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WO2011031998A1 - Modulation de l'expression du facteur de transcription pour le silençage de re1 (rest) - Google Patents

Modulation de l'expression du facteur de transcription pour le silençage de re1 (rest) Download PDF

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Publication number
WO2011031998A1
WO2011031998A1 PCT/US2010/048467 US2010048467W WO2011031998A1 WO 2011031998 A1 WO2011031998 A1 WO 2011031998A1 US 2010048467 W US2010048467 W US 2010048467W WO 2011031998 A1 WO2011031998 A1 WO 2011031998A1
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Prior art keywords
compound
rest
modified
modified oligonucleotide
seq
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PCT/US2010/048467
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English (en)
Inventor
Yalda Sedaghat
Breet P. Monia
Huynh-Hoa Bui
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Isis Pharmaceuticals, Inc.
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Publication of WO2011031998A1 publication Critical patent/WO2011031998A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C12N2310/3341—5-Methylcytosine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications

Definitions

  • Embodiments of the present invention provide methods, compounds, and compositions for reducing expression of REl Silencing Transcription Factor (REST) mRNA and protein in an animal. Such methods, compounds, and compositions are useful to treat, prevent, or ameliorate neurodegenerative diseases, such as, Huntington's Disease (HD).
  • REST REl Silencing Transcription Factor
  • REl silencing transcription factor also known as neuron-restrictive silencer factor (NRSF), (Schoenherr and Anderson, Science 1995, 267: 1360-1363) blocks transcription of its target genes by binding to a specific consensus 21 bp REl binding site/neuron-restrictive silencer element (RE1/NRSE) that is present in the target genes' regulatory regions.
  • REST functions very effectively as a transcriptional repressor at a distance and is able to repress transcription despite location or orientation of the binding site within a gene.
  • the wild-type huntingtin protein was found to bind to REST and thereby sequester REST in the cytoplasm (Zuccato et al., Nat. Genet. 2003, 35: 76-83). It is postulated that in the pathology of Huntington's disease, the REST- huntingtin protein interaction is lost, causing REST to enter the nucleus and repress its target genes.
  • REST antisense inhibitors modulate expression of REST mRNA and protein.
  • REST antisense inhibitors are useful for treating HD.
  • the present invention provides a method of inhibiting REST mRNA or protein expression in an animal, comprising administering to an animal in need thereof a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified oligonucleotide is at least 90% complementary over its entire length to a REST nucleic acid.
  • the modified oligonucleotide comprises at least 12 contiguous nucleobases of a nucleobase sequence selected from any one of nucleobase sequences of SEQ ID NOs: 4 to 81.
  • the modified oligonucleotide has a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of nucleobases 3965-3984, 5469-5488, 5487-5506, 5531-5550, 5622-5641, 5862-5881, 6690-6709, 6732-6751, 6780-6799, 6804-6823, 6817-6836, 6844-6863, 6961-6980, 6988-7007, 7061-7080, 7143-7162, 7158-7177, 7210-7229, 7281-7300, 7337-7356, 7563-7582, 15841- 15860, 15884-15903, 15906-15925, 15910-15929, 23651-23670, 23661-23680, 23671-23690, 23688-23707, 23700-23719, 23711-23730, 25904-25923, 2594
  • the modified oligonucleotide has a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of nucleobases 5469-5506, 6780-6863, 7143-7177, 15841-15929, 23651-23730, 25978- 26013, 26050-26082, 26135-26169, 27665-27731, 2786127891, 27956-27986, 28044-28074, and 28133-28164, wherein the nucleobase sequence of the modified oligonucleotide is at least 90% complementary over its entire length to SEQ ID NO: 1.
  • the present invention provides a method of increasing DMN1 mRNA or protein expression in an animal, comprising administering to an animal in need thereof a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified oligonucleotide is at least 90% complementary over its entire length to a REST nucleic acid.
  • the present invention provides a method of increasing BDNF mRNA or protein expression in an animal, comprising administering to an animal in need thereof a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified oligonucleotide is at least 90% complementary over its entire length to a REST nucleic acid.
  • the present invention provides a method of increasing synapsinl mRNA or protein expression in an animal, comprising administering to an animal in need thereof a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified oligonucleotide is at least 90% complementary over its entire length to a REST nucleic acid.
  • the present invention provides compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 12 contiguous nucleobases of a nucleobase sequence selected from any one of nucleobase sequences of SEQ ID NOs: 4 to 81.
  • the compound consists of a single-stranded modified oligonucleotide.
  • the modified oligonucleotide has a nucleobase sequence that is 100% complementary to a nucleobase sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
  • the present invention provides compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 12 contiguous nucleobases of a nucleobase sequence selected from any one of nucleobase sequences of SEQ ID NOs: 4 to 81 for use in treating an animal having a disease or condition associated with REST by administering to the animal a therapeutically effective amount of the compound so that expression of REST is inhibited.
  • the present invention provides compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 12 contiguous nucleobases of a nucleobase sequence selected from any one of nucleobase sequences of SEQ ID NOs: 4 to 81 for use in treating an animal having a disease or condition associated with REST by administering to the animal a therapeutically effective amount of the compound so that expression of BDNF, synapsin 1, or DNM1 is increased.
  • at least one internucleoside linkage of the oligonucleotide is a modified internucleoside linkage.
  • each internucleoside linkage is a phosphorothioate internucleoside linkage.
  • the compound comprises at least one nucleoside comprising a modified sugar.
  • at least one modified sugar is a bicyclic sugar.
  • at least one modified sugar comprises a 2'-0-methoxyethyl.
  • the compound comprises at least one nucleoside comprising a modified nucleobase.
  • the modified nucleobase is a 5- methylcytosine.
  • the modified oligonucleotide comprises:
  • a 5' wing segment consisting of linked nucleosides
  • a 3' wing segment consisting of linked nucleosides
  • each nucleoside of each wing segment comprises a modified sugar
  • the gap segment consists often linked deoxynucleosides; the 5' wing segment consists of five linked nucleosides; the 3' wing segment consisting of five linked nucleosides.
  • each nucleoside of each wing segment comprises a 2'-0- methoxyethyl sugar; and each internucleoside linkage is a phosphorothioate linkage.
  • the modified oligonucleotide consists of 20 linked nucleosides.
  • such compounds are useful for treating Huntington's Disease in an animal.
  • the animal is a human.
  • the invention provides a composition comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 12 contiguous nucleobases of a nucleobase sequence selected from any one of nucleobase sequences of SEQ ID NOs: 4 to 81 or a salt thereof and a pharmaceutically acceptable carrier or diluent.
  • the modified oligonucleotide is a single-stranded oligonucleotide.
  • the modified oligonucleotide of such a composition consists of 20 linked nucleosides.
  • 2'-0-methoxyethyl refers to an O-methoxy-ethyl modification of the 2' position of a furosyl ring.
  • a 2'-0-methoxyethyl modified sugar is a modified sugar.
  • cytosine means a cytosine modified with a methyl group attached to the 5' position.
  • a 5-methylcytosine is a modified nucleobase.
  • Active pharmaceutical agent means the substance or substances in a pharmaceutical composition that provide a therapeutic benefit when administered to an individual.
  • an antisense oligonucleotide targeted to REST is an active
  • Active target region or “target region” means a region to which one or more active antisense compounds is targeted.
  • Active antisense compounds means antisense compounds that reduce target nucleic acid levels or protein levels.
  • administering refers to the co-administration of two agents in any manner in which the pharmacological effects of both are manifest in the patient at the same time. Concomitant administration does not require that both agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of both agents need not manifest themselves at the same time. The effects need only be overlapping for a period of time and need not be coextensive.
  • administering means providing a pharmaceutical agent to an individual, and includes, but is not limited to administering by a medical professional and self-administering.
  • “Amelioration” refers to a lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition.
  • the severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art.
  • Animal refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.
  • Antibody refers to a molecule characterized by reacting specifically with an antigen in some way, where the antibody and the antigen are each defined in terms of the other. Antibody may refer to a complete antibody molecule or any fragment or region thereof, such as the heavy chain, the light chain, Fab region, and Fc region.
  • Antisense activity means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.
  • Antisense compound means an oligomeric compound that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
  • Antisense inhibition means reduction of target nucleic acid levels or target protein levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound.
  • Antisense oligonucleotide means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.
  • Bicyclic sugar means a furosyl ring modified by the bridging of two non-geminal ring atoms.
  • a bicyclic sugar is a modified sugar.
  • BNA Bicyclic nucleic acid
  • BNA a nucleoside or nucleotide wherein the furanose portion of the nucleoside or nucleotide includes a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system.
  • Cap structure or "terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an antisense compound.
  • “Chemically distinct region” refers to a region of an antisense compound that is in some way chemically different than another region of the same antisense compound. For example, a region having 2'-0-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2'-0-methoxyethyl modifications.
  • Chimeric antisense compound means an antisense compound that has at least two chemically distinct regions.
  • Co-administration means administration of two or more pharmaceutical agents to an individual.
  • the two or more pharmaceutical agents may be in a single pharmaceutical composition, or may be in separate pharmaceutical compositions.
  • Each of the two or more pharmaceutical agents may be administered through the same or different routes of
  • Co-administration encompasses parallel or sequential administration.
  • “Complementarity” means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.
  • Contiguous nucleobases means nucleobases immediately adjacent to each other.
  • diluent means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable.
  • the diluent in an injected composition may be a liquid, e.g. saline solution.
  • Dose means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose may be
  • the pharmaceutical agent is administered in one, two, or more boluses, tablets, or injections.
  • the desired dose requires a volume not easily accommodated by a single injection, therefore, two or more injections may be used to achieve the desired dose.
  • the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses may be stated as the amount of pharmaceutical agent per hour, day, week, or month.
  • Effective amount means the amount of active pharmaceutical agent sufficient to effectuate a desired physiological outcome in an individual in need of the agent.
  • the effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.
  • “Fully complementary” or “100% complementary” means each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid.
  • a first nucleic acid is an antisense compound and a target nucleic acid is a second nucleic acid.
  • Gapmer means a chimeric antisense compound in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions.
  • the internal region may be referred to as a "gap segment” and the external regions may be referred to as "wing segments.”
  • Gap-widened means a chimeric antisense compound having a gap segment of 12 or more contiguous 2'-deoxyribonucleosides positioned between and immediately adjacent to 5' and 3 ' wing segments having from one to six nucleosides.
  • Hybridization means the annealing of complementary nucleic acid molecules.
  • complementary nucleic acid molecules include an antisense compound and a target nucleic acid.
  • “Individual” means a human or non-human animal selected for treatment or therapy.
  • Internucleoside linkage refers to the chemical bond between nucleosides.
  • Linked nucleosides means adjacent nucleosides which are bonded together.
  • mismatch or “non-complementary nucleobase” refers to the case when a nucleobase of a first nucleic acid is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid.
  • Modified internucleoside linkage refers to a substitution or any change from a naturally occurring internucleoside bond (i.e. a phosphodiester internucleoside bond).
  • Modified nucleobase refers to any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil.
  • An "unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).
  • Modified nucleotide means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, or modified nucleobase.
  • a “modified nucleoside” means a nucleoside having, independently, a modified sugar moiety or modified nucleobase.
  • Modified oligonucleotide means an oligonucleotide comprising a modified
  • internucleoside linkage a modified sugar, or a modified nucleobase.
  • Modified sugar refers to a substitution or change from a natural sugar.
  • “Motif means the pattern of chemically distinct regions in an antisense compound.
  • “Naturally occurring internucleoside linkage” means a 3' to 5' phosphodiester linkage.
  • Natural sugar moiety means a sugar found in DNA (2'-H) or RNA (2'-OH).
  • Nucleic acid refers to molecules composed of monomelic nucleotides.
  • a nucleic acid includes ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acids (siRNA), and microRNAs (miRNA).
  • RNA ribonucleic acids
  • DNA deoxyribonucleic acids
  • siRNA small interfering ribonucleic acids
  • miRNA microRNAs
  • Nucleobase means a heterocyclic moiety capable of pairing with a base of another nucleic acid.
  • Nucleobase sequence means the order of contiguous nucleobases independent of any sugar, linkage, or nucleobase modification.
  • Nucleoside means a nucleobase linked to a sugar.
  • Nucleotide means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.
  • Oligomer means a polymer of linked monomelic subunits which is capable of hybridizing to at least a region of a nucleic acid molecule.
  • Oligomer means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another.
  • Parenteral administration means administration through injection or infusion.
  • Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g. intrathecal or intracerebroventricular administration.
  • Peptide means a molecule formed by linking at least two amino acids by amide bonds. Peptide refers to polypeptides and proteins.
  • “Pharmaceutical composition” means a mixture of substances suitable for administering to an individual.
  • a pharmaceutical composition may comprise one or more active pharmaceutical agents and a sterile aqueous solution.
  • “Pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.
  • Phosphorothioate linkage means a linkage between nucleosides where the
  • phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom.
  • a phosphorothioate linkage is a modified internucleoside linkage.
  • Portion means a defined number of contiguous (i.e. linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound.
  • Prevent refers to delaying or forestalling the onset or development of a disease, disorder, or condition for a period of time from minutes to indefinitely. Prevent also means reducing risk of developing a disease, disorder, or condition.
  • Prodrug means a therapeutic agent that is prepared in an inactive form that is converted to an active form within the body or cells thereof by the action of endogenous enzymes or other chemicals or conditions.
  • REST nucleic acid or "NRSF nucleic acid” means any nucleic acid encoding REST (aka NRSF).
  • a REST nucleic acid includes a DNA sequence encoding REST, an RNA sequence transcribed from DNA encoding REST (including genomic DNA comprising introns and exons), and an mRNA sequence encoding REST.
  • REST mRNA means an mRNA encoding a REST protein.
  • Side effects means physiological responses attributable to a treatment other than the desired effects.
  • side effects include injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, myopathies, and malaise. For example, increased
  • aminotransferase levels in serum may indicate liver toxicity or liver function abnormality.
  • increased bilirubin may indicate liver toxicity or liver function abnormality.
  • Single-stranded oligonucleotide means an oligonucleotide which is not hybridized to a complementary strand.
  • Specifically hybridizable refers to an antisense compound having a sufficient degree of complementarity between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e. under physiological conditions in the case of in vivo assays and therapeutic treatments.
  • Targeting or “targeted” means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.
  • Target nucleic acid “Target nucleic acid,” “target RNA,”.and “target RNA transcript” all refer to a nucleic acid capable of being targeted by antisense compounds.
  • Target segment means the sequence of nucleotides of a target nucleic acid to which an antisense compound is targeted.
  • 5' target site refers to the 5 '-most nucleotide of a target segment.
  • 3' target site refers to the 3 '-most nucleotide of a target segment.
  • “Therapeutically effective amount” means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual.
  • Treat refers to administering a pharmaceutical composition to effect an alteration or improvement of a disease, disorder, or condition.
  • Unmodified nucleotide means a nucleotide composed of naturally occuring
  • an unmodified nucleotide is an RNA nucleotide (i.e. ⁇ -D-ribonucleosides) or a DNA nucleotide (i.e. ⁇ -D-deoxyribonucleoside).
  • Embodiments of the present invention provide methods, compounds, and compositions for inhibiting REST mRNA and protein expression. Embodiments of the present invention provide methods, compounds, and compositions for the treatment, prevention, or amelioration of diseases, disorders, and conditions associated with REST in an individual in need thereof. Also contemplated are methods and compounds for the preparation of a medicament for the treatment, prevention, or amelioration of a disease, disorder, or condition associated with REST. REST associated diseases, disorders, and conditions include neurological disorders, such as Huntington's Disease.
  • Embodiments of the present invention provide antisense compounds targeted to a REST nucleic acid.
  • the REST nucleic acid is any of the sequences set forth in GENBANK Accession No. NT 022853.14 truncated at nucleotides 5110001 to 5141000 (incorporated herein as SEQ ID NO: 1 ), GENBANK Accession No. NT_109320.4 truncated at nucleotides 1166001 to 1187000, (incorporated herein as SEQ ID NO: 2), and GENBANK Accession No. NM 011263.1, incorporated herein as SEQ ID NO: 3).
  • Embodiments of the present invention provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 12 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 4-81.
  • the compound consists of a single-stranded modified
  • the modified oligonucleotide consists of 20 linked nucleosides.
  • the nucleobase sequence of the modified oligonucleotide is 100% complementary to a nucleobase sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3.
  • the compound has at least one modified internucleoside linkage.
  • the internucleoside linkage is a phosphorothioate internucleoside linkage.
  • the compound has at least one nucleoside comprising a modified sugar.
  • the at least one modified sugar is a bicyclic sugar.
  • the at least one modified sugar comprises a 2'-0-methoxyethyl.
  • the compound has at least one nucleoside comprising a modified nucleobase.
  • the modified nucleobase is a 5-methylcytosine.
  • the modified oligonucleotide of the compound comprises:
  • the modified oligonucleotide of the compound comprises:
  • each nucleoside of each wing segment comprises a 2'-0-methoxyethyl sugar; and wherein each internucleoside linkage is a phosphorothioate linkage.
  • the modified oligonucleotide of the compound comprises:
  • each nucleoside of each wing segment comprises a 2'-0-methoxyethyl sugar; and wherein each internucleoside linkage is a phosphorothioate linkage.
  • the modified oligonucleotide of the compound comprises:
  • each nucleoside of each wing segment comprises a 2'-0-methoxyethyl sugar; and wherein each internucleoside linkage is a phosphorothioate linkage.
  • Embodiments of the present invention provide a composition comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 12 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 3-81 or a salt thereof and a pharmaceutically acceptable carrier or diluent.
  • Embodiments of the present invention provide methods comprising administering to an animal a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 3- 81.
  • the animal is a human.
  • the administering is parenteral administration.
  • the parenteral administration is any of subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, intracranial administration, intrathecal administration, or intracerebroventricular administration. Parenteral administration may be by infusion or injection.
  • Oligomeric compounds include, but are not limited to, oligonucleotides,
  • oligonucleosides oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs.
  • An oligomeric compound may be "antisense" to a target nucleic acid, meaning that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
  • an antisense compound has a nucleobase sequence that, when ⁇ written in the 5' to 3' direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted. In certain such embodiments, an antisense
  • oligonucleotide has a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.
  • an antisense compound targeted to a REST nucleic acid is 12 to 30 subunits in length.
  • antisense compounds are from 12 to 30 linked subunits.
  • the antisense compound is 8 to 80, 12 to 50, 15 to 30, 18 to 24, 19 to 22, or 20 linked subunits.
  • the antisense compounds are 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a range defined by any two of the above values.
  • the antisense compound is an antisense oligonucleotide, and the linked subunits are nucleotides.
  • a shortened or truncated antisense compound targeted to a REST nucleic acid has a single subunit deleted from the 5' end (5' truncation), or alternatively from the 3' end (3' truncation).
  • a shortened or truncated antisense compound targeted to a REST nucleic acid may have two subunits deleted from the 5' end, or alternatively may have two subunits deleted from the 3' end, of the antisense compound.
  • the deleted nucleosides may be dispersed throughout the antisense compound, for example, in an antisense compound having one nucleoside deleted from the 5' end and one nucleoside deleted from the 3' end.
  • the additional subunit may be located at the 5' or 3' end of the antisense compound.
  • the added subunits may be adjacent to each other, for example, in an antisense compound having two subunits added to the 5' end (5' addition), or alternatively to the 3' end (3' addition), of the antisense compound.
  • the added subunits may be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit added to the 5' end and one subunit added to the 3' end.
  • an antisense compound such as an antisense oligonucleotide
  • an antisense oligonucleotide it is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity.
  • an antisense compound such as an antisense oligonucleotide
  • a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in:an oocyte injection model.
  • Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.
  • Gautschi et al demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo.
  • this oligonucleotide demonstrated potent anti-tumor activity in vivo.
  • antisense compounds targeted to a REST nucleic acid have chemically modified subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such as enhanced the inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.
  • Chimeric antisense compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased inhibitory activity.
  • a second region of a chimeric antisense compound may optionally serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.
  • Antisense compounds having a gapmer motif are considered chimeric antisense compounds.
  • a gapmer an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region.
  • the gap segment In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides;-
  • the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region.
  • each distinct region comprises uniform sugar moieties.
  • wing-gap-wing motif is frequently described as "X-Y-Z", where "X” represents the length of the 5' wing region, "Y” represents the length of the gap region, and “Z” represents the length of the 3' wing region.
  • a gapmer described as "X-Y-Z” has a configuration such that the gap segment is positioned immediately adjacent each of the 5' wing segment and the 3' wing segment. Thus, no intervening nucleotides exist between the 5' wing segment and gap segment, or the gap segement and the 3' wing segment.
  • Any of the antisense compounds described herein can have a gapmer motif.
  • X and Z are the same, in other embodiments they are different.
  • Y is between 8 and 15 nucleotides.
  • X, Y or Z can be any of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleotides.
  • gapmers of the present invention include, but are not limited to, for example 5-10-5, 4-8-4, 4-12-3, 4-12-4, 3- 14-3, 2-13-5, 2-16-2, 1-18-1 , 3-10-3, 2-10-2, 1-10-1 or 2-8-2.
  • the antisense compound as a "wingmer” motif, having a wing- gap or gap-wing configuration, i.e. an X-Y or Y-Z configuration as described above for the gapmer configuration.
  • wingmer configurations of the present invention include, but are not limited to, for example 5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1 , 10-3, 2-10, 1-10, 8-2, 2-13, or 5- 13.
  • antisense compounds targeted to a REST nucleic acid possess a 5-10-5 gapmer motif.
  • antisense compounds targeted to a REST nucleic acid possess a 3-14-3 gapmer motif.
  • antisense compounds targeted to a REST nucleic acid possess a 2-13-5 gapmer motif.
  • an antisense compound targeted to a REST nucleic acid has a gap-widened motif.
  • a gap-widened antisense oligonucleotide targeted to a REST nucleic acid has a gap segment of fourteen 2'-deoxyribonucleotides positioned immediately adjacent to and between wing segments of three chemically modified nucleosides.
  • the chemical modification comprises a 2 '-sugar modification.
  • the chemical modification comprises a 2'-MOE sugar modification.
  • a gap-widened antisense oligonucleotide targeted to a REST nucleic acid has a gap segment of thirteen 2'-deoxyribonucleotides positioned immediately adjacent to and between a 5' wing segment of two chemically modified nucleosides and a 3' wing segment of five chemically modified nucleosides.
  • the chemical modification comprises a 2 '-sugar modification.
  • the chemical modification comprises a 2'-MOE sugar modification.
  • Nucleotide sequences that encode REST include, without limitation, the following: GENBANK Accession No. NT_022853.14 truncated at nucleotides 5110001 to 5141000, first deposited with GENBANK® on November 29, 2000 incorporated herein as SEQ ID NO: 1 ; GENBANK Accession No. NT_109320.4 truncated at nucleotides 1166001 to 1187000, first deposited with GENBANK® on May 12, 2005, and incorporated herein as SEQ ID NO: 2; and Accession No. NM_011263.1 , first deposited with GENBANK® on August 19, 2003, incorporated herein as SEQ ID NO: 3.
  • antisense compounds defined by a SEQ ID NO may comprise,
  • Antisense compounds described by Isis Number indicate a combination of nucleobase sequence and motif.
  • a target region is a structurally defined region of the target nucleic acid.
  • a target region may encompass a 3' UTR, a 5' UTR, an exon, an intron, an exon/intron junction, a coding region, a translation initiation region, translation termination region, or other defined nucleic acid region.
  • the structurally defined regions for REST can be obtained by accession number from sequence databases such as NCBI and such information is incorporated herein by reference.
  • a target region may encompass the sequence from a 5' target site of one target segment within the target region to a 3 ' target site of another target segment within the target region.
  • Targeting includes determination of at least one target segment to which an antisense compound hybridizes, such that a desired effect occurs.
  • the desired effect is a reduction in mRNA target nucleic acid levels.
  • the desired effect is reduction of levels of protein encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid.
  • a target region may contain one or more target segments. Multiple target segments within a target region may be overlapping. Alternatively, they may be non-overlapping. In certain embodiments, target segments within a target region are separated by no more than about 300 nucleotides. In certain emodiments, target segments within a target region are separated by a number of nucleotides that is, is about, is no more than, is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on the target nucleic acid, or is a range defined by any two of the preceeding values.
  • target segments within a target region are separated by no more than, or no more than about, 5 nucleotides on the target nucleic acid. In certain embodiments, target segments are contiguous. Contemplated are target regions defined by a range having a starting nucleic acid that is any of the 5' target sites or 3' target sites listed herein.
  • Suitable target segments may be found within a 5' UTR, a coding region, a 3' UTR, an intron, an exon, or an exon/intron junction.
  • Target segments containing a start codon or a stop codon are also suitable target segments.
  • a suitable target segment may specifcally exclude a certain structurally defined region such as the start codon or stop codon.
  • the determination of suitable target segments may include a comparison of the sequence of a target nucleic acid to other sequences throughout the genome.
  • the BLAST algorithm may be used to identify regions of similarity amongst different nucleic acids. This comparison can prevent the selection of antisense compound sequences that may hybridize in a non-specific manner to sequences other than a selected target nucleic acid (i.e., non-target or off- target sequences).
  • hybridization occurs between an antisense compound disclosed herein and a REST nucleic acid.
  • the most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.
  • Hybridization can occur under varying conditions. Stringent conditions are sequence- dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.
  • the antisense compounds provided herein are specifically hybridizable with a REST nucleic acid.
  • An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., antisense inhibition of a target nucleic acid, such as a REST nucleic acid).
  • Non-complementary nucleobases between an antisense compound and a REST nucleic acid may be tolerated provided that the antisense compound remains able to specifically hybridize to a target nucleic acid.
  • an antisense compound may hybridize over one or more segments of a REST nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).
  • the antisense compounds provided herein, or a specified portion thereof are, or are at least, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a REST nucleic acid, a target region, target segment, or specified portion thereof. Percent complementarity of an antisense compound with a target nucleic acid can be determined using routine methods.
  • an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity.
  • the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases.
  • nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention.
  • Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410;
  • Percent homology, sequence identity or complementarity can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981 , 2, 482 489).
  • the antisense compounds provided herein, or specified portions thereof are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof.
  • antisense compound may be fully complementary to a REST nucleic acid, or a target region, or a target segment or target sequence thereof.
  • "fully complementary" means each nucleobase of an antisense compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid.
  • a 20 nucleobase antisense compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the antisense compound.
  • Fully complementary can also be used in reference to a specified portion of the first and /or the second nucleic acid.
  • a 20 nucleobase portion of a 30 nucleobase antisense compound can be "fully complementary" to a target sequence that is 400 nucleobases long.
  • the 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the antisense compound.
  • the entire 30 nucleobase antisense compound may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.
  • non-complementary nucleobase may be at the 5' end or 3' end of the antisense compound.
  • the non-complementary nucleobase or nucleobases may be at an internal position of the antisense compound.
  • two or more non-complementary nucleobases may be contiguous (i.e. linked) or non-contiguous.
  • a non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.
  • antisense compounds that are, or are up to 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a REST nucleic acid, or specified portion thereof.
  • antisense compounds that are, or are up to 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non- complementary nucleobase(s) relative to a target nucleic acid, such as a REST nucleic acid, or specified portion thereof.
  • the antisense compounds provided herein also include those which are complementary to a portion of a target nucleic acid.
  • portion refers to a defined number of contiguous (i.e. linked) nucleobases within a region or segment of a target nucleic acid.
  • a "portion" can also refer to a defined number of contiguous nucleobases of an antisense compound.
  • the antisense compounds are complementary to at least an 8 nucleobase portion of a target segment.
  • the antisense compounds are complementary to at least a 12 nucleobase portion of a target segment.
  • the antisense compounds are complementary to at least a 15 nucleobase portion of a target segment.
  • antisense compounds that are complementary to at least a 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values.
  • the antisense compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific Isis number, or portion thereof.
  • an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability.
  • a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine.
  • Shortened and lengthened versions of the antisense compounds described herein as well as compounds having non-identical bases relative to the antisense compounds provided herein also are contemplated.
  • the non-identical bases may be adjacent to each other or dispersed throughout the antisense compound. Percent identity of an antisense compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.
  • the antisense compounds, or portions thereof are at least 70%,
  • a nucleoside is a base-sugar combination.
  • the nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety.
  • Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentoiuranosyl sugar, the phosphate group can be linked to the 2', 3' or 5' hydroxyl moiety of the sugar.
  • Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the intemucleoside linkages of the oligonucleotide.
  • Modifications to antisense compounds encompass substitutions or changes to intemucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.
  • Chemically modified nucleosides may also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides.
  • RNA and DNA The naturally occuring intemucleoside linkage of RNA and DNA is a 3' to 5' phosphodiester linkage.
  • Antisense compounds having one or more modified, i.e. non-naturally occurring, intemucleoside linkages are often selected over antisense compounds having naturally occurring intemucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.
  • Oligonucleotides having modified intemucleoside linkages include intemucleoside linkages that retain a phosphorus atom as well as intemucleoside linkages that do not have a phosphorus atom.
  • Representative phosphorus containing intemucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous- containing linkages are well known.
  • antisense compounds targeted to a REST nucleic acid comprise one or more modified intemucleoside linkages.
  • the modified intemucleoside linkages are phosphorothioate linkages.
  • each intemucleoside linkage of an antisense compound is a phosphorothioate intemucleoside linkage.
  • Antisense compounds of the invention can optionally contain one or more nucleosides wherein the sugar group has been modified.
  • Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity or some other beneficial biological property to the antisense compounds.
  • nucleosides comprise a chemically modified ribofuranose ring moieties.
  • Examples of chemically modified sugars include 2'-F-5'- methyl substituted nucleoside (see PCT International Application WO 2008/101 157 Published on 8/21/08 for other disclosed 5',2'-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2'-position (see published U.S. Patent Application US2005-0130923, published on June 16, 2005) or alternatively 5'-substitution of a BNA (see PCT International Application WO 2007/134181 Published on 11/22/07 wherein LNA is substituted with for example a 5'-methyl or a 5'-vinyl group).
  • nucleosides having modified sugar moieties include without limitation nucleosides comprising 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3 and 2'-0(CH2)20CH3 substituent groups.
  • bicyclic nucleic acids examples include without limitation nucleosides comprising a bridge between the 4' and the 2' ribosyl ring atoms.
  • antisense compounds provided herein include one or more BNA nucleosides wherein the bridge comprises one of the formulas: 4'-(CH2)-0-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)-0-2' (LNA); 4'- (CH2)2-0-2' (ENA); 4'-C(CH3)2-0-2' (see PCT/US2008/068922); 4'-CH(CH3)--0-2' and 4'- C->H(CH20CH3)--0-2' (see U.S.
  • Patent 7,399,845, issued on July 15, 2008); 4'-CH2- N(OCH3)-2' (see PCT/US2008/ 064591); 4'-CH2-0-N(CH3)-2' (see published U.S. Patent Application US2004-0171570, published September 2, 2004 ); 4'-CH2-N(R)-0-2' (see U.S. Patent 7,427,672, issued on September 23, 2008); 4'-CH2-C(CH3)-2'and 4'-CH2-C-( CH2)-2' (see PCT/US2008/ 066154); and wherein R is, independently, H, C1-C12 alkyl, or a protecting group.
  • BNAs include various stereochemical sugar configurations including for example a-L-ribofuranose and ⁇ -D-ribofuranose (see PCT international application PCT/DK98/00393, published on March 25, 1999 as WO 99/14226).
  • nucleosides are modified by replacement of the ribosyl ring with a sugar surrogate.
  • modification includes without limitation, replacement of the ribosyl ring with a surrogate ring system (sometimes referred to as DNA analogs) such as a morpholino ring, a cyclohexenyl ring, a cyclohexyl ring or a tetrahydropyranyl ring such as one having one of the formula:
  • bicyclo and tricyclo sugar surrogate ring systems are also know in the art that can be m used to modify nucleosides for incorporation into antisense compounds (see for example review article: Leumann, Christian J., ). Such ring systems can undergo various additional substitutions to enhance activity.
  • nucleobase moieties In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.
  • antisense compounds targeted to a REST nucleic acid comprise one or more nucleotides having modified sugar moieties.
  • the modified sugar moiety is 2'-MOE.
  • the 2'-MOE modified nucleotides are arranged in a gapmer motif.
  • Nucleobase (or base) modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications may impart nuclease stability, binding affinity or some other beneficial biological property to antisense compounds. Modified nucleobases include synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of an antisense compound for a target nucleic acid.
  • 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).
  • Additional unmodified nucleobases include 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2- propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C ⁇ C-CH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil
  • Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2- aminopyridine and 2-pyridone.
  • Nucleobases that are particularly useful for increasing the binding affinity of antisense compounds include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5- propynylcytosine.
  • antisense compounds targeted to a REST nucleic acid comprise one or more modified nucleobases.
  • oligonucleotides targeted to a REST nucleic acid comprise one or more modified nucleobases.
  • the modified nucleobase is 5-methylcytosine.
  • each cytosine is a 5-methylcytosine.
  • Antisense oligonucleotides may be admixed with pharmaceutically acceptable active or inert substance for the preparation of pharmaceutical compositions or formulations.
  • compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
  • Antisense compound targeted to a REST nucleic acid can be utilized in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier.
  • a pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS).
  • PBS is a diluent suitable for use in compositions to be delivered parenterally.
  • a pharmaceutical composition comprising an antisense compound targeted to a REST nucleic acid and a pharmaceutically acceptable diluent.
  • the pharmaceutically acceptable diluent is PBS.
  • the antisense compound is an antisense oligonucleotide.
  • compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
  • a prodrug can include the incorporation of additional nucleosides at one or both ends of an antisense compound which are cleaved by endogenous nucleases within the body, to form the active antisense compound.
  • Antisense compounds may be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides.
  • Typical conjugate groups include cholesterol moieties and lipid moieties.
  • Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • Antisense compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of antisense compounds to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the antisense compound having terminal nucleic acid from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5'-terminus (5'-cap), or at the 3'-terminus (3'-cap), or can be present on both termini. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3' and 5'-stabilizing groups that can be used to cap one or both ends of an antisense compound to impart nuclease stability include those disclosed in WO 03/004602 published on January 16, 2003. Cell culture and antisense compounds treatment
  • REST nucleic acids The effects of antisense compounds on the level, activity or expression of REST nucleic acids can be tested in vitro in a variety of cell types.
  • Cell types used for such analyses are available from commerical vendors ⁇ e.g. American Type Culture Collection, Manassus, VA; Zen- Bio, Inc., Research Triangle Park, NC; Clonetics Corporation, Walkersville, MD) and cells are cultured according to the vendor's instructions using commercially available reagents (e.g.
  • Illustrative cell types include, but are not limited to, HepG2 cells, Hep3B cells, primary hepatocytes, HuVEC cells, and GM fibroblasts.
  • Described herein are methods for treatment of cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.
  • cells are treated with antisense oligonucleotides when the cells reach approximately 60-80% confluency in culture.
  • One reagent commonly used to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN® (Invitrogen, Carlsbad, CA).
  • Antisense oligonucleotides are mixed with LIPOFECTIN® in OPTI-MEM® 1 (Invitrogen,
  • LIPOFECTIN® concentration that typically ranges 2 to 12 ug mL per 100 nM antisense oligonucleotide.
  • Another reagent used to introduce antisense oligonucleotides into cultured cells includes
  • LIPOFECT AMINE® (Invitrogen, Carlsbad, CA). Antisense oligonucleotide is mixed with LIPOFECT AMINE® in OPTI-MEM® 1 reduced serum medium (Invitrogen, Carlsbad, CA) to achieve the desired concentration of antisense oligonucleotide and a LIPOFECT AMINE® concentration that typically ranges 2 to 12 ug/mL per 100 nM antisense oligonucleotide.
  • Another technique used to introduce antisense oligonucleotides into cultured cells includes electroporation.
  • Cells are treated with antisense oligonucleotides by routine methods. Cells are typically harvested 16-24 hours after antisense oligonucleotide treatment, at which time RNA or protein levels of target nucleic acids are measured by methods known in the art and described herein. In general, when treatments are performed in multiple replicates, the data are presented as the average of the replicate treatments.
  • the concentration of antisense oligonucleotide used varies from cell line to cell line.
  • Antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with LIPOFECT AMINE®. Antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation.
  • RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using the TRIZOL® Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's recommended protocols.
  • Target nucleic acid levels can be quantitated by, e.g.,
  • RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art.
  • Quantitative real-time PCR can be conveniently accomplished using the commercially available ABI PRISM® 7600, 7700, or 7900 Sequence Detection System, available from PE- Applied Biosystems, Foster City, CA and used according to manufacturer's instructions. Quantitative Real-Time PCR Analysis of Target RNA Levels
  • Quantitation of target RNA levels may be accomplished by quantitative real-time PCR using the ABI PRISM® 7600, 7700, or 7900 Sequence Detection System (PE-Applied
  • RNA Prior to real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction, which produces complementary DNA (cDNA) that is then used as the substrate for the real-time PCR amplification.
  • RT reverse transcriptase
  • cDNA complementary DNA
  • the RT and real-time PCR reactions are performed sequentially in the same sample well.
  • RT and real-time PCR reagents are obtained from Invitrogen (Carlsbad, CA). RT, real-time-PCR reactions are carried out by methods well known to those skilled in the art.
  • Gene (or RNA) target quantities obtained by real time PCR are normalized using either the expression level of a gene whose expression is constant, such as cyclophilin A, or by quantifying total RNA using RIBOGREEN® (Invitrogen, Inc. Carlsbad, CA). Cyclophilin A expression is quantified by real time PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RIBOGREEN® RNA quantification reagent (Invetrogen, Inc. Eugene, OR). Methods of RNA quantification by RIBOGREEN® are taught in Jones, L.J., et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR® 4000 instrument (PE Applied Biosystems) is used to measure RIBOGREEN® fluorescence.
  • Probes and primers are designed to hybridize to a REST nucleic acid.
  • Methods for designing real-time PCR probes and primers are well known in the art, and may include the use of software such as PRIMER EXPRESS® Software (Applied Biosystems, Foster City, CA).
  • Antisense inhibition of REST nucleic acids can be assessed by measuring REST protein levels. Protein levels of REST can be evaluated or quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (for example, caspase activity assays), immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS).
  • Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, MI), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art. Antibodies useful for the detection of human and rat REST are commercially available.
  • Antisense compounds for example, antisense oligonucleotides, are tested in animals to assess their ability to inhibit expression of REST.
  • antisense compounds for example, antisense oligonucleotides
  • oligonucleotides are tested for their ability to affect expression of other molecules, such as BDNF mRNA and BDNF protein. Other molecules, such as BDNF, may be upregulated or inhibited.
  • antisense oligonucleotides are tested for their ability to affect phenotype. Testing may be performed in wild type animals, or in experimental disease models. In certain embodiments, experimental disease models carry the human mutant huntingtin gene. Examples of such experimental disease models include the BACHD mouse, the YAC 128 mouse, and the R6/2 mouse. In the case of testing in disease model animals, amelioration of symptoms such as increased anxiety, decreased motor function, decreased brain weight, and decreased cognitive function may be tested.
  • oligonucleotides are formulated in a
  • Administration includes parenteral routes of administration.
  • the parenteral administration may be by infusion or injection.
  • Parental administration may be any of subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, intracranial administration, intrathecal administration, or intracerebroventricular administration.
  • RNA is isolated from tissue and changes in REST nucleic acid are measured. Changes in REST protein levels are also measured
  • the invention provides methods of treating an individual comprising administering one or more pharmaceutical compositions of the present invention.
  • the individual has a neurological disease.
  • the neurological disease is Huntington's Disease, Parkinson's Disease, Alzheimer's Disease,
  • the invention provides for a method of treating Huntington's disease (HD).
  • HD is a devastating autosomal dominant, neurodegenerative disease caused by a CAG trinucleotide repeat expansion encoding an abnormally long polyglutamine (PolyQ) tract in the huntingtin protein.
  • the Huntington disease gene was first mapped in 1993 (The Huntington's Disease Collaborative Research Group. Cell. 1993, 72:971-83), consisting of a gene, IT15, which contained a polymorphic trinucleotide repeat that is expanded and unstable on HD chromosomes.
  • CAG repeats in the normal size range are usually inherited as Mendelian alleles
  • expanded HD repeats are unstable through meiotic transmission and are found to be expanded beyond the normal size range (6-34 repeat units) in HD patients.
  • Both normal and variant huntingtin protein are localized chiefly in the cytoplasm of neurons (DiFiglia et al., Neuron 1995, 14: 1075-81). As a result of excessive polyglutamine length, huntingtin protein form aggregates in the cytoplasm and nucleus of CNS neurons (Davies et al., Cell 1997, 90:537-548). Both transgenic animals and genetically modified cell lines have been used to investigate the effects of expanded polyQ repeats on the localization and processing of huntingtin. However, it is still unclear whether the formation of aggregates per se is the essential cytotoxic step or a consequence of cellular dysfunction.
  • HD is characterized by progressive chorea, psychiatric changes and intellectual decline. This dominant disorder affects males and females equally, and occurs in all races (Gusella and MacDonald, Curr. Opin. Neurobiol. 1995 5:656-62). Symptoms of HD are due to the death of neurons in many brain regions, but is most apparent in the striatum, particularly in the caudate nucleus, which suffers a progressive gradient of cell loss that ultimately decimates the entire structure. Although the gene encoding huntingtin is expressed ubiquitously (Strong, T.V. et al., Nat. Genet.
  • Huntingtin is crucial for normal development and may be regarded as a cell survival gene (Nasir et al., Human Molecular Genetics, Vol 5, 1431-1435). The normal function of huntingtin remains incompletely characterized, but based upon protein-protein interactions, it appears to be associated with the cytoskeleton and required for neurogenesis (Walling et al., J. Neurosci Res. 1998, 54:301 -8). Huntingtin is specifically cleaved during apoptosis by a key cysteine protease, apopain, known to play a pivotal role in apoptotic cell death. The rate of cleavage is enhanced by longer polyglutamine tracts, suggesting that inappropriate apoptosis underlies HD.
  • Wild-type (but not mutant) huntingtin has been shown to facilitate production of cortical brain derived neurotrophic factor (BDNF) by acting at the level of BDNF gene transcription.
  • Wild-type huntingtin sustains the production of BDNF through interaction of wild-type huntingtin with REST in the cytoplasm of neuronal cells, therefore restricting its access to the nucleus, which leads to the transcription of target genes such as BDNF.
  • mutant huntingtin In the presence of mutant huntingtin, cytoplasmic retention of REST by huntingtin is impeded, resulting in the translocation of REST to the nucleus and transcriptional repression of target genes.
  • a major loss of BDNF protein in the striatum of HD patients may contribute to the clinical symptoms of HD.
  • BDNF is a member of a family of molecules with neurotropic activities. BDNF is widely expressed in the adult mammalian central nervous system and it is particularly abundant in the hippocampus and cerebral cortex where it is normally transported to its striatal target. Striatal neurons in the brain require BDNF for optimal activity and survival.
  • BDNF expression increases during brain development and expression appears to be maintained with age, thus suggesting that it plays an essential role in the adult nervous system.
  • BDNF controls a variety of brain processes, including the growth, development, differentiation and maintenance of neuronal systems, neuronal plasticity, synaptic activity and neurotransmitter- mediated activities.
  • Synapsin I is considered to be one of the major proteins involved in the regulation of
  • Synapsin I cross-links the synaptic vesicles to the cytoskeleton including actin microfilaments, microtubules, and brain spectrin. Studies suggest that an early impairment in synapsin phosphorylation-dephosphorylation may alter synaptic vesicle trafficking and lead to defective neurotransmission in HD. The 5 '-flanking region of synapsin 1 was observed to be sufficient for neuron-specific expression and the REST protein was discovered to bind to this region and act as a transcriptional repressor.
  • Dynamin is a GTPase involved in synaptic vesicle recycling.
  • the functional importance of dynamin in endocytosis has been illustrated further by the conformational change of the dynamin rings formed at the necks of invaginated coated pits that correlate with GTP hydrolysis, which represents a key step leading to vesicle fission.
  • Huntingtin (HD) protein is concentrated in the presynaptic termini, and enriched in the synaptosomal membrane fractions, together with other proteins, including dynamin 1 and the EEN family of proteins.
  • EEN proteins and dynamin 1 have been shown to have a role in the promotion the formation of insoluble polyglutamine-containing aggregates of the huntingtin by their interaction with the glutamine expansion region of the huntingtin protein.
  • the mRNA expression of dynamin 1 in cells expressing mutant huntingtin has been demonstrated to be significantly lower.
  • antisense oligonucleotides targeted to REST are useful for reversing transcriptional repression of target genes (e.g. BDNF, synapsin 1) and DNMI, which is caused by mutant hungtintin. That is, antisense mediated inhibition of REST results in specific suppression of REST with an associated increase in BDNF, synapsin 1 , and DNMI. In certain embodiments, antisense oligonucleotide inhibition of REST mitigates the physiologic damage caused to an animal by mutant hungtingin, and is, therefore, a useful therapeutic for the treatment of HD.
  • target genes e.g. BDNF, synapsin 1
  • DNMI mutant hungtintin
  • administering results in reduction of REST expression by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values.
  • compositions comprising an antisense compound targeted to REST are used for the preparation of a medicament for treating a patient suffering or susceptible to Huntington's Disease.
  • pharmaceutical compositions comprising an antisense compound targeted to REST ameliorate symptoms of HD including chorea, psychiatric changes, and intellectual decline.
  • compositions useful for the treatment, prevention, or amelioration of Huntington's Disease, and conditions associated with Huntington's disease are administered parenterally.
  • parenteral administration is by infusion.
  • Infused pharmaceutical agents may be delivered with a pump.
  • infused pharmaceutical agents may be delivered to the cerebrospinal fluid by intracranial administration, intrathecal administration, or intracerebroventricular administration. Broad 8467
  • distribution of pharmaceutical agents, including antisense oligonucleotides, within the central nervous system may be achieved with intracranial administration, intrathecal administration, or intracerebroventricular administration.
  • infused pharmaceutical agents are delivered directly to a tissue.
  • tissues include, the striatal tissue, the intracerebroventricular tissue, and the caudate tissue.
  • oligonucleotides may be achieved by direct infusion to a targeted tissue.
  • parenteral administration is by injection.
  • the injection may be delivered with a syringe or a pump.
  • the injection is a bolus administered directly to a tissue. Examples of such tissues include, the striatal tissue, the
  • intracerebroventricular tissue and the caudate tissue.
  • Specific localization of pharmaceutical agents, including antisense oligonucleotides, can be achieved via injection to a targeted tissue.
  • specific localization of a pharmaceutical agent such as an antisense oligonucleotide, to a targeted tissue improves the pharmacokinetic profile of a pharmaceutical agent as compared to broad diffusion of a pharmaceutical agent.
  • the specific localization of a pharmaceutical agent improves potencyscompared to broad diffusion of a pharmaceutical agent, requiring less pharmaceutical agent to achieve similar pharmacology.
  • similar pharmacology refers to the amount of time that a target mRNA and/or target protein is down-regulated/inhibited (e.g. duration of action).
  • methods of specifically localizing a pharmaceutical agent such as by bolus injection, decreases median effective concentration (EC50) by a factor of about 20 (e.g.
  • methods of specifically localizing a pharmaceutical agent decreases median effective concentration (EC50) by a factor of 15, 1 , 17, 18, 19, 20, 21 , 22, 23, 24, or 25.
  • the targeted tissue is brain tissue.
  • one or more pharmaceutical compositions of the present invention are co-administered with one or more other pharmaceutical agents.
  • such one or more other pharmaceutical agents are designed to treat the same disease, disorder, or condition as the one or more pharmaceutical compositions of the present invention.
  • such one or more other pharmaceutical agents are designed to treat a different disease, disorder, or condition as the one or more pharmaceutical compositions of the present invention.
  • such one or more other pharmaceutical agents are designed to treat an undesired side effect of one or more pharmaceutical compositions of the present invention.
  • one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to treat an undesired effect of that other pharmaceutical agent.
  • one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to produce a combinational effect. In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to produce a synergistic effect.
  • one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at the same time. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at different times. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared together in a single formulation. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other
  • pharmaceutical agents that may be co-administered with a pharmaceutical composition of include antipsychotic agents, such as, e.g., haloperidol, chlorpromazine, clozapine, quetapine, and olanzapine; antidepressant agents, such as, e.g., fluoxetine, sertraline hydrochloride, venlafaxine and nortriptyline; tranquilizing agents such as, e.g., benzodiazepines, clonazepam, paroxetine, venlafaxin, and beta-blockers; mood-stabilizing agents such as, e.g., lithium, valproate, lamotrigine, and carbamazepine; paralytic agents such as, e.g., Botulinum toxin; and/or other experimental agents including, but not limited to,
  • tetrabenazine (Xenazine), creatine, conezyme Q10, trehalose, docosahexanoic acids, ACR16, ethyl-EPA, atomoxetine, citalopram, dimebon, memantine, sodium phenylbutyrate, ramelteon, ursodiol, zyprexa, xenasine, tiapride, riluzole, amantadine, [123I]MNI-420, atomoxetine, tetrabenazine, digoxin, detromethorphan, warfarin, alprozam, ketoconazole, omeprazole, and minocycline.
  • Example 1 Antisense inhibition of human REST mRNA in HuVEC cells
  • Antisense oligonucleotides targeted to a REST nucleic acid were tested for their effects on REST mRNA in vitro.
  • Cultured HuVEC cells at a density of 5,000 cells per well were transfected using LipofectAMINE 2000 ® reagent with 10 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and REST mRNA levels were measured by quantitative real-time PCR. REST mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN ® . Results are presented as percent inhibition of REST, relative to untreated control cells.
  • the antisense oligonucleotides in Table 1 were designed asv;5-10-5 MOE gapmers.
  • Gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of ten 2'- deoxynucleotides and is flanked on both sides (in the 5' and 3' directions) by wings comprising 5 nucleotides each. Each nucleotide in the 5' wing segment and each nucleotide in the 3' wing segment has a 2' -MOE modification.
  • “Target start site” indicates the 5'-most nucleotide to which the gapmer is targeted.
  • “Target stop site” indicates the 3 '-most nucleotide to which the gapmer is targeted.
  • Each gapmer listed in Table 1 is targeted to SEQ ID NO: 1 (GENBANK Acession No.
  • NT_022853.14 truncated at nucleotides 5110001 to 5141000). 010 048467
  • Oligonucleotides listed in Table 2 are cross-reactive between the human REST sequence (GENBANK Accession No. NT 022853.14 truncated at nucleotides 5110001 to 5141000, incorporated as SEQ ID NO: 1) and the murine REST genomic sequence (GENBANK Accession No. NTJ 09320.4 truncated at nucleotides 1166001 to 1187000, incorporated herein at SEQ ID NO: 2).
  • "Murine start site” indicates the 5'-most nucleotide to which the gapmer is targeted on the murine sequence (e.g. SEQ ID NO: 2).
  • “Murine stop site” indicates the 3'-most nucleotide to which the gapmer is targeted on the murine sequence (e.g. SEQ ID NO: 2).
  • Oligonucleotides listed in Table 3 are murine REST oligonucleotides having significant homology with the human REST genomic sequence.
  • the oligonucleotides listed in Table 3 either have 1 , 2, or 3 mismatches with the human REST genomic sequence (GENBANK
  • “Human start site” indicates the 5'-most nucleotide to which the gapmer is targeted to SEQ ID NO: 1.
  • “Human stop site” indicates the 3'-most nucleotide to which the gapmer is targeted to SEQ ID NO: 1.
  • “Murine start site” indicates the 5 '-most nucleotide to which the gapmer is targeted on the murine sequence (e.g. SEQ ID NO: 2).
  • “Murine stop site” indicates the 3 '-most nucleotide to which the gapmer is targeted on the murine sequence (e.g. SEQ ID NO: 2).
  • Antisense oligonucleotides targeted to a REST nucleic acid were tested for their effects on human REST mRNA in vitro.
  • Cultured HuVEC cells at a density of 5,000 cells per well were transfected using LipofectAMINE2000® reagent with 1.875 nM, 3.75 nM, 7.5 nM, 15 nM, or 30 nM of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and REST mRNA levels were measured by quantitative real-time PCR.
  • Human primer probe set RTS 3188 forward sequence TGGAGCGGAGGACAAAGG, incorporated herein as SEQ ID NO: 82; reverse sequence TGCTTCATATTGGCATGGCTTA, incorporated herein as SEQ ID NO: 83; probe sequence
  • AGAGCTCGAAGACCAAACCCTTTCGCX was used to measure mRNA levels.
  • REST mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN ® . Results are presented as percent inhibition of REST mRNA, relative to untreated control cells.
  • GM04281 fibroblasts were obtained from a 20 year old female patient with 69 CAG repeats in her huntingtin gene; GM02173 fibroblasts were obtained from a 52 year old female patient with 44 CAG repeats in her huntingtin gene; and GM02171 fibroblasts were obtained from a 22 year old female with 17 CAG repeats in her huntingtin gene.
  • Each cell line was cultured at a density of 60,000 cells per well in 6-well plates and transfected after 24 hours using Lipofectin reagent with 30 nM, 50 nM, or 80 nM of antisense oligonucleotide.
  • a set of each cell line was also transfected using Lipofectin reagent with 30 nM, 50 nM, or 80 nM of ISIS 387916 (TCTCTATTGCACATTCCAAG, incorporated herein as SEQ ID NO: 85), an antisense oligonucleotide targeting huntingtin mRNA.
  • Control sets of each cell line were transfected using Lipofectin reagent with 30 nM, 50 nM, or 80 nM of ISIS 141923 (CCTTCCCTGAAGGTTCCTCC, incorporated herein as SEQ ID NO: 86), which has no known target. After a treatment period of approximately 16 hours, RNA was isolated from the cells. REST mRNA levels were measured by quantitative real-time PCR using primer probe set with forward primer sequence, 5'- TGGAGCGGAGGACAAAGG -3 * , incorporated herein as SEQ ID NO: 87; reverse primer sequence, 5'- TGCTTCATATTGGCATGGCTTA -3', incorporated herein as SEQ ID NO: 88; and fluorescent probe sequence, 5'-
  • AGAGCTCGAAGACCAAACCCTTTCGC -3' incorporated herein as SEQ ID NO: 89
  • Huntingtin mRNA levels were measured using the human primer probe set RTS 2617 (forward sequence CTCCGTCCGGTAGACATGCT , incorporated herein as SEQ ID NO: 90; reverse sequence GGAAATCAGAACCCTCAAAATGG , incorporated herein as SEQ ID NO:91 ; and probe sequence TGAGCACTGTTCAACTGTGGATATCGGGAX, incorporated herein as SEQ ID NO: 92) and mRNA levels were normalized to Cyclophilin levels. Results are presented in Table 5 as percent inhibition of REST mRNA, relative to untreated control cells and in Table 6 as percent inhibition of huntingtin mRNA, relative to untreated control cells. Table 5
  • Chimeric antisense oligonucleotides having 5-10-5 MOE wings and deoxy gap were designed to target murine REST genomic sequence (GENBANK Accession No. NT l 09320.4 truncated at nucleotides 1166001 to 1187000, incorporated herein as SEQ ID NO: 2) or murine REST mRNA (GENBANK Accession No. NM 01 1263.1 , incorporated herein as SEQ ID NO: 3).
  • the antisense oligonucleotides presented in Table 7 are 20 nucleotides in length, composed of a central 'gap' region consisting of ten 2'-deoxynucleotides, which is flanked on both sides (5' and 3' directions) by five-nucleotide 'wings'.
  • the wings are composed of 2'-methoxyefhyl (2'- MOE) nucleotides.
  • 'Target start site' indicates the 5'-most nucleotide to which the antisense oligonucleotide is targeted.
  • 'Target stop site' indicates the 3' -most nucleotide to which the antisense oligonucleotide is targeted.
  • the antisense oligonucleotides were evaluated for their ability to reduce murine REST mRNA in primary mouse hepatocytes.
  • Primary mouse hepatocytes were cultured at a density of 100,000 cells per well in 24-well plates and transfected using Cytofectin reagent with 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, or 200 nM of antisense oligonucleotides for a period of approximately 24 hours.
  • RNA was isolated from the cells and REST mRNA levels were measured by quantitative real-time PCR.
  • Murine REST primer probe set RTS3156 (forward sequence TGCACGAGCTCTCGAAAGC, incorporated herein as SEQ ID NO: 102; reverse sequence CAGGGCCACGTTGGCTAA, incorporated herein as SEQ ID NO: 103; probe sequence AACTGGCAGCCCCTCAGCTCATCAX, incorporated herein as SEQ ID NO: 104) was used to measure mRNA levels. REST mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN ® . The results are presented in Table 8 as percent inhibition compared to the control cells and demonstrate dose-dependent antisense inhibition of REST mRNA. Table 8
  • ISIS 447070 and ISIS 447052 displaying significant dose-dependent inhibition of REST mRNA from the previous assay (Example 4) were evaluated for their ability to reduce murine REST mRNA in R6/2 mouse primary hepatocytes.
  • Hepatocytes from R6/2 mice and their non- transgenic wild-type littermates were cultured at a density of 100,000 cells per well in 24- well plates and transfected using Cytofectin reagent with 11.1 nM, 33.3 nM, 100 nM, or 300 nM of antisense oligonucleotides for a period of approximately 24 hours.
  • REST primer probe set RTS3156 forward sequence
  • AACTGGCAGCCCCTCAGCTCATCAX incorporated herein as SEQ ID NO: 109 was used to measure REST mRNA levels. mRNA levels were normalized to Cyclophilin levels. The results are presented in Table 9 as percent inhibition of REST mRNA in wild-type primary hepatocytes compared to untreated control cells, and in Table 10 as percent inhibition of REST mRNA in R6/2 primary hepatocytes compared to untreated control cells. The data indicates that both ISIS 447070 and ISIS 447052 effect significant inhibition of REST mRNA compared to untreated cells in both wild-type and R6/2 primary hepatocytes. Table 9
  • Antisense oligonucleotides showing statistically significant dose-dependent inhibition from the in vitro study were evaluated for their ability to reduce REST mRNA in vivo as well to their tolerability in a mouse model.
  • mice Six week old BALB/c mice were injected intraperitoneally with 100 mg/kg of ISIS 447005, ISIS 447021, ISIS 447038, ISIS 447051 , ISIS 447052, and ISIS 447070 twice a week for 4 weeks.
  • a control mice group was injected with PBS twice a week for 4 weeks. Mice were sacrificed 48 hours after the last dose and their livers and spleens were harvested. Blood was also collected for analysis.
  • REST mRNA levels were measured using a murine primer probe set with forward primer sequence 5'- TGCACGAGCTCTCGAAAGC -3' incorporated herein as SEQ ID NO: 1 10; reverse primer sequence 5'- CAGGGCCACGTTGGCTAA -3' incorporated herein as SEQ ID NO: 11 1; and fluorescent probe, 5'- AACTGGCAGCCCCTCAGCTCATCA-3' incorporated herein as SEQ ID NO: 1 12.
  • mRNA levels of the REST target molecule, BDNF were also measured using a murine primer probe set with forward primer sequence 5'- AAGGCACTGGAACTCGCAAT -3 » incorporated herein as SEQ ID NO: 1 13 ; reverse primer sequence 5'- TTATGAATCGCCAGCCAATTC -3' incorporated herein as SEQ ID NO: 114; and fluorescent probe sequence 5'- CTACCCAATCGTATGTTCGGGCCCTT -3' incorporated herein as SEQ ID NO: 1 15. Both primer probe sets were synthesized by Integrated DNA
  • REST mRNA levels are presented in Table 11 and are expressed as percent inhibition compared to the PBS control group.
  • results of BDNF mRNA levels are presented in Table 12 and are expressed as percent over the PBS control group. It was expected that the inhibition of REST mRNA expression would lead to increase in target BDNF mRNA expression levels. All the antisense oligonucleotides effect significant inhibition of murine REST mRNA levels and corresponding increase in BDNF mRNA levels.
  • ALT and AST alanine transaminase and aspartate transaminase
  • Example 7 Bolus administration of antisense oligonucleotides against REST mRNA to the striatum of C57/BL6 mice
  • mice were treated with ISIS oligonucleotides via bolus administration to a defined mouse brain area, the striatum, for the purpose of screening the activity of the oligonucleotides in brain tissue against REST mRNA expression.
  • mice Groups of four C57/BL6 mice were administered with ISIS 447005, ISIS 447038, or ISIS 447070 delivered as a single bolus at 60 g concentrations. Groups of four C57/BL6 mice were administered with ISIS 447021 , ISIS 447051 , or ISIS 447052 delivered as a single bolus at 75 g concentrations.
  • a positive control group of four C57/BL6 mice were administered with 25 ⁇ g of ISIS 408737 (TCCTAGTGTTACATTACCGC, incorporated herein as SEQ ID NO: 1 16), an antisense oligonucleotide targeting murine huntingtin mRNA. A control group of mice were administered PBS.
  • mice were administered PBS or oligonucleotide in the following manner: Mice were individually anaesthetized with 3% isoflurane and were maintained throughout the surgical procedure in an ASI small animal stereotaxic system (ASI Instruments, SAS-4100) with a gas nose cone containing 2% isoflurane. The scalp of the animal was sterilized with iodine solution followed by 70% ethanol, and a longitudinal mid-saggital incision 1 cm length was then made to the scalp. Using a 10 ⁇ , Hamilton Gas Tight syringe for injecting each oligonucleotide, the tip of the syringe was placed +0.5 mm in the
  • mice Seven days after the bolus administration, the mice were euthanized using isoflurane and the organs were removed. The animals were decapitated and the brain was removed for dissection of the striatal tissue. Briefly, a pair of fine curved forceps was placed straight down into the brain just anterior to the hippocampus to make a transverse incision in the cortex and underlying tissues by blunt dissection. The tips of another pair of fine curved forceps were placed straight down along the midsaggital sinus midway between the hippocampus and the olfactory bulb to make a longitudinal incision, cutting the corpus callosum by blunt dissection.
  • the first pair of forceps were then used to reflect back the resultant corner of the cortex exposing the striatum and internal capsule, and then to dissect the internal capsule away from the striatum.
  • the second set of forceps was placed such that the curved ends were on either side of the striatum and were pressed down to isolate the tissue.
  • the first set of forceps was used to pinch off the posterior end of the striatum and to remove the striatum from the brain.
  • Murine REST mRNA levels were measured using murine primer probe set described in Example 6. The results for REST mRNA levels are presented in Table 15 and are expressed as percent inhibition compared to the PBS control group. All the antisense oligonucleotides effect dose- dependent inhibition of REST mRNA levels.
  • R6/2 mice were treated with ISIS 447070 administered via intracerebroventricular (ICV) to the right lateral ventricle of the brain.
  • R6/2 mice are transgenic for the 5' end of the human HD gene carrying (CAG)l 15-(CAG)150 repeat expansions (Mangiarini, L. et al., Cell. 1996. 87: 493-506).
  • the mice exhibit a progressive neurological phenotype that exhibits many of the features of Huntington's disease, including choreiform-like movements, involuntary stereotypic movements, tremor, and epileptic seizures.
  • mice were similarly treated with PBS. Pumps were surgically implanted into the mice. Mice were individually anaesthetized with 3% isoflurane and were maintained throughout the surgical procedure in an ASI small animal stereotaxic system (ASI Instruments, SAS-4100) with a gas nose cone containing 2% isoflurane. The scalp of the animal was sterilized with iodine solution followed by 70% ethanol. Then, a longitudinal mid- saggital incision 1 cm length was made to the scalp.
  • ASI Instruments, SAS-4100 ASI Instruments, SAS-4100
  • a 0.5 mm hole was drilled into the skull using a Wire gauge drill chuck (McMaster Carr, 30505A5) and 0.5 mm diameter cobalt steel drill bit (McMaster Carr, 8904A61). The hole was made -0.3 mm in the anterior/posterior direction and +1 mm to the right in the medial/lateral direction (using the Allen Brain Map atlas to determine the position). A 3 mm cannula connected to the pump was implanted into the right lateral ventricle. After two weeks, the mice were anesthetized again and the pump was surgically removed. The animals were allowed to recover for two weeks before being euthanized.
  • mice were euthanized at 12 weeks of age using isoflurane followed by decapitation. Brain tissue, including the striatum, was extracted for further analyses.
  • GTGGCCTCTAATCAGCATGAAGT designated herein as SEQ ID NO: 116; reverse sequence GCGGGCAATTAAGAGGTTTAGG, designated herein as SEQ ID NO: 1 17; probe sequence CCGACATGCAAGACAGGTTCACAACG, designated herein as SEQ ID NO: 1 18.
  • the mRNA levels of the REST target molecule, BDNF were also measured using a murine primer probe set with forward sequence AAGGCACTGGAACTCGCAAT, designated herein as SEQ ID NO: 113; reverse sequence TTATGAATCGCCAGCCAATTC, designated herein as SEQ ID NO: 114; probe sequence CTACCCAATCGTATGTTCGGGCCCTT, designated herein as SEQ ID NO: 1 15.
  • REST mRNA levels are presented in Table 16 and are expressed as percent inhibition compared to the PBS control group.
  • results of BDNF mRNA levels are also presented in Table 16 and are expressed as percent over the PBS control group. It was expected that the inhibition of REST mRNA expression would lead to increase in target BDNF mRNA expression levels. ISIS 447070 effected significant inhibition of REST mRNA levels and corresponding increase in BDNF mRNA levels.

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Abstract

L'invention concerne des composés antisens et des procédés visant à réduire l'expression de REST. Elle concerne également des procédés visant à augmenter l'expression de DMN1, BDNF, et de la synapsine 1 par réduction de l'expression de REST avec des composés antisens. L'invention concerne des procédés de traitement ou de prévention de la maladie de Huntington chez un individu le nécessitant par réduction de l'expression de REST et/ou augmentation de l'expression de DMN1, BDNF, et de la synapsine 1.
PCT/US2010/048467 2009-09-11 2010-09-10 Modulation de l'expression du facteur de transcription pour le silençage de re1 (rest) WO2011031998A1 (fr)

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WO2016089928A1 (fr) * 2014-12-01 2016-06-09 Weitz Andrew C Méthodes de traitement et d'évaluation d'invasion tumorale et de métastases
WO2019246112A1 (fr) 2018-06-18 2019-12-26 University Of Rochester Procédés de traitement de la schizophrénie et d'autres troubles neuropsychiatriques
EP4035659A1 (fr) 2016-11-29 2022-08-03 PureTech LYT, Inc. Exosomes destinés à l'administration d'agents thérapeutiques
WO2024181416A1 (fr) * 2023-02-28 2024-09-06 国立大学法人大阪大学 Oligonucléotide, et agent de suppression de l'expression de rest et composition pharmaceutique l'utilisant
EP4299744A4 (fr) * 2021-02-25 2024-10-23 Osaka University Oligonucléotide pour induire le saut de n-exons pendant le traitement des précurseurs de l'arnm rest

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