii (hsatii)
US9249468B2 (en) 2011-10-14 2016-02-02 The Ohio State University Methods and materials related to ovarian cancer
WO2016077689A1 (en) * 2014-11-14 2016-05-19 Voyager Therapeutics, Inc. Modulatory polynucleotides
US9388408B2 (en) 2012-06-21 2016-07-12 MiRagen Therapeutics, Inc. Oligonucleotide-based inhibitors comprising locked nucleic acid motif
US9428749B2 (en) 2011-10-06 2016-08-30 The Board Of Regents, The University Of Texas System Control of whole body energy homeostasis by microRNA regulation
WO2016164463A1 (en) 2015-04-07 2016-10-13 The General Hospital Corporation Methods for reactivating genes on the inactive x chromosome
WO2016210241A1 (en) 2015-06-26 2016-12-29 Beth Israel Deaconess Medical Center, Inc. Cancer therapy targeting tetraspanin 33 (tspan33) in myeloid derived suppressor cells
WO2017087708A1 (en) 2015-11-19 2017-05-26 The Brigham And Women's Hospital, Inc. Lymphocyte antigen cd5-like (cd5l)-interleukin 12b (p40) heterodimers in immunity
EP3210611A2 (en) 2010-03-12 2017-08-30 The Brigham and Women's Hospital, Inc. Methods of treating vascular inflammatory disorders
WO2017147087A1 (en) 2016-02-25 2017-08-31 The Brigham And Women's Hospital, Inc. Treatment methods for fibrosis targeting smoc2
US9790492B2 (en) 2012-08-20 2017-10-17 National Cancer Center Agent for treating cancer
EP3260540A1 (en) 2010-11-12 2017-12-27 The General Hospital Corporation Polycomb-associated non-coding rnas
US9885042B2 (en) 2015-01-20 2018-02-06 MiRagen Therapeutics, Inc. miR-92 inhibitors and uses thereof
WO2018080658A1 (en) * 2016-10-27 2018-05-03 Aalborg University Therapeutic targeting of a microrna to treat duchenne muscular dystrophy
WO2018081817A2 (en) 2016-10-31 2018-05-03 University Of Massachusetts Targeting microrna-101-3p in cancer therapy
US9994852B2 (en) * 2015-06-05 2018-06-12 MiRagen Therapeutics, Inc. Oligonucleotide compositions and uses thereof
CN108220427A (zh) * 2018-03-20 2018-06-29 南京大学 一种用于鉴别诊断BHD综合征与原发性自发性气胸的血浆microRNA标记物及应用
CN108272815A (zh) * 2017-12-06 2018-07-13 南方医科大学深圳医院 EB病毒miR-BART10-5p抑制剂的应用
WO2018195486A1 (en) 2017-04-21 2018-10-25 The Broad Institute, Inc. Targeted delivery to beta cells
WO2019089216A1 (en) 2017-11-01 2019-05-09 Dana-Farber Cancer Institute, Inc. Methods of treating cancers
CN109793897A (zh) * 2012-10-31 2019-05-24 洛克菲勒大学 结肠癌的治疗和诊断
WO2019178411A1 (en) * 2018-03-14 2019-09-19 Beth Israel Deaconess Medical Center Inhibitors of micro-rna 22
US10472626B2 (en) * 2014-07-31 2019-11-12 Agency For Science, Technology And Research Modified antimir-138 oligonucleotides
CN110468202A (zh) * 2019-01-18 2019-11-19 宁夏医科大学 一种靶向TIGIT的miR-206作为肝癌诊断和治疗新型分子的用途
WO2019232132A1 (en) * 2018-05-30 2019-12-05 The Regents Of The University Of California Methods of enhancing immunity
CN110548041A (zh) * 2019-08-30 2019-12-10 中国医科大学附属盛京医院 LNA-anti-miR-150在制备预防或治疗肾脏纤维化药物中的用途
WO2020047229A1 (en) 2018-08-29 2020-03-05 University Of Massachusetts Inhibition of protein kinases to treat friedreich ataxia
US10584337B2 (en) 2016-05-18 2020-03-10 Voyager Therapeutics, Inc. Modulatory polynucleotides
US10597660B2 (en) 2014-11-14 2020-03-24 Voyager Therapeutics, Inc. Compositions and methods of treating amyotrophic lateral sclerosis (ALS)
US10612021B2 (en) 2015-10-07 2020-04-07 Kyoto University Therapeutic or prophylactic composition for TDP-43 proteinopathy
US10758619B2 (en) 2010-11-15 2020-09-01 The Ohio State University Controlled release mucoadhesive systems
CN112301130A (zh) * 2020-11-12 2021-02-02 苏州京脉生物科技有限公司 一种肺癌早期检测的标志物、试剂盒及方法
US20210220387A1 (en) * 2018-05-18 2021-07-22 Hoffmann-La Roche, Inc. Pharmaceutical compositions for treatment of microrna related diseases
US11142800B2 (en) 2010-10-07 2021-10-12 The General Hospital Corporation Biomarkers of cancer
US11220689B2 (en) 2015-10-16 2022-01-11 Children's Medical Center Corporation Modulators of telomere disease
US11434502B2 (en) 2017-10-16 2022-09-06 Voyager Therapeutics, Inc. Treatment of amyotrophic lateral sclerosis (ALS)
US11603542B2 (en) 2017-05-05 2023-03-14 Voyager Therapeutics, Inc. Compositions and methods of treating amyotrophic lateral sclerosis (ALS)
US11752181B2 (en) 2017-05-05 2023-09-12 Voyager Therapeutics, Inc. Compositions and methods of treating Huntington's disease
US11931375B2 (en) 2017-10-16 2024-03-19 Voyager Therapeutics, Inc. Treatment of amyotrophic lateral sclerosis (ALS)
US11951121B2 (en) 2016-05-18 2024-04-09 Voyager Therapeutics, Inc. Compositions and methods for treating Huntington's disease

Families Citing this family (174)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2530157B1 (en) 2003-07-31 2016-09-28 Regulus Therapeutics Inc. Oligomeric compounds and compositions for use in modulation of miRNAs
CA2564503C (en) 2004-05-04 2015-12-29 The Board Of Trustees Of The Leland Stanford Junior University Inhibition of mir-122 in hepatitis c virus infected subjects and cells
EP1877557A2 (en) 2005-04-04 2008-01-16 The Board of Regents of The University of Texas System Micro-rna's that regulate muscle cells
ES2545383T3 (es) 2005-08-01 2015-09-10 The Ohio State University Research Foundation Métodos y composiciones basados en microARN para el diagnóstico, pronóstico y tratamiento de cáncer de mama
AU2006284855B2 (en) 2005-08-29 2011-10-13 Regulus Therapeutics Inc. Methods for use in modulating miR-122a
JP2009507918A (ja) 2005-09-12 2009-02-26 ジ・オハイオ・ステイト・ユニバーシティ・リサーチ・ファウンデイション Ｂｃｌ２関連癌の診断及び療法のための組成物及び方法
JP5490413B2 (ja) 2006-01-05 2014-05-14 ジ・オハイオ・ステイト・ユニバーシティ・リサーチ・ファウンデイション 膵内分泌腫瘍及び膵腺房腫瘍におけるマイクロｒｎａ発現異常
US7943318B2 (en) 2006-01-05 2011-05-17 The Ohio State University Research Foundation Microrna-based methods and compositions for the diagnosis, prognosis and treatment of lung cancer
WO2007143317A2 (en) 2006-05-05 2007-12-13 Isis Pharmaceuticals, Inc Compounds and methods for modulating expression of crp
EP2455494B1 (en) 2006-07-13 2013-12-18 The Ohio State University Research Foundation Micro-RNA-based methods and compositions for the diagnosis and treatment of colon related diseases
PT2056882E (pt) 2006-08-01 2012-11-19 Univ Texas Identificação de um micro-rna que ativa a expressão da cadeia pesada de beta-miosina
US8188255B2 (en) 2006-10-20 2012-05-29 Exiqon A/S Human microRNAs associated with cancer
EP2090665A2 (en) 2006-10-20 2009-08-19 Exiqon A/S Novel human microRNAs associated with cancer
CA2681568C (en) 2006-11-23 2019-01-08 Querdenker Aps Oligonucleotides for modulating target rna activity
CN105256004A (zh) 2007-01-31 2016-01-20 俄亥俄州立大学研究基金会 用于急性髓细胞白血病(aml)的诊断、预后和治疗的基于微rna的方法和组合物
CA2681406A1 (en) 2007-03-22 2008-09-25 Santaris Pharma A/S Rna antagonist compounds for the inhibition of apo-b100 expression
DK2149605T3 (da) 2007-03-22 2013-09-30 Santaris Pharma As Korte RNA antagonist forbindelser til modulering af det ønskede mRNA
AU2008232316A1 (en) * 2007-03-26 2008-10-02 Newcastle Innovation Limited Therapeutic targets and molecules
EP2714904B1 (en) 2007-06-14 2017-04-12 Mirx Therapeutics ApS Oligonucleotides for modulation of target rna activity
EP2167521A4 (en) * 2007-06-15 2011-11-23 Univ Ohio State Res Found ALL-1 ONCOGEN FUSION PROTEINS TO TARGE TREATMENT OF MICRO-RNA REGULATED BY DROSHA
EP2173911B1 (en) 2007-07-31 2014-06-18 The Ohio State University Research Foundation Methods for reverting methylation by targeting dnmt3a and dnmt3b
US8481507B2 (en) 2007-07-31 2013-07-09 The Board Of Regents, The University Of Texas System Micro-RNAs that control myosin expression and myofiber identity
ES2621161T3 (es) 2007-07-31 2017-07-03 The Board Of Regents Of The University Of Texas System Familia de micro-ARN que modula la fibrosis y usos de la misma
WO2009026487A1 (en) 2007-08-22 2009-02-26 The Ohio State University Research Foundation Methods and compositions for inducing deregulation of epha7 and erk phosphorylation in human acute leukemias
AU2013273821B2 (en) * 2007-10-04 2016-03-10 Roche Innovation Center Copenhagen A/S Micromirs
US8911998B2 (en) 2007-10-26 2014-12-16 The Ohio State University Methods for identifying fragile histidine triad (FHIT) interaction and uses thereof
WO2009062169A2 (en) * 2007-11-09 2009-05-14 The Board Of Regents Of The University Of Texas System Micro-rnas of the mir-15 family modulate cardiomyocyte survival and cardiac repair
GB0802754D0 (en) * 2008-02-14 2008-03-26 Inst Superiore Di Sanito Antisense RNA targetting CXCR4
WO2009105759A2 (en) * 2008-02-21 2009-08-27 The Board Of Regents Of The University Of Texas System Micro-rnas that modulate smooth muscle proliferation and differentiation and uses thereof
EP2096171A1 (en) 2008-02-27 2009-09-02 Julius-Maximilians-Universität Würzburg MicroRNA (miRNA) and down-stream targets for diagnostic and therapeutic purposes
JP5653899B2 (ja) 2008-03-17 2015-01-14 ボード・オブ・リージエンツ，ザ・ユニバーシテイ・オブ・テキサス・システム 神経筋シナプスの維持および再生に関与するマイクロｒｎａの同定
EP2105145A1 (en) 2008-03-27 2009-09-30 ETH Zürich Method for muscle-specific delivery lipid-conjugated oligonucleotides
JP5750710B2 (ja) * 2008-04-30 2015-07-22 日本電気株式会社 癌マーカー、それを用いた癌の評価方法および評価試薬
CN102149827B (zh) 2008-06-11 2014-08-20 由卫生与公众服务部代表的美利坚合众国政府 MiR-26家族作为肝细胞癌和对治疗的应答性的预测性标志物的用途
AU2009270005B2 (en) * 2008-06-16 2014-07-24 Academisch Ziekenhuis Maastricht Means and methods for counteracting, delaying and/or preventing heart disease
KR20110056482A (ko) 2008-06-27 2011-05-30 노파르티스 포르슝스티프퉁 쯔바이크니덜라쑹 프리드리히 미셔 인스티튜트 포 바이오메디칼 리서치 항바이러스 요법 반응의 예측
EP2191834A1 (en) * 2008-11-26 2010-06-02 Centre National De La Recherche Scientifique (Cnrs) Compositions and methods for treating retrovirus infections
JP6091752B2 (ja) * 2008-12-04 2017-03-08 クルナ・インコーポレーテッド Ｅｐｏに対する天然アンチセンス転写物の抑制によるエリスロポエチン（ｅｐｏ）関連疾患の治療
WO2010076248A1 (en) 2008-12-31 2010-07-08 Santaris Pharma A/S Use of lna apob antisense oligomers for the treatment of acute coronary syndromes
MX2011008190A (es) 2009-02-04 2011-10-06 Univ Texas Direccionamiento dual de mir-208 y mir-499 en el tratamiento de trastornos cardiacos.
EP2218458A1 (en) * 2009-02-13 2010-08-18 Fondazione Telethon Molecules able to modulate the expression of at least a gene involved in degradative pathways and uses thereof
EP2414549A4 (en) 2009-03-31 2013-08-28 Us Health DIFFERENTIAL EXPRESSION MICRO-RNA AS BIOMARKERS IN THE DIAGNOSIS OF SJÖGREN SYNDROME AND TREATMENT THEREOF
US8592388B2 (en) 2009-05-20 2013-11-26 Eth Zurich Targeting MicroRNAs for metabolic disorders
WO2010135714A2 (en) * 2009-05-22 2010-11-25 The Methodist Hospital Research Institute Methods for modulating adipocyte expression using microrna compositions
US8563528B2 (en) 2009-07-21 2013-10-22 Santaris Pharma A/S Antisense oligomers targeting PCSK9
WO2011022316A1 (en) * 2009-08-20 2011-02-24 The Regents Of The University Of Colorado, A Body Corporate Mirnas dysregulated in triple-negative breast cancer
CN102002493B (zh) * 2009-09-01 2013-04-10 中国科学院上海生命科学研究院 小rna-326制备药物的应用
WO2011032100A1 (en) * 2009-09-11 2011-03-17 Government Of The U.S.A., As Represented By The Secretary, Department Of Health And Human Services Inhibitors of kshv vil6 and human il6
EP2490699A1 (en) 2009-10-20 2012-08-29 Santaris Pharma A/S Oral delivery of therapeutically effective lna oligonucleotides
WO2011069100A2 (en) 2009-12-04 2011-06-09 Duke University Microrna and use thereof in identification of b cell malignancies
EP2536436A4 (en) 2010-01-20 2015-01-14 Univ Texas ANTIMIR-451 FOR THE TREATMENT OF POLYCYTHEMICS
WO2011111715A1 (ja) * 2010-03-09 2011-09-15 協和発酵キリン株式会社 細胞周期を制御する核酸
WO2011117353A1 (en) * 2010-03-24 2011-09-29 Mirrx Therapeutics A/S Bivalent antisense oligonucleotides
SG184821A1 (en) * 2010-04-21 2012-11-29 Amc Amsterdam Means and methods for determining risk of cardiovascular disease
EP2563935B1 (en) 2010-04-30 2014-04-16 Exiqon A/S In situ hybridization method and buffer.
US20130150256A1 (en) 2010-06-11 2013-06-13 Jane Synnergren Novel micrornas for the detection and isolation of human embryonic stem cell-derived cardiac cell types
CN103189511B (zh) * 2010-07-12 2016-10-12 国立大学法人鸟取大学 利用siRNA导入的新型hiPSC制作法
US8815826B2 (en) 2010-07-23 2014-08-26 Regulus Therapeutics, Inc. Targeting microRNAs for the treatment of fibrosis
CA2808889A1 (en) 2010-08-27 2012-03-01 New York University Mir-33 inhibitors and uses thereof
CN102031261B (zh) * 2010-10-27 2015-04-22 南京医科大学 一种与妊娠期糖尿病相关的血清/血浆miRNA标志物及其应用
EP2638159B1 (en) * 2010-11-11 2019-04-24 University of Miami Compositions, kits and methods for treatment of cardiovascular, immunological, and inflammatory diseases
US8889649B2 (en) 2010-11-12 2014-11-18 National University Corporation Ehime University Composition containing antisense oligonucleotide to micro RNA
US9920317B2 (en) 2010-11-12 2018-03-20 The General Hospital Corporation Polycomb-associated non-coding RNAs
CN102041316A (zh) * 2010-11-30 2011-05-04 华东师范大学 miRNA-219化合物作为脑胶质瘤标志物的应用
EP2654801B1 (en) * 2010-12-20 2017-11-01 Agency For Science, Technology And Research Targeting glioma stem cells by sequence-specific functional inhibition of pro-survival oncomir-138
CN102174516A (zh) * 2011-01-20 2011-09-07 中南大学 一种与ebv感染相关的鼻咽癌诊断和治疗的分子靶标及其应用
AU2012225506B2 (en) 2011-03-07 2016-11-17 The Ohio State University Mutator activity induced by microRNA-155 (miR-155) links inflammation and cancer
WO2012145374A1 (en) 2011-04-19 2012-10-26 Regulus Therapeutics Inc. TARGETING miR-378 FAMILY MEMBERS FOR THE TREATMENT OF METABOLIC DISORDERS
LT2702155T (lt) 2011-04-25 2017-05-10 Regulus Therapeutics Inc. Mikro rnr junginiai ir būdai mir-21 aktyvumo moduliavimui
WO2012149557A1 (en) 2011-04-28 2012-11-01 New York University miR-33 INHIBITORS AND USES THEREOF TO DECREASE INFLAMMATION
JP2014522814A (ja) 2011-06-23 2014-09-08 ステラ・アンパルトセルスカブ Ｈｃｖ併用療法
US20140194491A1 (en) * 2011-06-24 2014-07-10 Syddansk Universitet Modulation of microrna-138 for the treatment of bone loss
EP2726611A1 (en) 2011-06-30 2014-05-07 Stella ApS Hcv combination therapy
WO2013000855A1 (en) 2011-06-30 2013-01-03 Santaris Pharma A/S Hcv combination therapy
CN102908621A (zh) * 2011-08-02 2013-02-06 中国科学院上海生命科学研究院 miRNAs作为调节胰岛素敏感性的靶标的新用途
WO2013030362A1 (en) * 2011-08-31 2013-03-07 Universität Zürich Prorektorat Mnw Modulators of mir-323-3p for the prevention or treatment of rheumatoid arthritis
CN102978278B (zh) * 2011-09-07 2016-09-28 中国科学院动物研究所 内源性的非编码小RNAs及其应用
EP2756080B1 (en) 2011-09-14 2019-02-20 Translate Bio MA, Inc. Multimeric oligonucleotide compounds
GB201117482D0 (en) * 2011-10-11 2011-11-23 Univ Dundee Targetiing of miRNA precursors
WO2013068348A1 (en) 2011-11-07 2013-05-16 Santaris Pharma A/S Lna oligomers for improvement in hepatic function
AU2012334214A1 (en) 2011-11-07 2014-05-22 Roche Innovation Center Copenhagen A/S Prognostic method for checking efficacy of micro RNA-122 inhibitors in HCV+ patients
US9447471B2 (en) 2011-12-29 2016-09-20 Quest Diagnostics Investments Incorporated Microrna profiling for diagnosis of dysplastic nevi and melanoma
KR102118429B1 (ko) * 2012-04-25 2020-06-03 사노피 마이크로rna 화합물 및 mir-21 활성 조절 방법
WO2013170146A1 (en) 2012-05-10 2013-11-14 Uab Research Foundation Methods and compositions for modulating mir-204 activity
US10174323B2 (en) 2012-05-16 2019-01-08 The General Hospital Corporation Compositions and methods for modulating ATP2A2 expression
US10837014B2 (en) 2012-05-16 2020-11-17 Translate Bio Ma, Inc. Compositions and methods for modulating SMN gene family expression
WO2013173608A1 (en) 2012-05-16 2013-11-21 Rana Therapeutics, Inc. Compositions and methods for modulating mecp2 expression
CA2873797A1 (en) 2012-05-16 2013-11-21 Rana Therapeutics Inc. Compositions and methods for modulating utrn expression
CN104583398A (zh) 2012-05-16 2015-04-29 Rana医疗有限公司 用于调节基因表达的组合物和方法
AU2013262649A1 (en) 2012-05-16 2015-01-22 Rana Therapeutics, Inc. Compositions and methods for modulating smn gene family expression
US10174315B2 (en) 2012-05-16 2019-01-08 The General Hospital Corporation Compositions and methods for modulating hemoglobin gene family expression
WO2013190091A1 (en) * 2012-06-21 2013-12-27 Ruprecht-Karls-Universität Heidelberg CIRCULATING miRNAs AS MARKERS FOR BREAST CANCER
ES2708624T3 (es) 2012-08-15 2019-04-10 Univ Virginia Patent Foundation Composiciones y métodos para tratar la enfermedad arterial periférica
WO2014043544A1 (en) 2012-09-14 2014-03-20 Rana Therapeutics, Inc. Multimeric oligonucleotide compounds
UA116639C2 (uk) 2012-10-09 2018-04-25 Рег'Юлес Терап'Ютікс Інк. Способи лікування синдрому альпорта
RU2015119411A (ru) 2012-11-15 2017-01-10 Рош Инновейшен Сентер Копенгаген А/С Конъюгаты антисмысловых соединений, направленные на аполипопротеин в
WO2014082644A1 (en) * 2012-11-30 2014-06-05 WULFF, Peter, Samuel Circular rna for inhibition of microrna
WO2014118272A1 (en) 2013-01-30 2014-08-07 Santaris Pharma A/S Antimir-122 oligonucleotide carbohydrate conjugates
US20150368642A1 (en) 2013-01-30 2015-12-24 Hoffmann-La Roche Inc. Lna oligonucleotide carbohydrate conjugates
CN105189751B (zh) * 2013-03-15 2019-04-23 米拉根医疗股份有限公司 桥接双环核苷
KR102214740B1 (ko) 2013-05-01 2021-02-10 레굴루스 테라퓨틱스 인크 Mir-122를 조정하기 위한 마이크로rna 화합물 및 방법
EP2992096B1 (en) 2013-05-01 2021-03-03 Regulus Therapeutics Inc. Compounds and methods for enhanced cellular uptake
CN103293318B (zh) * 2013-05-22 2014-10-29 吉林大学 利用地高辛标记EDC交联桥连法检测miRNAs方法
WO2014201301A1 (en) 2013-06-12 2014-12-18 New York University Anti-mir-27b and anti-mir-148a oligonucleotides as therapeutic tools for treating dyslipidemias and cardiovascular diseases
US9976140B2 (en) 2013-06-14 2018-05-22 Joslin Diabetes Center, Inc. Microrna and uses in brown fat differentiation
CN105358692B (zh) 2013-06-27 2020-08-21 罗氏创新中心哥本哈根有限公司 靶向pcsk9的反义寡聚体和缀合物
EP3024933A4 (en) * 2013-07-24 2017-03-15 The General Hospital Corporation Agents and methods for inhibiting mir-148a for the modulation of cholesterol levels
JP6443889B2 (ja) * 2013-08-08 2018-12-26 国立大学法人大阪大学 尿路上皮癌の診断または治療剤
US9994846B2 (en) 2013-10-25 2018-06-12 Regulus Therapeutics Inc. MicroRNA compounds and methods for modulating miR-21 activity
JP2017511694A (ja) * 2014-02-12 2017-04-27 トーマス・ジェファーソン・ユニバーシティ マイクロｒｎａ阻害剤を使用するための組成物および方法
CA2948844C (en) 2014-05-12 2020-06-30 The Johns Hopkins University Engineering synthetic brain penetrating gene vectors
US10335500B2 (en) 2014-05-12 2019-07-02 The Johns Hopkins University Highly stable biodegradable gene vector platforms for overcoming biological barriers
RU2017105342A (ru) 2014-08-07 2018-09-13 Регулус Терапьютикс Инк. НАЦЕЛИВАНИЕ НА микроРНК ПРИ РАССТРОЙСТВАХ ОБМЕНА ВЕЩЕСТВ
US10174320B2 (en) 2014-09-21 2019-01-08 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Downregulating miR-132 for the treatment of lipid related disorders
CN104306988B (zh) * 2014-09-25 2017-02-15 中国医学科学院基础医学研究所 miR‑431在制备治疗肌肉疾病的药物中的用途
US10858650B2 (en) 2014-10-30 2020-12-08 The General Hospital Corporation Methods for modulating ATRX-dependent gene repression
WO2016077347A1 (en) 2014-11-10 2016-05-19 The Regents Of The University Of California Mir-214 as a diagnostic and prognostic biomarker specific for ulcerative colitis and a mir-214 inhibitor for treatment of same
EP3020813A1 (en) 2014-11-16 2016-05-18 Neurovision Pharma GmbH Antisense-oligonucleotides as inhibitors of TGF-R signaling
EP3256591A4 (en) 2015-02-13 2018-08-08 Translate Bio Ma, Inc. Hybrid oligonucleotides and uses thereof
US10900036B2 (en) 2015-03-17 2021-01-26 The General Hospital Corporation RNA interactome of polycomb repressive complex 1 (PRC1)
CA2986913A1 (en) * 2015-06-05 2016-12-08 MiRagen Therapeutics, Inc. Mir-155 inhibitors for treating amyotrophic lateral sclerosis (als)
EP3331546B1 (en) 2015-08-03 2023-10-04 Biokine Therapeutics Ltd. Cxcr4 inhibitor for the treatment of cancer
CN107922945A (zh) 2015-08-24 2018-04-17 罗氏创新中心哥本哈根有限公司 Lna‑g 方法
EP3352765A4 (en) * 2015-09-22 2019-05-22 Miragen Therapeutics, Inc. MIR-19 MODULATORS AND USES THEREOF
JP6893505B2 (ja) 2015-10-02 2021-06-23 ロシュ イノベーション センター コペンハーゲン エーエス オリゴヌクレオチドコンジュゲーション方法
EP3359553A4 (en) * 2015-10-06 2019-04-03 University Of Virginia Patent Foundation COMPOSITIONS AND METHODS OF TREATING DIABETIC RETINOPATHY
WO2017067970A1 (en) 2015-10-22 2017-04-27 Roche Innovation Center Copenhagen A/S In vitro toxicity screening assay
JOP20200228A1 (ar) 2015-12-21 2017-06-16 Novartis Ag تركيبات وطرق لخفض تعبير البروتين tau
CN107012199A (zh) * 2016-01-28 2017-08-04 上海市东方医院 一种在血浆和血清中检测miRNA的方法
JP7017517B2 (ja) 2016-03-18 2022-02-08 ロシュ イノベーション センター コペンハーゲン エーエス アシル保護ｌ－ｌｎａ－グアノシンモノマー
JP7097820B2 (ja) 2016-05-12 2022-07-08 ロシュ イノベーション センター コペンハーゲン エーエス ヌクレオシドまたはオリゴヌクレオチドへの、立体的に規定されたオキサザホスホリジンホスホルアミダイト単量体のカップリングの増大法
US11015196B2 (en) 2016-05-20 2021-05-25 The General Hospital Corporation Using microRNAs to control activation status of hepatic stellate cells and to prevent fibrosis in progressive liver diseases
CN109328236B (zh) 2016-06-17 2022-10-25 豪夫迈·罗氏有限公司 体外肾毒性筛选测定法
EP3472347B1 (en) 2016-06-17 2023-01-04 F. Hoffmann-La Roche AG In vitro nephrotoxicity screening assay
EP3481430A4 (en) 2016-07-11 2020-04-01 Translate Bio Ma, Inc. NUCLEIC ACID CONJUGATES AND USES THEREOF
US11566243B2 (en) 2016-07-18 2023-01-31 Jaan Biotherapeutics Llc Compositions and methods for treatment of cardiac diseases
EP3494219A1 (en) 2016-08-03 2019-06-12 Aalborg Universitet ANTISENSE OLIGONUCLEOTIDES (ASOs) DESIGNED TO INHIBIT IMMUNE CHECKPOINT PROTEINS
MX2019005101A (es) 2016-11-01 2019-08-22 Univ New York State Res Found Microarns modificados con 5-halouracilo y su uso en el tratamiento del cancer.
CN110753758A (zh) 2016-12-22 2020-02-04 俄亥俄州国家创新基金会 用于将体细胞重编程为诱导的血管生成细胞的组合物和方法
KR20190135494A (ko) 2017-03-29 2019-12-06 로슈 이노베이션 센터 코펜하겐 에이/에스 입체한정된 포스포로티오에이트 올리고뉴클레오티드의 제조를 위한 직교 보호기
JP7198768B2 (ja) 2017-03-29 2023-01-04 ロシュ イノベーション センター コペンハーゲン エーエス ＵｎｙＬｉｎｋｅｒ迅速切断方法
JP7492829B2 (ja) 2017-06-28 2024-05-30 ロシュ イノベーション センター コペンハーゲン エーエス 多重カップリングおよび酸化の方法
CN111226114A (zh) 2017-10-13 2020-06-02 罗氏创新中心哥本哈根有限公司 用部分立体限定的寡核苷酸子文库鉴定反义寡核苷酸改进的立体限定硫代磷酸酯寡核苷酸变体的方法
CN108220381A (zh) * 2017-12-07 2018-06-29 国家卫生计生委科学技术研究所 试剂在制备药物中的用途以及筛选药物的方法
EP4092118A1 (en) 2017-12-22 2022-11-23 Roche Innovation Center Copenhagen A/S Novel thiophosphoramidites
AU2018386527A1 (en) 2017-12-22 2020-04-30 Roche Innovation Center Copenhagen A/S Gapmer oligonucleotides comprising a phosphorodithioate internucleoside linkage
WO2019122279A1 (en) 2017-12-22 2019-06-27 Roche Innovation Center Copenhagen A/S Oligonucleotides comprising a phosphorodithioate internucleoside linkage
CN110777199B (zh) * 2018-07-24 2023-11-14 长庚医疗财团法人高雄长庚纪念医院 干癣性关节炎的诊断及治疗及其相应的试剂盒
US20210292358A1 (en) 2018-07-31 2021-09-23 Roche Innovation Center Copenhagen A/S Oligonucleotides comprising a phosphorotrithioate internucleoside linkage
CA3098267A1 (en) 2018-07-31 2020-02-06 Roche Innovation Center Copenhagen A/S Oligonucleotides comprising a phosphorotrithioate internucleoside linkage
EP3841220A1 (en) 2018-08-23 2021-06-30 Roche Innovation Center Copenhagen A/S Microrna-134 biomarker
EP3620519A1 (en) 2018-09-04 2020-03-11 F. Hoffmann-La Roche AG Use of isolated milk extracellular vesicles for delivering oligonucleotides orally
EP3914232A1 (en) 2019-01-25 2021-12-01 F. Hoffmann-La Roche AG Lipid vesicle for oral drug delivery
KR20210132681A (ko) 2019-02-20 2021-11-04 로슈 이노베이션 센터 코펜하겐 에이/에스 신규 포스포라미디트
CN113490742A (zh) 2019-02-20 2021-10-08 罗氏创新中心哥本哈根有限公司 膦酰基乙酸酯缺口聚物寡核苷酸
JP2022522430A (ja) 2019-02-26 2022-04-19 ロシュ イノベーション センター コペンハーゲン エーエス オリゴヌクレオチドの製剤化方法
KR20220024192A (ko) 2019-05-31 2022-03-03 알리고스 테라퓨틱스 인코포레이티드 변형된 갭머 올리고뉴클레오타이드 및 사용 방법
EP3990028A4 (en) 2019-06-26 2023-08-09 Biorchestra Co., Ltd. MICELLAR NANOPARTICLES AND ASSOCIATED USES
CN111057790B (zh) * 2019-12-11 2022-08-30 石河子大学 miRNA在制备用于检测KSHV潜伏感染的试剂盒中的用途
JP2023516142A (ja) 2020-02-28 2023-04-18 エフ・ホフマン－ラ・ロシュ・アクチェンゲゼルシャフト Ｃｄ７３エクソン７スプライシングを調節するためのオリゴヌクレオチド
JPWO2021177267A1 (es) * 2020-03-02 2021-09-10
KR102555878B1 (ko) * 2020-04-23 2023-07-17 주식회사 바이오오케스트라 하향조절된 mirna의 진단 및 치료를 위한 용도
WO2021251526A1 (ko) * 2020-06-11 2021-12-16 주식회사 프로스테믹스 신규한 mirna 유사체 및 이의 용도
JP2024506371A (ja) 2021-02-12 2024-02-13 メランド ファーマシューティカルズ，インコーポレイテッド 低酸素症及び虚血関連障害を処置する薬剤、組成物及び方法
WO2022254021A1 (en) 2021-06-04 2022-12-08 Neumirna Therapeutics Aps Antisense oligonucleotides targeting adenosine kinase
WO2022260162A1 (ja) * 2021-06-11 2022-12-15 国立大学法人京都大学 Ｎａｓｈの予防及び／又は治療剤
EP4105328A1 (en) 2021-06-15 2022-12-21 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Antisense-oligonucleotides for prevention of kidney dysfunction promoted by endothelial dysfunction by ephrin-b2 suppression
WO2023013818A1 (ko) * 2021-08-06 2023-02-09 주식회사 네오나 변형된 rt-let7을 유효성분으로 포함하는 간암의 예방 또는 치료용 조성물
KR102329524B1 (ko) * 2021-08-06 2021-11-23 주식회사 네오나 변형된 rt-let7을 유효성분으로 포함하는 간암의 예방 또는 치료용 조성물
AU2022329462A1 (en) 2021-08-19 2024-03-28 Neumirna Therapeutics Aps Antisense oligonucleotides targeting adenosine kinase
CN118043462A (zh) * 2021-10-08 2024-05-14 莱古路斯治疗法股份有限公司 用于避免脱靶效应的方法和组合物
WO2024026474A1 (en) 2022-07-29 2024-02-01 Regeneron Pharmaceuticals, Inc. Compositions and methods for transferrin receptor (tfr)-mediated delivery to the brain and muscle
WO2024098002A1 (en) 2022-11-04 2024-05-10 Regeneron Pharmaceuticals, Inc. Calcium voltage-gated channel auxiliary subunit gamma 1 (cacng1) binding proteins and cacng1-mediated delivery to skeletal muscle
US20240173426A1 (en) 2022-11-14 2024-05-30 Regeneron Pharmaceuticals, Inc. Compositions and methods for fibroblast growth factor receptor 3-mediated delivery to astrocytes

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6030785A (en) * 1997-03-05 2000-02-29 University Of Washington Screening methods to identify agents that selectively inhibit hepatitis C virus replication
US6284458B1 (en) * 1992-09-10 2001-09-04 Isis Pharmaceuticals, Inc. Compositions and methods for treatment of hepatitis C virus-associated diseases
US6423489B1 (en) * 1992-09-10 2002-07-23 Isis Pharmaceuticals, Inc. Compositions and methods for treatment of Hepatitis C virus-associated diseases
US6433159B1 (en) * 1992-09-10 2002-08-13 Isis Pharmaceuticals, Inc. Compositions and methods for treatment of Hepatitis C virus associated diseases
US20050069522A1 (en) * 2002-08-12 2005-03-31 Richard Colonno Combination pharmaceutical agents as inhibitors of HCV replication
US20050182005A1 (en) * 2004-02-13 2005-08-18 Tuschl Thomas H. Anti-microRNA oligonucleotide molecules
US20050227934A1 (en) * 2004-04-13 2005-10-13 Markus Stoffel Pancreatic islet microRNA and methods for inhibiting same
US20050261218A1 (en) * 2003-07-31 2005-11-24 Christine Esau Oligomeric compounds and compositions for use in modulation small non-coding RNAs
US20060035212A1 (en) * 2002-12-12 2006-02-16 Universte Joseph Fourier Molecules inhibiting hepatitis c virus protein synthesis and method for screening same
US20060185027A1 (en) * 2004-12-23 2006-08-17 David Bartel Systems and methods for identifying miRNA targets and for altering miRNA and target expression
US20060265771A1 (en) * 2005-05-17 2006-11-23 Lewis David L Monitoring microrna expression and function
US20070049547A1 (en) * 2003-07-31 2007-03-01 Christine Esau Methods for use in modulating miR-122a

Family Cites Families (87)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4962029A (en) 1987-10-02 1990-10-09 Cetus Corporation Covalent oligonucleotide-horseradish peroxidase conjugate
US4914210A (en) 1987-10-02 1990-04-03 Cetus Corporation Oligonucleotide functionalizing reagents
US4920115A (en) 1988-12-28 1990-04-24 Virginia Commonwealth University Method of lowering LDL cholesterol in blood
JPH06311885A (ja) 1992-08-25 1994-11-08 Mitsubishi Kasei Corp Ｃ型肝炎ウイルス遺伝子に相補的なアンチセンス化合物
EP0759979A4 (en) 1994-05-10 1999-10-20 Gen Hospital Corp ANTISENSE OLIGONUCLEOTIDES INHIBITION OF HEPATITIS C VIRUS
US5919795A (en) 1995-06-07 1999-07-06 Pfizer Inc. Biphenyl-2-carboxylic acid-tetrahydro-isoquinolin-6-yl amide derivatives, their preparation and their use as inhibitors of microsomal triglyceride transfer protein and/or apolipoprotein B (Apo B) secretion
AU716151B2 (en) 1996-11-27 2000-02-17 Pfizer Inc. Apo B-secretion/MTP inhibitory amides
DE69829760T3 (de) 1997-09-12 2016-04-14 Exiqon A/S Bi- und tri-zyklische - nukleosid, nukleotid und oligonukleotid-analoga
EP1161439B1 (en) 1999-03-18 2010-04-21 Exiqon A/S Xylo-lna analogues
AU3418800A (en) 1999-03-24 2000-10-09 Exiqon A/S Improved synthesis of (2.2.1)bicyclo nucleosides
US7098192B2 (en) * 1999-04-08 2006-08-29 Isis Pharmaceuticals, Inc. Antisense oligonucleotide modulation of STAT3 expression
NZ514348A (en) 1999-05-04 2004-05-28 Exiqon As L-ribo-LNA analogues
US6617442B1 (en) 1999-09-30 2003-09-09 Isis Pharmaceuticals, Inc. Human Rnase H1 and oligonucleotide compositions thereof
AU7406700A (en) 1999-10-04 2001-05-10 Exiqon A/S Design of high affinity rnase h recruiting oligonucleotide
IL139450A0 (en) 1999-11-10 2001-11-25 Pfizer Prod Inc Methods of administering apo b-secretion/mtp inhibitors
ATE325806T1 (de) 2000-10-04 2006-06-15 Santaris Pharma As Verbesserte synthese von purin-blockierten nukleinsäure-analoga
US20030068320A1 (en) 2001-03-02 2003-04-10 Christine Dingivan Methods of administering/dosing CD2 antagonists for the prevention and treatment of autoimmune disorders or inflammatory disorders
CA2442092A1 (en) 2001-03-26 2002-10-17 Ribozyme Pharmaceuticals, Inc. Oligonucleotide mediated inhibition of hepatitis b virus and hepatitis c virus replication
US20030125241A1 (en) 2001-05-18 2003-07-03 Margit Wissenbach Therapeutic uses of LNA-modified oligonucleotides in infectious diseases
DE60202681T2 (de) 2001-07-12 2006-01-12 Santaris Pharma A/S Verfahren zur herstellung des lna phosphoramidite
US7407943B2 (en) 2001-08-01 2008-08-05 Isis Pharmaceuticals, Inc. Antisense modulation of apolipoprotein B expression
WO2003020739A2 (en) * 2001-09-04 2003-03-13 Exiqon A/S Novel lna compositions and uses thereof
EP1430128B1 (en) 2001-09-28 2018-04-25 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Micro-rna molecules
EP1432724A4 (en) * 2002-02-20 2006-02-01 Sirna Therapeutics Inc RNA inhibition mediated inhibition of MAP KINASE GENES
JP2005517427A (ja) 2002-02-20 2005-06-16 サーナ・セラピューティクス・インコーポレイテッド 短干渉核酸（ｓｉＮＡ）を用いるＣ型肝炎ウイルス（ＨＣＶ）遺伝子発現のＲＮＡ干渉媒介性阻害
JP4476802B2 (ja) 2002-05-08 2010-06-09 サンタリス ファーマ アー／エス ロックト核酸誘導体の製造
UA79300C2 (en) 2002-08-12 2007-06-11 Janssen Pharmaceutica Nv N-aryl piperidine substituted biphenylcarboxamides as inhibitors of apolipoprotein b secretion
US7087229B2 (en) 2003-05-30 2006-08-08 Enzon Pharmaceuticals, Inc. Releasable polymeric conjugates based on aliphatic biodegradable linkers
WO2004044181A2 (en) 2002-11-13 2004-05-27 Isis Pharmaceuticals, Inc. Antisense modulation of apolipoprotein b expression
EP2141233B1 (en) * 2002-11-18 2016-10-19 Roche Innovation Center Copenhagen A/S Antisense design
US8124582B2 (en) 2002-12-06 2012-02-28 Fibrogen, Inc. Treatment of diabetes
CA2515623A1 (en) 2003-02-10 2004-08-19 Santaris Pharma A/S Oligomeric compounds for the modulation of survivin expression
CN1768139A (zh) 2003-02-10 2006-05-03 独立行政法人产业技术总合研究所 通过dna干扰调控基因的表达
AU2004209599A1 (en) * 2003-02-10 2004-08-19 Santaris Pharma A/S Oligomeric compounds for the modulation of ras expression
AU2004221760B2 (en) * 2003-03-21 2010-03-18 Roche Innovation Center Copenhagen A/S Short interfering RNA (siRNA) analogues
EP2669377A3 (en) * 2003-04-17 2015-10-14 Alnylam Pharmaceuticals Inc. Modified iRNA agents
CA2532795A1 (en) 2003-08-07 2005-02-17 Avi Biopharma, Inc. Sense antiviral compound and method for treating ssrna viral infection
US20050142581A1 (en) 2003-09-04 2005-06-30 Griffey Richard H. Microrna as ligands and target molecules
JP5654722B2 (ja) 2003-11-26 2015-01-14 ユニバーシティ オブ マサチューセッツ 短鎖ｒｎａ機能の配列特異的阻害法
UA83510C2 (en) 2003-12-09 2008-07-25 Янссен Фармацевтика Н.В. N-aryl piperidine substituted biphenylcarboxamides as inhibitors of apolipoprotein b
KR20070006709A (ko) * 2003-12-23 2007-01-11 산타리스 팔마 에이/에스 Ｂｃｌ-2의 조절을 위한 올리고머 화합물
EP1713938A2 (en) 2004-02-09 2006-10-25 Thomas Jefferson University DIAGNOSIS AND TREATMENT OF CANCERS WITH MicroRNA LOCATED IN OR NEAR CANCER-ASSOCIATED CHROMOSOMAL FEATURES
WO2005079397A2 (en) * 2004-02-13 2005-09-01 Rockefeller University Anti-microrna oligonucleotide molecules
CA2562390C (en) 2004-04-07 2014-12-02 Exiqon A/S Novel methods for quantification of micrornas and small interfering rnas
KR20070004957A (ko) * 2004-04-20 2007-01-09 제나코 바이오메디컬 프로덕츠, 인코포레이티드 ｎｃＲＮＡ 검출 방법
CA2564503C (en) 2004-05-04 2015-12-29 The Board Of Trustees Of The Leland Stanford Junior University Inhibition of mir-122 in hepatitis c virus infected subjects and cells
US20080213891A1 (en) 2004-07-21 2008-09-04 Alnylam Pharmaceuticals, Inc. RNAi Agents Comprising Universal Nucleobases
EP1828215A2 (en) 2004-07-21 2007-09-05 Alnylam Pharmaceuticals Inc. Oligonucleotides comprising a modified or non-natural nucleobase
FR2873694B1 (fr) 2004-07-27 2006-12-08 Merck Sante Soc Par Actions Si Nouveaux aza-indoles inhibiteurs de la mtp et apob
US7632932B2 (en) 2004-08-04 2009-12-15 Alnylam Pharmaceuticals, Inc. Oligonucleotides comprising a ligand tethered to a modified or non-natural nucleobase
WO2006020768A2 (en) 2004-08-10 2006-02-23 Alnylam Pharmaceuticals, Inc. Chemically modified oligonucleotides
US20060035858A1 (en) 2004-08-10 2006-02-16 Geary Richard S Methods for modulating lipoprotein and cholesterol levels in humans
EP1791954A1 (en) 2004-09-07 2007-06-06 Yissum Research Development Company Of The Hebrew University Of Jerusalem Agents, compositions and methods for treating pathologies in which regulating an ache-associated biological pathway is beneficial
AU2005289588B2 (en) 2004-09-24 2011-12-22 Alnylam Pharmaceuticals, Inc. RNAi modulation of ApoB and uses thereof
PL2302055T3 (pl) 2004-11-12 2015-02-27 Asuragen Inc Sposoby i kompozycje z wykorzystaniem miRNA oraz cząsteczek inhibitorowych miRNA
AU2005306533B2 (en) 2004-11-17 2012-05-31 Arbutus Biopharma Corporation siRNA silencing of apolipoprotein B
EP1838870A2 (en) * 2004-12-29 2007-10-03 Exiqon A/S NOVEL OLIGONUCLEOTIDE COMPOSITIONS AND PROBE SEQUENCES USEFUL FOR DETECTION AND ANALYSIS OF MICRORNAS AND THEIR TARGET MRNAs
CA2605510C (en) 2005-04-19 2013-12-24 Surface Logix, Inc. Inhibitors of microsomal triglyceride transfer protein and apo-b secretion
US20090209621A1 (en) 2005-06-03 2009-08-20 The Johns Hopkins University Compositions and methods for decreasing microrna expression for the treatment of neoplasia
EP1919512B1 (en) 2005-08-10 2014-11-12 Alnylam Pharmaceuticals Inc. Chemically modified oligonucleotides for use in modulating micro rna and uses thereof
US20070213292A1 (en) * 2005-08-10 2007-09-13 The Rockefeller University Chemically modified oligonucleotides for use in modulating micro RNA and uses thereof
EP1931780B1 (en) 2005-08-29 2016-01-06 Regulus Therapeutics Inc. Antisense compounds having enhanced anti-microrna activity
WO2007031091A2 (en) 2005-09-15 2007-03-22 Santaris Pharma A/S Rna antagonist compounds for the modulation of p21 ras expression
KR20080068019A (ko) 2005-09-15 2008-07-22 산타리스 팔마 에이/에스 아포지단백질-ｂ100 발현 억제용 ｒｎａ 길항제 화합물
EP2388328A1 (en) 2006-01-27 2011-11-23 Isis Pharmaceuticals, Inc. Oligomeric compounds and compositions for the use in modulation of micrornas
WO2007112753A2 (en) 2006-04-03 2007-10-11 Santaris Pharma A/S Pharmaceutical composition comprising anti-mirna antisense oligonucleotides
WO2007143317A2 (en) 2006-05-05 2007-12-13 Isis Pharmaceuticals, Inc Compounds and methods for modulating expression of crp
JP5441688B2 (ja) 2006-05-11 2014-03-12 アイシス ファーマシューティカルズ， インコーポレーテッド ５’修飾二環式核酸類似体
US20080199961A1 (en) 2006-08-25 2008-08-21 Avi Biopharma, Inc. ANTISENSE COMPOSITION AND METHOD FOR INHIBITION OF miRNA BIOGENESIS
CA2662520A1 (en) 2006-09-15 2008-03-20 Enzon Pharmaceuticals, Inc. Polymeric conjugates containing positively-charged moieties
JP2010503707A (ja) 2006-09-15 2010-02-04 エンゾン ファーマスーティカルズ インコーポレイテッド オリゴヌクレオチドの送達を目的としたヒンダードエステル系生体分解性リンカー
EP2090665A2 (en) 2006-10-20 2009-08-19 Exiqon A/S Novel human microRNAs associated with cancer
US7803784B2 (en) 2006-10-24 2010-09-28 The Board Of Trustees Of The Leland Stanford Junior University Modulation of T cell signaling threshold and T cell sensitivity to antigens
CA2681568C (en) 2006-11-23 2019-01-08 Querdenker Aps Oligonucleotides for modulating target rna activity
US20090137504A1 (en) 2006-12-21 2009-05-28 Soren Morgenthaler Echwald Microrna target site blocking oligos and uses thereof
DK2149605T3 (da) 2007-03-22 2013-09-30 Santaris Pharma As Korte RNA antagonist forbindelser til modulering af det ønskede mRNA
WO2008124384A2 (en) 2007-04-03 2008-10-16 Aegerion Pharmaceuticals, Inc. Combinations of mtp inhibitors with cholesterol absorption inhibitors or interferon for treating hepatitis c
ES2388590T3 (es) 2007-05-30 2012-10-16 Isis Pharmaceuticals, Inc. Análogos de ácidos nucleicos bicíclicos con puente aminometileno N-sustituido.
DK2173760T4 (en) 2007-06-08 2016-02-08 Isis Pharmaceuticals Inc Carbocyclic bicyclic nukleinsyreanaloge
US20090082297A1 (en) 2007-06-25 2009-03-26 Lioy Daniel T Compositions and Methods for Regulating Gene Expression
US20110009466A1 (en) 2007-08-29 2011-01-13 President And Fellows Of Harvard College Methods of increasing gene expression through rna protection
US8288356B2 (en) 2007-10-04 2012-10-16 Santaris Pharma A/S MicroRNAs
WO2009109665A1 (en) 2008-03-07 2009-09-11 Santaris Pharma A/S Pharmaceutical compositions for treatment of microrna related diseases
EP2310505B1 (en) 2008-06-30 2017-08-09 Roche Innovation Center Copenhagen A/S Antidote oligomers
US8398734B2 (en) 2008-08-01 2013-03-19 Twister B.V. Cyclonic separator with a volute outlet duct
ES2599979T3 (es) 2009-04-24 2017-02-06 Roche Innovation Center Copenhagen A/S Composiciones farmacéuticas para el tratamiento de pacientes de VHC que no responden al interferón
EP2490699A1 (en) 2009-10-20 2012-08-29 Santaris Pharma A/S Oral delivery of therapeutically effective lna oligonucleotides

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6284458B1 (en) * 1992-09-10 2001-09-04 Isis Pharmaceuticals, Inc. Compositions and methods for treatment of hepatitis C virus-associated diseases
US6423489B1 (en) * 1992-09-10 2002-07-23 Isis Pharmaceuticals, Inc. Compositions and methods for treatment of Hepatitis C virus-associated diseases
US6433159B1 (en) * 1992-09-10 2002-08-13 Isis Pharmaceuticals, Inc. Compositions and methods for treatment of Hepatitis C virus associated diseases
US6030785A (en) * 1997-03-05 2000-02-29 University Of Washington Screening methods to identify agents that selectively inhibit hepatitis C virus replication
US20050069522A1 (en) * 2002-08-12 2005-03-31 Richard Colonno Combination pharmaceutical agents as inhibitors of HCV replication
US20060035212A1 (en) * 2002-12-12 2006-02-16 Universte Joseph Fourier Molecules inhibiting hepatitis c virus protein synthesis and method for screening same
US20050261218A1 (en) * 2003-07-31 2005-11-24 Christine Esau Oligomeric compounds and compositions for use in modulation small non-coding RNAs
US20070049547A1 (en) * 2003-07-31 2007-03-01 Christine Esau Methods for use in modulating miR-122a
US20050182005A1 (en) * 2004-02-13 2005-08-18 Tuschl Thomas H. Anti-microRNA oligonucleotide molecules
US20050227934A1 (en) * 2004-04-13 2005-10-13 Markus Stoffel Pancreatic islet microRNA and methods for inhibiting same
US20060185027A1 (en) * 2004-12-23 2006-08-17 David Bartel Systems and methods for identifying miRNA targets and for altering miRNA and target expression
US20060265771A1 (en) * 2005-05-17 2006-11-23 Lewis David L Monitoring microrna expression and function

Cited By (132)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9017940B2 (en) 2006-01-05 2015-04-28 The Ohio State University Methods for diagnosing colon cancer using MicroRNA signatures
US9017939B2 (en) 2006-01-05 2015-04-28 The Ohio State University Methods for diagnosing breast, colon, lung, pancreatic and prostate cancer using miR-21 and miR-17-5p
US8865885B2 (en) 2006-03-20 2014-10-21 The Ohio State University Research Foundation MicroRNA fingerprints during human megakaryocytopoiesis
US20100286234A1 (en) * 2006-04-03 2010-11-11 Joacim Elmen Pharmaceutical Composition Comprising Anti-Mirna Antisense Oligonucleotides
US9133455B2 (en) * 2006-04-03 2015-09-15 Roche Innovation Center Copenhagen A/S Pharmaceutical composition comprising anti-miRNA antisense oligonucleotides
US8729250B2 (en) 2006-04-03 2014-05-20 Joacim Elmén Antisense oligonucleotides for inhibition of microRNA-21
US8163708B2 (en) 2006-04-03 2012-04-24 Santaris Pharma A/S Pharmaceutical composition comprising anti-mirna antisense oligonucleotide
US20140329883A1 (en) * 2006-04-03 2014-11-06 Santaris Pharma A/S Pharmaceutical Composition Comprising Anti-miRNA Antisense Oligonucleotides
US20100144850A1 (en) * 2007-04-30 2010-06-10 The Ohio State University Research Foundation Methods for Differentiating Pancreatic Cancer from Normal Pancreatic Function and/or Chronic Pancreatitis
US9085804B2 (en) 2007-08-03 2015-07-21 The Ohio State University Research Foundation Ultraconserved regions encoding ncRNAs
US8288356B2 (en) 2007-10-04 2012-10-16 Santaris Pharma A/S MicroRNAs
US8906871B2 (en) 2007-10-04 2014-12-09 Santaris Pharma A/S MicromiRs
US20100280099A1 (en) * 2007-10-04 2010-11-04 Santaris Pharma A/S Combination Treatment For The Treatment of Hepatitis C Virus Infection
US10450564B2 (en) 2007-10-04 2019-10-22 Roche Innovation Center Copenhagen A/S Micromirs
US20100298410A1 (en) * 2007-10-04 2010-11-25 Santaris Pharma A/S MICROMIRs
US8440637B2 (en) 2007-10-04 2013-05-14 Santaris Pharma A/S Combination treatment for the treatment of hepatitis C virus infection
US20090143326A1 (en) * 2007-10-04 2009-06-04 Santaris Pharma A/S MICROMIRs
US20100285471A1 (en) * 2007-10-11 2010-11-11 The Ohio State University Research Foundation Methods and Compositions for the Diagnosis and Treatment of Esphageal Adenocarcinomas
US20120295962A1 (en) * 2007-10-29 2012-11-22 Rosetta Genomics Ltd. Targeting micrornas for the treatment of liver cancer
US8680067B2 (en) * 2007-10-29 2014-03-25 Regulus Therapeutics, Inc. Targeting microRNAs for the treatment of liver cancer
US20110034538A1 (en) * 2008-02-28 2011-02-10 The Ohio State University Research Foundation MicroRNA-Based Methods and Compositions for the Diagnosis, Prognosis and Treatment of Gastric Cancer
US20110077288A1 (en) * 2008-03-07 2011-03-31 Santaris Pharma A/S Pharmaceutical Compositions for Treatment of MicroRNA Related Diseases
US20090298916A1 (en) * 2008-03-07 2009-12-03 Santaris Pharma A/S Pharmaceutical compositions for treatment of microRNA related diseases
US8404659B2 (en) 2008-03-07 2013-03-26 Santaris Pharma A/S Pharmaceutical compositions for treatment of MicroRNA related diseases
US8361980B2 (en) 2008-03-07 2013-01-29 Santaris Pharma A/S Pharmaceutical compositions for treatment of microRNA related diseases
US8492357B2 (en) 2008-08-01 2013-07-23 Santaris Pharma A/S Micro-RNA mediated modulation of colony stimulating factors
US9034837B2 (en) 2009-04-24 2015-05-19 Roche Innovation Center Copenhagen A/S Pharmaceutical compositions for treatment of HCV patients that are poor-responders to interferon
US20100330035A1 (en) * 2009-04-24 2010-12-30 Hildebrandt-Eriksen Elisabeth S Pharmaceutical Compositions for Treatment of HCV Patients that are Poor-Responders to Interferon
US20110190372A1 (en) * 2009-08-07 2011-08-04 New York University Compositions and methods for treating inflammatory disorders
US20150240232A1 (en) * 2009-10-19 2015-08-27 University Of Massachusetts Deducing Exon Connectivity by RNA-Templated DNA Ligation/Sequencing
US8916533B2 (en) 2009-11-23 2014-12-23 The Ohio State University Materials and methods useful for affecting tumor cell growth, migration and invasion
EP3210611A2 (en) 2010-03-12 2017-08-30 The Brigham and Women's Hospital, Inc. Methods of treating vascular inflammatory disorders
US20110281933A1 (en) * 2010-05-13 2011-11-17 Saint Louis University Methods and compositions for the management of cardiovascular disease with oligonucleotides
WO2011144831A1 (fr) 2010-05-21 2011-11-24 Sine Sileo Agent édulcorant contenant un extrait de stévia rebaudiana bertoni
US9212362B2 (en) 2010-05-21 2015-12-15 Universitat Fur Bodenkultur Wien Compositions for use in treating or diagnosing bone disorders and/or cardiovascular disorders
US9150855B2 (en) 2010-05-21 2015-10-06 Universität Für Bodenkultur Wien Methods for diagnosing bone or cardiovascular disorders
US9206115B2 (en) 2010-05-21 2015-12-08 Technische Universität Graz ATGListatin and pharmaceutical composition comprising the same
JP2013532141A (ja) * 2010-06-04 2013-08-15 ボード・オブ・リージエンツ，ザ・ユニバーシテイ・オブ・テキサス・システム ｍｉＲ−３７８による代謝調節
US8716258B2 (en) 2010-06-04 2014-05-06 The Board Of Regents, The University Of Texas System Regulation of metabolism by miR-378
US8859519B2 (en) 2010-08-25 2014-10-14 The General Hospital Corporation Methods targeting miR-33 microRNAs for regulating lipid metabolism
US11142800B2 (en) 2010-10-07 2021-10-12 The General Hospital Corporation Biomarkers of cancer
US8946187B2 (en) 2010-11-12 2015-02-03 The Ohio State University Materials and methods related to microRNA-21, mismatch repair, and colorectal cancer
EP3260540A1 (en) 2010-11-12 2017-12-27 The General Hospital Corporation Polycomb-associated non-coding rnas
US10758619B2 (en) 2010-11-15 2020-09-01 The Ohio State University Controlled release mucoadhesive systems
US11679157B2 (en) 2010-11-15 2023-06-20 The Ohio State University Controlled release mucoadhesive systems
US20120184596A1 (en) * 2010-12-15 2012-07-19 Miragen Therapeutics Microrna inhibitors comprising locked nucleotides
US8642751B2 (en) * 2010-12-15 2014-02-04 Miragen Therapeutics MicroRNA inhibitors comprising locked nucleotides
US20140187603A1 (en) * 2010-12-15 2014-07-03 Miragen Therapeutics Microrna inhibitors comprising locked nucleotides
WO2012097261A2 (en) 2011-01-14 2012-07-19 The General Hospital Corporation Methods targeting mir-128 for regulating cholesterol/lipid metabolism
US20150126579A1 (en) * 2011-04-12 2015-05-07 Beth Israel Deaconess Medical Center, Inc. Micro-rna inhibitors and their uses in disease
US11274301B2 (en) 2011-04-12 2022-03-15 Beth Israel Deaconess Medical Center Micro-RNA inhibitors and their uses in disease
US10131905B2 (en) * 2011-04-12 2018-11-20 Beth Israel Deaconess Medical Center Micro-RNA inhibitors and their uses in disease
US9428749B2 (en) 2011-10-06 2016-08-30 The Board Of Regents, The University Of Texas System Control of whole body energy homeostasis by microRNA regulation
WO2013055865A1 (en) 2011-10-11 2013-04-18 The Brigham And Women's Hospital, Inc. Micrornas in neurodegenerative disorders
US9249468B2 (en) 2011-10-14 2016-02-02 The Ohio State University Methods and materials related to ovarian cancer
WO2013090556A1 (en) * 2011-12-13 2013-06-20 The Ohio State University Methods and compositions related to mir-21 and mir-29a, exosome inhibition, and cancer metastasis
US9481885B2 (en) * 2011-12-13 2016-11-01 Ohio State Innovation Foundation Methods and compositions related to miR-21 and miR-29a, exosome inhibition, and cancer metastasis
US20140323553A1 (en) * 2011-12-13 2014-10-30 Ohio State Innovation Foundation Methods and Compositions Related to MiR-21 & MiR-29a, Exosome Inhibition, and Cancer Metastasis
CN104619353A (zh) * 2011-12-13 2015-05-13 俄亥俄州国家创新基金会 与miR-21和miR-29a相关的方法和组合物、外切体抑制和癌症转移
US20170002353A1 (en) * 2011-12-13 2017-01-05 Ohio State Innovation Foundation Methods and Compositions Related To miR-21 and miR-29A, Exosome Inhibition And Cancer Metastasis
US20140356459A1 (en) * 2011-12-15 2014-12-04 Oncostamen S.R.L. Micrornas and uses thereof
US8859202B2 (en) 2012-01-20 2014-10-14 The Ohio State University Breast cancer biomarker signatures for invasiveness and prognosis
US9434995B2 (en) 2012-01-20 2016-09-06 The Ohio State University Breast cancer biomarker signatures for invasiveness and prognosis
US9163235B2 (en) * 2012-06-21 2015-10-20 MiRagen Therapeutics, Inc. Inhibitors of the miR-15 family of micro-RNAs
US20130345288A1 (en) * 2012-06-21 2013-12-26 Miragen Therapeutics Inhibitors of the mir-15 family of micro-rnas
US10337005B2 (en) 2012-06-21 2019-07-02 MiRagen Therapeutics, Inc. Oligonucleotide-based inhibitors comprising locked nucleic acid motif
US9803202B2 (en) 2012-06-21 2017-10-31 MiRagen Therapeutics, Inc. Oligonucleotide-based inhibitors comprising locked nucleic acid motif
US9388408B2 (en) 2012-06-21 2016-07-12 MiRagen Therapeutics, Inc. Oligonucleotide-based inhibitors comprising locked nucleic acid motif
US9790492B2 (en) 2012-08-20 2017-10-17 National Cancer Center Agent for treating cancer
CN109793897A (zh) * 2012-10-31 2019-05-24 洛克菲勒大学 结肠癌的治疗和诊断
WO2014151835A1 (en) * 2013-03-15 2014-09-25 Miragen Therapeutics, Inc Locked nucleic acid inhibitor of mir-145 and uses thereof
US10633655B2 (en) 2013-03-15 2020-04-28 The Board Of Trustees Of The Leland Stanford Junior University tRNA derived small RNAs (tsRNAs) involved in cell viability
US9752143B2 (en) 2013-03-15 2017-09-05 MiRagen Therapeutics, Inc. Locked nucleic acid inhibitor of miR-145 and uses thereof
US9428537B2 (en) * 2013-03-15 2016-08-30 The Board Of Trustees Of The Leland Stanford Junior University tRNA derived small RNAs (tsRNAs) involved in cell viability
US20140323555A1 (en) * 2013-03-15 2014-10-30 The Board Of Trustees Of The Leland Stanford Junior University tRNA DERIVED SMALL RNAs (tsRNAs) INVOLVED IN CELL VIABILITY
US10982211B2 (en) 2013-03-15 2021-04-20 The Board Of Trustees Of The Leland Stanford Junior University tRNA derived small RNAs (tsRNAs) involved in cell viability
US9932585B2 (en) 2013-11-11 2018-04-03 Emory University Manipulating microRNA for the management of neurological diseases or conditions and compositions related thereto
US9458458B2 (en) * 2013-11-11 2016-10-04 Emory University Manipulating microRNA for the management of neurological diseases or conditions and compositions related thereto
US20150133522A1 (en) * 2013-11-11 2015-05-14 Emory University Manipulating microrna for the management of neurological diseases or conditions and compositions related thereto
WO2015200697A1 (en) 2014-06-25 2015-12-30 The General Hospital Corporation Targeting human satellite ii (hsatii)
EP3760208A1 (en) 2014-06-25 2021-01-06 The General Hospital Corporation Targeting human satellite ii (hsatii)
US10472626B2 (en) * 2014-07-31 2019-11-12 Agency For Science, Technology And Research Modified antimir-138 oligonucleotides
US11542506B2 (en) 2014-11-14 2023-01-03 Voyager Therapeutics, Inc. Compositions and methods of treating amyotrophic lateral sclerosis (ALS)
CN112410339A (zh) * 2014-11-14 2021-02-26 沃雅戈治疗公司 调节性多核苷酸
WO2016077689A1 (en) * 2014-11-14 2016-05-19 Voyager Therapeutics, Inc. Modulatory polynucleotides
US10570395B2 (en) 2014-11-14 2020-02-25 Voyager Therapeutics, Inc. Modulatory polynucleotides
AU2015346164B2 (en) * 2014-11-14 2020-01-30 Voyager Therapeutics, Inc. Modulatory polynucleotides
US10597660B2 (en) 2014-11-14 2020-03-24 Voyager Therapeutics, Inc. Compositions and methods of treating amyotrophic lateral sclerosis (ALS)
CN112410338A (zh) * 2014-11-14 2021-02-26 沃雅戈治疗公司 调节性多核苷酸
AU2020202530B2 (en) * 2014-11-14 2021-07-08 Voyager Therapeutics, Inc. Modulatory polynucleotides
US11198873B2 (en) 2014-11-14 2021-12-14 Voyager Therapeutics, Inc. Modulatory polynucleotides
US10920227B2 (en) 2014-11-14 2021-02-16 Voyager Therapeutics, Inc. Compositions and methods of treating amyotrophic lateral sclerosis (ALS)
US10280422B2 (en) 2015-01-20 2019-05-07 MiRagen Therapeutics, Inc. MiR-92 inhibitors and uses thereof
US9885042B2 (en) 2015-01-20 2018-02-06 MiRagen Therapeutics, Inc. miR-92 inhibitors and uses thereof
WO2016164463A1 (en) 2015-04-07 2016-10-13 The General Hospital Corporation Methods for reactivating genes on the inactive x chromosome
US11912994B2 (en) 2015-04-07 2024-02-27 The General Hospital Corporation Methods for reactivating genes on the inactive X chromosome
US20190359980A1 (en) * 2015-06-05 2019-11-28 MiRagen Therapeutics, Inc. Oligonucleotide compositions and uses thereof
US10316318B2 (en) 2015-06-05 2019-06-11 MiRagen Therapeutics, Inc. Oligonucleotide compositions and uses thereof
US9994852B2 (en) * 2015-06-05 2018-06-12 MiRagen Therapeutics, Inc. Oligonucleotide compositions and uses thereof
WO2016210241A1 (en) 2015-06-26 2016-12-29 Beth Israel Deaconess Medical Center, Inc. Cancer therapy targeting tetraspanin 33 (tspan33) in myeloid derived suppressor cells
US10612021B2 (en) 2015-10-07 2020-04-07 Kyoto University Therapeutic or prophylactic composition for TDP-43 proteinopathy
US11220689B2 (en) 2015-10-16 2022-01-11 Children's Medical Center Corporation Modulators of telomere disease
WO2017087708A1 (en) 2015-11-19 2017-05-26 The Brigham And Women's Hospital, Inc. Lymphocyte antigen cd5-like (cd5l)-interleukin 12b (p40) heterodimers in immunity
US11001622B2 (en) 2015-11-19 2021-05-11 The Brigham And Women's Hospital, Inc. Method of treating autoimmune disease with lymphocyte antigen CD5-like (CD5L) protein
US11884717B2 (en) 2015-11-19 2024-01-30 The Brigham And Women's Hospital, Inc. Method of treating autoimmune disease with lymphocyte antigen CD5-like (CD5L) protein
WO2017147087A1 (en) 2016-02-25 2017-08-31 The Brigham And Women's Hospital, Inc. Treatment methods for fibrosis targeting smoc2
US11193129B2 (en) 2016-05-18 2021-12-07 Voyager Therapeutics, Inc. Modulatory polynucleotides
US11951121B2 (en) 2016-05-18 2024-04-09 Voyager Therapeutics, Inc. Compositions and methods for treating Huntington's disease
US10584337B2 (en) 2016-05-18 2020-03-10 Voyager Therapeutics, Inc. Modulatory polynucleotides
US10626395B2 (en) 2016-10-27 2020-04-21 The General Hospital Corporation Therapeutic targeting of a microRNA to treat Duchenne muscular dystrophy
WO2018080658A1 (en) * 2016-10-27 2018-05-03 Aalborg University Therapeutic targeting of a microrna to treat duchenne muscular dystrophy
WO2018081817A2 (en) 2016-10-31 2018-05-03 University Of Massachusetts Targeting microrna-101-3p in cancer therapy
WO2018195486A1 (en) 2017-04-21 2018-10-25 The Broad Institute, Inc. Targeted delivery to beta cells
US11752181B2 (en) 2017-05-05 2023-09-12 Voyager Therapeutics, Inc. Compositions and methods of treating Huntington's disease
US11603542B2 (en) 2017-05-05 2023-03-14 Voyager Therapeutics, Inc. Compositions and methods of treating amyotrophic lateral sclerosis (ALS)
US11434502B2 (en) 2017-10-16 2022-09-06 Voyager Therapeutics, Inc. Treatment of amyotrophic lateral sclerosis (ALS)
US11931375B2 (en) 2017-10-16 2024-03-19 Voyager Therapeutics, Inc. Treatment of amyotrophic lateral sclerosis (ALS)
WO2019089216A1 (en) 2017-11-01 2019-05-09 Dana-Farber Cancer Institute, Inc. Methods of treating cancers
CN108272815A (zh) * 2017-12-06 2018-07-13 南方医科大学深圳医院 EB病毒miR-BART10-5p抑制剂的应用
WO2019178411A1 (en) * 2018-03-14 2019-09-19 Beth Israel Deaconess Medical Center Inhibitors of micro-rna 22
US11499152B2 (en) 2018-03-14 2022-11-15 Beth Israel Deaconess Medical Center Inhibitors of micro-RNA 22
JP2021518159A (ja) * 2018-03-14 2021-08-02 ベス イスラエル デアコネス メディカル センター マイクロｒｎａ２２の阻害剤
JP7318166B2 (ja) 2018-03-14 2023-08-01 ベス イスラエル デアコネス メディカル センター マイクロｒｎａ２２の阻害剤
US11753639B2 (en) 2018-03-14 2023-09-12 Beth Israel Deaconess Medical Center Micro-RNA and obesity
CN108220427A (zh) * 2018-03-20 2018-06-29 南京大学 一种用于鉴别诊断BHD综合征与原发性自发性气胸的血浆microRNA标记物及应用
US20210220387A1 (en) * 2018-05-18 2021-07-22 Hoffmann-La Roche, Inc. Pharmaceutical compositions for treatment of microrna related diseases
US11679100B2 (en) 2018-05-30 2023-06-20 The Regents Of The University Of California Methods of enhancing immunity
WO2019232132A1 (en) * 2018-05-30 2019-12-05 The Regents Of The University Of California Methods of enhancing immunity
WO2020047229A1 (en) 2018-08-29 2020-03-05 University Of Massachusetts Inhibition of protein kinases to treat friedreich ataxia
CN110468202A (zh) * 2019-01-18 2019-11-19 宁夏医科大学 一种靶向TIGIT的miR-206作为肝癌诊断和治疗新型分子的用途
CN110548041A (zh) * 2019-08-30 2019-12-10 中国医科大学附属盛京医院 LNA-anti-miR-150在制备预防或治疗肾脏纤维化药物中的用途
CN112301130A (zh) * 2020-11-12 2021-02-02 苏州京脉生物科技有限公司 一种肺癌早期检测的标志物、试剂盒及方法

Also Published As

Publication number Publication date
JP5198430B2 (ja) 2013-05-15
US8729250B2 (en) 2014-05-20
JP2013078317A (ja) 2013-05-02
US20120083596A1 (en) 2012-04-05
DK2666859T3 (en) 2019-04-08
JP2016073293A (ja) 2016-05-12
EA015570B1 (ru) 2011-10-31
US9133455B2 (en) 2015-09-15
US20180195062A1 (en) 2018-07-12
JP2009532392A (ja) 2009-09-10
CA3042781A1 (en) 2007-10-11
US20190071672A1 (en) 2019-03-07
EP2194129A2 (en) 2010-06-09
WO2007112754A2 (en) 2007-10-11
WO2007112753A8 (en) 2009-07-30
ES2715625T3 (es) 2019-06-05
CA3024953A1 (en) 2007-10-11
WO2007112753A2 (en) 2007-10-11
JP2009532044A (ja) 2009-09-10
US20160060627A1 (en) 2016-03-03
KR101407707B1 (ko) 2014-06-19
AU2007234191B2 (en) 2012-07-12
US20210071181A1 (en) 2021-03-11
CA2649045C (en) 2019-06-11
IL194007A0 (en) 2011-08-01
EP2194129A3 (en) 2012-12-26
AU2007234192A1 (en) 2007-10-11
CA3042781C (en) 2021-10-19
KR20080108154A (ko) 2008-12-11
MX2008012219A (es) 2008-10-02
US20140329883A1 (en) 2014-11-06
CA2648132C (en) 2019-05-28
CA2649045A1 (en) 2007-10-11
EP2007889A2 (en) 2008-12-31
JP2014128274A (ja) 2014-07-10
WO2007112753A3 (en) 2008-03-13
US20120238618A1 (en) 2012-09-20
JP5814505B2 (ja) 2015-11-17
WO2007112754A3 (en) 2008-04-24
AU2007234191A1 (en) 2007-10-11
EP2007888A2 (en) 2008-12-31
EA200870402A1 (ru) 2009-04-28
JP5872603B2 (ja) 2016-03-01
CA2648132A1 (en) 2007-10-11
JP6326025B2 (ja) 2018-05-16

Similar Documents

Publication Publication Date Title
US20210071181A1 (en) Pharmaceutical composition
EP2666859B1 (en) Pharmaceutical composition comprising anti-mirna antisense oligonucleotides
US10450564B2 (en) Micromirs
AU2013254923A1 (en) Pharmaceutical compositions comprising anti-miRNA antisense oligonucleotide
AU2007234192B2 (en) Pharmaceutical compositions comprising anti-miRNA antisense oligonucleotides
AU2012216487B2 (en) Pharmaceutical composition comprising anti-miRNA antisense oligonucleotides
AU2014208214A1 (en) Pharmaceutical composition comprising anti-miRNA antisense oligonucleotides
NZ571620A (en) Pharmaceutical composition comprising anti-miRNA antisense oligonucleotides

Legal Events

Date Code Title Description
AS Assignment

Owner name: SANTARIS PHARMA A/S, DENMARK

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:ELMEN, JOACIM;KEARNEY, PHIL;KAUPPINEN, SAKARI;REEL/FRAME:021650/0072

Effective date: 20060703

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION