WO2013173608A1 - Compositions and methods for modulating mecp2 expression - Google Patents

Compositions and methods for modulating mecp2 expression Download PDF

Info

Publication number
WO2013173608A1
WO2013173608A1 PCT/US2013/041394 US2013041394W WO2013173608A1 WO 2013173608 A1 WO2013173608 A1 WO 2013173608A1 US 2013041394 W US2013041394 W US 2013041394W WO 2013173608 A1 WO2013173608 A1 WO 2013173608A1
Authority
WO
WIPO (PCT)
Prior art keywords
oligonucleotide
single stranded
nucleotide
mecp2
nucleotides
Prior art date
Application number
PCT/US2013/041394
Other languages
French (fr)
Inventor
Arthur M. Krieg
Romesh Subramanian
James Mcswiggen
Jeannie T. Lee
Original Assignee
Rana Therapeutics, Inc.
The General Hospital Corporation D/B/A Massachusetts General Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Rana Therapeutics, Inc., The General Hospital Corporation D/B/A Massachusetts General Hospital filed Critical Rana Therapeutics, Inc.
Priority to JP2015512847A priority Critical patent/JP2015518712A/en
Priority to EA201492116A priority patent/EA201492116A1/en
Priority to CA2873779A priority patent/CA2873779A1/en
Priority to US14/401,237 priority patent/US20150152410A1/en
Priority to EP13791546.8A priority patent/EP2850190B1/en
Priority to AU2013262709A priority patent/AU2013262709A1/en
Priority to CN201380037614.0A priority patent/CN104583402A/en
Publication of WO2013173608A1 publication Critical patent/WO2013173608A1/en
Priority to US15/787,876 priority patent/US10655128B2/en
Priority to US15/872,684 priority patent/US10837014B2/en
Priority to US16/876,271 priority patent/US11788089B2/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/555—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • 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
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA

Definitions

  • the invention relates to oligonucleotide based compositions, as well as methods of using oligonucleotide based compositions for treating disease.
  • Rett syndrome is a developmental disorder of the brain occurring mostly in females characterized by normal early development, followed by a slowing of development resulting in loss of control of the hands, loss of speech, breathing problems, slowed brain and head growth, ambulatory problems, seizures, and mental retardation. Rett syndrome affects approximately 1 in 10,000 live female births. Most cases of Rett syndrome are caused by a mutation in the methyl CpG binding protein 2, or MECP2 gene, on the X chromosome that causes reduced activity or inactivation of MECP2.
  • MECP2 is a transcriptional repressor that binds to methylated DNA and is present in large quantities in mature nerve cells.
  • MECP2 represses transcription from methylated gene promoters through interaction with histone deacetylase and the corepressor SIN3A.
  • Many of the genes that are known to be regulated by the MECP2 protein play a role in normal brain function, particularly the maintenance of synapses.
  • Mouse studies have demonstrated MECP2 mutations cause defects in synaptic function, especially in synaptic plasticity.
  • aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating MECP2 in cells.
  • oligonucleotides are provided that target a PRC2- associated region of a MECP2 gene (e.g., human MECP2) and thereby cause upregulation of the gene.
  • a MECP2 gene e.g., human MECP2
  • single stranded oligonucleotides are provided that target a PRC2-associated region of the gene encoding MECP2.
  • these single stranded oligonucleotides activate or enhance expression of MECP2 by relieving or preventing PRC2 mediated repression of MECP2.
  • compositions that are useful for upregulating MECP2 for the treatment and/or prevention of Rett Syndrome are also associated with MECP2-related severe neonatal
  • certain aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating MECP2 for the treatment and/or prevention of MECP2-related severe neonatal encephalopathy, Angelman syndrome, and PPM-X syndrome.
  • Further aspects of the invention provide methods for selecting oligonucleotides for activating or enhancing expression of MECP2.
  • methods are provided for selecting a set of oligonucleotides that is enriched in candidates (e.g. , compared with a random selection of oligonucleotides) for activating or enhancing expression of MECP2. Accordingly, the methods may be used to establish sets of clinical candidates that are enriched in oligonucleotides that activate or enhance expression of MECP2.
  • Such libraries may be utilized, for example, to identify lead oligonucleotides for developing therapeutics to treat MECP2.
  • oligonucleotide chemistries are provided that are useful for controlling the pharmacokinetics, biodistribution, bioavailability and/or efficacy of the single stranded oligonucleotides for activating expression of MECP2.
  • single stranded oligonucleotides that have a region of complementarity that is complementarty with (e.g. , at least 8 consecutive nucleotides of ) a PRC2-associated region of a MECP2 gene, e.g., a PRC2- associated region of the nucleotide sequence set forth as SEQ ID NO: 1 or 2.
  • the oligonucleotide has at least one of the following features: a) a sequence that is 5'X-Y-Z, in which X is any nucleotide and in which X is at the 5' end of the oligonucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a human seed sequence of a microRNA, and Z is a nucleotide sequence of 1 to 23 nucleotides in length; b) a sequence that does not comprise three or more consecutive guanosine nucleotides; c) a sequence that has less than a threshold level of sequence identity with every sequence of nucleotides, of equivalent length to the second nucleotide sequence, that are between 50 kilobases upstream of a 5 '-end of an off-target gene and 50 kilobases downstream of a 3 '-end of the off-target gene; d) a sequence that is complementary to a P
  • the single stranded oligonucleotide has at least two of features a), b), c), d), and e), each independently selected. In some embodiments, the single stranded
  • oligonucleotide has at least three of features a), b), c), d), and e), each independently selected. In some embodiments, the single stranded oligonucleotide has at least four of features a), b), c), d), and e), each independently selected. In some embodiments, the single stranded oligonucleotide has each of features a), b), c), d), and e). In certain embodiments, the oligonucleotide has the sequence 5'X-Y-Z, in which the oligonucleotide is 8-50 nucleotides in length.
  • single stranded oligonucleotides have a sequence X-Y-Z, in which X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1 to 23 nucleotides in length, in which the single stranded oligonucleotide is complementary with a PRC2- associated region of a MECP2 gene, e.g., a PRC2-associated region of the nucleotide sequence set forth as SEQ ID NO: 1 or 2.
  • single stranded oligonucleotides have a sequence 5' -X-Y-Z, in which X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1 to 23 nucleotides in length, in which the single stranded oligonucleotide is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of a MECP2 gene, e.g., a PRC2- associated region of the nucleotide sequence set forth as SEQ ID NO: 1 or 2.
  • Y is a sequence selected from Table 1.
  • the PRC2- associated region is a sequence listed in any one of SEQ ID NOS: 5 to 118.
  • the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 119 to 83660, or a fragment thereof that is at least 8 nucleotides.
  • the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 119 to 83660, in which the 5' end of the nucleotide sequence provided is the 5' end of the oligonucleotide.
  • the region of complementarity e.g. , the at least 8 consecutive nucleotides is also present within the nucleotide sequence set forth as SEQ ID NO: 3 or 4.
  • the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 119 to 83660. In some embodiments, the single stranded oligonucleotide comprises a fragment of at least 8 nucleotides of a nucleotide sequence as set forth in any one of SEQ ID NOS: 119 to 83660.
  • the PRC2-associated region is a sequence listed in any one of SEQ ID NOS: 5 to 84.
  • the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 119 to 47653 or a fragment thereof that is at least 8 nucleotides. In some embodiments, the single stranded
  • oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 119 to 47653, wherein the 5' end of the nucleotide sequence provided in any one of SEQ ID NOS: 119 to 47653 is the 5' end of the oligonucleotide.
  • the at least 8 consecutive nucleotides are also present within the nucleotide sequence set forth as SEQ ID NO: 3.
  • the PRC2-associated region is a sequence listed in any one of SEQ ID NOS: 85 to 118.
  • the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 47462 to 83660 or a fragment thereof that is at least 8 nucleotides.
  • the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 47462 to 83660, wherein the 5' end of the nucleotide sequence provided in any one of SEQ ID NOS: 47462 to 83660 is the 5' end of the oligonucleotide.
  • the at least 8 consecutive nucleotides are present within the nucleotide sequence set forth as SEQ ID NO: 4.
  • a single stranded oligonucleotide comprises a nucleotide sequence as set forth in Table 3. In some embodiments, the single stranded oligonucleotide comprises a fragment of at least 8 nucleotides of a nucleotide sequence as set forth in Table 3. In some embodiments, a single stranded oligonucleotide consists of a nucleotide sequence as set forth in Table 3.
  • the single stranded oligonucleotide does not comprise three or more consecutive guanosine nucleotides. In some embodiments, the single stranded oligonucleotide does not comprise four or more consecutive guanosine nucleotides.
  • the single stranded oligonucleotide is 8 to 30 nucleotides in length. In some embodiments, the single stranded oligonucleotide is up to 50 nucleotides in length. In some embodiments, the single stranded oligonucleotide is 8 to 10 nucleotides in length and all but 1, 2, or 3 of the nucleotides of the complementary sequence of the PRC2- associated region are cytosine or guanosine nucleotides.
  • the single stranded oligonucleotide is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of a MECP2 gene, e.g., a PRC2- associated region of a nucleotide sequence set forth as SEQ ID NO: 1 or 2, in which the nucleotide sequence of the single stranded oligonucleotide comprises one or more of a nucleotide sequence selected from the group consisting of
  • XXXXXX XxXXXXX, XXxXXX, XXXxXX, XXXXxX and XXXXXx
  • X denotes a nucleotide analogue
  • X denotes an optional nucleotide analogue
  • x denotes a DNA or RNA nucleotide unit.
  • at least one nucleotide of the oligonucleotide is a nucleotide analogue.
  • the at least one nucleotide analogue results in an increase in Tm of the oligonucleotide in a range of 1 to 5 °C compared with an oligonucleotide that does not have the at least one nucleotide analogue.
  • At least one nucleotide of the oligonucleotide comprises a 2'
  • each nucleotide of the oligonucleotide comprises a 2' O- methyl.
  • the oligonucleotide comprises at least one ribonucleotide, at least one deoxyribonucleotide, or at least one bridged nucleotide.
  • the bridged nucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide.
  • each nucleotide of the oligonucleotide is a LNA nucleotide.
  • the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2'-fluoro-deoxyribonucleotides. In some embodiments, the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2'-0- methyl nucleotides. In some embodiments, the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and ENA nucleotide analogues. In some embodiments, the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and LNA nucleotides. In some embodiments, the 5' nucleotide of the oligonucleotide is a
  • the nucleotides of the oligonucleotide comprise alternating LNA nucleotides and 2' -O-methyl nucleotides.
  • the 5' nucleotide of the oligonucleotide is a LNA nucleotide.
  • the nucleotides of the oligonucleotide comprise deoxyribonucleotides flanked by at least one LNA nucleotide on each of the 5' and 3' ends of the deoxyribonucleotides.
  • the single stranded oligonucleotide comprises modified internucleotide linkages (e.g. , phosphorothioate internucleotide linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides. In some embodiments, the single stranded oligonucleotide comprises modified internucleotide linkages (e.g. , phosphorothioate internucleotide linkages or other linkages) between between all nucleotides.
  • modified internucleotide linkages e.g. , phosphorothioate internucleotide linkages or other linkages
  • the nucleotide at the 3' position of the oligonucleotide has a 3' hydroxyl group. In some embodiments, the nucleotide at the 3' position of the
  • the oligonucleotide has a 3' thiophosphate.
  • the single stranded oligonucleotide has a biotin moiety or other moiety conjugated to its 5' or 3' nucleotide.
  • the single stranded oligonucleotide has cholesterol, Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR or dynamic polyconjugates and variants thereof at its 5' or 3' end.
  • compositions are provided that comprise any of the oligonucleotides disclosed herein, and a carrier.
  • compositions are provided that comprise any of the oligonucleotides in a buffered solution.
  • the oligonucleotide is conjugated to the carrier.
  • the carrier is a peptide.
  • the carrier is a steroid.
  • pharmaceutical compositions are provided that comprise any of the oligonucleotides disclosed herein, and a pharmaceutically acceptable carrier.
  • kits that comprise a container housing any of the compositions disclosed herein.
  • methods of increasing expression of MECP2 in a cell involve delivering any one or more of the single stranded oligonucleotides disclosed herein into the cell.
  • delivery of the single stranded oligonucleotide into the cell results in a level of expression of MECP2 that is greater (e.g. , at least 50% greater) than a level of expression of MECP2 in a control cell that does not comprise the single stranded oligonucleotide.
  • methods of increasing levels of MECP2 in a subject are provided.
  • methods of treating a condition e.g. , Rett Syndrome, MECP2-related severe neonatal encephalopathy, Angelman syndrome, PPM-X syndrome
  • the methods involve administering any one or more of the single stranded oligonucleotides disclosed herein to the subject.
  • Table 3 Formatted oligonucleotide sequences made for testing showing nucleotide modifications.
  • the table shows the sequence of the modified nucleotides, where InaX represents an LNA nucleotide with 3' phosphorothioate linkage, omeX is a 2'-0-methyl nucleotide, dX is a deoxy nucleotide.
  • An s at the end of a nucleotide code indicates that the nucleotide had a 3' phosphorothioate linkage.
  • the "-Sup" at the end of the sequence marks the fact that the 3' end lacks either a phosphate or thiophosphate on the 3' linkage.
  • the Formatted Sequence column shows the sequence of the oligonucleotide, including modified nucleotides.
  • Polycomb repressive complex 2 (PRC2) is a histone methyltransferase and a known epigenetic regulator involved in silencing of genomic regions through methylation of histone H3.
  • PRC2 interacts with long noncoding RNAs (IncRNAs), such as Rep A, Xist, and Tsix, to catalyze
  • PRC2 contains four subunits, Eed, Suzl2, RbAp48, and Ezh2. Aspects of the invention relate to the recognition that single stranded
  • oligonucleotides that bind to PRC2-associated regions of RNAs that are expressed from within a genomic region that encompasses or that is in functional proximity to the MECP2 gene can induce or enhance expression of MECP2. In some embodiments, this upregulation is believed to result from inhibition of PRC2 mediated repression of MECP2.
  • PRC2-associated region refers to a region of a nucleic acid that comprises or encodes a sequence of nucleotides that interact directly or indirectly with a component of PRC2.
  • a PRC2-associated region may be present in a RNA (e.g. , a long non- coding RNA (IncRNA)) that that interacts with a PRC2.
  • a PRC2-associated region may be present in a DNA that encodes an RNA that interacts with PRC2. In some cases, the PRC2- associated region is equivalently referred to as a PRC2-interacting region.
  • a PRC2-associated region is a region of an RNA that crosslinks to a component of PRC2 in response to in situ ultraviolet irradiation of a cell that expresses the RNA, or a region of genomic DNA that encodes that RNA region.
  • a PRC2-associated region is a region of an RNA that immunoprecipitates with an antibody that targets a component of PRC2, or a region of genomic DNA that encodes that RNA region.
  • a PRC2-associated region is a region of an RNA that immunoprecipitates with an antibody that binds specifically to SUZ12, EED, EZH2 or RBBP4 (which as noted above are components of PRC2), or a region of genomic DNA that encodes that RNA region.
  • a PRC2-associated region is a region of an RNA that is protected from nucleases (e.g. , RNases) in an RNA-immunoprecipitation assay that employs an antibody that targets a component of PRC2, or a region of genomic DNA that encodes that protected RNA region.
  • a PRC2-associated region is a region of an RNA that is protected from nucleases (e.g. , RNases) in an RNA-immunoprecipitation assay that employs an antibody that targets SUZ12, EED, EZH2 or RBBP4, or a region of genomic DNA that encodes that protected RNA region.
  • a PRC2-associated region is a region of an RNA within which occur a relatively high frequency of sequence reads in a sequencing reaction of products of an RNA-immunoprecipitation assay that employs an antibody that targets a component of PRC2, or a region of genomic DNA that encodes that RNA region.
  • a PRC2- associated region is a region of an RNA within which occur a relatively high frequency of sequence reads in a sequencing reaction of products of an RNA-immunoprecipitation assay that employs an antibody that binds specifically to SUZ12, EED, EZH2 or RBBP4, or a region of genomic DNA that encodes that protected RNA region.
  • the PRC2-associated region may be referred to as a "peak.”
  • a PRC2-associated region comprises a sequence of 40 to 60 nucleotides that interact with PRC2 complex. In some embodiments, a PRC2-associated region comprises a sequence of 40 to 60 nucleotides that encode an RNA that interacts with PRC2. In some embodiments, a PRC2-associated region comprises a sequence of up to 5kb in length that comprises a sequence (e.g. , of 40 to 60 nucleotides) that interacts with
  • a PRC2-associated region comprises a sequence of up to 5kb in length within which an RNA is encoded that has a sequence (e.g. , of 40 to 60 nucleotides) that is known to interact with PRC2. In some embodiments, a PRC2-associated region comprises a sequence of about 4kb in length that comprise a sequence (e.g. , of 40 to 60 nucleotides) that interacts with PRC2. In some embodiments, a PRC2-associated region comprises a sequence of about 4kb in length within which an RNA is encoded that includes a sequence (e.g., of 40 to 60 nucleotides) that is known to interact with PRC2. In some embodiments, a PRC2-associated region has a sequence as set forth in any one of SEQ ID NOS: 5 to 118.
  • single stranded oligonucleotides are provided that specifically bind to, or are complementary to, a PRC2-associated region in a genomic region that encompasses or that is in proximity to the MECP2 gene. In some embodiments, single stranded oligonucleotides are provided that specifically bind to, or are complementary to, a PRC2-associated region that has a sequence as set forth in any one of SEQ ID NOS: 5 to 118.
  • single stranded oligonucleotides are provided that specifically bind to, or are complementary to, a PRC2-associated region that has a sequence as set forth in any one of SEQ ID NOS: 5 to 118 combined with up to 2kb, up to 5kb, or up to lOkb of flanking sequences from a corresponding genomic region to which these SEQ IDs map (e.g., in a human genome).
  • single stranded oligonucleotides have a sequence as set forth in any one of SEQ ID NOS: 119 to 83660.
  • single stranded oligonucleotides have a sequence as set forth in Table 3.
  • these oligonucleotides are able to interfere with the binding of and function of PRC2, by preventing recruitment of PRC2 to a specific chromosomal locus.
  • a single administration of single stranded oligonucleotides designed to specifically bind a PRC2-associated region IncRNA can stably displace not only the IncRNA, but also the PRC2 that binds to the IncRNA, from binding chromatin. After displacement, the full complement of PRC2 is not recovered for up to 24 hours.
  • IncRNA can recruit PRC2 in a cis fashion, repressing gene expression at or near the specific chromosomal locus from which the IncRNA was transcribed.
  • Methods of modulating gene expression are provided, in some embodiments, that may be carried out in vitro, ex vivo, or in vivo. It is understood that any reference to uses of compounds throughout the description contemplates use of the compound in preparation of a pharmaceutical composition or medicament for use in the treatment of condition (e.g., Rett Syndrome, MECP2-related severe neonatal encephalopathy, Angelman syndrome, PPM-X syndrome) associated with decreased levels or activity of MECP2.
  • condition e.g., Rett Syndrome, MECP2-related severe neonatal encephalopathy, Angelman syndrome, PPM-X syndrome
  • this aspect of the invention includes use of such single stranded oligonucleotides in the preparation of a medicament for use in the treatment of disease, wherein the treatment involves upregulating expression of MECP2.
  • methods are provided for selecting a candidate oligonucleotide for activating expression of MECP2.
  • the methods generally involve selecting as a candidate oligonucleotide, a single stranded oligonucleotide comprising a nucleotide sequence that is complementary to a PRC2-associated region (e.g., a nucleotide sequence as set forth in any one of SEQ ID NOS: 5 to 118).
  • sets of oligonucleotides may be selected that are enriched (e.g., compared with a random selection of oligonucleotides) in oligonucleotides that activate expression of MECP2.
  • single stranded oligonucleotides complementary to the PRC2-associated regions are provided for modulating expression of MECP2 in a cell.
  • expression of MECP2 is upregulated or increased.
  • single stranded oligonucleotides complementary to these PRC2-associated regions inhibit the interaction of PRC2 with long RNA transcripts such that gene expression is upregulated or increased.
  • single stranded oligonucleotides complementary to these PRC2-associated regions inhibit the interaction of PRC2 with long RNA transcripts, resulting in reduced methylation of histone H3 and reduced gene inactivation, such that gene expression is upregulated or increased. In some embodiments, this interaction may be disrupted or inhibited due to a change in the structure of the long RNA that prevents or reduces binding to PRC2.
  • the oligonucleotide may be selected using any of the methods disclosed herein for selecting a candidate oligonucleotide for activating expression of MECP2.
  • the single stranded oligonucleotide may comprise a region of complementarity that is complementary with a PRC2-associated region of a nucleotide sequence set forth in any one of SEQ ID NOS: 1 to 4.
  • oligonucleotide may be complementary with at least 6, e.g., at least 7, at least 8, at least 9, at least 10, at least 15 or more consecutive nucleotides of the PRC2-associated region.
  • the PRC2-associated region may map to a position in a chromosome between 50 kilobases upstream of a 5 '-end of the MECP2 gene and 50 kilobases downstream of a 3 '-end of the MECP2 gene.
  • the PRC2-associated region may map to a position in a chromosome between 25 kilobases upstream of a 5 '-end of the MECP2 gene and 25 kilobases downstream of a 3 '-end of the MECP2 gene.
  • the PRC2-associated region may map to a position in a chromosome between 12 kilobases upstream of a 5'-end of the MECP2 gene and 12 kilobases downstream of a 3 '-end of the MECP2 gene.
  • the PRC2-associated region may map to a position in a chromosome between 5 kilobases upstream of a 5'-end of the MECP2 gene and 5 kilobases downstream of a 3 '-end of the MECP2 gene.
  • the genomic position of the selected PRC2-associated region relative to the MECP2 gene may vary.
  • the PRC2-associated region may be upstream of the 5' end of the MECP2 gene.
  • the PRC2-associated region may be downstream of the 3' end of the
  • the PRC2-associated region may be within an intron of the MECP2 gene.
  • the PRC2-associated region may be within an exon of the MECP2 gene.
  • the PRC2-associated region may traverse an intron-exon junction, a 5 '-UTR-exon junction or a 3'-UTR-exon junction of the MECP2 gene.
  • the single stranded oligonucleotide may comprise a sequence having the formula X-
  • Y-Z in which X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a human seed sequence of a microRNA, and Z is a nucleotide sequence of varying length.
  • X is the 5' nucleotide of the oligonucleotide.
  • the oligonucleotide when X is anchored at the 5' end of the oligonucleotide, the oligonucleotide does not have any nucleotides or nucleotide analogs linked 5' to X.
  • the single stranded oligonucleotide has a sequence 5'X-Y-Z and is 8-50 nucleotides in length.
  • the Y sequence may be a nucleotide sequence of 6 nucleotides in length set forth in Table 1.
  • the single stranded oligonucleotide may have a sequence that does not contain guanosine nucleotide stretches (e.g. , 3 or more, 4 or more, 5 or more, 6 or more consecutive guanosine nucleotides).
  • guanosine nucleotide stretches e.g. 3 or more, 4 or more, 5 or more, 6 or more consecutive guanosine nucleotides.
  • oligonucleotides having guanosine nucleotide stretches have increased non-specific binding and/or off-target effects, compared with oligonucleotides that do not have guanosine nucleotide stretches.
  • the single stranded oligonucleotide may have a sequence that has less than a threshold level of sequence identity with every sequence of nucleotides, of equivalent length, that map to a genomic position encompassing or in proximity to an off-target gene.
  • an oligonucleotide may be designed to ensure that it does not have a sequence that maps to genomic positions encompassing or in proximity with all known genes (e.g. , all known protein coding genes) other than MECP2.
  • an oligonucleotide may be designed to ensure that it does not have a sequence that maps to genomic positions encompassing or in proximity with all known genes (e.g. , all known protein coding genes) other than MECP2.
  • an oligonucleotide may be designed to ensure that it does not have a sequence that maps to genomic positions encompassing or in proximity with all known genes (e.g. , all known protein coding genes) other than MECP2.
  • an oligonucleotide may be designed to ensure that it does not
  • oligonucleotide may be designed to ensure that it does not have a sequence that maps to any other known PRC2-associated region, particularly PRC2-associated regions that are functionally related to any other known gene (e.g. , any other known protein coding gene). In either case, the oligonucleotide is expected to have a reduced likelihood of having off-target effects.
  • the threshold level of sequence identity may be 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity.
  • the single stranded oligonucleotide may have a sequence that is complementary to a
  • oligonucleotides that are complementary to a PRC2-associated region that encodes an RNA that forms a secondary structure comprising one or more single stranded loops have a greater likelihood of being active (e.g. , of being capable of activating or enhancing expression of a target gene) than a randomly selected
  • the secondary structure may comprise a double stranded stem between the at least two single stranded loops. Accordingly, the region of
  • complementarity between the oligonucleotide and the PRC2-associated region may be at a location of the PRC2 associated region that encodes at least a portion of at least one of the loops. In some cases, the region of complementarity between the oligonucleotide and the PRC2-associated region may be at a location of the PRC2-associated region that encodes at least a portion of at least two of the loops. In some cases, the region of complementarity between the oligonucleotide and the PRC2-associated region may be at a location of the PRC2 associated region that encodes at least a portion of the double stranded stem. In some embodiments, a PRC2-associated region (e.g.
  • the predicted secondary structure RNA (e.g. , IncRNA) containing the PRC2-associated region is determined using RNA secondary structure prediction algorithms, e.g. , RNAfold, mfold.
  • oligonucleotides are designed to target a region of the RNA that forms a secondary structure comprising one or more single stranded loop (e.g. , at least two single stranded loops) structures which may comprise a double stranded stem between the at least two single stranded loops.
  • the single stranded oligonucleotide may have a sequence that is has greater than 30% G-C content, greater than 40% G-C content, greater than 50% G-C content, greater than 60% G-C content, greater than 70% G-C content, or greater than 80% G-C content.
  • the single stranded oligonucleotide may have a sequence that has up to 100% G-C content, up to 95% G-C content, up to 90% G-C content, or up to 80% G-C content.
  • the oligonucleotide is 8 to 10 nucleotides in length, all but 1, 2, 3, 4, or 5 of the nucleotides of the complementary sequence of the PRC2-associated region are cytosine or guanosine nucleotides.
  • the sequence of the PRC2- associated region to which the single stranded oligonucleotide is complementary comprises no more than 3 nucleotides selected from adenine and uracil.
  • the single stranded oligonucleotide may be complementary to a chromosome of a different species (e.g. , a mouse, rat, rabbit, goat, monkey, etc.) at a position that encompasses or that is in proximity to that species' homolog of MECP2.
  • a different species e.g. , a mouse, rat, rabbit, goat, monkey, etc.
  • oligonucleotide may be complementary to a human genomic region encompassing or in proximity to the MECP2 gene and also be complementary to a mouse genomic region encompassing or in proximity to the mouse homolog of MECP2.
  • the single stranded oligonucleotide may be complementary to a sequence as set forth in SEQ ID NO: 1 or 2, which is a human genomic region encompassing or in proximity to the MECP2 gene, and also be complementary to a sequence as set forth in SEQ ID NO: 3 or 4, which is a mouse genomic region encompassing or in proximity to the mouse homolog of the MECP2 gene.
  • Oligonucleotides having these characteristics may be tested in vivo or in vitro for efficacy in multiple species (e.g. , human and mouse).
  • the region of complementarity of the single stranded oligonucleotide is complementary with at least 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 bases, e.g. , 5, 6, 7, 8, 9, 10, 11, 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, or 50 consecutive nucleotides of a PRC2-associated region.
  • the region of complementarity is complementary with at least 8 consecutive nucleotides of a PRC2-associated region.
  • sequence of the single stranded oligonucleotide is based on an RNA sequence that binds to PRC2, or a portion thereof, said portion having a length of from 5 to 40 contiguous base pairs, or about 8 to 40 bases, or about 5 to 15, or about 5 to 30, or about 5 to 40 bases, or about 5 to 50 bases.
  • Complementary refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an
  • oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of PRC2-associated region, then the single stranded nucleotide and PRC2-associated region are considered to be complementary to each other at that position.
  • the single stranded nucleotide and PRC2-associated region are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond with each other through their bases.
  • “complementary” is a term which is used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the single stranded nucleotide and PRC2-associated region.
  • a base at one position of a single stranded nucleotide is capable of hydrogen bonding with a base at the corresponding position of a PRC2-associated region, then the bases are considered to be complementary to each other at that position. 100% complementarity is not required.
  • the single stranded oligonucleotide may be at least 80% complementary to
  • the single stranded oligonucleotide may contain 1, 2 or 3 base mismatches compared to the portion of the consecutive nucleotides of a PRC2-associated region.
  • the single stranded oligonucleotide may have up to 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases. It is understood in the art that a complementary nucleotide sequence need not be 100% complementary to that of its target to be specifically hybridizable.
  • a complementary nucleic acid sequence for purposes of the present disclosure is specifically hybridizable when binding of the sequence to the target molecule (e.g. , IncRNA) interferes with the normal function of the target (e.g. , IncRNA) to cause a loss of activity (e.g. , inhibiting PRC2-associated repression with consequent up-regulation of gene expression) and there is a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target sequences under conditions in which avoidance of non-specific binding is desired, e.g. , under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under suitable conditions of stringency.
  • the target molecule e.g. , IncRNA
  • the single stranded oligonucleotide is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more nucleotides in length. In a preferred embodiment, the oligonucleotide is 8 to 30 nucleotides in length.
  • the PRC2-associated region occurs on the same DNA strand as a gene sequence (sense). In some embodiments, the PRC2-associated region occurs on the opposite DNA strand as a gene sequence (anti-sense). Oligonucleotides complementary to a PRC2-associated region can bind either sense or anti-sense sequences.
  • Base pairings may include both canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g. , Wobble base pairing and Hoogsteen base pairing).
  • adenosine-type bases are complementary to thymidine-type bases (T) or uracil-type bases (U), that cytosine-type bases (C) are complementary to guanosine-type bases (G), and that universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize to and are considered complementary to any A, C, U, or T.
  • Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U or T.
  • any one or more thymidine (T) nucleotides (or modified nucleotide thereof) or uridine (U) nucleotides (or a modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be replaced with any other nucleotide suitable for base pairing (e.g., via a Watson-Crick base pair) with an adenosine nucleotide.
  • any one or more thymidine (T) nucleotides (or modified nucleotide thereof) or uridine (U) nucleotides (or a modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be suitably replaced with a different pyrimidine nucleotide or vice versa.
  • any one or more thymidine (T) nucleotides (or modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing may be suitably replaced with a uridine (U) nucleotide (or a modified nucleotide thereof) or vice versa.
  • GC content of the single stranded oligonucleotide is preferably between about 30-60 %. Contiguous runs of three or more Gs or Cs may not be preferable in some embodiments. Accordingly, in some embodiments, the oligonucleotide does not comprise a stretch of three or more guanosine nucleotides.
  • the single stranded oligonucleotide specifically binds to, or is complementary to an RNA that is encoded in a genome (e.g., a human genome) as a single contiguous transcript (e.g., a non-spliced RNA).
  • a genome e.g., a human genome
  • a single contiguous transcript e.g., a non-spliced RNA
  • the single stranded oligonucleotide specifically binds to, or is complementary to an RNA that is encoded in a genome (e.g., a human genome), in which the distance in the genome between the 5'end of the coding region of the RNA and the 3' end of the coding region of the RNA is less than 1 kb, less than 2 kb, less than 3 kb, less than 4 kb, less than 5 kb, less than 7 kb, less than 8 kb, less than 9 kb, less than 10 kb, or less than 20 kb.
  • a genome e.g., a human genome
  • any oligonucleotide provided herein can be excluded.
  • single stranded oligonucleotides disclosed herein may increase expression of mRNA corresponding to the gene by at least about 50% (i.e. 150% of normal or 1.5 fold), or by about 2 fold to about 5 fold.
  • expression may be increased by at least about 15 fold, 20 fold, 30 fold, 40 fold, 50 fold or 100 fold, or any range between any of the foregoing numbers. It has also been found that increased mRNA expression has been shown to correlate to increased protein expression.
  • the oligonucleotides will upregulate gene expression and may specifically bind or specifically hybridize or be complementary to the PRC2 binding RNA that is transcribed from the same strand as a protein coding reference gene.
  • the oligonucleotide may bind to a region of the PRC2 binding RNA that originates within or overlaps an intron, exon, intron exon junction, 5' UTR, 3' UTR, a translation initiation region, or a translation termination region of a protein coding sense strand of a reference gene (refGene).
  • the oligonucleotides will upregulate gene expression and may specifically bind or specifically hybridize or be complementary to a PRC2 binding RNA that transcribed from the opposite strand (the antisense strand) of a protein coding reference gene.
  • the oligonucleotide may bind to a region of the PRC2 binding RNA that originates within or overlaps an intron, exon, intron exon junction, 5' UTR, 3' UTR, a translation initiation region, or a translation termination region of a protein coding antisense strand of a reference gene
  • oligonucleotides described herein may be modified, e.g., comprise a modified sugar moiety, a modified internucleoside linkage, a modified nucleotide and/or combinations thereof.
  • the oligonucleotides can exhibit one or more of the following properties: do not induce substantial cleavage or degradation of the target RNA; do not cause
  • RNAse H pathway do not activate RNAse H pathway; do not activate RISC; do not recruit any Argonaute family protein; are not cleaved by Dicer; do not mediate alternative splicing; are not immune stimulatory; are nuclease resistant; have improved cell uptake compared to unmodified oligonucleotides; are not toxic to cells or mammals; may have improved endosomal exit; do interfere with interaction of IncRNA with PRC2, preferably the Ezh2 subunit but optionally the Suzl2, Eed, RbAp46/48 subunits or accessory factors such as Jarid2; do decrease histone H3 lysine27 methylation and/or do upregulate gene expression.
  • PRC2 preferably the Ezh2 subunit but optionally the Suzl2, Eed, RbAp46/48 subunits or accessory factors such as Jarid2; do decrease histone H3 lysine27 methylation and/or do upregulate gene expression.
  • Oligonucleotides that are designed to interact with RNA to modulate gene expression are a distinct subset of base sequences from those that are designed to bind a DNA target (e.g., are complementary to the underlying genomic DNA sequence from which the RNA is transcribed).
  • any of the oligonucleotides disclosed herein may be linked to one or more other oligonucleotides disclosed herein by a linker, e.g., a cleavable linker.
  • Method for Selecting Candidate Oligonucleotides for Activating Expression ofMECP2 Methods are provided herein for selecting a candidate oligonucleotide for activating or enhancing expression of MECP2.
  • the target selection methods may generally involve steps for selecting single stranded oligonucleotides having any of the structural and functional characteristics disclosed herein.
  • the methods involve one or more steps aimed at identifying oligonucleotides that target a PRC2-associated region that is functionally related to MECP2, for example a PRC2-associated region of a IncRNA that regulates expression of MECP2 by facilitating (e.g. , in a -regulatory manner) the recruitment of PRC2 to the MECP2 gene.
  • oligonucleotides are expected to be candidates for activating expression of MECP2 because of their ability to hybridize with the PRC2-associated region of a nucleic acid (e.g. , a IncRNA).
  • this hybridization event is understood to disrupt interaction of PRC2 with the nucleic acid (e.g. , a IncRNA) and as a result disrupt recruitment of PRC2 and its associated co-repressors (e.g. , chromatin remodeling factors) to the MECP2 gene locus.
  • Methods of selecting a candidate oligonucleotide may involve selecting a PRC2- associated region (e.g. , a nucleotide sequence as set forth in any one of SEQ ID NOS: 5 to 118) that maps to a chromosomal position encompassing or in proximity to the MECP2 gene (e.g. , a chromosomal position having a sequence as set forth in any one of SEQ ID NOS: 1 to 4).
  • the PRC2-associated region may map to the strand of the chromosome comprising the sense strand of the MECP2 gene, in which case the candidate oligonucleotide is
  • the PRC2-associated region may map to the strand of the first chromosome comprising the antisense strand of the MECP2 gene, in which case the oligonucleotide is complementary to the antisense strand (the template strand) of the MECP2 gene (i.e. , is sense to the MECP2 gene).
  • Methods for selecting a set of candidate oligonucleotides that is enriched in oligonucleotides that activate expression of MECP2 may involve selecting one or more PRC2-associated regions that map to a chromosomal position that encompasses or that is in proximity to the MECP2 gene and selecting a set of oligonucleotides, in which each oligonucleotide in the set comprises a nucleotide sequence that is complementary with the one or more PRC2-associated regions.
  • a set of oligonucleotides that is enriched in oligonucleotides that activate expression of refers to a set of oligonucleotides that has a greater number of oligonucleotides that activate expression of a target gene (e.g. , MECP2) compared with a random selection of oligonucleotides of the same physicochemical properties (e.g. , the same GC content, T m , length etc.) as the enriched set.
  • design and/or synthesis of a single stranded oligonucleotide involves design and/or synthesis of a sequence that is complementary to a nucleic acid or PRC2- associated region described by such sequence information
  • the skilled person is readily able to determine the complementary sequence, e.g., through understanding of Watson Crick base pairing rules which form part of the common general knowledge in the field.
  • design and/or synthesis of a single stranded oligonucleotide involves manufacture of an oligonucleotide from starting materials by techniques known to those of skill in the art, where the synthesis may be based on a sequence of a PRC2- associated region, or portion thereof.
  • Methods of design and/or synthesis of a single stranded oligonucleotide may involve one or more of the steps of:
  • Single stranded oligonucleotides so designed and/or synthesized may be useful in method of modulating gene expression as described herein.
  • oligonucleotides of the invention are synthesized chemically.
  • Oligonucleotides used to practice this invention can be synthesized in vitro by well-known chemical synthesis techniques.
  • Oligonucleotides of the invention can be stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g. , a nucleotide modification.
  • nucleic acid sequences of the invention include a phosphorothioate at least the first, second, or third internucleotide linkage at the 5' or 3' end of the nucleotide sequence.
  • the nucleic acid sequence can include a 2'-modified nucleotide, e.g.
  • the nucleic acid sequence can include at least one 2'-0-methyl-modified nucleotide, and in some embodiments, all of the nucleotides include a 2'-0-methyl modification.
  • the nucleic acids are "locked,” i.e., comprise nucleic acid analogues in which the ribose ring is "locked” by a methylene bridge connecting the 2'- O atom and the 4'-C atom.
  • any of the modified chemistries or formats of single stranded oligonucleotides described herein can be combined with each other, and that one, two, three, four, five, or more different types of modifications can be included within the same molecule.
  • the method may further comprise the steps of amplifying the synthesized single stranded oligonucleotide, and/or purifying the single stranded
  • oligonucleotide (or amplified single stranded oligonucleotide), and/or sequencing the single stranded oligonucleotide so obtained.
  • the process of preparing a single stranded oligonucleotide may be a process that is for use in the manufacture of a pharmaceutical composition or medicament for use in the treatment of disease, optionally wherein the treatment involves modulating expression of a gene associated with a PRC2-associated region.
  • a PRC2-associated region may be, or have been, identified, or obtained, by a method that involves identifying RNA that binds to PRC2.
  • Such methods may involve the following steps: providing a sample containing nuclear ribonucleic acids, contacting the sample with an agent that binds specifically to PRC2 or a subunit thereof, allowing complexes to form between the agent and protein in the sample, partitioning the complexes, synthesizing nucleic acid that is complementary to nucleic acid present in the complexes.
  • single stranded oligonucleotide is based on a PRC2-associated region, or a portion of such a sequence, it may be based on information about that sequence, e.g. , sequence information available in written or electronic form, which may include sequence information contained in publicly available scientific publications or sequence databases.
  • Nucleotide Analogues are examples of nucleotide Analogues.
  • the oligonucleotide may comprise at least one ribonucleotide, at least one deoxyribonucleotide, and/or at least one bridged nucleotide.
  • the oligonucleotide may comprise a bridged nucleotide, such as a locked nucleic acid (LNA) nucleotide, a constrained ethyl (cEt) nucleotide, or an ethylene bridged nucleic acid (ENA) nucleotide.
  • LNA locked nucleic acid
  • cEt constrained ethyl
  • ENA ethylene bridged nucleic acid
  • the oligonucleotide comprises a nucleotide analog disclosed in one of the following United States Patent or Patent Application Publications: US 7,399,845, US 7,741,457, US 8,022,193, US 7,569,686, US 7,335,765, US 7,314,923, US 7,335,765, and US 7,816,333, US 20110009471, the entire contents of each of which are incorporated herein by reference for all purposes.
  • the oligonucleotide may have one or more 2' O-methyl nucleotides.
  • the oligonucleotide may consist entirely of 2' O-methyl nucleotides.
  • the single stranded oligonucleotide has one or more nucleotide analogues.
  • the single stranded oligonucleotide may have at least one nucleotide analogue that results in an increase in T m of the oligonucleotide in a range of 1°C, 2 °C, 3°C, 4 °C, or 5°C compared with an oligonucleotide that does not have the at least one nucleotide analogue.
  • the single stranded oligonucleotide may have a plurality of nucleotide analogues that results in a total increase in T m of the oligonucleotide in a range of 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C or more compared with an oligonucleotide that does not have the nucleotide analogue.
  • the oligonucleotide may be of up to 50 nucleotides in length in which 2 to 10, 2 to 15 5 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, 2 to 45, or more nucleotides of the oligonucleotide are nucleotide analogues.
  • the oligonucleotide may be of 8 to 30 nucleotides in length in which 2 to 10, 2 to 15 5 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30 nucleotides of the oligonucleotide are nucleotide analogues.
  • the oligonucleotide may be of 8 to 15 nucleotides in length in which 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14 nucleotides of the oligonucleotide are nucleotide analogues.
  • the oligonucleotides may have every nucleotide except 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides modified.
  • the oligonucleotide may consist entirely of bridged nucleotides (e.g. , LNA nucleotides, cEt nucleotides, ENA nucleotides).
  • the oligonucleotide may comprise alternating deoxyribonucleotides and 2'-fluoro-deoxyribonucleotides.
  • the oligonucleotide may comprise alternating deoxyribonucleotides and 2'-0-methyl nucleotides.
  • the oligonucleotide may comprise alternating deoxyribonucleotides and ENA nucleotide analogues.
  • the oligonucleotide may comprise alternating deoxyribonucleotides and LNA nucleotides.
  • the oligonucleotide may comprise alternating LNA nucleotides and 2'-0- methyl nucleotides.
  • the oligonucleotide may have a 5' nucleotide that is a bridged nucleotide (e.g. , a LNA nucleotide, cEt nucleotide, ENA nucleotide).
  • the oligonucleotide may have a 5' nucleotide that is a deoxyribonucleotide.
  • the oligonucleotide may comprise deoxyribonucleotides flanked by at least one bridged nucleotide (e.g. , a LNA nucleotide, cEt nucleotide, ENA nucleotide) on each of the 5' and 3' ends of the deoxyribonucleotides.
  • the oligonucleotide may comprise
  • deoxyribonucleotides flanked by 1, 2, 3, 4, 5, 6, 7, 8 or more bridged nucleotides (e.g. , LNA nucleotides, cEt nucleotides, ENA nucleotides) on each of the 5' and 3' ends of the deoxyribonucleotides.
  • the 3' position of the oligonucleotide may have a 3' hydroxyl group.
  • the 3' position of the oligonucleotide may have a 3' thiophosphate.
  • the oligonucleotide may be conjugated with a label.
  • the oligonucleotide may be conjugated with a label.
  • oligonucleotide may be conjugated with a biotin moiety, cholesterol, Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR or dynamic polyconjugates and variants thereof at its 5' or 3' end.
  • a biotin moiety cholesterol, Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR or dynamic polyconjugates and variants thereof at its 5' or 3' end.
  • the single stranded oligonucleotide comprises one or more modifications comprising: a modified sugar moiety, and/or a modified internucleoside linkage, and/or a modified nucleotide and/or combinations thereof. It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the
  • modifications described herein may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide.
  • the single stranded oligonucleotides are chimeric
  • oligonucleotides that contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Chimeric single stranded oligonucleotides of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides,
  • oligonucleosides and/or oligonucleotide mimetics as described above.
  • Such compounds have also been referred to in the art as hybrids or gapmers.
  • Representative United States patents that teach the preparation of such hybrid structures comprise, but are not limited to, US patent nos. 5,013,830; 5,149,797; 5, 220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133;
  • the single stranded oligonucleotide comprises at least one nucleotide modified at the 2' position of the sugar, most preferably a 2'-0-alkyl, 2'-0-alkyl-0- alkyl or 2'-fluoro-modified nucleotide.
  • RNA modifications include 2'-fluoro, 2'-amino and 2' O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA.
  • modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are oligonucleotides with
  • phosphorothioate backbones and those with heteroatom backbones particularly CH 2 -NH-O- CH 2 , CH, ⁇ N(CH 3 ) ⁇ 0 ⁇ CH 2 (known as a methylene(methylimino) or MMI backbone, CH 2 - O-N (CH 3 )-CH 2 , CH 2 -N (CH 3 )-N (CH 3 )-CH 2 and O-N (CH 3 )- CH 2 -CH 2 backbones, wherein the native phosphodiester backbone is represented as O- P— O- CH,); amide backbones (see De Mesmaeker et al. Ace. Chem. Res.
  • PNA peptide nucleic acid
  • Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3 -5' to 5'-3' or 2 -5' to 5'-2'; see US patent nos. 3,687,808; 4,469,863;
  • Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and
  • the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g. , as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001 ; and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated herein by reference in their entireties).
  • PMO phosphorodiamidate morpholino oligomer
  • Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc, 2000, 122, 8595-8602.
  • Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts; see US patent nos.
  • Modified oligonucleotides are also known that include oligonucleotides that are based on or constructed from arabinonucleotide or modified arabinonucleotide residues.
  • Arabinonucleosides are stereoisomers of ribonucleosides, differing only in the configuration at the 2'-position of the sugar ring.
  • a 2'-arabino modification is 2'-F arabino.
  • the modified oligonucleotide is 2' -fluoro-D-arabinonucleic acid (FANA) (as described in, for example, Lon et al., Biochem., 41 :3457-3467, 2002 and Min et al., Bioorg. Med. Chem. Lett., 12:2651-2654, 2002; the disclosures of which are incorporated herein by reference in their entireties). Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on a 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
  • WO 99/67378 discloses arabinonucleic acids (ANA) oligomers and their analogues for improved sequence specific inhibition of gene expression via association to complementary messenger RNA.
  • ENAs ethylene-bridged nucleic acids
  • Preferred ENAs include, but are not limited to, 2'-0,4'-C-ethylene -bridged nucleic acids. Examples of LNAs are described in WO/2008/043753 and include compounds of the following general formula.
  • -CH 2 -0-, -CH 2 -S-, -CH 2 -N(H)-, -CH 2 -N(R)-, -CH 2 -CH 2 - or -CH 2 -CH- (if part of a double bond), -CH CH-, where R is selected from hydrogen and Ci-4-alkyl; Z and Z* are independently selected among an intemucleoside linkage, a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety; and the asymmetric groups may be found in either orientation.
  • the LNA used in the oligonucleotides described herein comprises at least one LNA unit according any of the formulas
  • the Locked Nucleic Acid (LNA) used in the oligonucleotides described herein comprises at least one Locked Nucleic Acid (LNA) unit according any of the formulas shown in Scheme 2 of PCT/DK2006/000512.
  • the LNA used in the oligomer of the invention comprises internucleoside linkages selected from -0-P(O) 2 -O-, -0-P(0,S)-0-, -0-P(S) 2 -O-, -S-P(0) 2 -0-, -S-P(0,S)-0-, -S-P(S) 2 -0-, -0-P(O) 2 -S-, -0-P(0,S)-S-, -S-P(0) 2 -S-, -0-PO(R H )-0-, o- PO(OCH 3 )-0-, -0-PO(NR H )-0-, -0-PO(OCH 2 CH 2 S-R)-O-, -0-PO(BH 3 )-0-, -0-PO(NHR H )- 0-, -0-P(0) 2 -NR H -, -NR H -P(0) 2 -0-, -NR H -CO
  • thio-LNA comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from S or -CH 2 -S-.
  • Thio-LNA can be in both beta-D and alpha-L-configuration.
  • amino-LNA comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from -N(H)-, N(R)-, CH 2 -N(H)-, and -CH 2 -N(R)- where R is selected from hydrogen and Ci-4-alkyl.
  • Amino-LNA can be in both beta-D and alpha- L-configuration.
  • Oxy-LNA comprises a locked nucleotide in which at least one of X or Y in the general formula above represents -O- or -CH 2 -0-. Oxy-LNA can be in both beta-D and alpha- L-configuration.
  • ena-LNA comprises a locked nucleotide in which Y in the general formula above is -CH 2 -0- (where the oxygen atom of -CH 2 -0- is attached to the 2'-position relative to the base B).
  • LNAs are described in additional detail herein.
  • One or more substituted sugar moieties can also be included, e.g. , one of the following at the 2' position: OH, SH, SCH 3 , F, OCN, OCH 3 OCH 3 , OCH 3 0(CH 2 )n CH 3 , 0(CH 2 )n NH 2 or 0(CH 2 )n CH 3 where n is from 1 to about 10; CI to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF ; OCF ; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH 3 ; S0 2 CH 3 ; ON0 2 ; N0 2 ; N 3 ; NH2; heterocycloalkyl; heterocyclo alkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter
  • a preferred modification includes 2'-methoxyethoxy [2'-0-CH 2 CH 2 OCH 3 , also known as 2'-0-(2-methoxyethyl)] (Martin et al, Helv. Chim. Acta, 1995, 78, 486).
  • Other preferred modifications include 2'- methoxy (2'-0-CH 3 ), 2'-propoxy (2'-OCH 2 CH 2 CH 3 ) and 2'-fluoro (2'-F). Similar
  • Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
  • Single stranded oligonucleotides can also include, additionally or alternatively, nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobase often referred to in the art simply as “base”
  • “unmodified” or “natural” nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g. , hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2'
  • deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, isocytosine, pseudoisocytosine, as well as synthetic nucleobases, e.g.
  • 2-aminoadenine 2- (methylamino)adenine, 2-(imidazolylalkyl)adenine, 2- (aminoalklyamino)adenine or other hetero substituted alkyladenines
  • 2-thiouracil 2- thiothymine
  • 5-bromouracil 5-hydroxymethyluracil, 5-propynyluracil
  • 8-azaguanine 7- deazaguanine
  • N6 (6-aminohexyl)adenine
  • 6-aminopurine 2-aminopurine, 2-chloro-6- aminopurine and 2,6-diaminopurine or other diaminopurines. See, e.g.
  • both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • an oligomeric compound an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar- backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an aminoethylglycine backbone.
  • the nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • PNA compounds include, but are not limited to, US patent nos. 5,539,082; 5,714,331 ; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500.
  • Single stranded oligonucleotides can also include one or more nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobases comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases comprise other synthetic and natural nucleobases such as 5-methylcytosine (5- me-C), 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 uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8- substituted adenines and guanines, 5-halo particularly 5- bromo, 5-trifluoromethyl and other 5-substitute
  • nucleobases comprise those disclosed in United States Patent No. 3,687,808, those disclosed in "The Concise Encyclopedia of Polymer Science And Engineering", pages 858-859, Kroschwitz, ed. John Wiley & Sons, 1990;, those disclosed by Englisch et al., Angewandle Chemie, International Edition, 1991, 30, page 613, and those disclosed by Sanghvi, Chapter 15, Antisense Research and Applications," pages 289- 302, Crooke, and Lebleu, eds., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention.
  • 5-substituted pyrimidines 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine.
  • 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 ⁇ 0>C (Sanghvi, et al., eds, "Antisense Research and Applications," CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications. Modified nucleobases are described in US patent nos.
  • the single stranded oligonucleotides are chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.
  • one or more single stranded oligonucleotides, of the same or different types, can be conjugated to each other; or single stranded
  • oligonucleotides can be conjugated to targeting moieties with enhanced specificity for a cell type or tissue type.
  • moieties include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g. , hexyl-S- tritylthiol (Manoharan et al, Ann. N. Y. Acad.
  • Acids Res., 1990, 18, 3777-3783 a polyamine or a polyethylene glycol chain (Mancharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-t oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp.
  • conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers.
  • Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence- specific hybridization with the target nucleic acid.
  • Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention.
  • Representative conjugate groups are disclosed in International Patent Application No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, which are incorporated herein by reference.
  • Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g.
  • hexyl-5-tritylthiol a thiocholesterol
  • an aliphatic chain e.g. , dodecandiol or undecyl residues
  • a phospholipid e.g. , di-hexadecyl-rac- glycerol or triethylammonium 1,2- di-O-hexadecyl-rac-glycero-3-H-phosphonate
  • a polyamine or a polyethylene glycol chain or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety. See, e.g.
  • single stranded oligonucleotide modification include modification of the 5' or 3' end of the oligonucleotide.
  • the 3' end of the oligonucleotide comprises a hydroxyl group or a thiophosphate.
  • additional molecules e.g. a biotin moiety or a fluorophor
  • the single stranded oligonucleotide comprises a biotin moiety conjugated to the 5' nucleotide.
  • the single stranded oligonucleotide comprises locked nucleic acids (LNA), ENA modified nucleotides, 2'-0-methyl nucleotides, or 2'-fluoro- deoxyribonucleotides. In some embodiments, the single stranded oligonucleotide comprises alternating deoxyribonucleotides and 2'-fluoro-deoxyribonucleotides. In some embodiments, the single stranded oligonucleotide comprises alternating deoxyribonucleotides and 2'-0- methyl nucleotides.
  • the single stranded oligonucleotide comprises alternating deoxyribonucleotides and ENA modified nucleotides. In some embodiments, the single stranded oligonucleotide comprises alternating deoxyribonucleotides and locked nucleic acid nucleotides. In some embodiments, the single stranded oligonucleotide comprises alternating locked nucleic acid nucleotides and 2'-0-methyl nucleotides.
  • the 5' nucleotide of the oligonucleotide is a
  • the 5' nucleotide of the oligonucleotide is a locked nucleic acid nucleotide.
  • the nucleotides of the oligonucleotide comprise deoxyribonucleotides flanked by at least one locked nucleic acid nucleotide on each of the 5' and 3' ends of the deoxyribonucleotides.
  • the nucleotide at the 3' position of the oligonucleotide has a 3' hydroxyl group or a 3' thiophosphate.
  • the single stranded oligonucleotide comprises
  • the single stranded oligonucleotide comprises phosphorothioate internucleotide linkages between at least two nucleotides. In some embodiments, the single stranded oligonucleotide comprises phosphorothioate internucleotide linkages between all nucleotides.
  • the single stranded oligonucleotide can have any combination of modifications as described herein.
  • the oligonucleotide may comprise a nucleotide sequence having one or more of the following modification patterns.
  • the invention relates to methods for modulating gene expression in a cell (e.g., a cell for which MECP2 levels are reduced) for research purposes (e.g., to study the function of the gene in the cell).
  • the invention relates to methods for modulating gene expression in a cell (e.g., a cell for which MECP2 levels are reduced) for gene or epigenetic therapy.
  • the cells can be in vitro, ex vivo, or in vivo (e.g., in a subject who has a disease resulting from reduced expression or activity of MECP2.
  • methods for modulating gene expression in a cell comprise delivering a single stranded oligonucleotide as described herein.
  • delivery of the single stranded oligonucleotide to the cell results in a level of expression of gene that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more greater than a level of expression of gene in a control cell to which the single stranded oligonucleotide has not been delivered.
  • methods comprise administering to a subject (e.g. a human) a composition comprising a single stranded oligonucleotide as described herein to increase protein levels in the subject.
  • a subject e.g. a human
  • the increase in protein levels is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more, higher than the amount of a protein in the subject before administering.
  • the methods include introducing into the cell a single stranded oligonucleotide that is sufficiently complementary to a PRC2-associated region (e.g., of a long non-coding RNA) that maps to a genomic position encompassing or in proximity to the MECP2 gene.
  • a PRC2-associated region e.g., of a long non-coding RNA
  • a condition e.g., Rett Syndrome, MECP2-related severe neonatal encephalopathy, Angelman syndrome, PPM-X syndrome
  • a condition e.g., Rett Syndrome, MECP2-related severe neonatal encephalopathy, Angelman syndrome, PPM-X syndrome
  • administering a single stranded oligonucleotide as described herein.
  • a subject can include a non-human mammal, e.g. mouse, rat, guinea pig, rabbit, cat, dog, goat, cow, or horse.
  • a subject is a human.
  • Single stranded oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals, including humans.
  • Single stranded oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.
  • an animal preferably a human, suspected of having Rett Syndrome, MECP2-related severe neonatal encephalopathy, Angelman syndrome, or PPM-X syndrome is treated by administering single stranded oligonucleotide in accordance with this invention.
  • the methods comprise the step of
  • oligonucleotides described herein can be formulated for administration to a subject for treating a condition (e.g., Rett Syndrome, MECP2-related severe neonatal encephalopathy, Angelman syndrome, PPM-X syndrome) associated with decreased levels of MECP2. It should be understood that the formulations, compositions and methods can be practiced with any of the oligonucleotides disclosed herein.
  • a condition e.g., Rett Syndrome, MECP2-related severe neonatal encephalopathy, Angelman syndrome, PPM-X syndrome
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient e.g. , an oligonucleotide or compound of the invention
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration, e.g. , intradermal or inhalation.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect, e.g. tumor regression.
  • compositions of this invention can be prepared according to any method known to the art for the manufacture of pharmaceuticals. Such formulations can contain sweetening agents, flavoring agents, coloring agents and preserving agents. A formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture. Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.
  • a formulated single stranded oligonucleotide composition can assume a variety of states.
  • the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g. , less than 80, 50, 30, 20, or 10% water).
  • the single stranded oligonucleotide is in an aqueous phase, e.g. , in a solution that includes water.
  • the aqueous phase or the crystalline compositions can, e.g. , be incorporated into a delivery vehicle, e.g. , a liposome (particularly for the aqueous phase) or a particle (e.g. , a microparticle as can be appropriate for a crystalline composition).
  • the single stranded oligonucleotide composition is formulated in a manner that is compatible with the intended method of administration.
  • the composition is prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and other self-assembly.
  • a single stranded oligonucleotide preparation can be formulated or administered (together or separately) in combination with another agent, e.g. , another therapeutic agent or an agent that stabilizes a single stranded oligonucleotide, e.g. , a protein that complexes with single stranded oligonucleotide.
  • Still other agents include chelators, e.g. , EDTA (e.g. , to remove divalent cations such as Mg 2+ ), salts, RNAse inhibitors (e.g. , a broad specificity RNAse inhibitor such as RNAsin) and so forth.
  • the single stranded oligonucleotide preparation includes another single stranded oligonucleotide, e.g. , a second single stranded oligonucleotide that modulates expression of a second gene or a second single stranded oligonucleotide that modulates expression of the first gene. Still other preparation can include at least 3, 5, ten, twenty, fifty, or a hundred or more different single stranded oligonucleotide species. Such single stranded oligonucleotides can mediated gene expression with respect to a similar number of different genes.
  • the single stranded oligonucleotide preparation includes at least a second therapeutic agent (e.g. , an agent other than an oligonucleotide).
  • a composition that includes a single stranded oligonucleotide can be delivered to a subject by a variety of routes.
  • routes include: intravenous, intradermal, topical, rectal, parenteral, anal, intravaginal, intranasal, pulmonary, ocular.
  • therapeutically effective amount is the amount of oligonucleotide present in the composition that is needed to provide the desired level of MECP2 expression in the subject to be treated to give the anticipated physiological response.
  • physiologically effective amount is that amount delivered to a subject to give the desired palliative or curative effect.
  • pharmaceutically acceptable carrier means that the carrier can be administered to a subject with no significant adverse toxicological effects to the subject.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically include one or more species of single stranded oligonucleotide and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or
  • the route and site of administration may be chosen to enhance targeting.
  • intramuscular injection into the muscles of interest would be a logical choice.
  • Lung cells might be targeted by administering the single stranded oligonucleotide in aerosol form.
  • the vascular endothelial cells could be targeted by coating a balloon catheter with the single stranded oligonucleotide and mechanically introducing the oligonucleotide.
  • Topical administration refers to the delivery to a subject by contacting the formulation directly to a surface of the subject.
  • the most common form of topical delivery is to the skin, but a composition disclosed herein can also be directly applied to other surfaces of the body, e.g. , to the eye, a mucous membrane, to surfaces of a body cavity or to an internal surface.
  • Topical administration can be used as a means to penetrate the epidermis and dermis and ultimately achieve systemic delivery of the composition. Topical administration can also be used as a means to selectively deliver oligonucleotides to the epidermis or dermis of a subject, or to specific strata thereof, or to an underlying tissue.
  • Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Coated condoms, gloves and the like may also be useful.
  • Transdermal delivery is a valuable route for the administration of lipid soluble therapeutics.
  • the dermis is more permeable than the epidermis and therefore absorption is much more rapid through abraded, burned or denuded skin.
  • Inflammation and other physiologic conditions that increase blood flow to the skin also enhance transdermal adsorption. Absorption via this route may be enhanced by the use of an oily vehicle
  • transdermal route provides a potentially effective means to deliver a composition disclosed herein for systemic and/or local therapy.
  • iontophoresis transfer of ionic solutes through biological membranes under the influence of an electric field
  • phonophoresis or sonophoresis use of ultrasound to enhance the absorption of various therapeutic agents across biological membranes, notably the skin and the cornea
  • optimization of vehicle characteristics relative to dose position and retention at the site of administration may be useful methods for enhancing the transport of topically applied compositions across skin and mucosal sites.
  • oligonucleotides administered through these membranes may have a rapid onset of action, provide therapeutic plasma levels, avoid first pass effect of hepatic metabolism, and avoid exposure of the oligonucleotides to the hostile gastrointestinal (GI) environment. Additional advantages include easy access to the membrane sites so that the oligonucleotide can be applied, localized and removed easily.
  • GI gastrointestinal
  • compositions can be targeted to a surface of the oral cavity, e.g. , to sublingual mucosa which includes the membrane of ventral surface of the tongue and the floor of the mouth or the buccal mucosa which constitutes the lining of the cheek.
  • the sublingual mucosa is relatively permeable thus giving rapid absorption and acceptable bioavailability of many agents. Further, the sublingual mucosa is convenient, acceptable and easily accessible.
  • a pharmaceutical composition of single stranded oligonucleotide may also be administered to the buccal cavity of a human being by spraying into the cavity, without inhalation, from a metered dose spray dispenser, a mixed micellar pharmaceutical
  • the dispenser is first shaken prior to spraying the pharmaceutical formulation and propellant into the buccal cavity.
  • compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, slurries, emulsions, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches.
  • carriers that can be used include lactose, sodium citrate and salts of phosphoric acid.
  • Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets.
  • useful diluents are lactose and high molecular weight polyethylene glycols.
  • the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added.
  • Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, intrathecal or intraventricular administration.
  • parental administration involves administration directly to the site of disease (e.g. injection into a tumor).
  • Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
  • Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir.
  • the total concentration of solutes should be controlled to render the preparation isotonic.
  • any of the single stranded oligonucleotides described herein can be administered to ocular tissue.
  • the compositions can be applied to the surface of the eye or nearby tissue, e.g. , the inside of the eyelid.
  • ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers.
  • Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives such as sorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers.
  • the single stranded oligonucleotide can also be administered to the interior of the eye, and can be introduced by a needle or other delivery device which can introduce it to a selected area or structure.
  • Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the composition, preferably single stranded oligonucleotides, within the dispersion can reach the lung where it can be readily absorbed through the alveolar region directly into blood circulation. Pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs.
  • Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellular and dry powder-based formulations. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose devices are preferred. One of the benefits of using an atomizer or inhaler is that the potential for contamination is minimized because the devices are self-contained. Dry powder dispersion devices, for example, deliver agents that may be readily formulated as dry powders. A single stranded oligonucleotide composition may be stably stored as lyophilized or spray-dried powders by itself or in combination with suitable powder carriers.
  • the delivery of a composition for inhalation can be mediated by a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.
  • a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.
  • the term “powder” means a composition that consists of finely dispersed solid particles that are free flowing and capable of being readily dispersed in an inhalation device and subsequently inhaled by a subject so that the particles reach the lungs to permit penetration into the alveoli.
  • the powder is said to be "respirable.”
  • the average particle size is less than about 10 ⁇ in diameter preferably with a relatively uniform spheroidal shape distribution. More preferably the diameter is less than about 7.5 ⁇ m and most preferably less than about 5.0 ⁇ m.
  • the particle size distribution is between about 0.1 ⁇ m and about 5 ⁇ m in diameter, particularly about 0.3 ⁇ m to about 5 ⁇ m.
  • dry means that the composition has a moisture content below about 10% by weight (% w) water, usually below about 5% w and preferably less it than about 3% w.
  • a dry composition can be such that the particles are readily dispersible in an inhalation device to form an aerosol.
  • the types of pharmaceutical excipients that are useful as carrier include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two.
  • HSA human serum albumin
  • bulking agents such as carbohydrates, amino acids and polypeptides
  • pH adjusters or buffers such as sodium chloride
  • salts such as sodium chloride
  • Suitable pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred.
  • Pulmonary administration of a micellar single stranded oligonucleotide formulation may be achieved through metered dose spray devices with propellants such as tetrafluoroethane, heptafluoroethane, dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFC and CFC propellants.
  • Exemplary devices include devices which are introduced into the vasculature, e.g. , devices inserted into the lumen of a vascular tissue, or which devices themselves form a part of the vasculature, including stents, catheters, heart valves, and other vascular devices. These devices, e.g. , catheters or stents, can be placed in the vasculature of the lung, heart, or leg.
  • Other devices include non-vascular devices, e.g. , devices implanted in the
  • the device can release a therapeutic substance in addition to a single stranded oligonucleotide, e.g. , a device can release insulin.
  • unit doses or measured doses of a composition that includes single stranded oligonucleotide are dispensed by an implanted device.
  • the device can include a sensor that monitors a parameter within a subject.
  • the device can include pump, e.g. , and, optionally, associated electronics.
  • Tissue e.g. , cells or organs can be treated with a single stranded oligonucleotide, ex vivo and then administered or implanted in a subject.
  • the tissue can be autologous, allogeneic, or xenogeneic tissue.
  • tissue can be treated to reduce graft v. host disease .
  • the tissue is allogeneic and the tissue is treated to treat a disorder characterized by unwanted gene expression in that tissue.
  • tissue e.g. , hematopoietic cells, e.g. , bone marrow hematopoietic cells, can be treated to inhibit unwanted cell proliferation.
  • the single stranded oligonucleotide treated cells are insulated from other cells, e.g. , by a semi-permeable porous barrier that prevents the cells from leaving the implant, but enables molecules from the body to reach the cells and molecules produced by the cells to enter the body.
  • the porous barrier is formed from alginate.
  • a contraceptive device is coated with or contains a single stranded oligonucleotide.
  • exemplary devices include condoms, diaphragms, IUD
  • the invention features a method of administering a single stranded oligonucleotide (e.g., as a compound or as a component of a composition) to a subject (e.g. , a human subject).
  • a subject e.g. , a human subject.
  • the unit dose is between about 10 mg and 25 mg per kg of bodyweight. In one embodiment, the unit dose is between about 1 mg and 100 mg per kg of bodyweight. In one embodiment, the unit dose is between about 0.1 mg and 500 mg per kg of bodyweight. In some embodiments, the unit dose is more than 0.001, 0.005, 0.01, 0.05,
  • the defined amount can be an amount effective to treat or prevent a disease or disorder, e.g. , a disease or disorder associated with the MECP2.
  • the unit dose for example, can be administered by injection (e.g. , intravenous or intramuscular), an inhaled dose, or a topical application.
  • the unit dose is administered daily. In some embodiments, less frequently than once a day, e.g. , less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g. , not a regular frequency). For example, the unit dose may be administered a single time. In some embodiments, the unit dose is administered more than once a day, e.g. , once an hour, two hours, four hours, eight hours, twelve hours, etc.
  • a subject is administered an initial dose and one or more maintenance doses of a single stranded oligonucleotide.
  • the maintenance dose or doses are generally lower than the initial dose, e.g. , one-half less of the initial dose.
  • a maintenance regimen can include treating the subject with a dose or doses ranging from 0.0001 to 100 mg/kg of body weight per day, e.g. , 100, 10, 1, 0.1, 0.01, 0.001, or 0.0001 mg per kg of bodyweight per day.
  • the maintenance doses may be administered no more than once every
  • the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient.
  • the dosage may be delivered no more than once per day, e.g. , no more than once per 24, 36, 48, or more hours, e.g. , no more than once for every 5 or 8 days.
  • the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state.
  • the dosage of the oligonucleotide may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.
  • the effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g. , a pump, semipermanent stent (e.g. , intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.
  • a delivery device e.g. , a pump, semipermanent stent (e.g. , intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.
  • the oligonucleotide pharmaceutical composition includes a plurality of single stranded oligonucleotide species.
  • the single stranded oligonucleotide species has sequences that are non- overlapping and non-adjacent to another species with respect to a naturally occurring target sequence (e.g. , a PRC2-associated region).
  • the plurality of single stranded oligonucleotide species is specific for different PRC2-associated regions.
  • the single stranded oligonucleotide is allele specific. In some cases, a patient is treated with a single stranded oligonucleotide in conjunction with other therapeutic modalities.
  • the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the compound of the invention is administered in maintenance doses, ranging from 0.0001 mg to 100 mg per kg of body weight.
  • the concentration of the single stranded oligonucleotide composition is an amount sufficient to be effective in treating or preventing a disorder or to regulate a physiological condition in humans.
  • concentration or amount of single stranded oligonucleotide administered will depend on the parameters determined for the agent and the method of administration, e.g. nasal, buccal, pulmonary.
  • nasal formulations may tend to require much lower concentrations of some ingredients in order to avoid irritation or burning of the nasal passages. It is sometimes desirable to dilute an oral formulation up to 10- 100 times in order to provide a suitable nasal formulation.
  • treatment of a subject with a therapeutically effective amount of a single stranded oligonucleotide can include a single treatment or, preferably, can include a series of treatments.
  • the effective dosage of a single stranded oligonucleotide used for treatment may increase or decrease over the course of a particular treatment.
  • the subject can be monitored after administering a single stranded oligonucleotide composition. Based on information from the monitoring, an additional amount of the single stranded
  • oligonucleotide composition can be administered.
  • Dosing is dependent on severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved.
  • Optimal dosing schedules can be calculated from measurements of MECP2 expression levels in the body of the patient.
  • Optimum dosages may vary depending on the relative potency of individual compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models.
  • the animal models include transgenic animals that express a human MECP2.
  • the composition for testing includes a single stranded oligonucleotide that is complementary, at least in an internal region, to a sequence that is conserved between MECP2 in the animal model and the MECP2 in a human.
  • the administration of the single stranded oligonucleotide composition is parenteral, e.g. intravenous (e.g. , as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular.
  • Administration can be provided by the subject or by another person, e.g. , a health care provider.
  • the composition can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.
  • kits comprising a container housing a composition comprising a single stranded oligonucleotide.
  • the composition is a pharmaceutical composition comprising a single stranded oligonucleotide and a pharmaceutically acceptable carrier.
  • the individual components of the pharmaceutical composition may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical composition separately in two or more containers, e.g. , one container for single stranded oligonucleotides, and at least another for a carrier compound.
  • the kit may be packaged in a number of different configurations such as one or more containers in a single box.
  • the different components can be combined, e.g. , according to instructions provided with the kit.
  • the components can be combined according to a method described herein, e.g. , to prepare and administer a pharmaceutical composition.
  • the kit can also include a delivery device.
  • RNA is harvested from the cells using Promega SV 96 Total RNA Isolation system or Trizol omitting the DNAse step. RNA harvested from cells is normalized so that equal RNA is input to each reverse transcription reaction. Reverse transcriptase reactions are performed using the Superscript II kit and real time PCR performed on cDNA samples using icycler SYBR green chemistry (Biorad). A baseline level of mRNA expression for MECP2 is determined through quantitative PCR as outlined above. Baseline levels are also determined for mRNA of various housekeeping genes which are constitutively expressed. A "control" housekeeping gene with approximately the same level of baseline expression as the target gene is chosen for comparison purposes.
  • Human hepatocyte Hep3B, human hepatocyte HepG2 cells, mouse hepatoma Hepal-6 cells, and human renal proximal tubule epithelial cells (RPTEC) are cultured using conditions known in the art (see, e.g. Current Protocols in Cell Biology).
  • Oligonucleotides were designed within PRC2-interacting regions in order to upregulate MECP2.
  • the sequence and structure of each oligonucleotide is shown in Table 3.
  • the following table provides a description of the nucleotide analogs, modifications and intranucleotide linkages used for certain oligonucleotides tested and described in Table 2.
  • Oligonucleotides are designed within PRC2-interacting regions in order to upregulate MECP2.
  • Cells are seeded into each well of 24-well plates at a density of 25,000 cells per 500uL and transfections are performed with Lipofectamine and the single stranded oligonucleotides.
  • Control wells contain Lipofectamine alone.
  • approximately 200 uL of cell culture supernatants are stored at -80 C for ELISA.
  • RNA is harvested from the cells and quantitative PCR is carried out as outlined above.
  • the percent induction of target mRNA expression by each oligonucleotide is determined by normalizing mRNA levels in the presence of the oligonucleotide to the mRNA levels in the presence of control (Lipofectamine alone). This is compared side-by- side with the increase in mRNA expression of the "control" housekeeping gene.
  • Oligonucleotides are designed as candidates for upregulating MECP2 expression.
  • the single stranded oligonucleotides are designed to be complementary to a PRC2- interacting region within a sequence as set forth in SEQ ID NO: 1 or 2.
  • the oligonucleotides are tested in at least duplicate. Briefly, cells are transfected in vitro with each of the oligonucleotides as described above. MECP2 expression in cells following treatment is evaluated by qRT-PCR. Oligonucleotides that upregulate MECP2 expression are identified.
  • GGGGGU GGGGUA, GGGUAC, GGGUAU, GGGUCA, GGGUCC, GGGUCG, GGGUGA, GGGUGC, GGGU UA, GGGU UG, GGUAAA, GGUAAC, GGUAAG, GGUAAU, GGUACA, GGUACC, GGUACG,
  • GGUACU GGUAGC, GGUAGG, GGUAGU, GGUAUA, GGUAUC, GGUAUG, GGUCAA, GGUCAC, GGUCAG, GGUCAU, GGUCCA, GGUCCG, GGUCCU, GGUCGA, GGUCGC, GG UCGG, GGUCGU, GGUCUC, GGUCU U, GGUGAA, GGUGAC, GGUGAU, GGUGCA, GGUGCC, GGUGGC, GGUGUA, GGUGUC, GGU UAA, GGU UAG, GGU UAU, GGUUCA, GGU UCC, GGU UCG, GGU UGC, GGU UUC, GGUU UU, GUAAAA, GUAAAG, GUAAAU, GUAACC, GUAACG, GUAACU, GUAAGA, GUAAGC, GUAAGG, GUAAGU, GUAAUA, GUAAUC, GUAAUG, GUAAUU
  • GUAGGU GUAGUA, GUAGUC, GUAUAA, GUAUAC, GUAUAG, GUAUAU, GUAUCA, GUAUCG, GUAUCU, GUAUGA, GUAUGC, GUAUGG, GUAUUA, GUAU UG, G UAU UU, GUCAAA, GUCAAG,
  • GUCAAU GUCACA, GUCACC, GUCACG, GUCAGA, GUCAGC, GUCAGG, GUCAUA, GUCAUC, GUCAUG, GUCCAA, GUCCAC, GUCCAU, GUCCCC, GUCCCU, GUCCGG, GUCCGU, GUCCUA, GUCCUG, GUCCU U, GUCGAA, GUCGAC, GUCGAG, GUCGAU, GUCGCA, GUCGCC, GUCGCG, GUCGCU, GUCGGA, GUCGGC, GUCGGG, GUCGGU, GUCGUA, GUCGUC, GUCGU U, GUCUAA, GUCUAG, GUCUCA, GUCUCC, GUCUCG, GUCUGA, GUCUGG, GUCUGU, GUCUCG, GUCUCG, GUCUGA, GUCUGG, GUCUGU, GUCUUC, GUCU UU, GUGAAA,
  • GUGCAU GUGCCC
  • GUGCCG GUGCGA
  • GUGCGG GUGCGU
  • GUGCUA GUGCUC
  • GUGCUG GUGCAU
  • GUGGAG GUGGCG, GUGGCU, GUGGGU, GUGGUC, GUGGUG, GUGUAA, GUGUAG, GUGUCG, GUGUGA, GUGUGC, GUGUGU, GUGUUG, GUGU UU, GU UAAA, GUUAAC, GUUAAG, GU UACA, GU UACC, GUUACG, GU UACU, GU UAGA, GUUAGC, GUUAGU, GUUAUA, GUUAUC, GUUAUG,
  • GU UAUU GUUCAA, GUUCAC, GUUCAG, GU UCCA, GUUCCG, GUUCGA, GU UCGC, GU UCGG, GU UCGU, GUUCUA, GUUCUG, GUUGAA, GU UGAC, GUUGAG, GUUGAU, GUUGCG, GUUGCU, GUUGGA, GU UGGC, GU UGGU, GU UGUC, GUUGUG, GUUGU U, GUU UAA, GU UUAC, GU UUAG, GUU UAU, GU UUCA, GUUUCC, GU UUCU, GU UUGA, GUU UGC, GUU UGG, GUU UGU, GU UUUA, GU UUUA, GU UU UC, GU UU UU, UAAAAA, UAAAAC, UAAAAG, UAAAAU, UAAACA, UAAACC, UAAACG,
  • UGUAUU UGUCAA, UGUCAC, UGUCAG, UGUCAU, UGUCCA, UGUCCC, UG UCCG, UGUCGA, UGUCGC, UGUCGG, UGUCGU, UGUCUA, UGUCUC, UGUGAC, UGUGAG, UGUGAU, UGUGCA, UGUGGU, UGUGUA, UGUGUU, UGUUAC, UGUUAG, UGUUAU, UGUUCA, UGUUCC, UGUUCG, UGUUGG, UGUUGU, UGUUUA, UGUUUC, UGUUUG, UGUUUU, UUAAAA, UUAAAC, UUAAAG, UUAAAU, UUAACC, UUAACG, UUAACU, UUAAGU, UUAAUA, UUAAUC, UUAAUG, UUAAUU, UUACAA, UUACAC, UUACAG, UUACAU, UUACCA, UUACCC
  • Table 3 Formatted oligonucleotide sequences made for testing showing nucleotide modifications.
  • SeqID range 47462- 83660

Landscapes

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

Abstract

Aspects of the invention provide single stranded oligonucleotides for activating or enhancing expression of MECP2. Further aspects provide compositions and kits comprising single stranded oligonucleotides for activating or enhancing expression of MECP2. Methods for modulating expression of MECP2 using the single stranded oligonucleotides are also provided. Further aspects of the invention provide methods for selecting a candidate oligonucleotide for activating or enhancing expression of MECP2.

Description

COMPOSITIONS AND METHODS FOR MODULATING MECP2 EXPRESSION
CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional
Application No. 61/648,051, entitled "COMPOSITIONS AND METHODS FOR
MODULATING MECP2 EXPRESSION", filed May 16, 2012, which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION
The invention relates to oligonucleotide based compositions, as well as methods of using oligonucleotide based compositions for treating disease.
BACKGROUND OF THE INVENTION
Rett syndrome is a developmental disorder of the brain occurring mostly in females characterized by normal early development, followed by a slowing of development resulting in loss of control of the hands, loss of speech, breathing problems, slowed brain and head growth, ambulatory problems, seizures, and mental retardation. Rett syndrome affects approximately 1 in 10,000 live female births. Most cases of Rett syndrome are caused by a mutation in the methyl CpG binding protein 2, or MECP2 gene, on the X chromosome that causes reduced activity or inactivation of MECP2.
MECP2 is a transcriptional repressor that binds to methylated DNA and is present in large quantities in mature nerve cells. MECP2 represses transcription from methylated gene promoters through interaction with histone deacetylase and the corepressor SIN3A. Many of the genes that are known to be regulated by the MECP2 protein play a role in normal brain function, particularly the maintenance of synapses. Mouse studies have demonstrated MECP2 mutations cause defects in synaptic function, especially in synaptic plasticity.
SUMMARY OF THE INVENTION
Aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating MECP2 in cells. In some embodiments, single stranded
oligonucleotides are provided that target a PRC2- associated region of a MECP2 gene (e.g., human MECP2) and thereby cause upregulation of the gene. In some embodiments, single stranded oligonucleotides are provided that target a PRC2-associated region of the gene encoding MECP2. In some embodiments, these single stranded oligonucleotides activate or enhance expression of MECP2 by relieving or preventing PRC2 mediated repression of MECP2.
Upregulation of MECP2 offers a treatment for diseases associated with decreased
MECP2 activity. Aspects of the invention disclosed herein provide methods and
compositions that are useful for upregulating MECP2 for the treatment and/or prevention of Rett Syndrome. MECP2 is also associated with MECP2-related severe neonatal
encephalopathy, Angelman syndrome, and PPM-X syndrome. Accordingly, certain aspects of the invention disclosed herein provide methods and compositions that are useful for upregulating MECP2 for the treatment and/or prevention of MECP2-related severe neonatal encephalopathy, Angelman syndrome, and PPM-X syndrome.
Further aspects of the invention provide methods for selecting oligonucleotides for activating or enhancing expression of MECP2. In some embodiments, methods are provided for selecting a set of oligonucleotides that is enriched in candidates (e.g. , compared with a random selection of oligonucleotides) for activating or enhancing expression of MECP2. Accordingly, the methods may be used to establish sets of clinical candidates that are enriched in oligonucleotides that activate or enhance expression of MECP2. Such libraries may be utilized, for example, to identify lead oligonucleotides for developing therapeutics to treat MECP2. Furthermore, in some embodiments, oligonucleotide chemistries are provided that are useful for controlling the pharmacokinetics, biodistribution, bioavailability and/or efficacy of the single stranded oligonucleotides for activating expression of MECP2.
According to some aspects of the invention single stranded oligonucleotides are provided that have a region of complementarity that is complementarty with (e.g. , at least 8 consecutive nucleotides of ) a PRC2-associated region of a MECP2 gene, e.g., a PRC2- associated region of the nucleotide sequence set forth as SEQ ID NO: 1 or 2. In some embodiments, the oligonucleotide has at least one of the following features: a) a sequence that is 5'X-Y-Z, in which X is any nucleotide and in which X is at the 5' end of the oligonucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a human seed sequence of a microRNA, and Z is a nucleotide sequence of 1 to 23 nucleotides in length; b) a sequence that does not comprise three or more consecutive guanosine nucleotides; c) a sequence that has less than a threshold level of sequence identity with every sequence of nucleotides, of equivalent length to the second nucleotide sequence, that are between 50 kilobases upstream of a 5 '-end of an off-target gene and 50 kilobases downstream of a 3 '-end of the off-target gene; d) a sequence that is complementary to a PRC2-associated region that encodes an RNA that forms a secondary structure comprising at least two single stranded loops; and e) a sequence that has greater than 60% G-C content. In some
embodiments, the single stranded oligonucleotide has at least two of features a), b), c), d), and e), each independently selected. In some embodiments, the single stranded
oligonucleotide has at least three of features a), b), c), d), and e), each independently selected. In some embodiments, the single stranded oligonucleotide has at least four of features a), b), c), d), and e), each independently selected. In some embodiments, the single stranded oligonucleotide has each of features a), b), c), d), and e). In certain embodiments, the oligonucleotide has the sequence 5'X-Y-Z, in which the oligonucleotide is 8-50 nucleotides in length.
According to some aspects of the invention, single stranded oligonucleotides are provided that have a sequence X-Y-Z, in which X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1 to 23 nucleotides in length, in which the single stranded oligonucleotide is complementary with a PRC2- associated region of a MECP2 gene, e.g., a PRC2-associated region of the nucleotide sequence set forth as SEQ ID NO: 1 or 2. In some aspects of the invention, single stranded oligonucleotides are provided that have a sequence 5' -X-Y-Z, in which X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1 to 23 nucleotides in length, in which the single stranded oligonucleotide is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of a MECP2 gene, e.g., a PRC2- associated region of the nucleotide sequence set forth as SEQ ID NO: 1 or 2. In some embodiments, Y is a sequence selected from Table 1. In some embodiments, the PRC2- associated region is a sequence listed in any one of SEQ ID NOS: 5 to 118.
In some embodiments, the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 119 to 83660, or a fragment thereof that is at least 8 nucleotides. In some embodiments, the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 119 to 83660, in which the 5' end of the nucleotide sequence provided is the 5' end of the oligonucleotide. In some embodiments, the region of complementarity (e.g. , the at least 8 consecutive nucleotides) is also present within the nucleotide sequence set forth as SEQ ID NO: 3 or 4.
In some embodiments, the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 119 to 83660. In some embodiments, the single stranded oligonucleotide comprises a fragment of at least 8 nucleotides of a nucleotide sequence as set forth in any one of SEQ ID NOS: 119 to 83660.
In some embodiments, the PRC2-associated region is a sequence listed in any one of SEQ ID NOS: 5 to 84. In some embodiments, the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 119 to 47653 or a fragment thereof that is at least 8 nucleotides. In some embodiments, the single stranded
oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 119 to 47653, wherein the 5' end of the nucleotide sequence provided in any one of SEQ ID NOS: 119 to 47653 is the 5' end of the oligonucleotide. In some embodiments, the at least 8 consecutive nucleotides are also present within the nucleotide sequence set forth as SEQ ID NO: 3.
In some embodiments, the PRC2-associated region is a sequence listed in any one of SEQ ID NOS: 85 to 118. In some embodiments, the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 47462 to 83660 or a fragment thereof that is at least 8 nucleotides. In some embodiments, the single stranded oligonucleotide comprises a nucleotide sequence as set forth in any one of SEQ ID NOS: 47462 to 83660, wherein the 5' end of the nucleotide sequence provided in any one of SEQ ID NOS: 47462 to 83660 is the 5' end of the oligonucleotide. In some embodiments, the at least 8 consecutive nucleotides are present within the nucleotide sequence set forth as SEQ ID NO: 4.
In some embodiments, a single stranded oligonucleotide comprises a nucleotide sequence as set forth in Table 3. In some embodiments, the single stranded oligonucleotide comprises a fragment of at least 8 nucleotides of a nucleotide sequence as set forth in Table 3. In some embodiments, a single stranded oligonucleotide consists of a nucleotide sequence as set forth in Table 3.
In some embodiments, the single stranded oligonucleotide does not comprise three or more consecutive guanosine nucleotides. In some embodiments, the single stranded oligonucleotide does not comprise four or more consecutive guanosine nucleotides.
In some embodiments, the single stranded oligonucleotide is 8 to 30 nucleotides in length. In some embodiments, the single stranded oligonucleotide is up to 50 nucleotides in length. In some embodiments, the single stranded oligonucleotide is 8 to 10 nucleotides in length and all but 1, 2, or 3 of the nucleotides of the complementary sequence of the PRC2- associated region are cytosine or guanosine nucleotides.
In some embodiments, the single stranded oligonucleotide is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of a MECP2 gene, e.g., a PRC2- associated region of a nucleotide sequence set forth as SEQ ID NO: 1 or 2, in which the nucleotide sequence of the single stranded oligonucleotide comprises one or more of a nucleotide sequence selected from the group consisting of
(a) (X)Xxxxxx, (X)xXxxxx, (X)xxXxxx, (X)xxxXxx, (X)xxxxXx and (X)xxxxxX,
(b) (X)XXxxxx, (X)XxXxxx, (X)XxxXxx, (X)XxxxXx, (X)XxxxxX, (X)xXXxxx, (X)xXxXxx, (X)xXxxXx, (X)xXxxxX, (X)xxXXxx, (X)xxXxXx, (X)xxXxxX, (X)xxxXXx, (X)xxxXxX and (X)xxxxXX,
(c) (X)XXXxxx, (X)xXXXxx, (X)xxXXXx, (X)xxxXXX, (X)XXxXxx, (X)XXxxXx,
(X)XXxxxX, (X)xXXxXx, (X)xXXxxX, (X)xxXXxX, (X)XxXXxx, (X)XxxXXx
(X)XxxxXX, (X)xXxXXx, (X)xXxxXX, (X)xxXxXX, (X)xXxXxX and (X)XxXxXx,
(d) (X)xxXXX, (X)xXxXXX, (X)xXXxXX, (X)xXXXxX, (X)xXXXXx,
(X)XxxXXXX, (X)XxXxXX, (X)XxXXxX, (X)XxXXx, (X)XXxxXX, (X)XXxXxX, (X)XXxXXx, (X)XXXxxX, (X)XXXxXx, and (X)XXXXxx,
(e) (X)xXXXXX, (X)XxXXXX, (X)XXxXXX, (X)XXXxXX, (X)XXXXxX and (X)XXXXXx, and
(f) XXXXXX, XxXXXXX, XXxXXXX, XXXxXXX, XXXXxXX, XXXXXxX and XXXXXXx, wherein "X" denotes a nucleotide analogue, (X) denotes an optional nucleotide analogue, and "x" denotes a DNA or RNA nucleotide unit. In some embodiments, at least one nucleotide of the oligonucleotide is a nucleotide analogue. In some embodiments, the at least one nucleotide analogue results in an increase in Tm of the oligonucleotide in a range of 1 to 5 °C compared with an oligonucleotide that does not have the at least one nucleotide analogue.
In some embodiments, at least one nucleotide of the oligonucleotide comprises a 2'
O-methyl. In some embodiments, each nucleotide of the oligonucleotide comprises a 2' O- methyl. In some embodiments, the oligonucleotide comprises at least one ribonucleotide, at least one deoxyribonucleotide, or at least one bridged nucleotide. In some embodiments, the bridged nucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide. In some embodiments, each nucleotide of the oligonucleotide is a LNA nucleotide.
In some embodiments, the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2'-fluoro-deoxyribonucleotides. In some embodiments, the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2'-0- methyl nucleotides. In some embodiments, the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and ENA nucleotide analogues. In some embodiments, the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and LNA nucleotides. In some embodiments, the 5' nucleotide of the oligonucleotide is a
deoxyribonucleotide. In some embodiments, the nucleotides of the oligonucleotide comprise alternating LNA nucleotides and 2' -O-methyl nucleotides. In some embodiments, the 5' nucleotide of the oligonucleotide is a LNA nucleotide. In some embodiments, the nucleotides of the oligonucleotide comprise deoxyribonucleotides flanked by at least one LNA nucleotide on each of the 5' and 3' ends of the deoxyribonucleotides.
In some embodiments, the single stranded oligonucleotide comprises modified internucleotide linkages (e.g. , phosphorothioate internucleotide linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides. In some embodiments, the single stranded oligonucleotide comprises modified internucleotide linkages (e.g. , phosphorothioate internucleotide linkages or other linkages) between between all nucleotides.
In some embodiments, the nucleotide at the 3' position of the oligonucleotide has a 3' hydroxyl group. In some embodiments, the nucleotide at the 3' position of the
oligonucleotide has a 3' thiophosphate. In some embodiments, the single stranded oligonucleotide has a biotin moiety or other moiety conjugated to its 5' or 3' nucleotide. In some embodiments, the single stranded oligonucleotide has cholesterol, Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR or dynamic polyconjugates and variants thereof at its 5' or 3' end.
According to some aspects of the invention compositions are provided that comprise any of the oligonucleotides disclosed herein, and a carrier. In some embodiments, compositions are provided that comprise any of the oligonucleotides in a buffered solution. In some embodiments, the oligonucleotide is conjugated to the carrier. In some embodiments, the carrier is a peptide. In some embodiments, the carrier is a steroid. According to some aspects of the invention pharmaceutical compositions are provided that comprise any of the oligonucleotides disclosed herein, and a pharmaceutically acceptable carrier.
According to other aspects of the invention, kits are provided that comprise a container housing any of the compositions disclosed herein.
According to some aspects of the invention, methods of increasing expression of MECP2 in a cell are provided. In some embodiments, the methods involve delivering any one or more of the single stranded oligonucleotides disclosed herein into the cell. In some embodiments, delivery of the single stranded oligonucleotide into the cell results in a level of expression of MECP2 that is greater (e.g. , at least 50% greater) than a level of expression of MECP2 in a control cell that does not comprise the single stranded oligonucleotide.
According to some aspects of the invention, methods of increasing levels of MECP2 in a subject are provided. According to some aspects of the invention, methods of treating a condition (e.g. , Rett Syndrome, MECP2-related severe neonatal encephalopathy, Angelman syndrome, PPM-X syndrome) associated with decreased levels of MECP2 in a subject are provided. In some embodiments, the methods involve administering any one or more of the single stranded oligonucleotides disclosed herein to the subject.
BRIEF DESCRIPTION OF TABLES
Table 1: Hexamers that are not seed sequences of human miRNAs
Table 2: A listing of oligonucleotide modifications
Table 3: Formatted oligonucleotide sequences made for testing showing nucleotide modifications. The table shows the sequence of the modified nucleotides, where InaX represents an LNA nucleotide with 3' phosphorothioate linkage, omeX is a 2'-0-methyl nucleotide, dX is a deoxy nucleotide. An s at the end of a nucleotide code indicates that the nucleotide had a 3' phosphorothioate linkage. The "-Sup" at the end of the sequence marks the fact that the 3' end lacks either a phosphate or thiophosphate on the 3' linkage. The Formatted Sequence column shows the sequence of the oligonucleotide, including modified nucleotides.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION
Aspects of the invention provided herein relate to the discovery of polycomb repressive complex 2 (PRC2)-interacting RNAs. Polycomb repressive complex 2 (PRC2) is a histone methyltransferase and a known epigenetic regulator involved in silencing of genomic regions through methylation of histone H3. Among other functions, PRC2 interacts with long noncoding RNAs (IncRNAs), such as Rep A, Xist, and Tsix, to catalyze
trimethylation of histone H3-lysine27. PRC2 contains four subunits, Eed, Suzl2, RbAp48, and Ezh2. Aspects of the invention relate to the recognition that single stranded
oligonucleotides that bind to PRC2-associated regions of RNAs (e.g. , IncRNAs) that are expressed from within a genomic region that encompasses or that is in functional proximity to the MECP2 gene can induce or enhance expression of MECP2. In some embodiments, this upregulation is believed to result from inhibition of PRC2 mediated repression of MECP2.
As used herein, the term "PRC2-associated region" refers to a region of a nucleic acid that comprises or encodes a sequence of nucleotides that interact directly or indirectly with a component of PRC2. A PRC2-associated region may be present in a RNA (e.g. , a long non- coding RNA (IncRNA)) that that interacts with a PRC2. A PRC2-associated region may be present in a DNA that encodes an RNA that interacts with PRC2. In some cases, the PRC2- associated region is equivalently referred to as a PRC2-interacting region.
In some embodiments, a PRC2-associated region is a region of an RNA that crosslinks to a component of PRC2 in response to in situ ultraviolet irradiation of a cell that expresses the RNA, or a region of genomic DNA that encodes that RNA region. In some embodiments, a PRC2-associated region is a region of an RNA that immunoprecipitates with an antibody that targets a component of PRC2, or a region of genomic DNA that encodes that RNA region. In some embodiments, a PRC2-associated region is a region of an RNA that immunoprecipitates with an antibody that binds specifically to SUZ12, EED, EZH2 or RBBP4 (which as noted above are components of PRC2), or a region of genomic DNA that encodes that RNA region.
In some embodiments, a PRC2-associated region is a region of an RNA that is protected from nucleases (e.g. , RNases) in an RNA-immunoprecipitation assay that employs an antibody that targets a component of PRC2, or a region of genomic DNA that encodes that protected RNA region. In some embodiments, a PRC2-associated region is a region of an RNA that is protected from nucleases (e.g. , RNases) in an RNA-immunoprecipitation assay that employs an antibody that targets SUZ12, EED, EZH2 or RBBP4, or a region of genomic DNA that encodes that protected RNA region.
In some embodiments, a PRC2-associated region is a region of an RNA within which occur a relatively high frequency of sequence reads in a sequencing reaction of products of an RNA-immunoprecipitation assay that employs an antibody that targets a component of PRC2, or a region of genomic DNA that encodes that RNA region. In some embodiments, a PRC2- associated region is a region of an RNA within which occur a relatively high frequency of sequence reads in a sequencing reaction of products of an RNA-immunoprecipitation assay that employs an antibody that binds specifically to SUZ12, EED, EZH2 or RBBP4, or a region of genomic DNA that encodes that protected RNA region. In such embodiments, the PRC2-associated region may be referred to as a "peak."
In some embodiments, a PRC2-associated region comprises a sequence of 40 to 60 nucleotides that interact with PRC2 complex. In some embodiments, a PRC2-associated region comprises a sequence of 40 to 60 nucleotides that encode an RNA that interacts with PRC2. In some embodiments, a PRC2-associated region comprises a sequence of up to 5kb in length that comprises a sequence (e.g. , of 40 to 60 nucleotides) that interacts with
PRC2. In some embodiments, a PRC2-associated region comprises a sequence of up to 5kb in length within which an RNA is encoded that has a sequence (e.g. , of 40 to 60 nucleotides) that is known to interact with PRC2. In some embodiments, a PRC2-associated region comprises a sequence of about 4kb in length that comprise a sequence (e.g. , of 40 to 60 nucleotides) that interacts with PRC2. In some embodiments, a PRC2-associated region comprises a sequence of about 4kb in length within which an RNA is encoded that includes a sequence (e.g., of 40 to 60 nucleotides) that is known to interact with PRC2. In some embodiments, a PRC2-associated region has a sequence as set forth in any one of SEQ ID NOS: 5 to 118.
In some embodiments, single stranded oligonucleotides are provided that specifically bind to, or are complementary to, a PRC2-associated region in a genomic region that encompasses or that is in proximity to the MECP2 gene. In some embodiments, single stranded oligonucleotides are provided that specifically bind to, or are complementary to, a PRC2-associated region that has a sequence as set forth in any one of SEQ ID NOS: 5 to 118. In some embodiments, single stranded oligonucleotides are provided that specifically bind to, or are complementary to, a PRC2-associated region that has a sequence as set forth in any one of SEQ ID NOS: 5 to 118 combined with up to 2kb, up to 5kb, or up to lOkb of flanking sequences from a corresponding genomic region to which these SEQ IDs map (e.g., in a human genome). In some embodiments, single stranded oligonucleotides have a sequence as set forth in any one of SEQ ID NOS: 119 to 83660. In some embodiments, single stranded oligonucleotides have a sequence as set forth in Table 3.
Without being bound by a theory of invention, these oligonucleotides are able to interfere with the binding of and function of PRC2, by preventing recruitment of PRC2 to a specific chromosomal locus. For example, a single administration of single stranded oligonucleotides designed to specifically bind a PRC2-associated region IncRNA can stably displace not only the IncRNA, but also the PRC2 that binds to the IncRNA, from binding chromatin. After displacement, the full complement of PRC2 is not recovered for up to 24 hours. Further, IncRNA can recruit PRC2 in a cis fashion, repressing gene expression at or near the specific chromosomal locus from which the IncRNA was transcribed.
Methods of modulating gene expression are provided, in some embodiments, that may be carried out in vitro, ex vivo, or in vivo. It is understood that any reference to uses of compounds throughout the description contemplates use of the compound in preparation of a pharmaceutical composition or medicament for use in the treatment of condition (e.g., Rett Syndrome, MECP2-related severe neonatal encephalopathy, Angelman syndrome, PPM-X syndrome) associated with decreased levels or activity of MECP2. Thus, as one nonlimiting example, this aspect of the invention includes use of such single stranded oligonucleotides in the preparation of a medicament for use in the treatment of disease, wherein the treatment involves upregulating expression of MECP2.
In further aspects of the invention, methods are provided for selecting a candidate oligonucleotide for activating expression of MECP2. The methods generally involve selecting as a candidate oligonucleotide, a single stranded oligonucleotide comprising a nucleotide sequence that is complementary to a PRC2-associated region (e.g., a nucleotide sequence as set forth in any one of SEQ ID NOS: 5 to 118). In some embodiments, sets of oligonucleotides may be selected that are enriched (e.g., compared with a random selection of oligonucleotides) in oligonucleotides that activate expression of MECP2.
Single Stranded Oligonucleotides for Modulating Expression ofMECP2
In one aspect of the invention, single stranded oligonucleotides complementary to the PRC2-associated regions are provided for modulating expression of MECP2 in a cell. In some embodiments, expression of MECP2 is upregulated or increased. In some
embodiments, single stranded oligonucleotides complementary to these PRC2-associated regions inhibit the interaction of PRC2 with long RNA transcripts such that gene expression is upregulated or increased. In some embodiments, single stranded oligonucleotides complementary to these PRC2-associated regions inhibit the interaction of PRC2 with long RNA transcripts, resulting in reduced methylation of histone H3 and reduced gene inactivation, such that gene expression is upregulated or increased. In some embodiments, this interaction may be disrupted or inhibited due to a change in the structure of the long RNA that prevents or reduces binding to PRC2. The oligonucleotide may be selected using any of the methods disclosed herein for selecting a candidate oligonucleotide for activating expression of MECP2.
The single stranded oligonucleotide may comprise a region of complementarity that is complementary with a PRC2-associated region of a nucleotide sequence set forth in any one of SEQ ID NOS: 1 to 4. The region of complementarity of the single stranded
oligonucleotide may be complementary with at least 6, e.g., at least 7, at least 8, at least 9, at least 10, at least 15 or more consecutive nucleotides of the PRC2-associated region.
The PRC2-associated region may map to a position in a chromosome between 50 kilobases upstream of a 5 '-end of the MECP2 gene and 50 kilobases downstream of a 3 '-end of the MECP2 gene. The PRC2-associated region may map to a position in a chromosome between 25 kilobases upstream of a 5 '-end of the MECP2 gene and 25 kilobases downstream of a 3 '-end of the MECP2 gene. The PRC2-associated region may map to a position in a chromosome between 12 kilobases upstream of a 5'-end of the MECP2 gene and 12 kilobases downstream of a 3 '-end of the MECP2 gene. The PRC2-associated region may map to a position in a chromosome between 5 kilobases upstream of a 5'-end of the MECP2 gene and 5 kilobases downstream of a 3 '-end of the MECP2 gene.
The genomic position of the selected PRC2-associated region relative to the MECP2 gene may vary. For example, the PRC2-associated region may be upstream of the 5' end of the MECP2 gene. The PRC2-associated region may be downstream of the 3' end of the
MECP2 gene. The PRC2-associated region may be within an intron of the MECP2 gene. The PRC2-associated region may be within an exon of the MECP2 gene. The PRC2-associated region may traverse an intron-exon junction, a 5 '-UTR-exon junction or a 3'-UTR-exon junction of the MECP2 gene.
The single stranded oligonucleotide may comprise a sequence having the formula X-
Y-Z, in which X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a human seed sequence of a microRNA, and Z is a nucleotide sequence of varying length. In some embodiments X is the 5' nucleotide of the oligonucleotide. In some embodiments, when X is anchored at the 5' end of the oligonucleotide, the oligonucleotide does not have any nucleotides or nucleotide analogs linked 5' to X. In some embodiments, other compounds such as peptides or sterols may be linked at the 5' end in this embodiment as long as they are not nucleotides or nucleotide analogs. In some embodiments, the single stranded oligonucleotide has a sequence 5'X-Y-Z and is 8-50 nucleotides in length.
Oligonucleotides that have these sequence characteristics are predicted to avoid the miRNA pathway. Therefore, in some embodiments, oligonucleotides having these sequence characteristics are unlikely to have an unintended consequence of functioning in a cell as a miRNA molecule. The Y sequence may be a nucleotide sequence of 6 nucleotides in length set forth in Table 1.
The single stranded oligonucleotide may have a sequence that does not contain guanosine nucleotide stretches (e.g. , 3 or more, 4 or more, 5 or more, 6 or more consecutive guanosine nucleotides). In some embodiments, oligonucleotides having guanosine nucleotide stretches have increased non-specific binding and/or off-target effects, compared with oligonucleotides that do not have guanosine nucleotide stretches.
The single stranded oligonucleotide may have a sequence that has less than a threshold level of sequence identity with every sequence of nucleotides, of equivalent length, that map to a genomic position encompassing or in proximity to an off-target gene. For example, an oligonucleotide may be designed to ensure that it does not have a sequence that maps to genomic positions encompassing or in proximity with all known genes (e.g. , all known protein coding genes) other than MECP2. In a similar embodiment, an
oligonucleotide may be designed to ensure that it does not have a sequence that maps to any other known PRC2-associated region, particularly PRC2-associated regions that are functionally related to any other known gene (e.g. , any other known protein coding gene). In either case, the oligonucleotide is expected to have a reduced likelihood of having off-target effects. The threshold level of sequence identity may be 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity.
The single stranded oligonucleotide may have a sequence that is complementary to a
PRC2-associated region that encodes an RNA that forms a secondary structure comprising at least two single stranded loops. In has been discovered that, in some embodiments, oligonucleotides that are complementary to a PRC2-associated region that encodes an RNA that forms a secondary structure comprising one or more single stranded loops (e.g. , at least two single stranded loops) have a greater likelihood of being active (e.g. , of being capable of activating or enhancing expression of a target gene) than a randomly selected
oligonucleotide. In some cases, the secondary structure may comprise a double stranded stem between the at least two single stranded loops. Accordingly, the region of
complementarity between the oligonucleotide and the PRC2-associated region may be at a location of the PRC2 associated region that encodes at least a portion of at least one of the loops. In some cases, the region of complementarity between the oligonucleotide and the PRC2-associated region may be at a location of the PRC2-associated region that encodes at least a portion of at least two of the loops. In some cases, the region of complementarity between the oligonucleotide and the PRC2-associated region may be at a location of the PRC2 associated region that encodes at least a portion of the double stranded stem. In some embodiments, a PRC2-associated region (e.g. , of an IncRNA) is identified (e.g. , using RIP- Seq methodology or information derived therefrom). In some embodiments, the predicted secondary structure RNA (e.g. , IncRNA) containing the PRC2-associated region is determined using RNA secondary structure prediction algorithms, e.g. , RNAfold, mfold. In some embodiments, oligonucleotides are designed to target a region of the RNA that forms a secondary structure comprising one or more single stranded loop (e.g. , at least two single stranded loops) structures which may comprise a double stranded stem between the at least two single stranded loops.
The single stranded oligonucleotide may have a sequence that is has greater than 30% G-C content, greater than 40% G-C content, greater than 50% G-C content, greater than 60% G-C content, greater than 70% G-C content, or greater than 80% G-C content. The single stranded oligonucleotide may have a sequence that has up to 100% G-C content, up to 95% G-C content, up to 90% G-C content, or up to 80% G-C content. In some embodiments in which the oligonucleotide is 8 to 10 nucleotides in length, all but 1, 2, 3, 4, or 5 of the nucleotides of the complementary sequence of the PRC2-associated region are cytosine or guanosine nucleotides. In some embodiments, the sequence of the PRC2- associated region to which the single stranded oligonucleotide is complementary comprises no more than 3 nucleotides selected from adenine and uracil.
The single stranded oligonucleotide may be complementary to a chromosome of a different species (e.g. , a mouse, rat, rabbit, goat, monkey, etc.) at a position that encompasses or that is in proximity to that species' homolog of MECP2. The single stranded
oligonucleotide may be complementary to a human genomic region encompassing or in proximity to the MECP2 gene and also be complementary to a mouse genomic region encompassing or in proximity to the mouse homolog of MECP2. For example, the single stranded oligonucleotide may be complementary to a sequence as set forth in SEQ ID NO: 1 or 2, which is a human genomic region encompassing or in proximity to the MECP2 gene, and also be complementary to a sequence as set forth in SEQ ID NO: 3 or 4, which is a mouse genomic region encompassing or in proximity to the mouse homolog of the MECP2 gene. Oligonucleotides having these characteristics may be tested in vivo or in vitro for efficacy in multiple species (e.g. , human and mouse). This approach also facilitates development of clinical candidates for treating human disease by selecting a species in which an appropriate animal exists for the disease. In some embodiments, the region of complementarity of the single stranded oligonucleotide is complementary with at least 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 bases, e.g. , 5, 6, 7, 8, 9, 10, 11, 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, or 50 consecutive nucleotides of a PRC2-associated region. In some embodiments, the region of complementarity is complementary with at least 8 consecutive nucleotides of a PRC2-associated region. In some embodiments the sequence of the single stranded oligonucleotide is based on an RNA sequence that binds to PRC2, or a portion thereof, said portion having a length of from 5 to 40 contiguous base pairs, or about 8 to 40 bases, or about 5 to 15, or about 5 to 30, or about 5 to 40 bases, or about 5 to 50 bases.
Complementary, as the term is used in the art, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an
oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of PRC2-associated region, then the single stranded nucleotide and PRC2-associated region are considered to be complementary to each other at that position. The single stranded nucleotide and PRC2-associated region are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond with each other through their bases. Thus, "complementary" is a term which is used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the single stranded nucleotide and PRC2-associated region. For example, if a base at one position of a single stranded nucleotide is capable of hydrogen bonding with a base at the corresponding position of a PRC2-associated region, then the bases are considered to be complementary to each other at that position. 100% complementarity is not required.
The single stranded oligonucleotide may be at least 80% complementary to
(optionally one of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to) the consecutive nucleotides of a PRC2-associated region. In some embodiments the single stranded oligonucleotide may contain 1, 2 or 3 base mismatches compared to the portion of the consecutive nucleotides of a PRC2-associated region. In some embodiments the single stranded oligonucleotide may have up to 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases. It is understood in the art that a complementary nucleotide sequence need not be 100% complementary to that of its target to be specifically hybridizable. In some
embodiments, a complementary nucleic acid sequence for purposes of the present disclosure is specifically hybridizable when binding of the sequence to the target molecule (e.g. , IncRNA) interferes with the normal function of the target (e.g. , IncRNA) to cause a loss of activity (e.g. , inhibiting PRC2-associated repression with consequent up-regulation of gene expression) and there is a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target sequences under conditions in which avoidance of non-specific binding is desired, e.g. , under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under suitable conditions of stringency.
In some embodiments, the single stranded oligonucleotide is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more nucleotides in length. In a preferred embodiment, the oligonucleotide is 8 to 30 nucleotides in length.
In some embodiments, the PRC2-associated region occurs on the same DNA strand as a gene sequence (sense). In some embodiments, the PRC2-associated region occurs on the opposite DNA strand as a gene sequence (anti-sense). Oligonucleotides complementary to a PRC2-associated region can bind either sense or anti-sense sequences. Base pairings may include both canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g. , Wobble base pairing and Hoogsteen base pairing). It is understood that for complementary base pairings, adenosine-type bases (A) are complementary to thymidine-type bases (T) or uracil-type bases (U), that cytosine-type bases (C) are complementary to guanosine-type bases (G), and that universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize to and are considered complementary to any A, C, U, or T. Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U or T.
In some embodiments, any one or more thymidine (T) nucleotides (or modified nucleotide thereof) or uridine (U) nucleotides (or a modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be replaced with any other nucleotide suitable for base pairing (e.g., via a Watson-Crick base pair) with an adenosine nucleotide. In some embodiments, any one or more thymidine (T) nucleotides (or modified nucleotide thereof) or uridine (U) nucleotides (or a modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be suitably replaced with a different pyrimidine nucleotide or vice versa. In some embodiments, any one or more thymidine (T) nucleotides (or modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be suitably replaced with a uridine (U) nucleotide (or a modified nucleotide thereof) or vice versa. In some embodiments, GC content of the single stranded oligonucleotide is preferably between about 30-60 %. Contiguous runs of three or more Gs or Cs may not be preferable in some embodiments. Accordingly, in some embodiments, the oligonucleotide does not comprise a stretch of three or more guanosine nucleotides.
In some embodiments, the single stranded oligonucleotide specifically binds to, or is complementary to an RNA that is encoded in a genome (e.g., a human genome) as a single contiguous transcript (e.g., a non-spliced RNA). In some embodiments, the single stranded oligonucleotide specifically binds to, or is complementary to an RNA that is encoded in a genome (e.g., a human genome), in which the distance in the genome between the 5'end of the coding region of the RNA and the 3' end of the coding region of the RNA is less than 1 kb, less than 2 kb, less than 3 kb, less than 4 kb, less than 5 kb, less than 7 kb, less than 8 kb, less than 9 kb, less than 10 kb, or less than 20 kb.
It is to be understood that any oligonucleotide provided herein can be excluded. In some embodiments, it has been found that single stranded oligonucleotides disclosed herein may increase expression of mRNA corresponding to the gene by at least about 50% (i.e. 150% of normal or 1.5 fold), or by about 2 fold to about 5 fold. In some embodiments, expression may be increased by at least about 15 fold, 20 fold, 30 fold, 40 fold, 50 fold or 100 fold, or any range between any of the foregoing numbers. It has also been found that increased mRNA expression has been shown to correlate to increased protein expression.
In some or any of the embodiments of the oligonucleotides described herein, or processes for designing or synthesizing them, the oligonucleotides will upregulate gene expression and may specifically bind or specifically hybridize or be complementary to the PRC2 binding RNA that is transcribed from the same strand as a protein coding reference gene. The oligonucleotide may bind to a region of the PRC2 binding RNA that originates within or overlaps an intron, exon, intron exon junction, 5' UTR, 3' UTR, a translation initiation region, or a translation termination region of a protein coding sense strand of a reference gene (refGene).
In some or any of the embodiments of oligonucleotides described herein, or processes for designing or synthesizing them, the oligonucleotides will upregulate gene expression and may specifically bind or specifically hybridize or be complementary to a PRC2 binding RNA that transcribed from the opposite strand (the antisense strand) of a protein coding reference gene. The oligonucleotide may bind to a region of the PRC2 binding RNA that originates within or overlaps an intron, exon, intron exon junction, 5' UTR, 3' UTR, a translation initiation region, or a translation termination region of a protein coding antisense strand of a reference gene
The oligonucleotides described herein may be modified, e.g., comprise a modified sugar moiety, a modified internucleoside linkage, a modified nucleotide and/or combinations thereof. In addition, the oligonucleotides can exhibit one or more of the following properties: do not induce substantial cleavage or degradation of the target RNA; do not cause
substantially complete cleavage or degradation of the target RNA; do not activate the RNAse H pathway; do not activate RISC; do not recruit any Argonaute family protein; are not cleaved by Dicer; do not mediate alternative splicing; are not immune stimulatory; are nuclease resistant; have improved cell uptake compared to unmodified oligonucleotides; are not toxic to cells or mammals; may have improved endosomal exit; do interfere with interaction of IncRNA with PRC2, preferably the Ezh2 subunit but optionally the Suzl2, Eed, RbAp46/48 subunits or accessory factors such as Jarid2; do decrease histone H3 lysine27 methylation and/or do upregulate gene expression.
Oligonucleotides that are designed to interact with RNA to modulate gene expression are a distinct subset of base sequences from those that are designed to bind a DNA target (e.g., are complementary to the underlying genomic DNA sequence from which the RNA is transcribed).
Any of the oligonucleotides disclosed herein may be linked to one or more other oligonucleotides disclosed herein by a linker, e.g., a cleavable linker. Method for Selecting Candidate Oligonucleotides for Activating Expression ofMECP2 Methods are provided herein for selecting a candidate oligonucleotide for activating or enhancing expression of MECP2. The target selection methods may generally involve steps for selecting single stranded oligonucleotides having any of the structural and functional characteristics disclosed herein. Typically, the methods involve one or more steps aimed at identifying oligonucleotides that target a PRC2-associated region that is functionally related to MECP2, for example a PRC2-associated region of a IncRNA that regulates expression of MECP2 by facilitating (e.g. , in a -regulatory manner) the recruitment of PRC2 to the MECP2 gene. Such oligonucleotides are expected to be candidates for activating expression of MECP2 because of their ability to hybridize with the PRC2-associated region of a nucleic acid (e.g. , a IncRNA). In some embodiments, this hybridization event is understood to disrupt interaction of PRC2 with the nucleic acid (e.g. , a IncRNA) and as a result disrupt recruitment of PRC2 and its associated co-repressors (e.g. , chromatin remodeling factors) to the MECP2 gene locus.
Methods of selecting a candidate oligonucleotide may involve selecting a PRC2- associated region (e.g. , a nucleotide sequence as set forth in any one of SEQ ID NOS: 5 to 118) that maps to a chromosomal position encompassing or in proximity to the MECP2 gene (e.g. , a chromosomal position having a sequence as set forth in any one of SEQ ID NOS: 1 to 4). The PRC2-associated region may map to the strand of the chromosome comprising the sense strand of the MECP2 gene, in which case the candidate oligonucleotide is
complementary to the sense strand of the MECP2 gene (i.e. , is antisense to the MECP2 gene). Alternatively, the PRC2-associated region may map to the strand of the first chromosome comprising the antisense strand of the MECP2 gene, in which case the oligonucleotide is complementary to the antisense strand (the template strand) of the MECP2 gene (i.e. , is sense to the MECP2 gene).
Methods for selecting a set of candidate oligonucleotides that is enriched in oligonucleotides that activate expression of MECP2 may involve selecting one or more PRC2-associated regions that map to a chromosomal position that encompasses or that is in proximity to the MECP2 gene and selecting a set of oligonucleotides, in which each oligonucleotide in the set comprises a nucleotide sequence that is complementary with the one or more PRC2-associated regions. As used herein, the phrase, "a set of oligonucleotides that is enriched in oligonucleotides that activate expression of refers to a set of oligonucleotides that has a greater number of oligonucleotides that activate expression of a target gene (e.g. , MECP2) compared with a random selection of oligonucleotides of the same physicochemical properties (e.g. , the same GC content, Tm, length etc.) as the enriched set.
Where the design and/or synthesis of a single stranded oligonucleotide involves design and/or synthesis of a sequence that is complementary to a nucleic acid or PRC2- associated region described by such sequence information, the skilled person is readily able to determine the complementary sequence, e.g., through understanding of Watson Crick base pairing rules which form part of the common general knowledge in the field.
In some embodiments design and/or synthesis of a single stranded oligonucleotide involves manufacture of an oligonucleotide from starting materials by techniques known to those of skill in the art, where the synthesis may be based on a sequence of a PRC2- associated region, or portion thereof.
Methods of design and/or synthesis of a single stranded oligonucleotide may involve one or more of the steps of:
Identifying and/or selecting PRC2-associated region;
Designing a nucleic acid sequence having a desired degree of sequence identity or complementarity to a PRC2-associated region or a portion thereof;
Synthesizing a single stranded oligonucleotide to the designed sequence;
Purifying the synthesized single stranded oligonucleotide; and
Optionally mixing the synthesized single stranded oligonucleotide with at least one pharmaceutically acceptable diluent, carrier or excipient to form a pharmaceutical composition or medicament.
Single stranded oligonucleotides so designed and/or synthesized may be useful in method of modulating gene expression as described herein.
Preferably, single stranded oligonucleotides of the invention are synthesized chemically. Oligonucleotides used to practice this invention can be synthesized in vitro by well-known chemical synthesis techniques.
Oligonucleotides of the invention can be stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g. , a nucleotide modification. For example, nucleic acid sequences of the invention include a phosphorothioate at least the first, second, or third internucleotide linkage at the 5' or 3' end of the nucleotide sequence. As another example, the nucleic acid sequence can include a 2'-modified nucleotide, e.g. , a 2'-deoxy, 2'- deoxy-2'-fluoro, 2'-0-methyl, 2'-0-methoxyethyl (2'-0-MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE), 2'-0-dimethylaminopropyl (2'-0-DMAP), 2'-0- dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0--N-methylacetamido (2'-0--NMA). As another example, the nucleic acid sequence can include at least one 2'-0-methyl-modified nucleotide, and in some embodiments, all of the nucleotides include a 2'-0-methyl modification. In some embodiments, the nucleic acids are "locked," i.e., comprise nucleic acid analogues in which the ribose ring is "locked" by a methylene bridge connecting the 2'- O atom and the 4'-C atom.
It is understood that any of the modified chemistries or formats of single stranded oligonucleotides described herein can be combined with each other, and that one, two, three, four, five, or more different types of modifications can be included within the same molecule.
In some embodiments, the method may further comprise the steps of amplifying the synthesized single stranded oligonucleotide, and/or purifying the single stranded
oligonucleotide (or amplified single stranded oligonucleotide), and/or sequencing the single stranded oligonucleotide so obtained.
As such, the process of preparing a single stranded oligonucleotide may be a process that is for use in the manufacture of a pharmaceutical composition or medicament for use in the treatment of disease, optionally wherein the treatment involves modulating expression of a gene associated with a PRC2-associated region.
In the methods described above a PRC2-associated region may be, or have been, identified, or obtained, by a method that involves identifying RNA that binds to PRC2.
Such methods may involve the following steps: providing a sample containing nuclear ribonucleic acids, contacting the sample with an agent that binds specifically to PRC2 or a subunit thereof, allowing complexes to form between the agent and protein in the sample, partitioning the complexes, synthesizing nucleic acid that is complementary to nucleic acid present in the complexes.
Where the single stranded oligonucleotide is based on a PRC2-associated region, or a portion of such a sequence, it may be based on information about that sequence, e.g. , sequence information available in written or electronic form, which may include sequence information contained in publicly available scientific publications or sequence databases. Nucleotide Analogues
In some embodiments, the oligonucleotide may comprise at least one ribonucleotide, at least one deoxyribonucleotide, and/or at least one bridged nucleotide. In some
embodiments, the oligonucleotide may comprise a bridged nucleotide, such as a locked nucleic acid (LNA) nucleotide, a constrained ethyl (cEt) nucleotide, or an ethylene bridged nucleic acid (ENA) nucleotide. Examples of such nucleotides are disclosed herein and known in the art. In some embodiments, the oligonucleotide comprises a nucleotide analog disclosed in one of the following United States Patent or Patent Application Publications: US 7,399,845, US 7,741,457, US 8,022,193, US 7,569,686, US 7,335,765, US 7,314,923, US 7,335,765, and US 7,816,333, US 20110009471, the entire contents of each of which are incorporated herein by reference for all purposes. The oligonucleotide may have one or more 2' O-methyl nucleotides. The oligonucleotide may consist entirely of 2' O-methyl nucleotides.
Often the single stranded oligonucleotide has one or more nucleotide analogues. For example, the single stranded oligonucleotide may have at least one nucleotide analogue that results in an increase in Tm of the oligonucleotide in a range of 1°C, 2 °C, 3°C, 4 °C, or 5°C compared with an oligonucleotide that does not have the at least one nucleotide analogue. The single stranded oligonucleotide may have a plurality of nucleotide analogues that results in a total increase in Tm of the oligonucleotide in a range of 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C or more compared with an oligonucleotide that does not have the nucleotide analogue.
The oligonucleotide may be of up to 50 nucleotides in length in which 2 to 10, 2 to 155 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, 2 to 45, or more nucleotides of the oligonucleotide are nucleotide analogues. The oligonucleotide may be of 8 to 30 nucleotides in length in which 2 to 10, 2 to 155 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30 nucleotides of the oligonucleotide are nucleotide analogues.
The oligonucleotide may be of 8 to 15 nucleotides in length in which 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14 nucleotides of the oligonucleotide are nucleotide analogues. Optionally, the oligonucleotides may have every nucleotide except 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides modified. The oligonucleotide may consist entirely of bridged nucleotides (e.g. , LNA nucleotides, cEt nucleotides, ENA nucleotides). The oligonucleotide may comprise alternating deoxyribonucleotides and 2'-fluoro-deoxyribonucleotides. The oligonucleotide may comprise alternating deoxyribonucleotides and 2'-0-methyl nucleotides. The oligonucleotide may comprise alternating deoxyribonucleotides and ENA nucleotide analogues. The oligonucleotide may comprise alternating deoxyribonucleotides and LNA nucleotides. The oligonucleotide may comprise alternating LNA nucleotides and 2'-0- methyl nucleotides. The oligonucleotide may have a 5' nucleotide that is a bridged nucleotide (e.g. , a LNA nucleotide, cEt nucleotide, ENA nucleotide). The oligonucleotide may have a 5' nucleotide that is a deoxyribonucleotide.
The oligonucleotide may comprise deoxyribonucleotides flanked by at least one bridged nucleotide (e.g. , a LNA nucleotide, cEt nucleotide, ENA nucleotide) on each of the 5' and 3' ends of the deoxyribonucleotides. The oligonucleotide may comprise
deoxyribonucleotides flanked by 1, 2, 3, 4, 5, 6, 7, 8 or more bridged nucleotides (e.g. , LNA nucleotides, cEt nucleotides, ENA nucleotides) on each of the 5' and 3' ends of the deoxyribonucleotides. The 3' position of the oligonucleotide may have a 3' hydroxyl group. The 3' position of the oligonucleotide may have a 3' thiophosphate.
The oligonucleotide may be conjugated with a label. For example, the
oligonucleotide may be conjugated with a biotin moiety, cholesterol, Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR or dynamic polyconjugates and variants thereof at its 5' or 3' end.
Preferably the single stranded oligonucleotide comprises one or more modifications comprising: a modified sugar moiety, and/or a modified internucleoside linkage, and/or a modified nucleotide and/or combinations thereof. It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the
modifications described herein may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide.
In some embodiments, the single stranded oligonucleotides are chimeric
oligonucleotides that contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Chimeric single stranded oligonucleotides of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides,
oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures comprise, but are not limited to, US patent nos. 5,013,830; 5,149,797; 5, 220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133;
5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference.
In some embodiments, the single stranded oligonucleotide comprises at least one nucleotide modified at the 2' position of the sugar, most preferably a 2'-0-alkyl, 2'-0-alkyl-0- alkyl or 2'-fluoro-modified nucleotide. In other preferred embodiments, RNA modifications include 2'-fluoro, 2'-amino and 2' O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA. Such modifications are routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than 2'-deoxyoligonucleotides against a given target.
A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide; these modified oligos survive intact for a longer time than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are oligonucleotides with
phosphorothioate backbones and those with heteroatom backbones, particularly CH2 -NH-O- CH2, CH,~N(CH3)~0~CH2 (known as a methylene(methylimino) or MMI backbone, CH2 - O-N (CH3)-CH2, CH2 -N (CH3)-N (CH3)-CH2 and O-N (CH3)- CH2 -CH2 backbones, wherein the native phosphodiester backbone is represented as O- P— O- CH,); amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbone structures (see Summerton and Weller, U.S. Pat. No. 5,034,506); peptide nucleic acid (PNA) backbone (wherein the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497). Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3 -5' to 5'-3' or 2 -5' to 5'-2'; see US patent nos. 3,687,808; 4,469,863;
4,476,301 ; 5,023,243; 5, 177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321, 131 ; 5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677; 5,476,925; 5,519, 126; 5,536,821 ; 5,541,306; 5,550, 111 ; 5,563, 253; 5,571,799; 5,587,361 ; and 5,625,050.
Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and
David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis, volume 30, issue 3, 2001 ; Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216- 220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991. In some embodiments, the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g. , as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001 ; and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated herein by reference in their entireties).
Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc, 2000, 122, 8595-8602.
Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts; see US patent nos. 5,034,506; 5, 166,315; 5,185,444; 5,214,134; 5,216, 141 ; 5,235,033; 5,264, 562; 5, 264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596, 086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623, 070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.
Modified oligonucleotides are also known that include oligonucleotides that are based on or constructed from arabinonucleotide or modified arabinonucleotide residues.
Arabinonucleosides are stereoisomers of ribonucleosides, differing only in the configuration at the 2'-position of the sugar ring. In some embodiments, a 2'-arabino modification is 2'-F arabino. In some embodiments, the modified oligonucleotide is 2' -fluoro-D-arabinonucleic acid (FANA) (as described in, for example, Lon et al., Biochem., 41 :3457-3467, 2002 and Min et al., Bioorg. Med. Chem. Lett., 12:2651-2654, 2002; the disclosures of which are incorporated herein by reference in their entireties). Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on a 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
PCT Publication No. WO 99/67378 discloses arabinonucleic acids (ANA) oligomers and their analogues for improved sequence specific inhibition of gene expression via association to complementary messenger RNA.
Other preferred modifications include ethylene-bridged nucleic acids (ENAs) (e.g. , International Patent Publication No. WO 2005/042777, Morita et al., Nucleic Acid Res., Suppl 1 :241-242, 2001 ; Surono et al., Hum. Gene Ther., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther., 8: 144- 149, 2006 and Horie et al., Nucleic Acids Symp. Ser (Oxf), 49: 171- 172, 2005; the disclosures of which are incorporated herein by reference in their entireties). Preferred ENAs include, but are not limited to, 2'-0,4'-C-ethylene -bridged nucleic acids. Examples of LNAs are described in WO/2008/043753 and include compounds of the following general formula.
Figure imgf000028_0001
where X and Y are independently selected among the groups -0-,
-S-, -N(H)-, N(R)-, -CH2- or -CH- (if part of a double bond),
-CH2-0-, -CH2-S-, -CH2-N(H)-, -CH2-N(R)-, -CH2-CH2- or -CH2-CH- (if part of a double bond), -CH=CH-, where R is selected from hydrogen and Ci-4-alkyl; Z and Z* are independently selected among an intemucleoside linkage, a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety; and the asymmetric groups may be found in either orientation.
Preferably, the LNA used in the oligonucleotides described herein comprises at least one LNA unit according any of the formulas
Figure imgf000028_0002
wherein Y is -0-, -S-, -NH-, or N(R ); Z and Z* are independently selected among an intemucleoside linkage, a terminal group or a protecting group; B constitutes a natural or non-natural nucleotide base moiety, and RH is selected from hydrogen and Ci-4-alkyl. In some embodiments, the Locked Nucleic Acid (LNA) used in the oligonucleotides described herein comprises at least one Locked Nucleic Acid (LNA) unit according any of the formulas shown in Scheme 2 of PCT/DK2006/000512.
In some embodiments, the LNA used in the oligomer of the invention comprises internucleoside linkages selected from -0-P(O)2-O-, -0-P(0,S)-0-, -0-P(S)2-O-, -S-P(0)2-0-, -S-P(0,S)-0-, -S-P(S)2-0-, -0-P(O)2-S-, -0-P(0,S)-S-, -S-P(0)2-S-, -0-PO(RH)-0-, o- PO(OCH3)-0-, -0-PO(NRH)-0-, -0-PO(OCH2CH2S-R)-O-, -0-PO(BH3)-0-, -0-PO(NHRH)- 0-, -0-P(0)2-NRH-, -NRH-P(0)2-0-, -NRH-CO-0-, where RH is selected from hydrogen and Ci-4-alkyl.
Specifically preferred LNA units are shown in scheme 2:
Figure imgf000029_0001
Scheme 2
The term "thio-LNA" comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from S or -CH2-S-. Thio-LNA can be in both beta-D and alpha-L-configuration. The term "amino-LNA" comprises a locked nucleotide in which at least one of X or Y in the general formula above is selected from -N(H)-, N(R)-, CH2-N(H)-, and -CH2-N(R)- where R is selected from hydrogen and Ci-4-alkyl. Amino-LNA can be in both beta-D and alpha- L-configuration. The term "oxy-LNA" comprises a locked nucleotide in which at least one of X or Y in the general formula above represents -O- or -CH2-0-. Oxy-LNA can be in both beta-D and alpha- L-configuration.
The term "ena-LNA" comprises a locked nucleotide in which Y in the general formula above is -CH2-0- (where the oxygen atom of -CH2-0- is attached to the 2'-position relative to the base B).
LNAs are described in additional detail herein.
One or more substituted sugar moieties can also be included, e.g. , one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3 OCH3, OCH3 0(CH2)n CH3, 0(CH2)n NH2 or 0(CH2)n CH3 where n is from 1 to about 10; CI to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF ; OCF ; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH3; S02 CH3; ON02; N02; N3; NH2; heterocycloalkyl; heterocyclo alkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy [2'-0-CH2CH2OCH3, also known as 2'-0-(2-methoxyethyl)] (Martin et al, Helv. Chim. Acta, 1995, 78, 486). Other preferred modifications include 2'- methoxy (2'-0-CH3), 2'-propoxy (2'-OCH2 CH2CH3) and 2'-fluoro (2'-F). Similar
modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
Single stranded oligonucleotides can also include, additionally or alternatively, nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g. , hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2'
deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, isocytosine, pseudoisocytosine, as well as synthetic nucleobases, e.g. , 2-aminoadenine, 2- (methylamino)adenine, 2-(imidazolylalkyl)adenine, 2- (aminoalklyamino)adenine or other hetero substituted alkyladenines, 2-thiouracil, 2- thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 5-propynyluracil, 8-azaguanine, 7- deazaguanine, N6 (6-aminohexyl)adenine, 6-aminopurine, 2-aminopurine, 2-chloro-6- aminopurine and 2,6-diaminopurine or other diaminopurines. See, e.g. , Kornberg, "DNA Replication," W. H. Freeman & Co., San Francisco, 1980, pp75-77; and Gebeyehu, G., et al. Nucl. Acids Res., 15:4513 (1987)). A "universal" base known in the art, e.g. , inosine, can also be included. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6- 1.2°C. (Sanghvi, in Crooke, and Lebleu, eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and may be used as base substitutions.
It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the modifications described herein may be
incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide.
In some embodiments, both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar- backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, US patent nos. 5,539,082; 5,714,331 ; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500.
Single stranded oligonucleotides can also include one or more nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases comprise other synthetic and natural nucleobases such as 5-methylcytosine (5- me-C), 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 uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8- substituted adenines and guanines, 5-halo particularly 5- bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3- deazaguanine and 3-deazaadenine.
Further, nucleobases comprise those disclosed in United States Patent No. 3,687,808, those disclosed in "The Concise Encyclopedia of Polymer Science And Engineering", pages 858-859, Kroschwitz, ed. John Wiley & Sons, 1990;, those disclosed by Englisch et al., Angewandle Chemie, International Edition, 1991, 30, page 613, and those disclosed by Sanghvi, Chapter 15, Antisense Research and Applications," pages 289- 302, Crooke, and Lebleu, eds., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine. 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2<0>C (Sanghvi, et al., eds, "Antisense Research and Applications," CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications. Modified nucleobases are described in US patent nos. 3,687,808, as well as 4,845,205; 5,130,302; 5,134,066; 5,175, 273; 5, 367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711 ; 5,552,540; 5,587,469; 5,596,091 ; 5,614,617; 5,750,692, and 5,681,941, each of which is herein incorporated by reference.
In some embodiments, the single stranded oligonucleotides are chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. For example, one or more single stranded oligonucleotides, of the same or different types, can be conjugated to each other; or single stranded
oligonucleotides can be conjugated to targeting moieties with enhanced specificity for a cell type or tissue type. Such moieties include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g. , hexyl-S- tritylthiol (Manoharan et al, Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g. , dodecandiol or undecyl residues (Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49- 54), a phospholipid, e.g. , di-hexadecyl-rac-glycerol or triethylammonium 1,2- di-O-hexadecyl- rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Mancharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-t oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). See also US patent nos. 4,828,979; 4,948,882; 5,218, 105; 5,525,465; 5,541,313; 5,545,730; 5,552, 538; 5,578,717, 5,580,731 ; 5,580,731 ; 5,591,584; 5,109, 124; 5, 118,802; 5,138,045; 5,414,077; 5,486, 603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762, 779; 4,789,737; 4,824,941 ; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082, 830; 5,112,963; 5,214,136; 5,082,830; 5, 112,963; 5,214, 136; 5, 245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391, 723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5, 565,552; 5,567,810; 5,574, 142; 5,585,481 ; 5,587,371 ; 5,595,726; 5,597,696; 5,599,923; 5,599, 928 and 5,688,941, each of which is herein incorporated by reference. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence- specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, which are incorporated herein by reference. Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g. , hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g. , dodecandiol or undecyl residues, a phospholipid, e.g. , di-hexadecyl-rac- glycerol or triethylammonium 1,2- di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety. See, e.g. , U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218, 105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731 ; 5,580,731 ; 5,591,584; 5,109, 124; 5,118,802; 5, 138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941 ; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5, 112,963; 5,214, 136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574, 142; 5,585,481 ; 5,587,371 ; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.
In some embodiments, single stranded oligonucleotide modification include modification of the 5' or 3' end of the oligonucleotide. In some embodiments, the 3' end of the oligonucleotide comprises a hydroxyl group or a thiophosphate. It should be appreciated that additional molecules (e.g. a biotin moiety or a fluorophor) can be conjugated to the 5' or 3' end of the single stranded oligonucleotide. In some embodiments, the single stranded oligonucleotide comprises a biotin moiety conjugated to the 5' nucleotide.
In some embodiments, the single stranded oligonucleotide comprises locked nucleic acids (LNA), ENA modified nucleotides, 2'-0-methyl nucleotides, or 2'-fluoro- deoxyribonucleotides. In some embodiments, the single stranded oligonucleotide comprises alternating deoxyribonucleotides and 2'-fluoro-deoxyribonucleotides. In some embodiments, the single stranded oligonucleotide comprises alternating deoxyribonucleotides and 2'-0- methyl nucleotides. In some embodiments, the single stranded oligonucleotide comprises alternating deoxyribonucleotides and ENA modified nucleotides. In some embodiments, the single stranded oligonucleotide comprises alternating deoxyribonucleotides and locked nucleic acid nucleotides. In some embodiments, the single stranded oligonucleotide comprises alternating locked nucleic acid nucleotides and 2'-0-methyl nucleotides.
In some embodiments, the 5' nucleotide of the oligonucleotide is a
deoxyribonucleotide. In some embodiments, the 5' nucleotide of the oligonucleotide is a locked nucleic acid nucleotide. In some embodiments, the nucleotides of the oligonucleotide comprise deoxyribonucleotides flanked by at least one locked nucleic acid nucleotide on each of the 5' and 3' ends of the deoxyribonucleotides. In some embodiments, the nucleotide at the 3' position of the oligonucleotide has a 3' hydroxyl group or a 3' thiophosphate.
In some embodiments, the single stranded oligonucleotide comprises
phosphorothioate internucleotide linkages. In some embodiments, the single stranded oligonucleotide comprises phosphorothioate internucleotide linkages between at least two nucleotides. In some embodiments, the single stranded oligonucleotide comprises phosphorothioate internucleotide linkages between all nucleotides.
It should be appreciated that the single stranded oligonucleotide can have any combination of modifications as described herein.
The oligonucleotide may comprise a nucleotide sequence having one or more of the following modification patterns.
(a) (X)Xxxxxx, (X)xXxxxx, (X)xxXxxx, (X)xxxXxx, (X)xxxxXx and (X)xxxxxX,
(b) (X)XXxxxx, (X)XxXxxx, (X)XxxXxx, (X)XxxxXx, (X)XxxxxX, (X)xXXxxx, (X)xXxXxx, (X)xXxxXx, (X)xXxxxX, (X)xxXXxx, (X)xxXxXx, (X)xxXxxX, (X)xxxXXx,
(X)xxxXxX and (X)xxxxXX, (c) (X)XXXxxx, (X)xXXXxx, (X)xxXXXx, (X)xxxXXX, (X)XXxXxx, (X)XXxxXx, (X)XXxxxX, (X)xXXxXx, (X)xXXxxX, (X)xxXXxX, (X)XxXXxx, (X)XxxXXx
(X)XxxxXX, (X)xXxXXx, (X)xXxxXX, (X)xxXxXX, (X)xXxXxX and (X)XxXxXx,
(d) (X)xxXXX, (X)xXxXXX, (X)xXXxXX, (X)xXXXxX, (X)xXXXXx,
(X)XxxXXXX, (X)XxXxXX, (X)XxXXxX, (X)XxXXx, (X)XXxxXX, (X)XXxXxX, (X)XXxXXx, (X)XXXxxX, (X)XXXxXx, and (X)XXXXxx,
(e) (X)xXXXXX, (X)XxXXXX, (X)XXxXXX, (X)XXXxXX, (X)XXXXxX and (X)XXXXXx, and
(f) XXXXXX, XxXXXXX, XXxXXXX, XXXxXXX, XXXXxXX, XXXXXxX and XXXXXXx, in which "X" denotes a nucleotide analogue, (X) denotes an optional nucleotide analogue, and "x" denotes a DNA or RNA nucleotide unit. Each of the above listed patterns may appear one or more times within an oligonucleotide, alone or in combination with any of the other disclosed modification patterns. Methods for Modulating Gene Expression
In one aspect, the invention relates to methods for modulating gene expression in a cell (e.g., a cell for which MECP2 levels are reduced) for research purposes (e.g., to study the function of the gene in the cell). In another aspect, the invention relates to methods for modulating gene expression in a cell (e.g., a cell for which MECP2 levels are reduced) for gene or epigenetic therapy. The cells can be in vitro, ex vivo, or in vivo (e.g., in a subject who has a disease resulting from reduced expression or activity of MECP2. In some
embodiments, methods for modulating gene expression in a cell comprise delivering a single stranded oligonucleotide as described herein. In some embodiments, delivery of the single stranded oligonucleotide to the cell results in a level of expression of gene that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more greater than a level of expression of gene in a control cell to which the single stranded oligonucleotide has not been delivered. In certain embodiments, delivery of the single stranded oligonucleotide to the cell results in a level of expression of gene that is at least 50% greater than a level of expression of gene in a control cell to which the single stranded oligonucleotide has not been delivered. In another aspect of the invention, methods comprise administering to a subject (e.g. a human) a composition comprising a single stranded oligonucleotide as described herein to increase protein levels in the subject. In some embodiments, the increase in protein levels is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more, higher than the amount of a protein in the subject before administering.
As another example, to increase expression of MECP2 in a cell, the methods include introducing into the cell a single stranded oligonucleotide that is sufficiently complementary to a PRC2-associated region (e.g., of a long non-coding RNA) that maps to a genomic position encompassing or in proximity to the MECP2 gene.
In another aspect of the invention provides methods of treating a condition (e.g., Rett Syndrome, MECP2-related severe neonatal encephalopathy, Angelman syndrome, PPM-X syndrome) associated with decreased levels of expression of MECP2 in a subject, the method comprising administering a single stranded oligonucleotide as described herein.
A subject can include a non-human mammal, e.g. mouse, rat, guinea pig, rabbit, cat, dog, goat, cow, or horse. In preferred embodiments, a subject is a human. Single stranded oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Single stranded oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.
For therapeutics, an animal, preferably a human, suspected of having Rett Syndrome, MECP2-related severe neonatal encephalopathy, Angelman syndrome, or PPM-X syndrome is treated by administering single stranded oligonucleotide in accordance with this invention. For example, in one non-limiting embodiment, the methods comprise the step of
administering to the animal in need of treatment, a therapeutically effective amount of a single stranded oligonucleotide as described herein.
Formulation, Delivery, And Dosing
The oligonucleotides described herein can be formulated for administration to a subject for treating a condition (e.g., Rett Syndrome, MECP2-related severe neonatal encephalopathy, Angelman syndrome, PPM-X syndrome) associated with decreased levels of MECP2. It should be understood that the formulations, compositions and methods can be practiced with any of the oligonucleotides disclosed herein.
The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient (e.g. , an oligonucleotide or compound of the invention) which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration, e.g. , intradermal or inhalation. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect, e.g. tumor regression.
Pharmaceutical formulations of this invention can be prepared according to any method known to the art for the manufacture of pharmaceuticals. Such formulations can contain sweetening agents, flavoring agents, coloring agents and preserving agents. A formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture. Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.
A formulated single stranded oligonucleotide composition can assume a variety of states. In some examples, the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g. , less than 80, 50, 30, 20, or 10% water). In another example, the single stranded oligonucleotide is in an aqueous phase, e.g. , in a solution that includes water. The aqueous phase or the crystalline compositions can, e.g. , be incorporated into a delivery vehicle, e.g. , a liposome (particularly for the aqueous phase) or a particle (e.g. , a microparticle as can be appropriate for a crystalline composition). Generally, the single stranded oligonucleotide composition is formulated in a manner that is compatible with the intended method of administration.
In some embodiments, the composition is prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and other self-assembly. A single stranded oligonucleotide preparation can be formulated or administered (together or separately) in combination with another agent, e.g. , another therapeutic agent or an agent that stabilizes a single stranded oligonucleotide, e.g. , a protein that complexes with single stranded oligonucleotide. Still other agents include chelators, e.g. , EDTA (e.g. , to remove divalent cations such as Mg2+), salts, RNAse inhibitors (e.g. , a broad specificity RNAse inhibitor such as RNAsin) and so forth.
In one embodiment, the single stranded oligonucleotide preparation includes another single stranded oligonucleotide, e.g. , a second single stranded oligonucleotide that modulates expression of a second gene or a second single stranded oligonucleotide that modulates expression of the first gene. Still other preparation can include at least 3, 5, ten, twenty, fifty, or a hundred or more different single stranded oligonucleotide species. Such single stranded oligonucleotides can mediated gene expression with respect to a similar number of different genes. In one embodiment, the single stranded oligonucleotide preparation includes at least a second therapeutic agent (e.g. , an agent other than an oligonucleotide).
Route of Delivery
A composition that includes a single stranded oligonucleotide can be delivered to a subject by a variety of routes. Exemplary routes include: intravenous, intradermal, topical, rectal, parenteral, anal, intravaginal, intranasal, pulmonary, ocular. The term "therapeutically effective amount" is the amount of oligonucleotide present in the composition that is needed to provide the desired level of MECP2 expression in the subject to be treated to give the anticipated physiological response. The term "physiologically effective amount" is that amount delivered to a subject to give the desired palliative or curative effect. The term "pharmaceutically acceptable carrier" means that the carrier can be administered to a subject with no significant adverse toxicological effects to the subject.
The single stranded oligonucleotide molecules of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more species of single stranded oligonucleotide and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or
intraventricular administration.
The route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. Lung cells might be targeted by administering the single stranded oligonucleotide in aerosol form. The vascular endothelial cells could be targeted by coating a balloon catheter with the single stranded oligonucleotide and mechanically introducing the oligonucleotide.
Topical administration refers to the delivery to a subject by contacting the formulation directly to a surface of the subject. The most common form of topical delivery is to the skin, but a composition disclosed herein can also be directly applied to other surfaces of the body, e.g. , to the eye, a mucous membrane, to surfaces of a body cavity or to an internal surface.
As mentioned above, the most common topical delivery is to the skin. The term encompasses several routes of administration including, but not limited to, topical and transdermal. These modes of administration typically include penetration of the skin's permeability barrier and efficient delivery to the target tissue or stratum. Topical administration can be used as a means to penetrate the epidermis and dermis and ultimately achieve systemic delivery of the composition. Topical administration can also be used as a means to selectively deliver oligonucleotides to the epidermis or dermis of a subject, or to specific strata thereof, or to an underlying tissue.
Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.
Transdermal delivery is a valuable route for the administration of lipid soluble therapeutics. The dermis is more permeable than the epidermis and therefore absorption is much more rapid through abraded, burned or denuded skin. Inflammation and other physiologic conditions that increase blood flow to the skin also enhance transdermal adsorption. Absorption via this route may be enhanced by the use of an oily vehicle
(inunction) or through the use of one or more penetration enhancers. Other effective ways to deliver a composition disclosed herein via the transdermal route include hydration of the skin and the use of controlled release topical patches. The transdermal route provides a potentially effective means to deliver a composition disclosed herein for systemic and/or local therapy. In addition, iontophoresis (transfer of ionic solutes through biological membranes under the influence of an electric field), phonophoresis or sonophoresis (use of ultrasound to enhance the absorption of various therapeutic agents across biological membranes, notably the skin and the cornea), and optimization of vehicle characteristics relative to dose position and retention at the site of administration may be useful methods for enhancing the transport of topically applied compositions across skin and mucosal sites.
Both the oral and nasal membranes offer advantages over other routes of
administration. For example, oligonucleotides administered through these membranes may have a rapid onset of action, provide therapeutic plasma levels, avoid first pass effect of hepatic metabolism, and avoid exposure of the oligonucleotides to the hostile gastrointestinal (GI) environment. Additional advantages include easy access to the membrane sites so that the oligonucleotide can be applied, localized and removed easily.
In oral delivery, compositions can be targeted to a surface of the oral cavity, e.g. , to sublingual mucosa which includes the membrane of ventral surface of the tongue and the floor of the mouth or the buccal mucosa which constitutes the lining of the cheek. The sublingual mucosa is relatively permeable thus giving rapid absorption and acceptable bioavailability of many agents. Further, the sublingual mucosa is convenient, acceptable and easily accessible.
A pharmaceutical composition of single stranded oligonucleotide may also be administered to the buccal cavity of a human being by spraying into the cavity, without inhalation, from a metered dose spray dispenser, a mixed micellar pharmaceutical
formulation as described above and a propellant. In one embodiment, the dispenser is first shaken prior to spraying the pharmaceutical formulation and propellant into the buccal cavity.
Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, slurries, emulsions, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches. In the case of tablets, carriers that can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added.
Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, intrathecal or intraventricular administration. In some embodiments, parental administration involves administration directly to the site of disease (e.g. injection into a tumor).
Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic.
Any of the single stranded oligonucleotides described herein can be administered to ocular tissue. For example, the compositions can be applied to the surface of the eye or nearby tissue, e.g. , the inside of the eyelid. For ocular administration, ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers. Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives such as sorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers. The single stranded oligonucleotide can also be administered to the interior of the eye, and can be introduced by a needle or other delivery device which can introduce it to a selected area or structure. Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the composition, preferably single stranded oligonucleotides, within the dispersion can reach the lung where it can be readily absorbed through the alveolar region directly into blood circulation. Pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs.
Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellular and dry powder-based formulations. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose devices are preferred. One of the benefits of using an atomizer or inhaler is that the potential for contamination is minimized because the devices are self-contained. Dry powder dispersion devices, for example, deliver agents that may be readily formulated as dry powders. A single stranded oligonucleotide composition may be stably stored as lyophilized or spray-dried powders by itself or in combination with suitable powder carriers. The delivery of a composition for inhalation can be mediated by a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.
The term "powder" means a composition that consists of finely dispersed solid particles that are free flowing and capable of being readily dispersed in an inhalation device and subsequently inhaled by a subject so that the particles reach the lungs to permit penetration into the alveoli. Thus, the powder is said to be "respirable." Preferably the average particle size is less than about 10 μιη in diameter preferably with a relatively uniform spheroidal shape distribution. More preferably the diameter is less than about 7.5 μ m and most preferably less than about 5.0 μ m. Usually the particle size distribution is between about 0.1 μ m and about 5 μ m in diameter, particularly about 0.3 μ m to about 5 μ m.
The term "dry" means that the composition has a moisture content below about 10% by weight (% w) water, usually below about 5% w and preferably less it than about 3% w. A dry composition can be such that the particles are readily dispersible in an inhalation device to form an aerosol.
The types of pharmaceutical excipients that are useful as carrier include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two.
Suitable pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred. Pulmonary administration of a micellar single stranded oligonucleotide formulation may be achieved through metered dose spray devices with propellants such as tetrafluoroethane, heptafluoroethane, dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFC and CFC propellants.
Exemplary devices include devices which are introduced into the vasculature, e.g. , devices inserted into the lumen of a vascular tissue, or which devices themselves form a part of the vasculature, including stents, catheters, heart valves, and other vascular devices. These devices, e.g. , catheters or stents, can be placed in the vasculature of the lung, heart, or leg.
Other devices include non- vascular devices, e.g. , devices implanted in the
peritoneum, or in organ or glandular tissue, e.g. , artificial organs. The device can release a therapeutic substance in addition to a single stranded oligonucleotide, e.g. , a device can release insulin.
In one embodiment, unit doses or measured doses of a composition that includes single stranded oligonucleotide are dispensed by an implanted device. The device can include a sensor that monitors a parameter within a subject. For example, the device can include pump, e.g. , and, optionally, associated electronics.
Tissue, e.g. , cells or organs can be treated with a single stranded oligonucleotide, ex vivo and then administered or implanted in a subject. The tissue can be autologous, allogeneic, or xenogeneic tissue. E.g. , tissue can be treated to reduce graft v. host disease . In other embodiments, the tissue is allogeneic and the tissue is treated to treat a disorder characterized by unwanted gene expression in that tissue. E.g. , tissue, e.g. , hematopoietic cells, e.g. , bone marrow hematopoietic cells, can be treated to inhibit unwanted cell proliferation. Introduction of treated tissue, whether autologous or transplant, can be combined with other therapies. In some implementations, the single stranded oligonucleotide treated cells are insulated from other cells, e.g. , by a semi-permeable porous barrier that prevents the cells from leaving the implant, but enables molecules from the body to reach the cells and molecules produced by the cells to enter the body. In one embodiment, the porous barrier is formed from alginate.
In one embodiment, a contraceptive device is coated with or contains a single stranded oligonucleotide. Exemplary devices include condoms, diaphragms, IUD
(implantable uterine devices, sponges, vaginal sheaths, and birth control devices.
Dosage
In one aspect, the invention features a method of administering a single stranded oligonucleotide (e.g., as a compound or as a component of a composition) to a subject (e.g. , a human subject). In one embodiment, the unit dose is between about 10 mg and 25 mg per kg of bodyweight. In one embodiment, the unit dose is between about 1 mg and 100 mg per kg of bodyweight. In one embodiment, the unit dose is between about 0.1 mg and 500 mg per kg of bodyweight. In some embodiments, the unit dose is more than 0.001, 0.005, 0.01, 0.05,
0.1, 0.5, 1, 2, 5, 10, 25, 50 or 100 mg per kg of bodyweight.
The defined amount can be an amount effective to treat or prevent a disease or disorder, e.g. , a disease or disorder associated with the MECP2. The unit dose, for example, can be administered by injection (e.g. , intravenous or intramuscular), an inhaled dose, or a topical application.
In some embodiments, the unit dose is administered daily. In some embodiments, less frequently than once a day, e.g. , less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g. , not a regular frequency). For example, the unit dose may be administered a single time. In some embodiments, the unit dose is administered more than once a day, e.g. , once an hour, two hours, four hours, eight hours, twelve hours, etc.
In one embodiment, a subject is administered an initial dose and one or more maintenance doses of a single stranded oligonucleotide. The maintenance dose or doses are generally lower than the initial dose, e.g. , one-half less of the initial dose. A maintenance regimen can include treating the subject with a dose or doses ranging from 0.0001 to 100 mg/kg of body weight per day, e.g. , 100, 10, 1, 0.1, 0.01, 0.001, or 0.0001 mg per kg of bodyweight per day. The maintenance doses may be administered no more than once every
1, 5, 10, or 30 days. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient. In some embodiments the dosage may be delivered no more than once per day, e.g. , no more than once per 24, 36, 48, or more hours, e.g. , no more than once for every 5 or 8 days. Following treatment, the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state. The dosage of the oligonucleotide may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.
The effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g. , a pump, semipermanent stent (e.g. , intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.
In some embodiments, the oligonucleotide pharmaceutical composition includes a plurality of single stranded oligonucleotide species. In another embodiment, the single stranded oligonucleotide species has sequences that are non- overlapping and non-adjacent to another species with respect to a naturally occurring target sequence (e.g. , a PRC2-associated region). In another embodiment, the plurality of single stranded oligonucleotide species is specific for different PRC2-associated regions. In another embodiment, the single stranded oligonucleotide is allele specific. In some cases, a patient is treated with a single stranded oligonucleotide in conjunction with other therapeutic modalities.
Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the compound of the invention is administered in maintenance doses, ranging from 0.0001 mg to 100 mg per kg of body weight.
The concentration of the single stranded oligonucleotide composition is an amount sufficient to be effective in treating or preventing a disorder or to regulate a physiological condition in humans. The concentration or amount of single stranded oligonucleotide administered will depend on the parameters determined for the agent and the method of administration, e.g. nasal, buccal, pulmonary. For example, nasal formulations may tend to require much lower concentrations of some ingredients in order to avoid irritation or burning of the nasal passages. It is sometimes desirable to dilute an oral formulation up to 10- 100 times in order to provide a suitable nasal formulation.
Certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a single stranded oligonucleotide can include a single treatment or, preferably, can include a series of treatments. It will also be appreciated that the effective dosage of a single stranded oligonucleotide used for treatment may increase or decrease over the course of a particular treatment. For example, the subject can be monitored after administering a single stranded oligonucleotide composition. Based on information from the monitoring, an additional amount of the single stranded
oligonucleotide composition can be administered.
Dosing is dependent on severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved. Optimal dosing schedules can be calculated from measurements of MECP2 expression levels in the body of the patient.
Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In some embodiments, the animal models include transgenic animals that express a human MECP2. In another embodiment, the composition for testing includes a single stranded oligonucleotide that is complementary, at least in an internal region, to a sequence that is conserved between MECP2 in the animal model and the MECP2 in a human.
In one embodiment, the administration of the single stranded oligonucleotide composition is parenteral, e.g. intravenous (e.g. , as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular. Administration can be provided by the subject or by another person, e.g. , a health care provider. The composition can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.
Kits
In certain aspects of the invention, kits are provided, comprising a container housing a composition comprising a single stranded oligonucleotide. In some embodiments, the composition is a pharmaceutical composition comprising a single stranded oligonucleotide and a pharmaceutically acceptable carrier. In some embodiments, the individual components of the pharmaceutical composition may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical composition separately in two or more containers, e.g. , one container for single stranded oligonucleotides, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g. , according to instructions provided with the kit. The components can be combined according to a method described herein, e.g. , to prepare and administer a pharmaceutical composition. The kit can also include a delivery device.
The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.
EXAMPLES
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
MATERIALS AND METHODS:
Real Time PCR RNA is harvested from the cells using Promega SV 96 Total RNA Isolation system or Trizol omitting the DNAse step. RNA harvested from cells is normalized so that equal RNA is input to each reverse transcription reaction. Reverse transcriptase reactions are performed using the Superscript II kit and real time PCR performed on cDNA samples using icycler SYBR green chemistry (Biorad). A baseline level of mRNA expression for MECP2 is determined through quantitative PCR as outlined above. Baseline levels are also determined for mRNA of various housekeeping genes which are constitutively expressed. A "control" housekeeping gene with approximately the same level of baseline expression as the target gene is chosen for comparison purposes.
Cell Culture
Human hepatocyte Hep3B, human hepatocyte HepG2 cells, mouse hepatoma Hepal-6 cells, and human renal proximal tubule epithelial cells (RPTEC) are cultured using conditions known in the art (see, e.g. Current Protocols in Cell Biology).
Oligonucleotide design
Oligonucleotides were designed within PRC2-interacting regions in order to upregulate MECP2. The sequence and structure of each oligonucleotide is shown in Table 3. The following table provides a description of the nucleotide analogs, modifications and intranucleotide linkages used for certain oligonucleotides tested and described in Table 2.
In vitro transfection of cells with oligonucleotides
Oligonucleotides are designed within PRC2-interacting regions in order to upregulate MECP2. Cells are seeded into each well of 24-well plates at a density of 25,000 cells per 500uL and transfections are performed with Lipofectamine and the single stranded oligonucleotides. Control wells contain Lipofectamine alone. At 48 hours post-transfection, approximately 200 uL of cell culture supernatants are stored at -80 C for ELISA. At 48 hours post-transfection, RNA is harvested from the cells and quantitative PCR is carried out as outlined above. The percent induction of target mRNA expression by each oligonucleotide is determined by normalizing mRNA levels in the presence of the oligonucleotide to the mRNA levels in the presence of control (Lipofectamine alone). This is compared side-by- side with the increase in mRNA expression of the "control" housekeeping gene.
In vitro delivery of single stranded oligonucleotides Oligonucleotides are designed as candidates for upregulating MECP2 expression.
The single stranded oligonucleotides are designed to be complementary to a PRC2- interacting region within a sequence as set forth in SEQ ID NO: 1 or 2. The oligonucleotides are tested in at least duplicate. Briefly, cells are transfected in vitro with each of the oligonucleotides as described above. MECP2 expression in cells following treatment is evaluated by qRT-PCR. Oligonucleotides that upregulate MECP2 expression are identified.
TABLE
Table 1: Hexamers that are not seed sequences of human miRNAs
AAAAAA, AAAAAG, AAAACA, AAAAGA, AAAAGC, AAAAGG, AAAAUA, AAACAA, AAACAC, AAACAG, AAACAU, AAACCC, AAACCU, AAACGA, AAACGC, AAACGU, AAACUA, AAACUC, AAACUU, AAAGAU, AAAGCC, AAAGGA, AAAGGG, AAAGUC, AAAUAC, AAAUAU, AAAUCG, AAAUCU, AAAUGC, AAAUGU, AAAUUA, AAAUUG, AACAAC, AACAAG, AACAAU, AACACA, AACACG, AACAGA, AACAGC, AACAGG, AACAUC, AACAUG, AACCAA, AACCAC, AACCAG, AACCAU, AACCCC, AACCCG, AACCGA, AACCGC, AACCGG, AACCUA, AACCUU, AACGAA, AACGAC, AACGAG, AACGAU, AACGCU, AACGGG, AACGGU, AACGUA, AACGUC, AACGUG, AACGUU, AACUAU, AACUCA, AACUCC, AACUCG, AACUGA, AACUGC, AACUGU, AACU UA, AACUUC, AACU UG, AACUUU, AAGAAA, AAGAAG, AAGAAU, AAGACG, AAGAGA, AAGAGC, AAGAGG, AAGAGU, AAGAUU, AAGCAA, AAGCAC, AAGCAG, AAGCAU, AAGCCA, AAGCCC, AAGCCG, AAGCCU, AAGCGA, AAGCGG, AAGCGU, AAGCUA, AAGGAA, AAGGAC, AAGGCU, AAGGGC, AAGGGU, AAGGUU, AAGUAA, AAGUAC, AAGUAU, AAGUCC, AAGUCG, AAGUGA, AAGUGG, AAGUUA, AAGU UU, AAUAAA, AAUAAC, AAUAAG, AAUAAU, AAUACA, AAUACC, AAUACG, AAUAGA, AAUAGC, AAUAGG, AAUAGU, AAUAUC, AAUAU U, AAUCAA, AAUCAU, AAUCCA, AAUCCC, AAUCCG, AAUCGA, AAUCGC, AAUCGU, AAUCUA, AAUCUG, AAUCUU, AAUGAA, AAUGAC, AAUGAG, AAUGAU, AAUGCG, AAUGCU, AAUGGA, AAUGGU, AAUGUA, AAUGUC, AAUGUG, AAUUAA, AAUUAC, AAUUAG, AAU UCC, AAU UCG, AAUUGA, AAUUGG, AAU UGU, AAUUUC, AAU UUG, ACAAAA, ACAAAC, ACAAAG, ACAAAU, ACAACC, ACAACG, ACAACU, ACAAGA, ACAAGC, ACAAGU, ACAAUC, ACAAUG, ACAAUU, ACACAG, ACACCA, ACACCC, ACACCG, ACACCU, ACACGA, ACACGC, ACACGU, ACACUC, ACACUG, ACACUU, ACAGAA, ACAGAC, ACAGCC, ACAGCG, ACAGCU, ACAGGG, ACAGUC, ACAGUG, ACAGU U, ACAUAA, ACAUAC, ACAUCC, ACAUCG, ACAUCU, ACAUGA, ACAUGC, ACAUGU, ACAU UG, ACAU UU, ACCAAA, ACCAAC, ACCAAG, ACCAAU, ACCACC, ACCACG, ACCAGA, ACCAGU, ACCAUA, ACCAUG, ACCAU U, ACCCAA, ACCCAC, ACCCCA, ACCCCG, ACCCGA, ACCCGC, ACCCUA, ACCCUC, ACCCUU, ACCGAA, ACCGAC, ACCGAU, ACCGCA, ACCGCC, ACCGCG, ACCGCU, ACCGGA, ACCGGC, ACCGGU, ACCGUA, ACCGUC, ACCGUG, ACCGUU, ACCUAA, ACCUAC, ACCUAG, ACCUAU, ACCUCA, ACCUCC, ACCUCG, ACCUCU, ACCUGA, ACCUGC, ACCUGU, ACCUUA, ACCUUC, ACCUU U, ACGAAA, ACGAAC, ACGAAG, ACGAAU, ACGACA, ACGACC, ACGACG, ACGACU, ACGAGA, ACGAGC, ACGAGG, ACGAGU, ACGAUA, ACGAUC, ACGAUG, ACGAUU, ACGCAA, ACGCAG, ACGCAU, ACGCCC, ACGCCG, ACGCCU, ACGCGA, ACGCGG, ACGCGU, ACGCUA, ACGCUG, ACGCUU, ACGGAA, ACGGAC, ACGGAG, ACGGAU, ACGGCC, ACGGCG, ACGGCU, ACGGGC, ACGGGG, ACGGGU, ACGGUA, ACGGUC, ACGGUG, ACGGUU, ACGUAA, ACGUAC, ACGUAU, ACGUCC, ACGUCG, ACGUCU, ACGUGA, ACGUGC, ACGUGG, ACGUGU, ACGUUA, ACGU UC, ACGUUG, ACGUU U, AC U AAA, ACUAAG, ACUAAU, ACUACA, ACUACC, ACUACG, ACUACU, ACUAGG, ACUAUC, ACUAUG, ACUAUU, ACUCAU, ACUCCC, ACUCCG, ACUCCU, ACUCGA, ACUCGC, ACUCGG, ACUCUC, ACUCUU, ACUGAG, ACUGAU, ACUGCC, ACUGCG, ACUGCU, ACUGGG, ACUGGU, ACUGUC, ACUUAA, ACU UAC, ACUUAU, ACU UCA, ACUUCC, ACUUCG, ACUUCU, ACUUGA, ACU UGC, ACU UGU, ACUUUA, ACU UUC, ACUU UG, AGAAAA, AGAAAC, AGAAAG, AGAACC, AGAACG, AGAACU, AGAAGC, AGAAGU, AGAAUA, AGAAUC, AGAAUG, AGAAUU, AGACAA, AGACAC, AGACAU, AGACCA, AGACCC, AGACCG, AGACCU, AGACGA, AGACGC, AGACGU, AGACUA, AGACUC, AGACUU, AGAGAC,
AGAGAG, AGAGAU, AGAGCC, AGAGCG, AGAGCU, AGAGGC, AGAGGG, AGAGGU, AGAGUA, AGAGUU, AGAUAC, AGAUAG, AGAUAU, AGAUCC, AGAUCG, AGAUCU, AGAUGA, AGAUGC, AGAUGG, AGAU UA, AGAU UC, AGAUUG, AGAUU U, AGCAAC, AGCACA, AGCACG, AGCACU, AGCAGA, AGCAUA, AGCAUC, AGCAUG, AGCCAA, AGCCAU, AGCCCA, AGCCGA, AGCCGC, AGCCGG, AGCCGU, AGCCUA, AGCCUC, AGCGAA, AGCGAG, AGCGAU, AGCGCA, AGCGCC, AGCGCG, AGCGCU, AGCGGA, AGCGGC, AGCGGU, AGCGUA, AGCGUC, AGCGUG, AGCGUU, AGCUAA, AGCUAC, AGCUAG, AGCUAU, AGCUCA, AGCUCC, AGCUCG, AGCUCU, AGCUGA, AGCUGG, AGCUGU, AGCU UC, AGCUU U, AGGAAU, AGGACC, AGGACG, AGGAGA, AGGAGU, AGGAUA, AGGCAA, AGGCAU, AGGCCG, AGGCGA, AGGCGC, AGGCGG, AGGCUA, AGGCUC, AGGCUU, AGGGAC, AGGGAU, AGGGGA, AGGGGU, AGGGUA, AGGGUG, AGGUAA, AGGUAC, AGGUCA, AGGUCC, AGGUCU, AGGUGA, AGGUGC, AGGUGG, AGGUGU, AGGU UC,
AGGUUG, AGUAAA, AGUAAG, AGUAAU, AGUACA, AGUACG, AGUAGC, AGUAGG, AGUAUA, AGUAUC, AGUAUG, AGUAUU, AGUCAA, AGUCAC, AGUCAG, AGUCAU, AGUCCA, AGUCCG, AGUCCU, AGUCGA, AGUCGC, AGUCGG, AGUCGU, AGUCUA, AGUCUC, AGUCUG, AGUCU U, AGUGAA, AGUGAC, AGUGCG, AGUGGG, AGUGUC, AGU UAA, AGU UAC, AGUUAG, AGUUCC, AGU UCG, AGUUGA, AGUUGC, AGUUGU, AGUUUA, AGUUUC, AGUU UG, AGU UU U, AUAAAC, AUAAAU, AUAACA, AUAACC, AUAACG, AUAACU, AUAAGA, AUAAGC, AUAAGG, AUAAGU, AUAAUC, AUAAUG, AUAAUU, AUACAC, AUACAG, AUACAU, AUACCA, AUACCC, AUACCG, AUACGA, AUACGC, AUACGG, AUACGU, AUACUA, AUACUC, AUACUG, AUACUU, AUAGAA, AUAGAC, AUAGAU, AUAGCA, AUAGCG, AUAGCU, AUAGGA, AUAGGU, AUAGUA, AUAGUC, AUAGUG, AUAGUU, AUAUAC, AUAUAG, AUAUCC, AUAUCG, AUAUCU, AUAUGA, AUAUGC, AUAUGG, AUAUGU, AUAUUC, AUAU UG, AUAUU U, AUCAAA, AUCAAC, AUCAAG, AUCAAU, AUCACA, AUCACC, AUCACG, AUCAGC, AUCAGG, AUCCAA, AUCCAU, AUCCCC, AUCCCG, AUCCGA, AUCCGC, AUCCGG, AUCCUA, AUCCUC, AUCCUG, AUCGAA, AUCGAC, AUCGAG, AUCGAU, AUCGCA, AUCGCC, AUCGCG, AUCGCU, AUCGGC, AUCGGG, AUCGGU, AUCGUC, AUCGUG, AUCGU U, AUCUAA, AUCUAC, AUCUAG, AUCUAU, AUCUCC, AUCUCG, AUCUGU, AUCUUG, AUCUU U, AUGAAA, AUGAAC, AUGAAG, AUGAAU, AUGACC, AUGACU, AUGAGG, AUGAGU, AUGAUA, AUGAUC, AUGAU U, AUGCAA, AUGCAG, AUGCCA, AUGCCC, AUGCCG, AUGCGA, AUGCGG, AUGCGU, AUGCUC, AUGCUU, AUGGAC, AUGGCC, AUGGGA, AUGGGC, AUGGGU, AUGGUC, AUGGUG, AUGUAC, AUGUAU, AUGUCA, AUGUCC, AUGUCG, AUGUGU, AUGUUA, AUGUUC, AU UAAA, AUUAAC, AU UAAG, AU UAAU, AU UACA, AUUACC, AUUACG, AUUACU, AU UAGA, AUUAGC, AUUAGG, AUUAGU, AU UAUA, AUUAUC, AUUAUG, AUUCAC, AUUCCA, AU UCCG, AU UCCU, AUUCGA, AUUCGC, AUUCGG, AU UCGU, AUUCUA, AUUCUC, AUUCU U, AUUGAA, AUUGAC, AUUGAU, AU UGCC, AUUGCG, AU UGCU, AUUGGA, AUUGGC,
AUUGGG, AU UGGU, AUUGUA, AUUGUC, AUUGUG, AU UGU U, AU UUAA, AU UUAG, AU UUAU, AUU UCC, AUU UCG, AUU UCU, AU UUGA, AU UUGC, AU UUGU, AU UUUA, AU UU UC, AUU UUG, AUU UU U, CAAAAG, CAAACA, CAAACC, CAAACG, CAAACU, CAAAGA, CAAAGG, CAAAUA, CAAAUU, CAACAC, CAACAU, CAACCA, CAACCC, CAACCG, CAACGA, CAACGC, CAACGG, CAACGU, CAACUA, CAACUC, CAACUG, CAACUU, CAAGAA, CAAGAC, CAAGAU, CAAGCA, CAAGCC, CAAGCG, CAAGCU, CAAGGA, CAAGGG, CAAGUC, CAAGUG, CAAGU U, CAAUAA, CAAUAC, CAAUAG, CAAUCC, CAAUCG, CAAUCU, CAAUGA, CAAUGC, CAAUGG, CAAUGU, CAAU UC, CAAU UG, CAAU UU, CACAAU, CACACA, CACACG, CACACU, CACAGA, CACAGC, CACAGG, CACAUA, CACAUC, CACAUU, CACCAA, CACCAC, CACCAU, CACCCA, CACCCC, CACCCG, CACCGA, CACCGC, CACCGG, CACCGU, CACCUA, CACCU U, CACGAA, CACGAC, CACGAG, CACGAU, CACGCA, CACGCC, CACGCU, CACGGA, CACGGC, CACGGG, CACGG U, CACGUA, CACGUC, CACGUG, CACGU U, CACUAA, CACUAG, CACUAU, CACUCA, CACUCG, CACUGA, CACUGC, CACUGG, CACUUA, CACU UC, CACU UU, CAGAAA, CAGAAG, CAGAAU, CAGACC, CAGACG, CAGAGC, CAGAUA, CAGAUC, CAGCCG, CAGCCU, CAGCGA, CAGCGC, CAGCGG, CAGCGU, CAGCUC, CAGCUU, CAGGAU, CAGGGG, CAGGGU, CAGGUA, CAGGUC, CAGGUU, CAGUAC, CAGUCG, CAGU UG, CAUAAA, CAUAAC, CAUAAG, CAUAAU, CAUACA, CAUACC, CAUACG, CAUACU, CAUAGA, CAUAGG, CAUAGU, CAUAUA, CAUAUC, CAUAUG, CAUCAA, CAUCAC, CAUCAG, CAUCAU, CAUCCA, CAUCCC, CAUCCG, CAUCGA, CAUCGC, CAUCGG, CAUCGU, CAUCUA, CAUCUC, CAUCUG, CAUCUU, CAUGAA, CAUGAC, CAUGAG, CAUGAU, CAUGCA, CAUGCC, CAUGCG, CAUGCU, CAUGGC, CAUGGG, CAUGGU, CAUGUA, CAUGUC, CAUGUU, CAU UAA, CAUUAC, CAUUAG, CAUUCA, CAU UCC, CAU UCG, CAU UCU, CAU UGA, CAU UGG, CAUU UC, CAU UUG, CAUU UU, CCAAAA, CCAAAC, CCAAAG, CCAAAU, CCAACA, CCAACC, CCAACG, CCAACU, CCAAGA, CCAAGC, CCAAGG, CCAAUC, CCAAUG, CCAAU U, CCACAA, CCACAC, CCACAG, CCACAU, CCACCA, CCACCC, CCACCG, CCACCU, CCACGA, CCACGC, CCACGG, CCACGU, CCACUA, CCACUC, CCACUU, CCAGAA, CCAGAC, CCAGAG, CCAGCC, CCAGGU, CCAGUC, CCAGUU, CCAUAA, CCAUAC, CCAUAG, CCAUAU, CCAUCA, CCAUCC, CCAUCU, CCAUGA, CCAUGC, CCAUGG, CCAUUC, CCAUUG, CCAUU U, CCCAAC, CCCAAG, CCCAAU, CCCACA, CCCAGA, CCCAGC, CCCAGU, CCCAUA, CCCAUC, CCCAUG, CCCAUU, CCCCAA, CCCCAG, CCCCAU, CCCCCC, CCCCCG, CCCCCU, CCCCGA, CCCCGC, CCCCGU, CCCCUA, CCCCUC, CCCGAA, CCCGAC, CCCGAU, CCCGCA, CCCGCU, CCCGGA, CCCGGC, CCCGUA, CCCGUG, CCCGU U, CCCUAA, CCCUAG, CCCUCA, CCCUCU, CCCUGC, CCCUUA, CCCU UC, CCCU UU, CCGAAA, CCGAAC, CCGAAU, CCGACA, CCGACC, CCGACG, CCGACU, CCGAGA, CCGAGG, CCGAGU, CCGAUA, CCGAUC, CCGAUG, CCGAU U, CCGCAA, CCGCAC, CCGCAG, CCGCAU, CCGCCA, CCGCCC, CCGCCG, CCGCCU, CCGCGA, CCGCGC, CCGCGG, CCGCGU, CCGCUA, CCGCUC, CCGCUG, CCGCU U, CCGGAA, CCGGAU, CCGGCA, CCGGCC, CCGGCG, CCGGCU, CCGGGA, CCGGGC, CCGGGG, CCGGGU, CCGGUA, CCGGUC, CCGGUG, CCGUAA, CCGUAG, CCGUAU, CCGUCA, CCGUCC, CCGUCG, CCGUGA, CCGUGU, CCGUUA, CCGUUC, CCGU UG, CCGUU U, CCUAAC, CCUAAG, CCUAAU, CCUACA, CCUACC, CCUACG, CCUACU, CCUAGA, CCUAGC, CCUAGG, CCUAGU, CCUAUA, CCUAUC, CCUAUG, CCUAUU, CCUCAA, CCUCAC, CCUCAG, CCUCAU, CCUCCA, CCUCCC, CCUCCG, CCUCGA, CCUCGC, CCUCGG, CCUCGU, CCUCUA, CCUCUG, CCUGAC, CCUGAU, CCUGCA, CCUGGG, CCUGGU, CCUGU U, CCUUAA, CCUUAC, CCUUAG, CCUUAU, CCUUCG, CCUUGA, CCUUGU, CCU UUA, CCUU UC, CCU UU U, CGAAAA, CGAAAC, CGAAAG, CGAAAU, CGAACA, CGAACC, CGAACG, CGAACU, CGAAGA, CGAAGC, CGAAGG, CGAAGU, CGAAUA, CGAAUC, CGAAUG, CGAAUU, CGACAA, CGACAC, CGACAU, CGACCA, CGACCU, CGACGA, CGACGC, CGACGG, CGACGU, CGACUA, CGACUG, CGACU U, CGAGAA, CGAGAC, CGAGAG, CGAGAU, CGAGCA, CGAGCC, CGAGCG, CGAGCU, CGAGGC, CGAGGG, CGAGGU, CGAGUA, CGAGUC, CGAGUG, CGAGUU, CGAUAA, CGAUAC, CGAUAG, CGAUAU, CGAUCA, CGAUCC, CGAUCG, CGAUCU, CGAUGA, CGAUGC, CGAUGG, CGAUGU, CGAUUA, CGAU UC, CGAUUG, CGAUU U, CGCAAA, CGCAAC, CGCAAG, CGCAAU, CGCACA, CGCACC, CGCACG, CGCAGA, CGCAGC, CGCAGG, CGCAGU, CGCAUA, CGCAUC, CGCAUG, CGCAU U, CGCCAA, CGCCAC, CGCCAG, CGCCAU, CGCCCA, CGCCCC, CGCCCG, CGCCGA, CGCCGC, CGCCGG, CGCCGU, CGCCUA, CGCCUG, CGCCUU, CGCGAA, CGCGAC, CGCGAG, CGCGAU, CGCGCA, CGCGCC, CGCGCG, CGCGCU, CGCGGA, CGCGGC, CGCGGG, CGCGGU, CGCGUA, CGCGUC, CGCGUG, CGCGU U, CGCUAA, CGCUAC, CGCUAG, CGCUAU, CGCUCA, CGCUCC, CGCUCG, CGCUCU, CGCUGA, CGCUGC, CGCUGG, CGCUGU, CGCUUA, CGCU UC, CGCU UG, CGGAAA, CGGAAC, CGGAAG, CGGACA, CGGACC, CGGACG, CGGACU, CGGAGC, CGGAGG, CGGAGU, CGGAUA, CGGAU U, CGGCAA, CGGCAC, CGGCAG, CGGCCA, CGGCCC, CGGCCG, CGGCGC, CGGCGG, CGGCGU, CGGCUA, CGGCUC, CGGCUG, CGGCU U, CGGGAA, CGGGAC, CGGGAG, CGGGAU, CGGGCA, CGGGCC, CGGGCG, CGGGCU, CGGGGU, CGGGUA, CGGGUC, CGGGUG, CGGUAA, CGGUAC, CGGUAG, CGGUAU, CGGUCA, CGGUCG, CGGUCU, CGGUGA, CGGUGG, CGGUGU, CGGU UA, CGGU UC, CGGUUG, CGGUUU, CGUAAA, CGUAAC, CGUAAG, CGUAAU, CGUACA, CGUACG, CGUACU, CGUAGA, CGUAGC, CGUAGG, CGUAGU, CGUAUA, CGUAUC, CGUAUG, CGUAUU, CGUCAA, CGUCAC, CGUCAG, CGUCAU, CGUCCA, CGUCCC, CGUCCG, CGUCCU, CG UCGA, CGUCGG, CGUCGU, CGUCUA, CGUCUC, CGUCUG, CGUCU U, CGUGAA, CGUGAC, CGUGAG, CGUGAU, CGUGCC, CGUGCG, CGUGCU, CGUGGA, CGUGGG, CGUGGU, CGUGUA, CGUGUG, CGUUAA, CGUUAC, CGU UAG,
CGU UAU, CGUUCA, CGUUCC, CGUUCG, CGU UCU, CGUUGA, CGUUGC, CGU UGU, CGU UUA, CGUUUC, CGU U UU, CUAAAA, CUAAAC, CUAAAU, CUAACA, CUAACC, CUAACG, CUAACU, CUAAGA, CUAAGC, CUAAGU, CUAAUA, CUAAUC, CUAAUG, CUACAC, CUACAU, CUACCA, CUACCC, CUACCG, CUACCU, CUACGA, CUACGC, CUACGG, CUACGU, CUACUA, CUACUC, CUACUG, CUAGAA, CUAGAG, CUAGAU, CUAGCA, CUAGCC, CUAGCG, CUAGCU, CUAGGA, CUAGGG, CUAGGU, CUAGUG, CUAGUU, CUAUAA, CUAUAG, CUAUAU, CUAUCA, CUAUCC, CUAUCG, CUAUCU, CUAUGA, CUAUGC, CUAUGG, CUAUGU, CUAUUA, CUAUUG, CUCAAC, CUCAAG, CUCAAU, CUCACC, CUCACG, CUCAGC, CUCAUA, CUCAUC, CUCAUG, CUCAU U, CUCCAC, CUCCCC, CUCCCG, CUCCGA, CUCCGC, CUCCGG, CUCCUA, CUCCUC, CUCCU U, CUCGAA, CUCGAC, CUCGAG, CUCGAU, CUCGCA, CUCGCC, CUCGCG, CUCGGG, CUCGGU, CUCGUA, CUCGUC, CUCGUG, CUCGU U, CUCUAA, CUCUAC, CUCUAU, CUCUCA, CUCUCC, CUCUCU, CUCUGC, CUCUGU, CUCUUA, CUCU UG, CUGAAG, CUGACC, CUGACG, CUGAGC, CUGAUA, CUGAUC, CUGCCG, CUGCCU, CUGCGA, CUGCUA, CUGCUU, CUGGAG, CUGGAU, CUGGCG, CUGGGU, CUGUAC, CUGUCA, CUGUCC, CUGUCG, CUGUGG, CUGUGU, CUGUUA, CUGU UU, CUUAAC, CUUAAG, CU UAAU, CU UACC, CUUACG, CUUAGA, CU UAGC, CU UAGG, CUUAGU, CUUAUA, CU UAUC, CU UAUG, CUUAUU, CU UCAG, CU UCAU, CUUCCA, CUUCCC, CU UCCG, CU UCCU, CUUCGA, CUUCGC, CU UCGG, CUUCGU, CU UCUA, CUUGAC, CUUGAG, CUUGAU, CUUGCA, CUUGCC, CUUGCG, CU UGCU, CUUGGC, CUUGGU, CU UGUU, CUU UAC, CU UUAG, CU UUAU, CUU UCA, CUU UCG, CU UUCU, CUUUGA, CU UUGC, CU UUGU, CU U UUA, CUUU UC, CUU UUG, CUU UUU, GAAAAA, GAAAAG, GAAAAU, GAAACC, GAAACG, GAAAGA, GAAAGC, GAAAGU, GAAAUA, GAAAUC, GAAAUG, GAAAUU, GAACAA, GAACAC, GAACAG, GAACAU, GAACCA, GAACCC, GAACCG, GAACCU, GAACGA, GAACGC, GAACGG, GAACGU, GAACUA, GAACUG, GAACUU, GAAGAC, GAAGAG, GAAGCA, GAAGCG, GAAGCU, GAAGUC, GAAUAA, GAAUAC, GAAUAG, GAAUAU, GAAUCC, GAAUCG, GAAUCU, GAAUGA, GAAUGC, GAAUGU, GAAU UA, GAAU UC, GAAUU U, GACAAA, GACAAG, GACAAU, GACACC, GACAGA, GACAGG, GACAUA, GACAUG, GACAUU, GACCAA, GACCAC, GACCAG, GACCCA, GACCCC, GACCCG, GACCGC, GACCGG, GACCGU, GACCUA, GACCUC, GACCU U, GACGAA, GACGAC, GACGAG, GACGAU, GACGCA, GACGCC, GACGCG, GACGCU, GACGGA, GACGGC, GACGGG, GACGGU, GACGUA, GACGUC, GACGUG, GACGUU, GACUAA, GACUAC, GACUAG, GACUAU, GACUCA, GACUCC, GACUCG, GACUGG, GACUGU, GACUUA, GACUUG, GACUU U, GAGAAU, GAGAGA, GAGAGC, GAGAGG, GAGAUA, GAGAUC, GAGCAA, GAGCAU, GAGCCA, GAGCGA, GAGCGG, GAGCGU, GAGGGU, GAGGUC, GAGGUG, GAGUAA, GAGUAG, GAGUCC, GAGUUC, GAGU UU, GAUAAA, GAUAAC, GAUAAG, GAUAAU, GAUACA, GAUACC, GAUACG, GAUACU, GAUAGA, GAUAGC, GAUAGG, GAUAGU, GAUAUA, GAUCAA, GAUCAC, GAUCAU, GAUCCA, GAUCCC, GAUCCU, GAUCGC, GAUCGG, GAUCGU, GAUCUA, GAUCUG, GAUCU U, GAUGAA, GAUGAC, GAUGAG, GAUGCA, GAUGCC, GAUGCG, GAUGCU, GAUGGC, GAUGGG, GAUGGU, GAUGUG, GAUGUU, GAUUAA, GAUUAC, GAUUAG, GAUUAU, GAUUCA, GAUUCG, GAU UCU, GAUUGA, GAUUGC, GAU UUA, GAU UUC, GAUU UG, GAUUU U, GCAAAC, GCAAAG, GCAAAU, GCAACA, GCAACC, GCAAGC, GCAAGU, GCAAUA, GCAAUC, GCAAUG, GCAAUU, GCACAA, GCACAC, GCACAG, GCACCC, GCACCG, GCACCU, GCACGA, GCACGC, GCACGU, GCACUA, GCACUC, GCACUG, GCACUU, GCAGAU, GCAGCC, GCAGCG, GCAGGC, GCAGUA, GCAGUC, GCAGUG, GCAGUU, GCAUAA, GCAUAG, GCAUAU, GCAUCG, GCAUCU, GCAUGA, GCAUGC, GCAUGG, GCAUGU, GCAU UA, GCAU UC, GCAUUG, GCAUU U, GCCAAA, GCCAAC, GCCAAU, GCCACA, GCCACC, GCCACG, GCCAGA, GCCAGU, GCCAUA, GCCAUC, GCCAUG, GCCAUU, GCCCAA, GCCCAC, GCCCAG, GCCCCG, GCCCGA, GCCCGG, GCCCGU, GCCGAA, GCCGAC, GCCGAG, GCCGAU, GCCGCA, GCCGCU, GCCGGA, GCCGGC, GCCGGG, GCCGGU, GCCGUA, GCCGUC, GCCGUG, GCCGU U, GCCUAA, GCCUAU, GCCUCA, GCCUCC, GCCUCG, GCCUGA, GCCU UA, GCCU UU, GCGAAA, GCGAAC, GCGAAG, GCGAAU, GCGACC, GCGACG, GCGACU, GCGAGA, GCGAGC, GCGAGG, GCGAGU, GCGAUA, GCGAUC, GCGAUG, GCGAUU, GCGCAA, GCGCAC, GCGCAG, GCGCAU, GCGCCA, GCGCCC, GCGCCU, GCGCGA, GCGCGU, GCGCUA, GCGCUC, GCGCUG, GCGCUU, GCGGAA, GCGGAC, GCGGAU, GCGGCA, GCGGCC, GCGGCU, GCGGGA, GCGGUA, GCGGUC, GCGGUU, GCGUAA, GCGUAC, GCGUAG, GCGUAU, GCGUCA, GCGUCC, GCGUCG, GCGUCU, GCGUGA, GCGUGC, GCGUGG, GCGUGU, GCGU UA, GCGUUC, GCGU UG, GCGUU U, GCUAAA, GCUAAC, GCUAAG, GCUAAU, GCUACC, GCUACG, GCUACU, GCUAGA, GCUAGG, GCUAGU, GCUAUA, GCUAUC, GCUAUU, GCUCAA, GCUCAC, GCUCAG, GCUCAU, GCUCCA, GCUCCC, GCUCCG, GCUCGA, GCUCGC, GCUCGU, GCUCUA, GCUCUC, GCUCU U, GCUGAA, GCUGAC, GCUGAU, GCUGCA, GCUGCC, GCUGCG, GCUGCU, GCUGUG, GCUGUU, GCUUAC, GCUUAG, GCUUAU, GCU UCA, GCUUCG, GCU UGA, GCUUGG, GCUUGU, GCUU UA, GCU UUG, GGAAAG, GGAACA, GGAACC, GGAACG, GGAACU, GGAAGU, GGAAUA, GGAAUC, GGAAU U, GGACAA, GGACAC, GGACAG, GGACAU, GGACCG, GGACGA, GGACGC, GGACGU, GGACUA, GGACUC, GGACU U, GGAGAC, GGAGCA, GGAGCG, GGAGGG, GGAGUA, GGAUAA, GGAUAC, GGAUCA, GGAUCC, GGAUCG, GGAUCU, GGAUGC, GGAUUA, GGAUUG, GGCAAU, GGCACA, GGCACU, GGCAGA, GGCAUA, GGCAUC, GGCCAC, GGCCAG, GGCCCC, GGCCGA, GGCCGC, GGCCGU, GGCCUA, GGCCUG, GGCCU U, GGCGAA, GGCGAG, GGCGAU, GGCGCA, GGCGCU, GGCGGU, GGCGUA, GGCGUC, GGCGUG, GGCGU U, GGCUAA, GGCUAC, GGCUAG, GGCUAU, GGCUCC, GGCUCG, GGCUGA, GGCU UA, GGCUUC, GGCUUG, GGGAAU, GGGACA, GGGAGA, GGGAGU, GGGAUA, GGGAU U, GGGCAA, GGGCAC, GGGCAG, GGGCCG, GGGCGG, GGGGCC, GGGGGG,
GGGGGU, GGGGUA, GGGUAC, GGGUAU, GGGUCA, GGGUCC, GGGUCG, GGGUGA, GGGUGC, GGGU UA, GGGU UG, GGUAAA, GGUAAC, GGUAAG, GGUAAU, GGUACA, GGUACC, GGUACG,
GGUACU, GGUAGC, GGUAGG, GGUAGU, GGUAUA, GGUAUC, GGUAUG, GGUCAA, GGUCAC, GGUCAG, GGUCAU, GGUCCA, GGUCCG, GGUCCU, GGUCGA, GGUCGC, GG UCGG, GGUCGU, GGUCUC, GGUCU U, GGUGAA, GGUGAC, GGUGAU, GGUGCA, GGUGCC, GGUGGC, GGUGUA, GGUGUC, GGU UAA, GGU UAG, GGU UAU, GGUUCA, GGU UCC, GGU UCG, GGU UGC, GGU UUC, GGUU UU, GUAAAA, GUAAAG, GUAAAU, GUAACC, GUAACG, GUAACU, GUAAGA, GUAAGC, GUAAGG, GUAAGU, GUAAUA, GUAAUC, GUAAUG, GUAAUU, GUACAA, GUACAC, GUACAG, GUACAU, GUACCA, GUACCC, GUACCG, GUACCU, GUACGA, GUACGC, GUACGG, GUACGU, GUACUA, GUACUC, GUACUG, GUACUU, GUAGAA, GUAGAC, GUAGCA, GUAGCC, GUAGCG, GUAGCU, GUAGGA, GUAGGC, GUAGGG,
GUAGGU, GUAGUA, GUAGUC, GUAUAA, GUAUAC, GUAUAG, GUAUAU, GUAUCA, GUAUCG, GUAUCU, GUAUGA, GUAUGC, GUAUGG, GUAUUA, GUAU UG, G UAU UU, GUCAAA, GUCAAG,
GUCAAU, GUCACA, GUCACC, GUCACG, GUCAGA, GUCAGC, GUCAGG, GUCAUA, GUCAUC, GUCAUG, GUCCAA, GUCCAC, GUCCAU, GUCCCC, GUCCCU, GUCCGA, GUCCGC, GUCCGG, GUCCGU, GUCCUA, GUCCUG, GUCCU U, GUCGAA, GUCGAC, GUCGAG, GUCGAU, GUCGCA, GUCGCC, GUCGCG, GUCGCU, GUCGGA, GUCGGC, GUCGGG, GUCGGU, GUCGUA, GUCGUC, GUCGU U, GUCUAA, GUCUAG, GUCUCA, GUCUCC, GUCUCG, GUCUGA, GUCUGG, GUCUGU, GUCU UC, GUCU UU, GUGAAA, GUGAAC, GUGAAG, GUGACC, GUGACG, GUGAGA, GUGAGC, GUGAGU, GUGAUC, GUGAUG, GUGAUU, GUGCAC,
GUGCAU, GUGCCC, GUGCCG, GUGCGA, GUGCGG, GUGCGU, GUGCUA, GUGCUC, GUGCUG,
GUGGAG, GUGGCG, GUGGCU, GUGGGU, GUGGUC, GUGGUG, GUGUAA, GUGUAG, GUGUCG, GUGUGA, GUGUGC, GUGUGU, GUGUUG, GUGU UU, GU UAAA, GUUAAC, GUUAAG, GU UACA, GU UACC, GUUACG, GU UACU, GU UAGA, GUUAGC, GUUAGU, GUUAUA, GUUAUC, GUUAUG,
GU UAUU, GUUCAA, GUUCAC, GUUCAG, GU UCCA, GUUCCG, GUUCGA, GU UCGC, GU UCGG, GU UCGU, GU UCUA, GUUCUG, GUUGAA, GU UGAC, GUUGAG, GUUGAU, GUUGCG, GUUGCU, GUUGGA, GU UGGC, GU UGGU, GU UGUC, GUUGUG, GUUGU U, GUU UAA, GU UUAC, GU UUAG, GUU UAU, GU UUCA, GUUUCC, GU UUCU, GU UUGA, GUU UGC, GUU UGG, GUU UGU, GU UUUA, GU UU UC, GU UU UU, UAAAAA, UAAAAC, UAAAAG, UAAAAU, UAAACA, UAAACC, UAAACG, UAAACU, UAAAGA, UAAAGG, UAAAGU, UAAAUA, UAAAUC, UAAAUG, UAAAU U, U A AC A A, UAACAC, UAACAG, UAACCA, UAACCC, UAACCG, UAACCU, UAACGA, UAACGC, UAACGG, UAACGU, UAACUA, UAACUG, UAACUU, UAAGAG, UAAGAU, UAAGCA, UAAGCC, UAAGCG, UAAGCU, UAAGGA, UAAGGC, UAAGGG, UAAGGU, UAAGUA, UAAGUC, UAAGUG, UAAGUU, UAAUAA, UAAUCA, UAAUCC, UAAUCG, UAAUCU, UAAUGA, UAAUGG, UAAUGU, UAAUUA, UAAU UC, UAAUUG, UACAAC, UACAAG, UACAAU, UACACC, UACACG, UACACU, UACAGA, UACAGC, UACAUA, UACAUC, UACAU U, UACCAA, UACCAC, UACCAG, UACCAU, UACCCC, UACCCG, UACCCU, UACCGA, UACCGC, UACCGG, UACCGU, UACCUA, UACCUG, UACGAA, UACGAC, UACGAG, UACGAU, UACGCA, UACGCC, UACGCG, UACGCU, UACGGC, UACGGG, UACGGU, UACGUA, UACGUC, UACGUG, UACGUU, UACUAA, UACUAC, UACUAG, UACUAU, UACUCA, UACUCC, UACUCG, UACUCU, UACUGA, UACUGC, UACUGG, UACU UA, UACU UG, UACU UU, UAGAAA, UAGAAG, UAGAAU, UAGACA, UAGACG, UAGAGA, UAGAGC, UAGAGU, UAGAUA, UAGAUC, UAGAUG, UAGCAU, UAGCCC, UAGCCG, UAGCCU, UAGCGA, UAGCGC, UAGCGU, UAGCUA, UAGCUC, UAGCUG, UAGGAA, UAGGAU, UAGGCG, UAGGCU, UAGGGU, UAGGUC, UAGGUG, UAGGUU, UAGUAA, UAGUAC, UAGUAG, UAGUAU, UAGUCA, UAGUCG, UAGUGU, UAGUUA, UAGU UC, UAGU UG, UAGUU U, UAUAAC, UAUAAG, UAUACU, UAUAGA, UAUAGC, UAUAGG, UAUAGU, UAUAUA, UAUAUC, UAUAUG, UAUAU U, UAUCAA, UAUCAC, UAUCAU, UAUCCA, UAUCCC, UAUCCG, UAUCCU, UAUCGA, UAUCGC, UAUCGG, UAUCGU, UAUCUA, UAUCUC, UAUCUG, UAUCUU, UAUGAA, UAUGAC, UAUGAG,
UAUGAU, UAUGCA, UAUGCG, UAUGCU, UAUGGA, UAUGGC, UAUGUC, UAUGUG, UAUGU U, UAU UAG, UAUUCA, UAU UCC, UAUUCG, UAUUCU, UAUUGA, UAUUGG, UAUU UA, UAU UUC, UAU UUG, UAUUU U, UCAAAA, UCAAAC, UCAAAG, UCAACC, UCAACU, UCAAGA, UCAAGC, UCAAUA, UCAAUC, UCAAUG, UCAAUU, UCACCC, UCACCG, UCACCU, UCACGA, UCACGC, UCACGG, UCACGU, UCACUA, UCACUC, UCACUU, UCAGAA, UCAGAC, UCAGAG, UCAGCG, UCAGCU, UCAGGA, UCAGGC, UCAGGU, UCAGUC, UCAGU U, UCAUAA, UCAUCA, UCAUCC, UCAUCG, UCAUGC, UCAUGG, UCAUGU, UCAUUA, UCAUUG, UCCAAA, UCCAAC, UCCAAG, UCCAAU, UCCACA, UCCACC, UCCACG, UCCAGC, UCCAGG, UCCAUA, UCCAUC, UCCAU U, UCCCAA, UCCCAG, UCCCAU, UCCCCC, UCCCCG, UCCCCU, UCCCGA, UCCCGC, UCCCGG, UCCCGU, UCCCUA, UCCCUC, UCCGAA, UCCGAC, UCCGAG, UCCGAU, UCCGCA, UCCGCC, UCCGGA, UCCGGC, UCCGGU, UCCGUA, UCCGUC, UCCGUG, UCCUAA, UCCUCA, UCCUCG, UCCUCU, UCCUGC, UCCUGU, UCCU UA, UCCU UC, UCCUU U, UCGAAA, UCGAAC, UCGAAG, UCGAAU, UCGACA, UCGACC, UCGACG, UCGACU, UCGAGA, UCGAGC, UCGAGG, UCGAUA, UCGAUC, UCGAUG, UCGAU U, UCGCAA, UCGCAC, UCGCAG, UCGCAU, UCGCCA, UCGCCC, UCGCCG, UCGCCU, UCGCGA, UCGCGC, UCGCGU, UCGCUA, UCGCUC, UCGGAA, UCGGAC, UCGGAG, UCGGAU, UCGGCA, UCGGCU, UCGGGG, UCGGGU, UCGGUC, UCGGUG, UCGGU U, UCGUAA, UCGUAC, UCGUAG,
UCGUAU, UCGUCA, UCGUCC, UCGUCG, UCGUCU, UCGUGA, UCGUGU, UCGUUA, UCGU UC, UCGU UG, UCGU UU, UCUAAC, UCUAAG, UCUAAU, UCUACA, UCUACC, UCUACG, UCUACU, UCUAGC, UCUAGG, UCUAGU, UCUAUA, UCUAUC, UCUAUG, UCUAUU, UCUCAG, UCUCAU, UCUCCG, UCUCGC, UCUCGG, UCUCGU, UCUCUC, UCUGAA, UCUGAU, UCUGCA, UCUGCG, UCUGCU, UCUGGC, UCUGGU, UCUGUC, UCUGUG, UCUGU U, UCUUAA, UCU UAC, UCUUAG, UCU UAU, UCU UCA, UCUUCC, UCUUCG, UCUUCU, UCU UGC, UCU UGG, UCUUGU, UCUU UA, UCU UUC, UCU UUG, UCU UU U, UGAAAA, UGAAAC,
UGAACA, UGAACC, UGAAGG, UGAAUC, UGAAUG, UGACAA, UGACAC, UGACAG, UGACCA, UGACCC, UGACCG, UGACGA, UGACGC, UGACGG, UGACGU, UGACUA, UGACUC, UGACU U, UGAGAG, UGAGAU, UGAGCA, UGAGCC, UGAGCU, UGAGGC, UGAGGU, UGAGUA, UGAGUU, UGAUAC, UGAUAG,
UGAUAU, UGAUCA, UGAUCG, UGAUCU, UGAUGA, UGAUGC, UGAUGG, UGAUGU, UGAUUA, UGAU UC, UGAUUG, UGAUU U, UGCAAC, UGCAAG, UGCACA, UGCACG, UGCAGG, UGCAGU, UGCAUC, UGCCCA, UGCCCC, UGCCCG, UGCCGA, UGCCGC, UGCCGG, UGCCGU, UGCCUA, UGCCUC, UGCCUG, UGCCU U, UGCGAA, UGCGAC, UGCGAU, UGCGCC, UGCGCG, UGCGCU, UGCGGC, UGCGGG, UGCGGU, UGCGUA, UGCGUC, UGCGUG, UGCGUU, UGCUAC, UGCUAU, UGCUCC, UGCUCG, UGCUGC, UGCUGG, UGCUGU, UGCUUA, UGCUUU, UGGAAC, UGGAAG, UGGAGC, UGGAUC, UGGAUU, UGGCAA, UGGCAC, UGGCAG, UGGCCG, UGGCCU, UGGCGA, UGGCGC, UGGCGU, UGGCUA, UGGCUC, UGGCUU, UGGGAA, UGGGCA, UGGGCC, UGGGGC, UGGGUC, UGGUAA, UGGUAG, UGGUAU, UGGUCC, UGGUCG, UGGUCU, UGGUGA, UGGUGC, UGGUGG, UGGUGU, UGGU UA, UGGUUG, UGUAAA, UGUAAC, UGUAAG, UGUACC, UGUACG, UGUACU, UGUAGA, UGUAGC, UGUAGU, UGUAUC,
UGUAUU, UGUCAA, UGUCAC, UGUCAG, UGUCAU, UGUCCA, UGUCCC, UG UCCG, UGUCGA, UGUCGC, UGUCGG, UGUCGU, UGUCUA, UGUCUC, UGUGAC, UGUGAG, UGUGAU, UGUGCA, UGUGGU, UGUGUA, UGUGUU, UGUUAC, UGUUAG, UGUUAU, UGUUCA, UGUUCC, UGUUCG, UGUUGG, UGUUGU, UGUUUA, UGUUUC, UGUUUG, UGUUUU, UUAAAA, UUAAAC, UUAAAG, UUAAAU, UUAACC, UUAACG, UUAACU, UUAAGU, UUAAUA, UUAAUC, UUAAUG, UUAAUU, UUACAA, UUACAC, UUACAG, UUACAU, UUACCA, UUACCC, UUACCG, UUACCU, UUACGA, UUACGC, UUACGG, UUACGU, UUACUA, UUACUC, UUACUG, UUACUU, UUAGAA, UUAGAC, UUAGCC, UUAGCG, UUAGCU, UUAGGC, UUAGGU, UUAGUA, UUAGUC, UUAGUU, UUAUAA, UUAUAC, UUAUAG, UUAUAU, UUAUCC, UUAUCG, UUAUCU, UUAUGA, UUAUGG, UUAUGU, UUAUUA, UUAUUC, UUAUUG, UUAUUU, UUCAAC, UUCAAU, UUCACA, UUCACC, UUCACG, UUCACU, UUCAGC, UUCAGG, UUCAGU, UUCAUA, UUCAUC, UUCAUG, UUCAUU, UUCCAA, UUCCCA, UUCCCG, UUCCGA, UUCCGU, UUCCUU, UUCGAA, UUCGAC, UUCGAG, UUCGAU, UUCGCA, UUCGCC, UUCGCG, UUCGCU, UUCGGA, UUCGGC, UUCGGG, UUCGGU, UUCGUA, UUCGUC, UUCGUG, UUCGUU, UUCUAC, UUCUAG, UUCUCA, UUCUCG,
UUCUGG, UUCUUA, UUCUUU, UUGAAA, UUGAAC, UUGAAG, UUGAAU, UUGACC, UUGACG, UUGACU, UUGAGA, UUGAGC, UUGAGU, UUGAUA, UUGAUC, UUGAUG, UUGAUU, UUGCAA, UUGCAC, UUGCAG, UUGCAU, UUGCCC, UUGCCG, UUGCGA, UUGCGC, UUGCGG, UUGCGU, UUGCUA, UUGCUC, UUGCUG, UUGCUU, UUGGAA, UUGGAG, UUGGCC, UUGGCG, UUGGCU, UUGGGC, UUGGGU, UUGGUA, UUGGUG, UUGUAA, UUGUAC, UUGUCA, UUGUCG, UUGUCU, UUGUGC, UUGUGG, UUGUUA, UUGUUG, UUGUUU, UUUAAA, UUUAAC, UUUAAG, UUUAAU, UUUACA, UUUACC, UUUACG, UUUACU, UUUAGA, UUUAGC, UUUAGG, UUUAGU, UUUAUA, UUUAUC, UUUAUG, UUUAUU, UUUCAU, UUUCCA, UUUCCG, UUUCCU, UUUCGA, UUUCGC, UUUCGG, UUUCGU, UUUCUA, UUUCUC, UUUCUG, UUUCUU, UUUGAA, UUUGAC, UUUGAG, UUUGAU, UUUGCC, UUUGCU, UUUGGA, UUUGGC, UUUGGG, UUUGGU, UUUGUA, UUUGUC, UUUGUU, UUUUAA, UUUUAG, UUUUAU, UUUUCC, UUUUCG, UUUUCU, UUUUGA, UUUUGC, UUUUGG, UUUUGU, UUUUUA, UUUUUC, UUUUUU
Table 2: A listing of oligonucleotide modifications
Symbol Feature Description
bio 5' biotin
dAs DNA w/3' thiophosphate
dCs DNA w/3' thiophosphate
dGs DNA w/3' thiophosphate
dTs DNA w/3' thiophosphate
dG DNA
enaAs ENA w/3' thiophosphate
enaCs ENA w/3' thiophosphate
enaGs ENA w/3' thiophosphate
enaTs ENA w/3' thiophosphate
fluAs 2'-fluoro w/3' thiophosphate
fluCs 2'-fluoro w/3' thiophosphate
fluGs 2'-fluoro w/3' thiophosphate Symbol Feature Description
fluUs 2'-fluoro w/3' thiophosphate
InaAs LNA w/3' thiophosphate
InaCs LNA w/3' thiophosphate
InaGs LNA w/3' thiophosphate
InaTs LNA w/3' thiophosphate
omeAs 2'-Ome w/3' thiophosphate
omeCs 2'-Ome w/3' thiophosphate
omeGs 2'-Ome w/3' thiophosphate
omeTs 2'-Ome w/3' thiophosphate
InaAs-Sup LNA w/3' thiophosphate at 3' terminus
InaCs-Sup LNA w/3' thiophosphate at 3' terminus
InaGs-Sup LNA w/3' thiophosphate at 3' terminus
InaTs-Sup LNA w/3' thiophosphate at 3' terminus
InaA-Sup LNA w/3' OH at 3' terminus
InaC-Sup LNA w/3' OH at 3' terminus
InaG-Sup LNA w/3' OH at 3' terminus
InaT-Sup LNA w/3' OH at 3' terminus
omeA-Sup 2'-Ome w/3' OH at 3' terminus
omeC-Sup 2'-Ome w/3' OH at 3' terminus
omeG-Sup 2'-Ome w/3' OH at 3' terminus
omeU-Sup 2'-Ome w/3' OH at 3' terminus
dAs-Sup DNA w/3' thiophosphate at 3' terminus
dCs-Sup DNA w/3' thiophosphate at 3' terminus
dGs-Sup DNA w/3' thiophosphate at 3' terminus
dTs-Sup DNA w/3' thiophosphate at 3' terminus
dA-Sup DNA w/3' OH at 3' terminus
dC-Sup DNA w/3' OH at 3' terminus
dG-Sup DNA w/3' OH at 3' terminus
dT-Sup DNA w/3' OH at 3' terminus
Table 3: Formatted oligonucleotide sequences made for testing showing nucleotide modifications.
Oligo Base Sequence RaNA Sequence SeqID Name
MECP2- CGACGTCGCGC lnaCs;omeGs;lnaAs;omeCs;lnaGs;omeUs;lnaCs;ome 1438 01 mOl CCCG Gs;lnaCs;omeGs;lnaCs;omeCs;lnaCs;omeCs;lnaG-Sup
MECP2- CCGACGTCGCG lnaCs;omeCs;lnaGs;omeAs;lnaCs;omeGs;lnaTs;omeC 1439 02 mOl CCCC s;lnaGs;omeCs;lnaGs;omeCs;lnaCs;omeCs;lnaC-Sup
MECP2- CCATTTTCCGG lnaCs;omeCs;lnaAs;omeUs;lnaTs;omeUs;lnaTs;omeC 1521 Oligo Base Sequence RaNA Sequence SeqID
Name
03 mOl ACGG s;lnaCs;omeGs;lnaGs;omeAs;lnaCs;omeGs;lnaG-Sup
MECP2- GCCATTTTCCG lnaGs;omeCs;lnaCs;omeAs;lnaTs;omeUs;lnaTs;omeU 1522
04 mOl GACG s;lnaCs;omeCs;lnaGs;omeGs;lnaAs;omeCs;lnaG-Sup
MECP2- GCGGCCA I 1 1 1 lnaGs;omeCs;lnaGs;omeGs;lnaCs;omeCs;lnaAs;ome 1525
05 mOl CCGG Us;lnaTs;omeUs;lnaTs;omeCs;lnaCs;omeGs;lnaG-Sup
MECP2- GGCGGCCATTT lnaGs;omeGs;lnaCs;omeGs;lnaGs;omeCs;lnaCs;ome 1526
06 mOl TCCG As;lnaTs;omeUs;lnaTs;omeUs;lnaCs;omeCs;lnaG-Sup
MECP2- GCCAGCCGTGT lnaGs;omeCs;lnaCs;omeAs;lnaGs;omeCs;lnaCs;omeG 1683
07 mOl CGTC s;lnaTs;omeGs;lnaTs;omeCs;lnaGs;omeUs;lnaC-Sup
MECP2- ATCCGCCAGCC lnaAs;omeUs;lnaCs;omeCs;lnaGs;omeCs;lnaCs;omeA 1687
08 mOl GTGT s;lnaGs;omeCs;lnaCs;omeGs;lnaTs;omeGs;lnaT-Sup
MECP2- GATCGCCGGCG lnaGs;omeAs;lnaTs;omeCs;lnaGs;omeCs;lnaCs;omeG 2463
09 mOl AAGG s;lnaGs;omeCs;lnaGs;omeAs;lnaAs;omeGs;lnaG-Sup
MECP2- GATCCCGAGCG lnaGs;omeAs;lnaTs;omeCs;lnaCs;omeCs;lnaGs;omeA 2532
10 mOl CTCC s;lnaGs;omeCs;lnaGs;omeCs;lnaTs;omeCs;lnaC-Sup
MECP2- TGTGGCGACCA lnaTs;omeGs;lnaTs;omeGs;lnaGs;omeCs;lnaGs;omeA 7070
11 mOl AGTA s;lnaCs;omeCs;lnaAs;omeAs;lnaGs;omeUs;lnaA-Sup
MECP2- AGATCACCAGT lnaAs;omeGs;lnaAs;omeUs;lnaCs;omeAs;lnaCs;omeC 7117
12 mOl TCCG s;lnaAs;omeGs;lnaTs;omeUs;lnaCs;omeCs;lnaG-Sup
MECP2- TTGTACCTATA lnaTs;omeUs;lnaGs;omeUs;lnaAs;omeCs;lnaCs;ome 14460
13 mOl CGCA Us;lnaAs;omeUs;lnaAs;omeCs;lnaGs;omeCs;lnaA- Sup
MECP2- TTTGTACCTATA lnaTs;omeUs;lnaTs;omeGs;lnaTs;omeAs;lnaCs;omeC 14461
14 mOl CGC s;lnaTs;omeAs;lnaTs;omeAs;lnaCs;omeGs;lnaC-Sup
MECP2- CCCAAACAGCG lnaCs;omeCs;lnaCs;omeAs;lnaAs;omeAs;lnaCs;omeA 65064
15 mOl GCGC s;lnaGs;omeCs;lnaGs;omeGs;lnaCs;omeGs;lnaC-Sup
MECP2- CCAAACAGCGG lnaCs;omeCs;lnaAs;omeAs;lnaAs;omeCs;lnaAs;omeG 65065
16 mOl CGCT s;lnaCs;omeGs;lnaGs;omeCs;lnaGs;omeCs;lnaT-Sup
MECP2- CAAACAGCGGC lnaCs;omeAs;lnaAs;omeAs;lnaCs;omeAs;lnaGs;omeC 65066
17 mOl GCTC s;lnaGs;omeGs;lnaCs;omeGs;lnaCs;omeUs;lnaC-Sup
MECP2- AAACAGCGGC lnaAs;omeAs;lnaAs;omeCs;lnaAs;omeGs;lnaCs;omeG 65067
18 mOl GCTCC s;lnaGs;omeCs;lnaGs;omeCs;lnaTs;omeCs;lnaC-Sup
MECP2- AACAGCGGCG lnaAs;omeAs;lnaCs;omeAs;lnaGs;omeCs;lnaGs;ome 65068
19 mOl CTCCA Gs;lnaCs;omeGs;lnaCs;omeUs;lnaCs;omeCs;lnaA-Sup
MECP2- TCCATCATCCG lnaTs;omeCs;lnaCs;omeAs;lnaTs;omeCs;lnaAs;omeU 65079
20 mOl TGAC s;lnaCs;omeCs;lnaGs;omeUs;lnaGs;omeAs;lnaC-Sup
MECP2- GGACCCATGTA lnaGs;omeGs;lnaAs;omeCs;lnaCs;omeCs;lnaAs;omeU 65097
21 mOl TGAT s;lnaGs;omeUs;lnaAs;omeUs;lnaGs;omeAs;lnaT-Sup
MECP2- CTTTCGCTCTAA lnaCs;omeUs;lnaTs;omeUs;lnaCs;omeGs;lnaCs;omeU 65972
22 mOl AGT s;lnaCs;omeUs;lnaAs;omeAs;lnaAs;omeGs;lnaT-Sup
MECP2- TCGCTCTAAAG lnaTs;omeCs;lnaGs;omeCs;lnaTs;omeCs;lnaTs;omeAs 65975
23 mOl TGGA ;lnaAs;omeAs;lnaGs;omeUs;lnaGs;omeGs;lnaA-Sup
MECP2- GACTTCACGGT lnaGs;omeAs;lnaCs;omeUs;lnaTs;omeCs;lnaAs;omeC 66045
24 mOl AACT s;lnaGs;omeGs;lnaTs;omeAs;lnaAs;omeCs;lnaT-Sup Oligo Base Sequence RaNA Sequence SeqlD
Name
MECP2- CTTCACGGTAA lnaCs;omeUs;lnaTs;omeCs;lnaAs;omeCs;lnaGs;omeG 66047
25 mOl CTGG s;lnaTs;omeAs;lnaAs;omeCs;lnaTs;omeGs;lnaG-Sup
MECP2- CAAACGCCCCG lnaCs;omeAs;lnaAs;omeAs;lnaCs;omeGs;lnaCs;omeC 66323
26 mOl GCAG s;lnaCs;omeCs;lnaGs;omeGs;lnaCs;omeAs;lnaG-Sup
MECP2- GAAGCGAAAA lnaGs;omeAs;lnaAs;omeGs;lnaCs;omeGs;lnaAs;ome 66338
27 mOl GCTGA As;lnaAs;omeAs;lnaGs;omeCs;lnaTs;omeGs;lnaA-Sup
MECP2- AAGAAAGCCGT lnaAs;omeAs;lnaGs;omeAs;lnaAs;omeAs;lnaGs;ome 66432
28 mOl GAAG Cs;lnaCs;omeGs;lnaTs;omeGs;lnaAs;omeAs;lnaG-Sup
MECP2- CATCAAGAAGC lnaCs;omeAs;lnaTs;omeCs;lnaAs;omeAs;lnaGs;omeA 66485
29 mOl GCAA s;lnaAs;omeGs;lnaCs;omeGs;lnaCs;omeAs;lnaA-Sup
MECP2- CAATGTCTTTG lnaCs;omeAs;lnaAs;omeUs;lnaGs;omeUs;lnaCs;ome 27322
30 mOl CGCT Us;lnaTs;omeUs;lnaGs;omeCs;lnaGs;omeCs;lnaT-Sup
MECP2- GGCGTCCGGCT lnaGs;omeGs;lnaCs;omeGs;lnaTs;omeCs;lnaCs;omeG 27377
31 mOl GTCC s;lnaGs;omeCs;lnaTs;omeGs;lnaTs;omeCs;lnaC-Sup
MECP2- TTTGCAATCCG lnaTs;omeUs;lnaTs;omeGs;lnaCs;omeAs;lnaAs;omeU 27422
32 mOl CTCC s;lnaCs;omeCs;lnaGs;omeCs;lnaTs;omeCs;lnaC-Sup
MECP2- GCCACGAAACT lnaGs;omeCs;lnaCs;omeAs;lnaCs;omeGs;lnaAs;omeA 27548
33 mOl CTAA s;lnaAs;omeCs;lnaTs;omeCs;lnaTs;omeAs;lnaA-Sup
MECP2- AGCGCAGGTAT lnaAs;omeGs;lnaCs;omeGs;lnaCs;omeAs;lnaGs;ome 28519
34 mOl ATAT Gs;lnaTs;omeAs;lnaTs;omeAs;lnaTs;omeAs;lnaT-Sup
MECP2- TTCGGAGCTTC lnaTs;omeUs;lnaCs;omeGs;lnaGs;omeAs;lnaGs;omeC 28565
35 mOl GTGG s;lnaTs;omeUs;lnaCs;omeGs;lnaTs;omeGs;lnaG-Sup
MECP2- GACAAACACG lnaGs;omeAs;lnaCs;omeAs;lnaAs;omeAs;lnaCs;omeA 29761
36 mOl G l 1 1 1 s;lnaCs;omeGs;lnaGs;omeUs;lnaTs;omeUs;lnaT-Sup
MECP2- TTGCTACCACG lnaTs;omeUs;lnaGs;omeCs;lnaTs;omeAs;lnaCs;omeC 72740
37 mOl GCCT s;lnaAs;omeCs;lnaGs;omeGs;lnaCs;omeCs;lnaT-Sup
MECP2- TGCTACCACGG lnaTs;omeGs;lnaCs;omeUs;lnaAs;omeCs;lnaCs;omeA 72741
38 mOl CCTC s;lnaCs;omeGs;lnaGs;omeCs;lnaCs;omeUs;lnaC-Sup
MECP2- ATCAATAACAG lnaAs;omeUs;lnaCs;omeAs;lnaAs;omeUs;lnaAs;ome 72783
39 mOl CCGC As;lnaCs;omeAs;lnaGs;omeCs;lnaCs;omeGs;lnaC-Sup
MECP2- CAATAACAGCC lnaCs;omeAs;lnaAs;omeUs;lnaAs;omeAs;lnaCs;omeA 72785
40 mOl GCTC s;lnaGs;omeCs;lnaCs;omeGs;lnaCs;omeUs;lnaC-Sup
MECP2- ACTCAATGTGT lnaAs;omeCs;lnaTs;omeCs;lnaAs;omeAs;lnaTs;omeG 72848
41 mOl GCCG s;lnaTs;omeGs;lnaTs;omeGs;lnaCs;omeCs;lnaG-Sup
MECP2- CAATGTGTGCC lnaCs;omeAs;lnaAs;omeUs;lnaGs;omeUs;lnaGs;ome 72851
42 mOl GAGC Us;lnaGs;omeCs;lnaCs;omeGs;lnaAs;omeGs;lnaC- Sup
MECP2- CAAACGCGTCA lnaCs;omeAs;lnaAs;omeAs;lnaCs;omeGs;lnaCs;omeG 36117
43 mOl CTTA s;lnaTs;omeCs;lnaAs;omeCs;lnaTs;omeUs;lnaA-Sup
MECP2- ATCGAGAATGC lnaAs;omeUs;lnaCs;omeGs;lnaAs;omeGs;lnaAs;ome 36165
44 mOl TAAC As;lnaTs;omeGs;lnaCs;omeUs;lnaAs;omeAs;lnaC-Sup
MECP2- CGCACGAGGCC lnaCs;omeGs;lnaCs;omeAs;lnaCs;omeGs;lnaAs;omeG 76153
45 mOl GGCT s;lnaGs;omeCs;lnaCs;omeGs;lnaGs;omeCs;lnaT-Sup
MECP2- GAGTCAGTGTC lnaGs;omeAs;lnaGs;omeUs;lnaCs;omeAs;lnaGs;ome 76180
46 mOl CGCG Us;lnaGs;omeUs;lnaCs;omeCs;lnaGs;omeCs;lnaG- Oligo Base Sequence RaNA Sequence SeqID
Name
Sup
MECP2- GTGTCCGCGGA lnaGs;omeUs;lnaGs;omeUs;lnaCs;omeCs;lnaGs;ome 76186
47 mOl CCCG Cs;lnaGs;omeGs;lnaAs;omeCs;lnaCs;omeCs;lnaG-Sup
MECP2- AGCACTTCTTG lnaAs;omeGs;lnaCs;omeAs;lnaCs;omeUs;lnaTs;omeC 76316
48 mOl TACG s;lnaTs;omeUs;lnaGs;omeUs;lnaAs;omeCs;lnaG-Sup
MECP2- TTCTTGTACGA lnaTs;omeUs;lnaCs;omeUs;lnaTs;omeGs;lnaTs;omeA 76321
49 mOl GGTG s;lnaCs;omeGs;lnaAs;omeGs;lnaGs;omeUs;lnaG-Sup
MECP2- TGTGCCGCTTC lnaTs;omeGs;lnaTs;omeGs;lnaCs;omeCs;lnaGs;omeC 76349
50 mOl TACA s;lnaTs;omeUs;lnaCs;omeUs;lnaAs;omeCs;lnaA-Sup
MECP2- CCGCCGACTGG lnaCs;omeCs;lnaGs;omeCs;lnaCs;omeGs;lnaAs;omeC 76385
51 mOl TGCC s;lnaTs;omeGs;lnaGs;omeUs;lnaGs;omeCs;lnaC-Sup
MECP2- TGGTGCCAGTT lnaTs;omeGs;lnaGs;omeUs;lnaGs;omeCs;lnaCs;omeA 76393
52 mOl CGGT s;lnaGs;omeUs;lnaTs;omeCs;lnaGs;omeGs;lnaT-Sup
MECP2- TGATCGTGCGC lnaTs;omeGs;lnaAs;omeUs;lnaCs;omeGs;lnaTs;omeG 76490
53 mOl GACC s;lnaCs;omeGs;lnaCs;omeGs;lnaAs;omeCs;lnaC-Sup
MECP2- GATCGTGCGCG lnaGs;omeAs;lnaTs;omeCs;lnaGs;omeUs;lnaGs;omeC 76491
54 mOl ACCA s;lnaGs;omeCs;lnaGs;omeAs;lnaCs;omeCs;lnaA-Sup
MECP2- TCGTGCGCGAC lnaTs;omeCs;lnaGs;omeUs;lnaGs;omeCs;lnaGs;omeC 76493
55 mOl CAGA s;lnaGs;omeAs;lnaCs;omeCs;lnaAs;omeGs;lnaA-Sup
MECP2- GCGGCTGTGCG lnaGs;omeCs;lnaGs;omeGs;lnaCs;omeUs;lnaGs;ome 76515
56 mOl AGCG Us;lnaGs;omeCs;lnaGs;omeAs;lnaGs;omeCs;lnaG- Sup
MECP2- GGATCAACCGC lnaGs;omeGs;lnaAs;omeUs;lnaCs;omeAs;lnaAs;ome 76565
57 mOl AACG Cs;lnaCs;omeGs;lnaCs;omeAs;lnaAs;omeCs;lnaG-Sup
MECP2- GATCAACCGCA lnaGs;omeAs;lnaTs;omeCs;lnaAs;omeAs;lnaCs;omeC 76566
58 mOl ACGC s;lnaGs;omeCs;lnaAs;omeAs;lnaCs;omeGs;lnaC-Sup
MECP2- CGACTGGACAT lnaCs;omeGs;lnaAs;omeCs;lnaTs;omeGs;lnaGs;omeA 78887
59 mOl CCTT s;lnaCs;omeAs;lnaTs;omeCs;lnaCs;omeUs;lnaT-Sup
MECP2- CCCGAGGAGTA lnaCs;omeCs;lnaCs;omeGs;lnaAs;omeGs;lnaGs;omeA 79422
60 mOl CATC s;lnaGs;omeUs;lnaAs;omeCs;lnaAs;omeUs;lnaC-Sup
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
SEQ ID Chrom Gene Chr. Start Chr. End Strand
1 chrX MECP2 153275263 153375188 -
2 chrX MECP2 153275263 153375188 +
3 chrX Mecp2 71260160 71342932 -
4 chrX Mecp2 71260160 71342932 +
5 chrX MECP2 153278064 153278111 -
6 chrX MECP2 153278111 153278156 -
7 chrX MECP2 153278706 153278747 -
8 chrX MECP2 153279512 153279556 - SEQ ID Chrom Gene Chr. Start Chr. End Strand
9 chrX MECP2 153279613 153279658 -
10 chrX MECP2 153281486 153281531 -
11 chrX MECP2 153283707 153283737 -
12 chrX MECP2 153284059 153284105 -
13 chrX MECP2 153287944 153287992 -
14 chrX MECP2 153288681 153288722 -
15 chrX MECP2 153290087 153290134 -
16 chrX MECP2 153290216 153290263 -
17 chrX MECP2 153290364 153290414 -
18 chrX MECP2 153291585 153291633 -
19 chrX MECP2 153292312 153292362 -
20 chrX MECP2 153292731 153292774 -
21 chrX MECP2 153293138 153293185 -
22 chrX MECP2 153293331 153293377 -
23 chrX MECP2 153293427 153293469 -
24 chrX MECP2 153293568 153293614 -
25 chrX MECP2 153293715 153293764 -
26 chrX MECP2 153293792 153293878 -
27 chrX MECP2 153293901 153293948 -
28 chrX MECP2 153294420 153294467 -
29 chrX MECP2 153297927 153297972 -
30 chrX MECP2 153315466 153315571 -
31 chrX MECP2 153343401 153343447 -
32 chrX MECP2 153344298 153344339 -
33 chrX MECP2 153348654 153348702 -
34 chrX MECP2 153348997 153349021 -
35 chrX MECP2 153349179 153349222 -
36 chrX MECP2 153349694 153349734 -
37 chrX MECP2 153350493 153350518 -
38 chrX MECP2 153356667 153356713 -
39 chrX MECP2 153356742 153356795 -
40 chrX MECP2 153357047 153357106 -
41 chrX MECP2 153357161 153357204 -
42 chrX MECP2 153361085 153361163 -
43 chrX MECP2 153361423 153361467 -
44 chrX MECP2 153362464 153362527 -
45 chrX MECP2 153276064 153280111 -
46 chrX MECP2 153276111 153280156 - SEQ ID Chrom Gene Chr. Start Chr. End Strand
47 chrX MECP2 153276706 153280747 -
48 chrX MECP2 153277512 153281556 -
49 chrX MECP2 153277613 153281658 -
50 chrX MECP2 153279486 153283531 -
51 chrX MECP2 153281707 153285737 -
52 chrX MECP2 153282059 153286105 -
53 chrX MECP2 153285944 153289992 -
54 chrX MECP2 153286681 153290722 -
55 chrX MECP2 153288087 153292134 -
56 chrX MECP2 153288216 153292263 -
57 chrX MECP2 153288364 153292414 -
58 chrX MECP2 153289585 153293633 -
59 chrX MECP2 153290312 153294362 -
60 chrX MECP2 153290731 153294774 -
61 chrX MECP2 153291138 153295185 -
62 chrX MECP2 153291331 153295377 -
63 chrX MECP2 153291427 153295469 -
64 chrX MECP2 153291568 153295614 -
65 chrX MECP2 153291715 153295764 -
66 chrX MECP2 153291792 153295878 -
67 chrX MECP2 153291901 153295948 -
68 chrX MECP2 153292420 153296467 -
69 chrX MECP2 153295927 153299972 -
70 chrX MECP2 153313466 153317571 -
71 chrX MECP2 153341401 153345447 -
72 chrX MECP2 153342298 153346339 -
73 chrX MECP2 153346654 153350702 -
74 chrX MECP2 153346997 153351021 -
75 chrX MECP2 153347179 153351222 -
76 chrX MECP2 153347694 153351734 -
77 chrX MECP2 153348493 153352518 -
78 chrX MECP2 153354667 153358713 -
79 chrX MECP2 153354742 153358795 -
80 chrX MECP2 153355047 153359106 -
81 chrX MECP2 153355161 153359204 -
82 chrX MECP2 153359085 153363163 -
83 chrX MECP2 153359423 153363467 -
84 chrX MECP2 153360464 153364527 - SEQ ID Chrom Gene Chr. Start Chr. End Strand
85 chrX MECP2 153279614 153279660 +
86 chrX MECP2 153281662 153281720 +
87 chrX MECP2 153281946 153281988 +
88 chrX MECP2 153284367 153284448 +
89 chrX MECP2 153284489 153284534 +
90 chrX MECP2 153288786 153288832 +
91 chrX MECP2 153289895 153289940 +
92 chrX MECP2 153292315 153292365 +
93 chrX MECP2 153292496 153292548 +
94 chrX MECP2 153297642 153297688 +
95 chrX MECP2 153297723 153297765 +
96 chrX MECP2 153300816 153300879 +
97 chrX MECP2 153315579 153315621 +
98 chrX MECP2 153316595 153316640 +
99 chrX MECP2 153348783 153348830 +
100 chrX MECP2 153349199 153349250 +
101 chrX MECP2 153358221 153358285 +
102 chrX MECP2 153277614 153281660 +
103 chrX MECP2 153279662 153283720 +
104 chrX MECP2 153279946 153283988 +
105 chrX MECP2 153282367 153286448 +
106 chrX MECP2 153282489 153286534 +
107 chrX MECP2 153286786 153290832 +
108 chrX MECP2 153287895 153291940 +
109 chrX MECP2 153290315 153294365 +
110 chrX MECP2 153290496 153294548 +
111 chrX MECP2 153295642 153299688 +
112 chrX MECP2 153295723 153299765 +
113 chrX MECP2 153298816 153302879 +
114 chrX MECP2 153313579 153317621 +
115 chrX MECP2 153314595 153318640 +
116 chrX MECP2 153346783 153350830 +
117 chrX MECP2 153347199 153351250 +
118 chrX MECP2 153356221 153360285 +
Single Strand Oligonucleotides (Antisense Strand of Target Gene) SeqlD range: 119-47653
SeqIDs w/o G Runs:
119-142, 157-202, 217- 307-350, 364-544, 559-609, 623-
677, 691-711, 726-787, 803- 839, 854-915, 930 -1055 , 1070 -1081 , 1095
1103, 1118 -1149, 1163, 1186 -1189 , 1204 -1236, 1259 -1301, 1343 -1452,
1466- 1476, 1490- 1610, 1624- 1658, 1672- 1694, 1730- 1740, 1769 - 1790,
1804- 1814, 1843, 1881- 1910, 1924 -1947, 1978- 1980, 2038- 2052 r 2066-
2092, 2108 -2114, 2142- 2147, 2175 -2192, 2207- 2261, 2275- 2294 r 2308-
2336, 2350 -2379, 2394- 2431, 2463 -2515, 2529- 2631, 2645- 2771 r 2786-
2994, 3009 -3291, 3305- 3395, 3409 -3463, 3477- 3515, 3529- 3566 r 3581-
3599, 3613 -3686, 3701- 3718, 3732 -3830, 3844- 3861, 3875- 3970 r 3984-
4133, 4157 -4235, 4251- 4254, 4268 -4474, 4488- 4575, 4589- 4608 r 4627-
4739, 4745 -4756, 4780- 4812, 4826 -5007, 5034- 5123, 5137- 5239 r 5253-
5345, 5353 -5371, 5384- 5401, 5406 -5477, 5503- 5517, 5531- 5635 r 5649-
5867, 5881 -6171, 6185- 6214, 6228 -6231, 6245- 6265, 6280- 6406 r 6420-
6490, 6505 -6661, 6684- 6784, 6799 -6964, 6979- 7024, 7045- 7174 r 7188-
7310, 7337 -7388, 7402- 7410, 7424 -7474, 7489- 7729, 7756- 7797 r 7811-
7974, 7988 -8049, 8063- 8448, 8462 -8671, 8686- 8728, 8742- 8854 r 8869-
8872, 8893 -9071, 9076- 9363, 9384 -9399, 9423- 9613, 9619- 9706 r 9715-
9810, 9818 -9931, 9951- 9956, 9970 -10101 , 10116-10148, 10153- 10230,
10244- 10288, 10292- 10379, 10386- 10412, 10426 -10639, 10680- 10979,
11005- 11060, 11072- 11159, 11173- 11198, 11204 -11226, 11240- 11566,
11580- 11763, 11777- 12069, 12083- 12178, 12183 -12197, 12213- 12235,
12247- 12257, 12268- 12315, 12342- 12351, 12373 -12387, 12402- 12409,
12431- 12443, 12457- 12466, 12485, 12510 -12524 , 12547, 12561 -12802,
12817- 12828, 12843- 12854, 12868, 12882 -12936 , 12956- ■13001, 13015-
13044, 13058 -13087, 13106- ■13116, 13139 -13141 , 13155- ■13163, 13178-
13612, 13634 -13823, 13838- ■13847, 13857 -13866 , 13877- ■14524, 14538-
14555, 14569 -14754, 14768- ■14791, 14804 -14806 , 14820- ■14830, 14842-
14851, 14865 -14891, 14895- ■15008, 15022 -15295 , 15299- ■15317, 15332-
15643, 15656 -15658, 15670- ■15697, 15701 -15748 , 15765- ■15872, 15886-
15941, 15964 -15985, 15992- ■16007, 16021 -16058 , 16062- ■16084, 16098-
16202, 16216 -16428, 16439- ■16487, 16500 -16758 , 16772- ■16857, 16871-
16875, 16891 -16933, 16947- ■16990, 16999 -17030 , 17057- ■17072, 17096-
17143, 17149 -17150, 17165- ■17200, 17235 -17246 , 17260- ■17466, 17480-
17545, 17566 -17572, 17586- ■17592, 17638 -17675 , 17689- ■18203, 18217-
18417, 18431 -18540, 18554- ■18642, 18656 -18669 , 18683- ■18724, 18739-
18828, 18842 -19079, 19110- 19222, 19236 -19260 , 19275- ■19321, 19335-
19336, 19350 -19415, 19430- ■19530, 19555 -19666 , 19681, 19695 -19717,
19731- 19841, 19856- 19864, 19878- 19905, 19945 -20156, 20180- 20184,
20218- 20297, 20349- 20360, 20374- 20388, 20402 -20409, 20423- 20435,
20449- 20456, 20470- 20505, 20519- 20524, 20564 -20574, 20589- 20605,
20620- 20641, 20655- 20673, 20687- 20717, 20745 -20752, 20767- 20790,
20804- 20808, 20832- 20834, 20859- 20871, 20885 -20922, 20936- 20972,
20995- 21005, 21020- 21075, 21089- 21097, 21111 -21120, 21134- 21161,
21175- 21210, 21224- 21241, 21256- 21263, 21277 -21283, 21297- 21331,
21345- 21417, 21431- 21526, 21540- 21592, 21606 -21693, 21707- 21746,
21760- 21783, 21808- 21870, 21884- 21896, 21911 -21947, 21961- 22177,
22191- 22199, 22213, 22229- 22539, 22553 -22572 , 22587- ■22603, 22617-
22680, 22719 -22733, 22755- ■22777, 22791 -22804 , 22818- ■22848, 22862- 22877, 22891 -22909, 22923-■22938, 22959 -22995, 23009-■23017, 23034-
23037, 23066 -23070, 23084- ■23108, 23130 -23171, 23182- ■23237, 23261-
23263, 23274 -23279, 23301- ■23322, 23336 -23365, 23379, 23389 -23430,
23441- 23450, 23464- 23561, 23576- 23654, 23668- 23725, 23739- 23770,
23784- 23823, 23837- 23851, 23865- 23908, 23922- 24117, 24132- 24246,
24261- 24314, 24328- 24402, 24416- 24489, 24512- 24778, 24799- 24805,
24837- 24862, 24884- 25011, 25032- 25045, 25069- 25256, 25271- 25316,
25330- 25332, 25346- 25349, 25363- 25395, 25410- 25422, 25438- 25460,
25474, 25493 -25614, 25628- ■25641, 25655 -25722, 25736- ■25752, 25766-
25878, 25892 -26037, 26051- ■26076, 26090 -26250, 26264- ■26309, 26333-
26385, 26406 -26431, 26450- ■26465, 26488 -26509, 26524- ■26534, 26548-
26667, 26681 -26689, 26704- ■26719, 26746 -26842, 26857- ■26862, 26876-
26903, 26918 -26919, 26933- ■26962, 26976 -26989, 27004- ■27020, 27037-
27063, 27088 , 27114 -27116, 27130 -27133 , 27167 -27194, 27208 -27226,
27241- 27251, 27265- 27377, 27391- 27490, 27504- 27720, 27756- 27790,
27804- 27812, 27867- 27947, 27965- 27972, 27998- 28071, 28085- 28119,
28133- 28399, 28413- 28459, 28473- 28551, 28565- 28619, 28634- 28665,
28679- 28697, 28711- 28723, 28737- 28748, 28763- 28765, 28779- 28793,
28824- 28897, 28920- 28934, 28952- 28960, 28974- 28997, 29011- 29057,
29071- 29153, 29174- 29185, 29227- 29377, 29391- 29548, 29575- 29623,
29637- 29795, 29810- 29887, 29929- 29939, 29957- 29965, 29980- 30040,
30056- 30211, 30226- 30257, 30291- 30358, 30386- 30390, 30416- 30466,
30482- 30484, 30509- 30550, 30564- 30566, 30580- 30585, 30600- 30662,
30676- 30777, 30791- 30815, 30835- 30881, 30918- 31003, 31017- 31024,
31038- 31049, 31063- 31065, 31081- 31098, 31113- 31192, 31206- 31229,
31244- 31254, 31268- 31390, 31404- 31422, 31437- 31454, 31468- 31487,
31503- 31620, 31635- 31736, 31750- 31754, 31775- 31902, 31918- 32203,
32235- 32249, 32264- 32276, 32290- 32323, 32337- 32384, 32417- 32459,
32482- 32502, 32522- 32568, 32592- 32659, 32692- 32724, 32738- 32829,
32843- 33144, 33158- 33168, 33182- 33188, 33202- 33214, 33228- 33303,
33331- 33339, 33353- 33366, 33380- 33400, 33414- 33490, 33505- 33521,
33536- 33554, 33581- 33745, 33763- 33804, 33818- 33824, 33846- 33867,
33881- 33928, 33946- 34050, 34064- 34065, 34079- 34080, 34099- 34101,
34119- 34176, 34190- 34304, 34337- 34362, 34383, 34397- ■34701, 34721-
34727, 34741 -34742, 34765- ■34827, 34841 -34850, 34872- ■34878, 34892-
34978, 34996 -35092, 35107- ■35130, 35149 -35172, 35201- ■35238, 35252-
35271, 35291 -35317, 35331- ■35342, 35356 -35359, 35378- ■35449, 35465-
35474, 35500 -35532, 35546- ■35645, 35659 -35886, 35917- ■36221, 36237-
36277, 36291 -36354, 36369- ■36447, 36473 -36476, 36492- ■36513, 36535-
36563, 36587 -36631, 36658- ■36756, 36770 -36787, 36801- ■36836, 36850-
36884, 36900 -36955, 36969- ■37119, 37134 -37223, 37238- ■37280, 37294-
37359, 37373 -37525, 37539- ■37566, 37580 -37585, 37611, 37651 -37671,
37700- 37714, 37749, 37774- ■37797, 37832 -37874, 37945- ■37964, 38004-
38016, 38030 -38053, 38075- ■38146, 38178 -38238, 38252- ■38257, 38271-
38350, 38365 -38374, 38406- ■38426, 38470 , 38510 -38519, 38535 -38557,
38572, 38586 -38594, 38620- ■38635, 38649 -38693, 38713- ■38727, 38755-
38768, 38783 -38793, 38816- ■38858, 38873 -38942, 38957- ■38965, 38991-
38994, 39008 -39009, 39028- ■39032, 39047 -39048, 39071- ■39173, 39199-
39206, 39220 -39229, 39255- ■39259, 39273 -39279, 39300- ■39326, 39340-
39359, 39403 -39498, 39512- ■39536, 39550 -39562, 39576- ■39578, 39593,
39611- 39623, 39638- 39648, 39662- 39718, 39742- 39768, 39801- 39889, 39914- 39917, 39931- 39971, 39984- 39999, 40006- 40065, 40080- 40092,
40094- 40095, 40113- 40124, 40150- 40176, 40191- 40233, 40249- 40295,
40309- 40322, 40336- 40337, 40351- 40361, 40376- 40517, 40567- 40600,
40614- 40631, 40646- 40672, 40686- 40712, 40727- 40761, 40776- 40801,
40815- 40820, 40836- 40958, 40973- 40996, 41010- 41021, 41035- 41049,
41064- 41072, 41086- 41096, 41110, 41124 -41185, 41200- ■41275, 41290-
41300, 41341 -41381, 41408- 41425, 41450 -41466, 41480- ■41492, 41513-
41582, 41597 -41637, 41652- 41668, 41682 -41792, 41807- ■41826, 41840-
41841, 41855 -41905, 41920- 41924, 41939 -42053, 42068- ■42090, 42105-
42108, 42122 -42219, 42245- 42250, 42283 -42334, 42364- ■42379, 42394-
42413, 42433 -42434, 42466- 42471, 42485 -42530, 42548- ■42598, 42612-
42693, 42707 -42721, 42736- 42741, 42757 -42775, 42789- ■42800, 42822-
42824, 42838 -42930, 42939- 43001, 43015 -43127, 43142- ■43152, 43166-
43231, 43245 -43290, 43304- 43337, 43351 -43372, 43404- ■43470, 43484-
43516, 43530 -43545, 43560- 43599, 43613 -43624, 43639- ■43642, 43657-
43667, 43694 -43717, 43733- 43761, 43789 -43815, 43830- ■43911, 43925-
43949, 43963 -43977, 43991- 44009, 44035 -44054, 44078- ■44128, 44142-
44157, 44180 -44204, 44218- 44230, 44245 -44258, 44272- ■44286, 44310-
44336, 44350 -44367, 44398- 44445, 44472 -44493, 44507- ■44528, 44559-
44602, 44631 -44633, 44647- 44656, 44682 -44724, 44743- ■44832, 44858-
44896, 44915 -44928, 44942- 44946, 44960 -44979, 44997, 45010 -45037,
45052- 45059, 45073- 45121, 45184- 45202, 45216, 45231- ■45246, 45261-
45266, 45280 -45300, 45314- 45368, 45374 -45420, 45433- ■45453, 45487-
45495, 45509 -45535, 45551- 45603, 45617 -45652, 45668- ■45740, 45760-
45770, 45784 -45814, 45828- 45906, 45920 -45961, 45976- ■45990, 46004-
46016, 46030 -46039, 46053- 46070, 46084 -46126, 46140- ■46164, 46178-
46221, 46235 -46311, 46321- 46327, 46341 -46418, 46432- ■46504, 46518-
46569, 46582 -46600, 46614- 46617, 46631 -46632, 46646- ■46648, 46663-
46785, 46799 -46846, 46862- 47025, 47049 -47197, 47203- ■47255, 47269-
47274, 47288 -47314, 47328- 47419, 47426 -47461, 47463, 47468 -47483,
47485- 47551, 47554- 47559, 47561- 47569, 47577- 47608, 47612- 47623,
47625- 47631, 47633- 47638, 47643- 47653
SeqIDs w/o miR Seeds:
119, 121-123, 125, 130, 132-133, 137-140, 142, 149, 151-152, 161- 164, 166-167, 169, 172-176, 178-181, 183, 186-187, 189, 192, 194- 195, 198, 202-203, 210-213, 215-218, 220, 222-223, 225-228, 230-231, 233, 235-236, 238-239, 242-243, 247, 251-252, 254-255, 257-259, 261- 263, 265, 267, 272-277, 279-282, 285-287, 290-293, 297-300, 305-306, 308, 310, 313, 316, 318, 324-329, 331-332, 335, 337-339, 341-343,
345, 347-349, 353-359, 361-362, 366, 369-371, 378-381, 384-389, 391, 393-394, 401, 404, 406-407, 409, 411-412, 421-422, 424-425, 427-438, 441, 443-446, 449-451, 454-455, 458-459, 461-462, 464, 467-468, 470- 471, 473, 476-478, 483-489, 491, 493-498, 502, 504, 506-510, 512- 523, 525, 527, 529-530, 532-534, 536, 539-544, 548, 550, 552, 554- 558, 562, 565-566, 569-572, 575, 577, 579-588, 590, 594, 598-600, 602-606, 608-609, 612, 614-615, 617-619, 621-624, 630-633, 636, 639- 641, 643-647, 649-651, 653, 655, 658, 660-663, 666-667, 669-670, 676-677, 682, 684-688, 690, 699, 701, 703-708, 710-713, 717-720, 724, 726, 730, 732-735, 739-740, 745-746, 748-749, 755-756, 758,
760-762, 764-768, 773, 776-778, 780, 785-787, 796-797, 802, 805-809, 812, 818-824, 826, 831, 834, 836-840, 846, 848, 851, 853-856, 858- 859, 862, 864, 868-870, 876, 878-883, 885-886, 889, 893-895, 897- 899, 901, 904, 906, 909-910, 913, 916, 918, 924, 927-928, 930-931, 933-939, 943-944, 949, 951-953, 955, 959, 964-967, 969-974, 976-980, 982, 984, 986-987, 989-1000, 1002, 1005-1017, 1020-1023, 1025, 1027- 1028, 1030-1031, 1034-1035, 1038-1048, 1050-1055, 1068-1072, 1074- 1077, 1080-1082, 1087-1090, 1092, 1094, 1099-1101, 1103, 1105, 1110, 1112-1113, 1115, 1117-1118, 1121, 1125-1131, 1134, 1136, 1138, 1140- 1147, 1149, 1151, 1154, 1157-1158, 1162-1163, 1166, 1168-1169, 1171, 1176, 1178-1181, 1183, 1185-1186, 1188-1189, 1195-1196, 1198-1204, 1206-1208, 1210-1212, 1215-1216, 1218-1220, 1223-1224, 1229, 1231, 1233, 1235-1236, 1238, 1243-1245, 1252-1253, 1255-1263, 1265, 1267- 1270, 1272-1289, 1293-1301, 1309, 1314-1315, 1317, 1323-1324, 1332- 1334, 1336-1341, 1343, 1346-1349, 1351-1352, 1354-1356, 1361-1362, 1365-1369, 1372, 1375, 1379, 1381-1384, 1386, 1389-1390, 1393, 1396, 1398, 1401-1402, 1404-1407, 1409-1410, 1415-1416, 1418-1422, 1424, 1427, 1429-1432, 1434-1435, 1437-1445, 1447-1452, 1455-1465, 1467, 1471, 1473, 1475, 1477, 1481, 1485-1493, 1495, 1499-1501, 1503-1505, 1507, 1510-1512, 1514, 1516, 1518-1523, 1525-1526, 1528, 1531, 1534, 1537, 1539, 1542, 1546-1547, 1549-1550, 1552, 1555, 1558, 1561,
1563, 1565-1569, 1572, 1574-1575, 1577, 1579, 1584-1585, 1591-1602, 1605-1606, 1608-1610, 1612, 1614-1615, 1618-1620, 1622-1623, 1625- 1626, 1628-1629, 1631-1632, 1634, 1638-1639, 1641, 1643-1649, 1651, 1653-1655, 1657, 1659, 1661, 1663-1664, 1666-1668, 1670-1677, 1679- 1683, 1685-1686, 1688-1692, 1694-1695, 1697, 1699, 1703-1705, 1709- 1711, 1717, 1719-1720, 1722, 1725, 1728-1737, 1742, 1744, 1746-1760, 1762, 1764-1767, 1769-1776, 1778, 1781-1783, 1786, 1788-1789, 1796, 1798-1801, 1803, 1806-1809, 1811-1815, 1820-1822, 1828-1829, 1831, 1834-1841, 1843-1844, 1846-1848, 1850-1851, 1854-1855, 1857-1858, 1861-1862, 1865-1866, 1871-1873, 1878-1879, 1881-1887, 1890, 1892-
1895, 1898-1901, 1903, 1905, 1907, 1914, 1918-1919, 1921-1922, 1924- 1925, 1927, 1929, 1932-1934, 1936, 1939-1946, 1952-1953, 1955-1957, 1959-1960, 1962, 1964-1965, 1967, 1970, 1972-1977, 1979-1980, 1982, 1987, 1991, 1995, 1997-1999, 2007-2008, 2010-2011, 2013-2015, 2017- 2023, 2030-2033, 2037, 2040-2042, 2045, 2048-2051, 2055, 2057-2062, 2064-2065, 2067, 2069, 2071-2072, 2074, 2076, 2084, 2087, 2089-2092, 2094, 2096, 2099-2100, 2102, 2104-2109, 2111, 2113, 2121, 2127-2129, 2132, 2139-2140, 2144, 2146, 2148, 2157-2158, 2160-2161, 2166, 2168- 2178, 2183-2186, 2191, 2193, 2198-2202, 2204, 2206, 2209, 2211-2213, 2215-2218, 2222-2227, 2229-2230, 2234, 2236-2239, 2241, 2245-2247, 2249-2252, 2254-2255, 2258-2259, 2261, 2267-2269, 2271-2274, 2276- 2280, 2283, 2285-2291, 2293-2295, 2297-2300, 2302, 2306-2310, 2312, 2314-2315, 2317-2319, 2322, 2324-2325, 2327-2328, 2332, 2334-2336, 2338-2344, 2346-2349, 2351-2352, 2355-2357, 2359, 2361, 2363-2364, 2366-2375, 2377-2378, 2380-2381, 2389, 2392-2393, 2396-2397, 2399- 2400, 2403-2405, 2409-2410, 2413-2416, 2419-2424, 2426, 2429, 2431, 2433-2436, 2439-2440, 2443-2445, 2447-2449, 2452, 2455-2457, 2459- 2465, 2473-2475, 2478-2482, 2484, 2487, 2491-2506, 2509-2511, 2513- 2515, 2521, 2523-2526, 2528, 2531-2533, 2535-2542, 2545, 2547-2550, 2552-2557, 2559-2565, 2567-2573, 2575, 2580-2581, 2583-2584, 2586- 2589, 2592-2593, 2595-2603, 2608-2610, 2613, 2615-2621, 2623-2633, 2635-2638, 2640-2641, 2643-2646, 2650, 2652-2653, 2657, 2661-2663, 2666-2667, 2672, 2674, 2676, 2678-2682, 2684, 2694-2698, 2700-2705, 2707-2715, 2717, 2719-2722, 2724-2726, 2729-2730, 2737, 2740, 2742- 2743, 2745-2749, 2751-2760, 2764, 2766-2773, 2775-2778, 2780-2782, 2785-2787, 2789, 2791, 2793-2797, 2800-2803, 2805, 2809-2824, 2826- 2833, 2835-2837, 2839-2842, 2844-2846, 2848-2854, 2856-2857, 2861- 2865, 2867, 2870-2871, 2873-2876, 2878-2879, 2881-2884, 2886-2889, 2891-2893, 2896-2899, 2901, 2903-2904, 2906, 2909-2911, 2913-2925, 2927, 2929, 2937, 2939-2940, 2942-2945, 2947, 2949, 2952-2954, 2957- 2959, 2963, 2965-2970, 2974, 2976, 2979, 2982-2989, 2991-2997, 3001- 3002, 3004, 3010-3024, 3031-3039, 3041-3044, 3050, 3054-3055, 3058- 3059, 3063-3067, 3069-3072, 3077-3078, 3080, 3084-3091, 3093, 3095- 3097, 3100, 3102-3107, 3109-3110, 3112-3113, 3115-3116, 3119, 3121- 3125, 3129-3130, 3132-3134, 3136-3138, 3140-3149, 3151, 3153-3161, 3163-3164, 3170-3188, 3193, 3198, 3200, 3202-3204, 3207-3209, 3211, 3214-3215, 3218-3222, 3224-3230, 3234-3235, 3237-3238, 3242-3245, 3247-3250, 3252, 3254-3257, 3260-3267, 3269-3270, 3272-3273, 3276- 3278, 3282-3283, 3285-3286, 3289, 3291, 3294, 3297-3299, 3301-3303, 3311-3313, 3316, 3320-3330, 3332-3333, 3335, 3337, 3342-3344, 3350- 3351, 3353-3354, 3357-3358, 3360-3366, 3368-3370, 3372-3377, 3381, 3384, 3386-3388, 3390, 3392-3395, 3397, 3400-3403, 3405, 3407-3408, 3410, 3413, 3416-3419, 3423, 3425, 3427-3428, 3430, 3432-3436, 3439, 3441-3442, 3444, 3448, 3453, 3455-3457, 3459-3465, 3467-3468, 3470- 3474, 3476, 3478-3480, 3482, 3484-3486, 3489-3496, 3498-3500, 3502- 3504, 3506-3510, 3512, 3515-3517, 3519, 3522-3526, 3530, 3534-3542, 3548-3549, 3555-3556, 3558, 3561, 3563-3565, 3568, 3577, 3580-3587, 3589, 3592, 3595-3600, 3604, 3606-3610, 3612, 3614, 3621, 3623-3625, 3627-3634, 3636-3644, 3646, 3649-3650, 3653, 3655, 3660-3667, 3670- 3671, 3673-3676, 3679, 3682, 3684-3686, 3688, 3692-3693, 3695-3700, 3702-3707, 3709-3716, 3718, 3724-3727, 3732, 3735, 3738-3739, 3742, 3749-3751, 3754-3755, 3758, 3764, 3766-3770, 3776-3779, 3781, 3783- 3786, 3791-3793, 3797-3801, 3805-3806, 3808-3810, 3812-3813, 3817- 3820, 3825-3828, 3834-3835, 3837, 3841-3842, 3844, 3848, 3850-3852, 3854-3862, 3865-3867, 3870-3872, 3874-3884, 3886, 3890-3891, 3895- 3896, 3898-3899, 3901, 3906-3912, 3915-3916, 3918-3924, 3931, 3934, 3936-3938, 3940, 3943, 3948-3951, 3953-3954, 3956, 3958, 3960-3961, 3964-3965, 3967, 3969-3975, 3977-3981, 3984, 3986-3987, 3989, 3991, 3993, 4001-4008, 4010-4021, 4024-4030, 4034, 4036-4047, 4049-4051, 4054, 4057, 4059, 4065, 4067, 4069-4071, 4073-4075, 4077-4078, 4080- 4085, 4088-4092, 4094-4100, 4103, 4105-4106, 4109-4111, 4113, 4115- 4117, 4120-4121, 4123, 4125, 4130-4133, 4136, 4138, 4143, 4152, 4155-4158, 4160-4161, 4163, 4165-4166, 4170-4172, 4174-4177, 4179, 4181-4183, 4185, 4188-4193, 4195-4197, 4202-4210, 4212, 4214-4217, 4219-4225, 4227-4234, 4236, 4238-4239, 4242, 4244, 4246-4247, 4249, 4251, 4253, 4258-4259, 4262-4264, 4266-4267, 4269-4276, 4278-4280, 4282-4283, 4285, 4287, 4289-4292, 4294, 4296-4304, 4308-4309, 4311- 4312, 4315-4319, 4321-4325, 4328-4329, 4331-4335, 4337, 4339-4341, 4345, 4347, 4351-4353, 4357-4358, 4360-4364, 4367-4371, 4374, 4379- 4381, 4383, 4385-4386, 4390, 4392-4393, 4395, 4399, 4401-4406, 4409- 4414, 4417-4425, 4429-4431, 4436-4447, 4449-4456, 4459, 4462-4465, 4469-4471, 4473, 4477, 4479-4481, 4483-4484, 4487-4489, 4491, 4493, 4495-4496, 4498-4502, 4505, 4507-4510, 4512-4513, 4515-4516, 4518- 4520, 4522, 4525-4527, 4529, 4531-4533, 4535-4536, 4538, 4541-4542, 4545-4546, 4549-4555, 4558, 4562, 4564-4567, 4569-4571, 4575, 4580, 4582, 4584, 4586-4589, 4593-4594, 4597-4598, 4602-4607, 4618, 4621, 4623, 4625-4627, 4629-4631, 4636, 4638, 4640, 4643, 4645-4646, 4649, 4651-4655, 4658, 4664-4683, 4685-4687, 4690-4691, 4693-4694, 4697- 4698, 4700-4706, 4708, 4710, 4712-4715, 4719-4720, 4724, 4726-4727, 4734-4737, 4742, 4744, 4746-4748, 4752, 4757-4764, 4766, 4770-4774, 4776-4780, 4782-4783, 4785-4786, 4789-4790, 4792-4794, 4796, 4800- 4804, 4806-4808, 4810-4811, 4815, 4817-4819, 4821, 4823-4824, 4826- 4829, 4831-4833, 4835-4841, 4843-4844, 4847-4848, 4850, 4852-4853, 4855-4860, 4862-4866, 4869-4870, 4872, 4875-4876, 4880-4881, 4883, 4885, 4890, 4893, 4896-4899, 4901-4903, 4905, 4907, 4909-4912, 4914, 4916-4917, 4919, 4922, 4924-4926, 4931-4932, 4934-4935, 4937-4942, 4945-4948, 4950-4965, 4968-4970, 4973, 4976-4977, 4981-4985, 4987-
4992, 4994, 4996-4997, 4999-5000, 5003, 5006-5008, 5016, 5022, 5027- 5031, 5033, 5035-5036, 5039, 5042, 5044-5045, 5047-5054, 5057-5062, 5064, 5066, 5069-5070, 5073-5076, 5078, 5080-5081, 5084, 5086, 5092, 5094-5095, 5097, 5100, 5102-5103, 5106, 5110, 5112-5114, 5116-5118, 5120, 5122-5124, 5126-5127, 5129-5133, 5136-5140, 5143-5146, 5152-
5154, 5160, 5162, 5164, 5166, 5169-5171, 5173-5176, 5178-5180, 5182- 5189, 5191, 5193, 5195-5199, 5201-5202, 5204-5207, 5209-5214, 5216- 5218, 5220-5221, 5225-5226, 5230-5232, 5234, 5237-5239, 5243, 5245- 5253, 5255-5256, 5258-5260, 5264, 5266, 5268, 5272, 5274-5275, 5277- 5279, 5281-5285, 5287-5290, 5292-5297, 5299, 5303-5304, 5306, 5309- 5310, 5313, 5315-5318, 5320-5322, 5324-5326, 5328-5332, 5334-5335, 5339, 5345, 5347, 5353, 5357-5358, 5361, 5363-5364, 5368-5369, 5371, 5373-5374, 5376, 5379-5382, 5384-5388, 5393, 5396, 5399, 5404, 5406- 5407, 5411, 5413-5418, 5425-5428, 5431, 5433-5446, 5448-5449, 5452- 5457, 5459-5460, 5466-5468, 5471, 5474-5475, 5477, 5479, 5482-5483, 5485-5487, 5494, 5496-5498, 5501-5502, 5505-5507, 5513-5514, 5516- 5517, 5519, 5521, 5526, 5528-5529, 5531-5533, 5535-5538, 5540-5541, 5543-5550, 5552-5555, 5557-5566, 5569, 5571-5574, 5576-5577, 5579- 5580, 5583-5587, 5589, 5592-5593, 5596-5598, 5603-5604, 5606-5609, 5611-5612, 5614, 5616-5623, 5625-5632, 5636-5638, 5642-5643, 5645- 5648, 5651-5652, 5656, 5669-5670, 5672, 5676-5677, 5679-5680, 5682, 5685-5688, 5692, 5694-5695, 5699-5704, 5707-5708, 5710-5713, 5715- 5719, 5721, 5723-5725, 5727-5728, 5730-5735, 5737, 5739, 5741-5746, 5748-5752, 5754-5755, 5757, 5759-5760, 5762, 5764-5766, 5768-5769, 5773-5778, 5780, 5784, 5789-5792, 5794, 5796-5803, 5805, 5807-5808, 5810-5813, 5816, 5819, 5822-5825, 5827-5828, 5830-5831, 5835-5838, 5841-5842, 5844, 5846-5849, 5851-5854, 5856, 5858-5864, 5866-5867, 5869, 5871, 5874, 5877-5879, 5882, 5885-5888, 5891-5898, 5900-5901, 5903, 5905-5907, 5909-5911, 5913-5914, 5916, 5918-5921, 5923, 5925- 5926, 5929, 5932, 5935-5939, 5942-5953, 5955-5957, 5959-5963, 5965- 5967, 5972, 5974-5982, 5984-5986, 5989-5995, 5997-6003, 6005, 6007- 6008, 6010-6011, 6013-6016, 6018-6023, 6026-6028, 6031-6032, 6034- 6037, 6040, 6042-6044, 6046-6049, 6051-6054, 6056, 6058, 6061-6065, 6067-6068, 6070-6074, 6076, 6078-6082, 6085-6098, 6102-6118, 6121, 6124-6127, 6131-6138, 6140, 6142-6145, 6147, 6151, 6154-6155, 6158- 6160, 6162-6165, 6167, 6171-6173, 6177, 6180, 6182, 6184, 6186-6194, 6196-6200, 6202-6207, 6209, 6211-6213, 6216, 6223, 6228-6229, 6231, 6236-6239, 6249-6251, 6254-6255, 6259, 6261-6263, 6265, 6271-6272, 6274-6276, 6278-6279, 6281-6282, 6284-6285, 6287, 6290, 6292, 6295- 6299, 6301, 6303, 6307-6310, 6312-6313, 6317-6319, 6323-6326, 6329, 6333-6334, 6336, 6340, 6342-6348, 6354-6355, 6359, 6366-6367, 6369- 6373, 6375-6377, 6379-6385, 6387-6389, 6391, 6393, 6395-6399, 6401- 6404, 6413-6415, 6418, 6420-6427, 6429-6437, 6439-6444, 6446, 6448, 6450-6456, 6458-6459, 6463-6469, 6471-6472, 6474-6476, 6479, 6483- 6489, 6491-6492, 6494, 6496-6497, 6499-6500, 6502-6505, 6507, 6510- 6512, 6515-6519, 6522, 6524-6525, 6527-6534, 6536-6539, 6541-6546, 6548-6550, 6552, 6554-6557, 6559, 6562, 6564-6565, 6567-6568, 6570- 6573, 6575, 6578-6584, 6587, 6590-6594, 6596-6598, 6600, 6602-6603, 6605-6614, 6616, 6618-6620, 6622-6623, 6625-6628, 6630-6632, 6634, 6637-6642, 6644, 6646-6650, 6655-6663, 6666, 6668, 6670-6671, 6677- 6678, 6680-6690, 6692, 6695-6696, 6698-6701, 6705-6707, 6709-6711, 6713-6719, 6721-6723, 6725-6726, 6728-6731, 6733-6734, 6737, 6739- 6747, 6750-6751, 6754, 6756, 6758, 6760-6761, 6766, 6769-6771, 6773, 6775-6779, 6782-6783, 6786, 6791, 6793, 6795, 6797-6798, 6803-6808, 6810, 6813, 6816, 6819-6822, 6824-6826, 6828-6829, 6833-6838, 6841- 6842, 6844-6846, 6848-6850, 6854-6855, 6857-6864, 6867, 6871-6885, 6887-6888, 6893, 6896-6897, 6900-6902, 6905-6907, 6909-6926, 6929, 6931-6943, 6945-6947, 6949-6954, 6956-6961, 6963-6967, 6970, 6972- 6974, 6976-6980, 6983-6985, 6988, 6990-6992, 6994-6996, 6998, 7000- 7002, 7004-7006, 7008, 7010-7017, 7019-7020, 7022-7023, 7025, 7037, 7039-7042, 7046-7048, 7050, 7053-7054, 7056, 7058-7059, 7061, 7064- 7067, 7069-7070, 7072-7079, 7081, 7083-7084, 7087-7088, 7091-7095, 7098-7102, 7105-7107, 7109-7110, 7112-7113, 7116-7117, 7119-7120, 7122, 7124, 7128, 7130-7131, 7133-7135, 7141, 7143, 7145, 7149-7150, 7152-7153, 7155-7164, 7166-7168, 7178-7181, 7183-7187, 7194, 7196, 7198, 7200, 7202-7207, 7209, 7211-7212, 7215-7216, 7219-7222, 7224, 7227-7229, 7232, 7234-7238, 7241-7249, 7251-7254, 7258-7262, 7264, 7267-7268, 7271-7275, 7279, 7281-7282, 7284, 7286-7292, 7294, 7296- 7303, 7305-7311, 7315-7318, 7320-7321, 7324, 7326-7328, 7331-7332, 7334, 7336-7338, 7346, 7350-7351, 7354, 7356, 7358-7362, 7364-7366, 7368, 7371-7372, 7375-7378, 7381-7383, 7387, 7392-7404, 7407-7412, 7414, 7417-7420, 7422, 7427-7430, 7432-7435, 7438, 7440-7441, 7444- 7446, 7449, 7451-7455, 7457, 7460, 7462-7463, 7465-7467, 7469-7470, 7472, 7475, 7477, 7480-7481, 7483-7487, 7489-7496, 7498, 7503, 7505, 7509-7515, 7521, 7523, 7526, 7528, 7537-7543, 7545-7550, 7552, 7554, 7556, 7559, 7561-7562, 7564-7566, 7568, 7570-7571, 7573-7575, 7577- 7578, 7580-7583, 7587-7588, 7590-7591, 7594, 7596, 7599-7601, 7604, 7607-7610, 7612-7617, 7621, 7624, 7627, 7630-7632, 7634, 7636-7637, 7639-7641, 7644-7645, 7647-7651, 7653-7656, 7659, 7662-7664, 7667- 7668, 7670-7672, 7674-7679, 7681-7682, 7684, 7686, 7689, 7691, 7695- 7701, 7703-7709, 7713, 7716-7724, 7729, 7732-7733, 7738-7741, 7745, 7748-7753, 7755-7758, 7762-7763, 7766-7779, 7781-7783, 7785, 7788, 7790-7794, 7796-7798, 7801, 7803, 7805-7810, 7813-7814, 7819, 7821- 7825, 7827-7828, 7830, 7832-7837, 7839-7840, 7842, 7844-7847, 7849, 7851-7855, 7858, 7860, 7862, 7868-7875, 7877-7879, 7881, 7883-7888, 7890-7895, 7898-7899, 7902-7903, 7906-7907, 7909-7911, 7914-7915,
7917-7918, 7921-7922, 7924, 7926, 7928-7930, 7932-7933, 7935, 7937- 7939, 7941-7942, 7945-7949, 7951-7952, 7954, 7956, 7958-7963, 7970, 7972-7975, 7979, 7981, 7983, 7985-7986, 7988-7989, 7991, 7996-7997, 7999, 8001, 8003, 8005-8006, 8009-8016, 8018-8021, 8023, 8025-8028, 8030, 8034-8036, 8038-8040, 8042-8043, 8047-8049, 8055, 8057-8060, 8062-8063, 8065-8068, 8071-8072, 8074-8075, 8077, 8079-8083, 8085- 8090, 8095, 8099-8103, 8105-8108, 8110, 8112-8115, 8118, 8120-8122, 8125-8127, 8133-8138, 8140-8144, 8146-8148, 8150-8152, 8157-8158, 8160, 8162-8163, 8165-8169, 8172-8175, 8178-8179, 8182-8183, 8185- 8187, 8189-8192, 8196, 8198, 8200, 8202-8204, 8207, 8209, 8211, 8214-8218, 8220-8221, 8224-8227, 8233-8235, 8239-8253, 8255-8263,
8266-8269, 8271, 8274, 8276-8278, 8280, 8284, 8287, 8290, 8293-8301, 8303, 8305, 8307-8308, 8312-8313, 8315-8317, 8319-8320, 8322, 8324, 8326-8328, 8330-8331, 8334-8340, 8343-8361, 8364, 8366-8368, 8370- 8375, 8377-8380, 8382-8386, 8388-8392, 8395-8400, 8402-8403, 8410- 8411, 8413, 8415-8419, 8425-8426, 8428-8430, 8433-8436, 8438-8440, 8442-8443, 8445-8446, 8448-8453, 8455, 8457, 8459-8467, 8469, 8471- 8475, 8478-8481, 8483, 8485-8487, 8490, 8492-8496, 8499-8502, 8504- 8506, 8508-8517, 8520-8523, 8526-8527, 8529-8530, 8532-8533, 8535- 8543, 8548-8554, 8556-8563, 8565-8566, 8569-8574, 8577-8579, 8581- 8582, 8584, 8587, 8591, 8593-8596, 8598-8601, 8605, 8607, 8609-8610, 8615, 8617-8618, 8620-8623, 8625, 8627-8629, 8631-8634, 8636, 8638, 8641-8642, 8644-8646, 8648-8650, 8660-8662, 8665-8670, 8672, 8677- 8678, 8680-8681, 8683, 8685-8692, 8694-8695, 8697, 8701-8702, 8706, 8708-8714, 8716-8721, 8723-8728, 8734-8743, 8747, 8749-8753, 8755- 8758, 8760, 8762-8763, 8765, 8767-8769, 8772-8773, 8776, 8780-8785, 8787-8788, 8792, 8797, 8800-8806, 8809, 8813-8814, 8817, 8820-8822, 8824-8841, 8843-8847, 8849-8851, 8853, 8861-8862, 8864-8866, 8868, 8871, 8877, 8880, 8883, 8885, 8887-8889, 8892, 8896-8897, 8899, 8901-8905, 8907-8908, 8910-8915, 8920-8922, 8924-8925, 8927, 8929- 8932, 8934, 8936-8939, 8941, 8943, 8945-8947, 8950, 8952, 8955-8956, 8958-8959, 8961-8963, 8966-8968, 8972, 8974, 8978, 8980-8981, 8983, 8985-8986, 8988, 8990-8991, 8993-8996, 8998-9001, 9003, 9007-9008, 9010, 9017-9018, 9022-9025, 9033-9034, 9036-9039, 9041-9044, 9048- 9049, 9052, 9054, 9058-9059, 9061, 9067-9070, 9075-9076, 9079-9086, 9089-9096, 9098, 9100-9102, 9106, 9111-9113, 9120, 9123, 9127, 9129, 9131-9132, 9134-9136, 9138, 9140-9141, 9143, 9145, 9147-9149, 9152, 9154, 9157, 9162, 9164, 9166-9168, 9171, 9173-9175, 9177, 9180, 9183-9185, 9187, 9189, 9191-9196, 9198-9207, 9217-9218, 9220-9226, 9228, 9233-9239, 9241, 9243-9246, 9248, 9252-9261, 9263-9265, 9268- 9269, 9271-9273, 9276-9279, 9282-9284, 9287-9288, 9291, 9293-9294,
9298, 9301, 9303-9307, 9309, 9314-9316, 9318, 9320-9323, 9325, 9327- 9329, 9331-9332, 9334, 9336-9337, 9340-9342, 9344-9346, 9349, 9351, 9354-9360, 9363, 9365, 9368, 9370-9371, 9373, 9376-9378, 9381, 9384, 9386-9394, 9399, 9402-9403, 9405, 9407-9410, 9415, 9418-9425, 9427, 9431-9436, 9438-9441, 9444-9458, 9462-9468, 9472, 9475-9476, 9478- 9485, 9487-9488, 9490, 9492-9494, 9496-9497, 9499-9501, 9503-9504, 9506-9510, 9513-9514, 9516-9517, 9519-9522, 9524, 9528, 9530-9535, 9540-9542, 9544-9546, 9549-9558, 9560, 9562-9565, 9568-9571, 9575, 9577, 9579-9581, 9584-9587, 9590-9592, 9594, 9596-9600, 9603-9604, 9607, 9609, 9611-9613, 9618-9620, 9622-9626, 9628, 9631-9632, 9639, 9643-9650, 9656, 9658, 9662-9667, 9670, 9673-9674, 9676-9680, 9682- 9685, 9687-9690, 9694-9704, 9706-9707, 9709-9710, 9712-9713, 9715, 9717-9720, 9722-9724, 9727-9732, 9734, 9737-9740, 9743, 9746-9751, 9753-9754, 9757, 9759-9761, 9763, 9765-9766, 9768, 9770, 9773, 9777- 9783, 9785-9797, 9799, 9801-9802, 9809-9810, 9813, 9817-9820, 9822- 9829, 9831, 9833, 9835, 9837-9842, 9844, 9846-9849, 9851-9853, 9855- 9857, 9863, 9865, 9871, 9875, 9877-9881, 9883-9887, 9890-9892, 9894- 9896, 9898, 9900, 9902-9905, 9907, 9910-9911, 9916-9926, 9929, 9931- 9932, 9934-9935, 9942-9944, 9947-9950, 9952, 9954, 9956-9957, 9962, 9964-9968, 9971, 9974-9975, 9977-9980, 9982-9983, 9985-9986, 9988- 9997, 9999-10011, 10013-10016, 10018-10022, 10027, 10029-10030,
10032-10033, 10035, 10040, 10042, 10045-10049, 10051, 10053-10056, 10059-10060, 10062, 10064-10065, 10067-10068, 10070, 10074-10076, 10078-10083, 10085-10086, 10090-10092, 10094, 10096-10099, 10101- 10102, 10107-10108, 10110-10111, 10114-10118, 10120-10121, 10125- 10140, 10143-10144, 10147-10148, 10153-10155, 10157, 10159-10160,
10163, 10165, 10168-10173, 10175-10177, 10184, 10194, 10197, 10199- 10202, 10208-10212, 10214-10219, 10222-10225, 10227, 10230-10235, 10237, 10239-10240, 10242-10243, 10248, 10250-10251, 10254-10260, 10262, 10264, 10266-10269, 10272-10283, 10285-10290, 10292, 10294- 10297, 10301, 10305-10312, 10315, 10319, 10321-10323, 10325-10328, 10331, 10334-10340, 10342-10345, 10347-10348, 10351-10355, 10357- 10368, 10370-10371, 10373, 10378-10379, 10382-10383, 10385, 10387- 10389, 10391, 10393, 10395-10398, 10400, 10403-10407, 10409-10413, 10416, 10421, 10423, 10425-10427, 10429-10431, 10434, 10437, 10439- 10440, 10442-10449, 10452, 10458-10460, 10462-10467, 10470-10472, 10474, 10476-10482, 10484, 10486-10492, 10494, 10496, 10498-10506, 10509, 10512-10517, 10519, 10521, 10523-10524, 10526, 10528, 10530, 10532-10533, 10537, 10547-10553, 10557-10560, 10563-10564, 10566, 10572-10578, 10580, 10585, 10587-10588, 10590, 10594-10595, 10598- 10603, 10605-10606, 10608, 10610, 10612-10613, 10616, 10618, 10620- 10623, 10626-10627, 10629-10633, 10635, 10637-10638, 10641, 10644- 10645, 10647, 10650, 10652, 10654, 10656, 10669-10671, 10673-10674, 10678-10679, 10682-10688, 10690-10692, 10694-10697, 10699-10700, 10702, 10704-10709, 10711-10712, 10715, 10718, 10720-10723, 10725- 10727, 10729, 10733, 10735-10736, 10739-10743, 10745-10746, 10749, 10752, 10754-10759, 10761, 10764, 10766-10776, 10778-10779, 10782- 10784, 10786-10792, 10794-10804, 10807-10811, 10813-10814, 10816, 10818-10821, 10823-10827, 10829-10834, 10838-10843, 10845-10846, 10849-10859, 10863, 10866-10874, 10876-10881, 10883-10885, 10887- 10890, 10892-10897, 10899-10901, 10903-10915, 10918-10925, 10927- 10931, 10933, 10938-10941, 10946-10953, 10955, 10957-10963, 10966- 10967, 10969-10972, 10974, 10977-10980, 10984, 10990-10993, 10995, 10997-10998, 11000, 11005, 11007, 11011-11012, 11014-11015, 11017- 11021, 11023-11027, 11029-11030, 11032, 11035, 11039, 11041, 11043, 11045, 11047, 11052, 11054, 11056-11058, 11066, 11069, 11072-11075, 11077-11078, 11081, 11084-11085, 11093-11095, 11097-11099, 11103- 11104, 11106, 11112-11114, 11119-11120, 11122, 11124, 11126, 11128, 11130, 11134-11138, 11140, 11143-11152, 11154, 11156-11159, 11161- 11162, 11167-11168, 11170, 11173, 11175-11183, 11185-11189, 11199- 11202, 11204-11205, 11207-11208, 11210, 11213, 11215-11218, 11220, 11222-11223, 11226-11227, 11229, 11231-11233, 11235-11243, 11245- 11246, 11248, 11250-11251, 11254, 11257-11263, 11266-11269, 11271- 11272, 11274-11279, 11281-11282, 11284-11298, 11300-11302, 11308- 11309, 11312-11313, 11317-11319, 11322-11323, 11327-11329, 11331- 11332, 11334-11345, 11348-11349, 11352-11354, 11357-11359, 11361- 11366, 11368, 11372-11375, 11377-11380, 11382-11383, 11386-11392, 11394, 11397, 11399, 11401-11407, 11409-11410, 11412-11418, 11423- 11425, 11429-11431, 11435-11437, 11439, 11441-11444, 11447-11453, 11455-11457, 11459, 11462, 11465, 11467-11468, 11470, 11474-11475, 11477-11482, 11485-11489, 11494, 11496-11500, 11502-11507, 11510, 11512, 11514, 11516, 11518, 11521-11523, 11525-11526, 11528-11530, 11532, 11534, 11536-11538, 11540-11541, 11543-11548, 11551-11552, 11554-11559, 11563-11565, 11568, 11571, 11574, 11582-11583, 11587, 11589, 11596-11597, 11599, 11602-11603, 11608, 11611, 11613-11615, 11617, 11619-11621, 11624-11628, 11630, 11632, 11639, 11641, 11644- 11647, 11649-11653, 11656, 11660-11661, 11664-11665, 11667-11668, 11670, 11674-11675, 11677-11679, 11681, 11685-11692, 11694, 11697- 11700, 11704-11712, 11714-11716, 11718-11724, 11726-11728, 11731, 11734-11736, 11741-11742, 11744, 11746, 11754-11759, 11761-11764, 11766-11767, 11773, 11777, 11779-11782, 11785-11787, 11789-11790, 11792-11793, 11795-11797, 11799, 11806-11810, 11812-11813, 11817- 11818, 11821-11824, 11826-11827, 11830-11835, 11837-11840, 11842- 11845, 11848-11849, 11852-11856, 11861, 11865-11867, 11870-11872, 11875-11882, 11884, 11886-11888, 11890, 11892, 11894-11900, 11902, 11904-11907, 11909-11910, 11912-11915, 11918, 11922, 11924, 11926- 11927, 11929-11931, 11933-11934, 11938-11942, 11944-11947, 11949- 11950, 11952-11958, 11960-11963, 11966-11969, 11972-11980, 11982- 11984, 11986, 11989-11995, 11997-11998, 12003, 12005-12006, 12008- 12010, 12012-12017, 12020-12025, 12027-12030, 12033, 12035-12036, 12040-12043, 12045, 12047, 12049-12052, 12054, 12057-12058, 12060- 12062, 12064-12066, 12069-12070, 12073, 12076, 12078, 12081-12082, 12084, 12089, 12091-12092, 12094, 12096-12103, 12105-12107, 12109, 12111, 12113, 12121, 12123-12125, 12127-12129, 12131, 12133-12134, 12136-12139, 12142-12144, 12146-12152, 12154-12156, 12158-12160, 12162-12168, 12170-12171, 12176-12177, 12179-12180, 12182, 12184- 12186, 12188, 12190-12194, 12196-12199, 12201, 12204-12205, 12207, 12214-12216, 12218, 12220-12221, 12225, 12227, 12230, 12232-12234, 12241, 12244-12246, 12248, 12250-12251, 12258, 12260-12265, 12269, 12272, 12274, 12276-12278, 12280-12285, 12287, 12291, 12293-12296, 12298, 12300-12303, 12305, 12307-12314, 12318-12320, 12322-12327, 12337-12338, 12341-12342, 12344-12345, 12347, 12350-12351, 12358- 12360, 12363-12364, 12366, 12372, 12374-12380, 12382-12383, 12388, 12390, 12392-12397, 12401-12410, 12416, 12419, 12423, 12427-12436, 12438, 12444, 12446-12454, 12458, 12460-12463, 12474-12475, 12477, 12484-12485, 12488, 12490, 12493, 12501, 12505, 12510, 12514, 12516- 12518, 12520-12522, 12524-12526, 12528, 12531, 12533-12534, 12540- 12542, 12544-12549, 12556-12560, 12562, 12565-12566, 12569-12574, 12576, 12578, 12582-12583, 12586, 12593, 12595-12596, 12600-12601, 12603-12613, 12615, 12618-12624, 12627-12629, 12631-12632, 12636, 12639-12640, 12643, 12647, 12649-12651, 12653, 12657-12661, 12663- 12665, 12667-12668, 12670-12672, 12674-12687, 12689-12692, 12694-
12695, 12697, 12706-12707, 12709, 12711, 12713-12714, 12716, 12718, 12720-12722, 12725-12727, 12729, 12733-12736, 12738-12745, 12748- 12752, 12754, 12757, 12759-12760, 12763-12766, 12769, 12772, 12775- 12778, 12780-12784, 12787, 12797, 12799-12802, 12807-12814, 12816- 12817, 12824-12827, 12830, 12835-12837, 12841-12842, 12844-12845, 12850, 12852, 12854, 12860-12869, 12871, 12874, 12876-12878, 12886- 12887, 12891, 12893-12894, 12896-12898, 12903-12905, 12907, 12915, 12917-12918, 12920-12928, 12930, 12933, 12936, 12944, 12947-12949, 12952, 12960, 12962-12968, 12970, 12972, 12974-12978, 12980-12984, 12986-12990, 12992, 12994, 12996, 12999-13003, 13005, 13007-13008, 13010-13013, 13015-13018, 13021-13022, 13024-13034, 13036-13038, 13041-13042, 13044-13045, 13049-13058, 13060-13067, 13069, 13071, 13073-13078, 13080-13081, 13083-13084, 13087-13088, 13090, 13097- 13099, 13101-13104, 13109, 13111-13114, 13117, 13123-13124, 13134- 13137, 13139, 13147-13151, 13153-13162, 13164-13165, 13169-13170, 13172-13173, 13176-13177, 13181, 13185, 13187, 13189, 13191-13193, 13195-13200, 13202, 13206-13213, 13215-13216, 13220, 13222-13223, 13225-13227, 13232-13233, 13235, 13239-13245, 13247-13249, 13251- 13254, 13257-13258, 13262, 13264-13269, 13271-13275, 13278-13279, 13283, 13286-13287, 13291-13292, 13294-13298, 13300, 13302-13308, 13310, 13312-13314, 13316, 13318, 13321, 13323-13325, 13327-13334, 13338-13339, 13341, 13343-13344, 13347, 13349, 13352-13354, 13356- 13358, 13360-13362, 13364-13366, 13368-13372, 13374-13375, 13377, 13379-13380, 13383-13384, 13391, 13393-13395, 13397-13398, 13404, 13406-13411, 13413-13414, 13417-13422, 13426-13428, 13430-13432, 13435-13436, 13438-13441, 13443-13444, 13446, 13452-13453, 13455- 13456, 13461-13462, 13464-13470, 13472, 13475, 13477-13481, 13483, 13485, 13487, 13493-13501, 13503, 13506-13507, 13511, 13513, 13515- 13518, 13520-13523, 13526, 13529-13531, 13534-13535, 13537-13542, 13544-13545, 13550, 13555, 13558-13560, 13562, 13564, 13566, 13568- 13569, 13571-13573, 13578-13582, 13584-13589, 13591, 13593-13594, 13596-13598, 13600, 13602-13605, 13607-13608, 13610-13612, 13617- 13619, 13621-13623, 13625-13626, 13629-13630, 13632-13633, 13636, 13641, 13647-13649, 13656-13661, 13664-13670, 13672, 13676, 13678, 13680, 13682-13684, 13687-13692, 13694-13698, 13700-13702, 13704- 13705, 13708-13709, 13713-13719, 13722-13724, 13726-13730, 13732- 13739, 13741-13742, 13744, 13746-13748, 13750-13753, 13755, 13757- 13758, 13760-13765, 13767, 13771, 13775, 13778, 13782-13783, 13785, 13787-13794, 13796, 13799-13801, 13804, 13806-13807, 13809, 13811- 13817, 13819-13821, 13823-13824, 13829-13830, 13834, 13838-13843, 13845, 13847-13849, 13851-13857, 13859-13860, 13862-13864, 13866, 13869, 13872, 13874-13878, 13881-13882, 13885-13886, 13890, 13894- 13900, 13904, 13909-13911, 13913-13914, 13916-13924, 13926-13931, 13936, 13939-13940, 13942-13957, 13959-13960, 13962-13963, 13969- 13971, 13973-13974, 13976-13983, 13985-13987, 13990-13991, 13993, 13996-14010, 14012-14016, 14018-14020, 14023-14030, 14033, 14035- 14036, 14038-14041, 14043, 14045-14052, 14055-14057, 14059-14063, 14065-14067, 14069, 14071-14072, 14074-14075, 14078, 14081-14083, 14086, 14088-14094, 14100, 14103, 14105-14107, 14110-14112, 14115- 14119, 14121-14127, 14129, 14132-14134, 14136-14140, 14142-14143, 14145-14148, 14151-14157, 14159, 14167-14168, 14170-14173, 14175, 14177-14184, 14190-14192, 14195-14199, 14201-14202, 14204-14205, 14207-14208, 14210, 14212, 14216-14221, 14225, 14227, 14229-14234, 14237-14239, 14242-14243, 14246-14247, 14250-14256, 14258-14263, 14265-14266, 14269-14270, 14272-14274, 14276, 14279-14283, 14286- 14289, 14291-14294, 14298-14302, 14304, 14306-14310, 14312, 14314- 14315, 14317-14322, 14326, 14328-14330, 14333-14336, 14339-14342, 14345, 14348-14354, 14366-14370, 14372, 14375-14385, 14387-14390, 14392-14398, 14402-14409, 14413-14416, 14418-14421, 14423-14424, 14426-14427, 14429-14433, 14435-14437, 14439-14440, 14443, 14447- 14453, 14456-14472, 14476-14479, 14485-14486, 14490-14499, 14503- 14505, 14508-14509, 14513, 14515-14517, 14519-14522, 14524, 14532- 14533, 14536, 14540, 14544-14545, 14548-14549, 14552-14553, 14555- 14556, 14559, 14561-14565, 14567, 14569-14570, 14572-14574, 14576- 14588, 14590, 14592-14595, 14600-14607, 14612-14613, 14617-14618, 14620-14628, 14630, 14634-14635, 14637, 14640-14652, 14654-14657, 14660, 14662, 14664, 14667, 14672-14673, 14678-14683, 14685-14686, 14688, 14691-14695, 14697-14701, 14703-14705, 14708-14715, 14717- 14720, 14723-14724, 14726, 14729, 14731, 14733, 14735-14739, 14742- 14743, 14745-14746, 14748, 14750, 14754-14755, 14758, 14761-14762, 14765-14768, 14771-14783, 14789, 14791-14793, 14795-14799, 14802- 14805, 14807, 14809-14815, 14822-14824, 14826, 14829-14830, 14838- 14839, 14844-14845, 14847-14850, 14852, 14855-14857, 14861-14872, 14874, 14878-14880, 14882-14883, 14885, 14887-14888, 14890, 14893, 14896, 14899-14900, 14902-14906, 14910, 14913-14917, 14919, 14921- 14924, 14926-14928, 14930, 14932-14934, 14936-14939, 14944, 14946- 14950, 14952, 14955-14960, 14962, 14966-14968, 14970, 14972, 14974- 14975, 14977, 14979-14980, 14985-14986, 14988-14997, 14999-15008, 15011-15014, 15016-15018, 15020-15023, 15025-15029, 15031-15035, 15037, 15039-15041, 15044-15046, 15054-15055, 15059, 15061, 15065, 15067-15074, 15076, 15080-15081, 15085, 15087-15088, 15090-15093, 15095-15099, 15102, 15104-15112, 15114-15116, 15118, 15121-15122, 15126-15127, 15129-15131, 15134, 15136, 15141, 15145-15153, 15157, 15159-15160, 15165-15176, 15181, 15184-15185, 15188-15192, 15195, 15197-15198, 15201, 15204-15205, 15208-15209, 15211-15215, 15217- 15230, 15232-15235, 15237-15242, 15244-15245, 15247-15250, 15252, 15257, 15259-15261, 15264-15267, 15271-15272, 15274-15278, 15280, 15283, 15285-15289, 15291, 15294-15295, 15299-15300, 15308-15311, 15314-15316, 15318, 15323-15324, 15326-15328, 15330-15334, 15337- 15338, 15341, 15343, 15348-15350, 15353-15354, 15357-15359, 15361- 15362, 15366, 15369-15374, 15376-15380, 15383-15384, 15386-15396, 15399, 15401, 15404-15414, 15416-15418, 15420-15423, 15427-15432, 15435-15440, 15443-15451, 15454-15455, 15457, 15461, 15463, 15467- 15468, 15470, 15472-15477, 15479-15480, 15482, 15484-15490, 15492, 15494-15497, 15503, 15505-15509, 15511, 15513, 15515-15518, 15521- 15522, 15525-15527, 15531, 15533, 15535-15543, 15545-15547, 15549, 15552, 15557, 15559-15560, 15562, 15564, 15566, 15568, 15570-15571, 15573-15584, 15586-15588, 15590-15592, 15594-15596, 15598, 15600, 15603-15604, 15606-15607, 15609, 15611-15612, 15615, 15620-15621, 15625-15626, 15632-15638, 15641, 15643, 15649-15652, 15654-15655, 15657-15658, 15660-15662, 15664, 15666, 15672-15675, 15677-15678, 15681-15682, 15686-15688, 15691-15697, 15700, 15702-15703, 15706, 15708-15713, 15715, 15719, 15721, 15726, 15728-15730, 15732-15733, 15735- 15738, 15744- 15745, 15747- 15749, 15751, 15753-15756, 15761, 15763- 15764, 15766, 15768- 15769, 15774- 15778, 15780-15781, 15785, 15789, 15791- 15792, 15794, 15796 -15800, 15802, 15808, 15814, 15817 15819, 15821- 15822, 15824- 15825, 15827, 15831, 15833-15834, 15836- 15840, 15842- 15843, 15845- 15847, 15849, 15852, 15855, 15857, 15860 15862, 15864, 15866 -15870, 15876 , 15878 , 15880, 15884-15886, 15893 15896, 15898- 15901, 15903- 15904, 15906- 15907, 15911, 15914, 15916- 15920, 15922- 15924, 15926- 15930, 15932- 15934, 15936, 15938-15939, 15945, 15947, 15949 -15950, 15959 -15962, 15968-15969, 15972-15978, 15980- 15982, 15984- 15985, 15988, 15991- 15992, 15994, 15997-15998, 16001, 16003, 16005 -16007, 16009 -16010, 16013-16014, 16016-16018, 16022- 16026, 16028, 16030- 16032, 16034- 16038, 16040-16043, 16049, 16051, 16053- 16057, 16061, 16063 , 16065 , 16067-16073, 16075, 16078 16080, 16085, 16091 -16092, 16096 -16100, 16102-16103, 16105, 16107, 16109- 16110, 16113- 16117, 16119, 16121- 16124, 16127-16128, 16130- 16135, 16138- 16139, 16144, 16149 , 16151 -16156, 16158, 16160-16162, 16164- 16165, 16172- 16173, 16175- 16182, 16184, 16186-16189, 16191- 16193, 16197- 16199, 16202, 16208 -16211, 16213-16214, 16216-16219, 16221- 16230, 16232, 16234- 16236, 16238- 16240, 16242, 16244-16245, 16247- 16248, 16250, 16252, 16255 -16256, 16262-16263, 16267, 16271, 16273- 16277, 16279, 16281, 16286 -16287, 16290, 16295, 16297-16298, 16300- 16301, 16310, 16312, 16316 , 16321 -16322, 16324-16326, 16328- 16329, 16333- 16336, 16339, 16342 , 16347 , 16349-16358, 16360, 16362 16363, 16372- 16373, 16376- 16377, 16379- 16381, 16388-16389, 16391- 16400, 16402, 16407 , 16409 -16414 , 16416 , 16418, 16420-16421, 16423 16424, 16427- 16428, 16430- 16431, 16434, 16436, 16439, 16441-16443, 16445, 16450, 16453 , 16455 -16456 , 16468 , 16473-16479, 16481-16482, 16485, 16487- 16489, 16495, 16497 , 16499 , 16501-16503, 16505-16507, 16510- 16513, 16515- 16517, 16519- 16533, 16535-16536, 16538-16539, 16543- 16544, 16546, 16548- 16552, 16554, 16556, 16558-16561, 16563- 16564, 16566, 16568 -16570, 16572 -16573, 16575-16578, 16580-16586, 16589- 16590, 16592- 16594, 16596, 16599- 16608, 16610, 16612-16614, 16617- 16619, 16623- 16624, 16626- 16629, 16631, 16633-16634, 16636, 16640- 16649, 16651- 16655, 16657- 16664, 16666-16675, 16679-16680, 16682- 16690, 16693, 16696- 16697, 16701- 16704, 16706, 16708, 16711- 16718, 16720- 16721, 16725, 16728 , 16731 -16732, 16734, 16736, 16738 16739, 16742, 16745 , 16750 -16753 , 16755 -16756, 16760, 16762-16765, 16767- 16768, 16771, 16773- 16774, 16776- 16778, 16782-16787, 16789- 16792, 16795- 16796, 16799- 16800, 16803- 16806, 16810-16815, 16818- 16823, 16826, 16828 -16829, 16831 -16833, 16835-16840, 16842, 16845- 16848, 16850- 16852, 16854- 16858, 16862- 16863, 16866-16869, 16873- 16875, 16884- 16886, 16889- 16894, 16897- 16901, 16904, 16906-16907, 16909, 16911, 16915 -16921, 16923 , 16925 -16927, 16930, 16937-16938, 16940- 16941, 16943- 16944, 16946, 16951- 16953, 16955-16957, 16959, 16961- 16966, 16970, 16972- 16973, 16975- 16976, 16978, 16981, 16983- 16985, 16987- 16990, 16992, 16994 -16997, 16999-17004, 17006-17007, 17010, 17013- 17014, 17016- 17024, 17026, 17028-17030, 17033-17034, 17037- 17043, 17045, 17050- 17056, 17060, 17062-17066, 17069-17071, 17075, 17079- 17083, 17087, 17089 , 17091 -17092, 17094-17101, 17103- 17106, 17108- 17109, 17111- 17113, 17117- 17124, 17126, 17133, 17136- 17139, 17141- 17145, 17147, 17149 -17151, 17158, 17163, 17166-17170, 17178-17182, 17184, 17186-17188, 17190, 17192, 17194-17197, 17201, 17205, 17207, 17210, 17217-17220, 17223, 17227, 17230-17231, 17233, 17236-17237, 17240-17241, 17243-17244, 17249, 17252, 17255, 17257- 17258, 17260, 17262-17266, 17270-17278, 17280, 17283, 17285, 17292- 17293, 17295, 17302, 17305-17307, 17309-17311, 17313, 17318-17321, 17323, 17325, 17327-17328, 17330, 17332-17334, 17338-17339, 17341, 17343-17346, 17349-17353, 17356, 17360, 17363, 17368, 17370, 17372, 17374, 17377-17379, 17381-17396, 17398-17400, 17402-17403, 17406- 17407, 17409-17410, 17414-17419, 17421, 17423-17427, 17432-17439, 17443, 17445, 17447-17449, 17451-17454, 17457-17459, 17462-17463, 17465-17467, 17469-17470, 17472-17476, 17478-17479, 17481-17483, 17486, 17488-17489, 17491-17500, 17502-17510, 17512-17515, 17518- 17521, 17524, 17526, 17529, 17531-17532, 17534, 17537, 17540-17542, 17545-17547, 17549-17557, 17559, 17561-17565, 17571-17573, 17576- 17581, 17584-17586, 17589-17590, 17592-17595, 17600, 17602, 17605, 17611-17613, 17615, 17621-17624, 17629, 17631-17633, 17635-17639, 17641-17648, 17650, 17652-17653, 17656-17657, 17659-17661, 17665- 17667, 17669-17674, 17680-17689, 17692, 17695-17696, 17698-17702, 17704-17708, 17715, 17718, 17720, 17723, 17725, 17727, 17730-17735, 17737-17739, 17742-17745, 17747-17749, 17752-17754, 17764-17765, 17768, 17770-17773, 17778-17786, 17788-17793, 17795-17798, 17801, 17803-17807, 17809-17815, 17819-17820, 17822, 17824-17826, 17830- 17834, 17838, 17840-17841, 17844-17845, 17847-17850, 17854-17858, 17860-17861, 17863, 17865, 17867-17868, 17870, 17872-17874, 17876- 17878, 17880-17881, 17884, 17886, 17888, 17890-17891, 17893-17894, 17896-17900, 17905-17933, 17935, 17937-17940, 17943-17945, 17947- 17955, 17958-17961, 17963-17964, 17967, 17980, 17982, 17986, 17988- 17994, 17997, 17999-18000, 18002-18005, 18007-18014, 18016-18025, 18029, 18031, 18033-18035, 18038, 18041-18043, 18045-18049, 18051, 18053, 18056-18062, 18064-18065, 18067-18076, 18078, 18081-18082, 18084, 18086-18090, 18092-18098, 18100-18102, 18104-18107, 18109, 18112, 18119-18122, 18125-18126, 18128, 18133, 18137-18140, 18142, 18144-18145, 18147, 18149-18150, 18152, 18155, 18157-18160, 18163- 18165, 18170-18175, 18177-18178, 18180-18182, 18184-18185, 18188- 18193, 18195-18198, 18200, 18203-18204, 18208-18217, 18219, 18222- 18229, 18232-18236, 18238-18241, 18243-18249, 18251-18252, 18256- 18258, 18261-18262, 18264-18267, 18270, 18272-18277, 18279, 18282, 18284-18287, 18289-18295, 18298-18301, 18304-18306, 18308-18314, 18316-18317, 18319, 18323-18324, 18328-18333, 18335-18336, 18338, 18342-18345, 18350, 18352, 18359, 18362, 18365-18369, 18374-18387, 18389-18395, 18397-18398, 18401, 18403, 18405-18406, 18414-18415, 18417-18418, 18420-18421, 18423-18425, 18427-18430, 18432-18434, 18438-18439, 18441-18442, 18444-18446, 18450-18452, 18455-18460, 18463-18465, 18467-18471, 18474-18483, 18485-18488, 18490-18493, 18496, 18499, 18501, 18503, 18507, 18509, 18513-18515, 18518-18520, 18523-18525, 18528-18529, 18531, 18533, 18536, 18539-18541, 18549- 18551, 18553-18557, 18559-18567, 18570-18573, 18575-18578, 18582- 18586, 18588-18599, 18601-18603, 18605, 18609, 18611-18612, 18620, 18622, 18624, 18626, 18628-18629, 18631, 18633, 18635-18642, 18646- 18649, 18651, 18654-18657, 18664, 18666-18668, 18671-18672, 18674- 18675, 18678, 18681-18686, 18689-18690, 18692-18693, 18695, 18697, 18699-18700, 18702-18704, 18706, 18709, 18711-18712, 18714-18715, 18717, 18720, 18733, 18735-18736, 18739-18742, 18744-18747, 18749- 18750, 18754, 18756-18758, 18760, 18762-18766, 18768, 18771-18773, 18775-18778, 18781-18782, 18785-18786, 18788, 18790, 18792-18797, 18799-18802, 18807-18809, 18814, 18816, 18818-18820, 18823, 18825, 18829, 18835, 18839-18840, 18843-18847, 18849-18852, 18857-18861, 18865, 18868-18876, 18878, 18880-18887, 18889-18890, 18892, 18896- 18897, 18899-18900, 18903, 18905-18906, 18909-18911, 18913, 18916, 18918-18919, 18921-18922, 18925, 18931, 18934, 18936-18937, 18941- 18943, 18948, 18951, 18956, 18958-18960, 18962, 18965-18970, 18972- 18975, 18977-18980, 18982-18992, 18995, 18997, 19001, 19008, 19010, 19012-19018, 19020, 19022, 19024-19025, 19028-19033, 19036-19046, 19048-19054, 19057-19058, 19060-19066, 19068-19073, 19075, 19079, 19082-19083, 19089, 19102-19103, 19108, 19111-19115, 19117, 19120- 19124, 19126, 19128, 19130-19132, 19137-19139, 19141-19142, 19144- 19150, 19152-19156, 19160-19163, 19166-19181, 19183, 19187-19194, 19197, 19199, 19201, 19205, 19208-19209, 19212, 19214, 19216-19218, 19220-19221, 19224, 19228, 19231, 19233-19236, 19240-19241, 19244, 19246-19259, 19266, 19268-19269, 19271, 19274-19275, 19277-19279, 19281, 19286-19288, 19294-19295, 19297-19298, 19301-19305, 19307, 19311, 19313, 19315, 19317-19319, 19321-19323, 19325, 19327-19329, 19331, 19335-19338, 19340-19342, 19345, 19347-19350, 19353-19355, 19357, 19360-19364, 19366, 19368-19369, 19373, 19375, 19377-19378, 19382-19386, 19388-19390, 19395-19401, 19403-19404, 19407-19408, 19410, 19412-19414, 19419, 19421-19423, 19425-19429, 19431-19432, 19434, 19436-19437, 19439, 19441-19442, 19448-19452, 19454, 19456, 19458-19468, 19470, 19472, 19474, 19478, 19481-19483, 19485, 19487- 19488, 19492, 19496-19498, 19501, 19504, 19507-19514, 19516-19522, 19524-19526, 19528, 19530-19531, 19535, 19538, 19541, 19543, 19548- 19549, 19556-19558, 19560-19563, 19565, 19567-19570, 19573-19574, 19576, 19579-19581, 19583, 19585, 19593-19601, 19603-19616, 19618- 19621, 19624-19625, 19627-19628, 19630, 19632-19636, 19639-19644, 19646, 19649, 19653-19657, 19659, 19661-19664, 19666-19668, 19673- 19676, 19680-19683, 19687, 19689-19690, 19692-19696, 19698-19699, 19701, 19703, 19706-19707, 19709, 19712-19718, 19725-19726, 19728- 19730, 19732-19735, 19738, 19740, 19742-19745, 19748-19749, 19751, 19753-19758, 19760, 19762-19764, 19766, 19768, 19770-19771, 19773- 19782, 19784, 19787, 19789-19794, 19797, 19799, 19802-19804, 19806, 19809-19815, 19817-19827, 19829-19833, 19836-19837, 19839-19841, 19846, 19849, 19852, 19857, 19859, 19863, 19866, 19869-19871, 19873, 19881, 19885-19886, 19890-19893, 19895-19896, 19898-19902, 19904, 19915, 19919, 19926, 19928-19930, 19933, 19943-19944, 19947-19949, 19951-19952, 19954-19955, 19960-19961, 19963-19965, 19967-19968, 19970-19975, 19977-19979, 19982-19983, 19986-19987, 19989-19991, 19993, 19995-19997, 20002, 20004, 20006-20009, 20013-20027, 20029- 20030, 20032, 20036-20041, 20043-20044, 20047-20051, 20053-20066, 20070-20077, 20079-20080, 20083-20094, 20096-20098, 20100-20101, 20104-20108, 20110-20112, 20115, 20117-20121, 20125-20126, 20128- 20130, 20134-20136, 20138, 20140, 20143, 20148-20149, 20152-20153, 20155, 20157, 20161-20162, 20164-20165, 20168, 20175, 20177, 20183- 20184, 20186, 20188-20189, 20192-20195, 20200-20202, 20207-20210, 20214, 20218, 20221-20226, 20228-20234, 20237-20240, 20242, 20244, 20246, 20248-20253, 20257, 20259-20260, 20265, 20267-20268, 20270- 20274, 20276, 20279-20287, 20289-20292, 20294-20295, 20298-20299, 20301, 20303, 20305, 20309-20311, 20314, 20316, 20318, 20320, 20322, 20324, 20329, 20332-20333, 20338, 20342, 20351-20358, 20360-20361, 20366, 20368-20374, 20376-20381, 20383, 20385-20391, 20393-20394, 20396-20400, 20402, 20404-20407, 20409, 20412, 20416-20417, 20419- 20423, 20425, 20430-20438, 20441-20443, 20445, 20448, 20451-20453, 20456, 20458, 20460-20462, 20464, 20466, 20468-20472, 20474, 20477- 20479, 20484, 20486, 20488-20491, 20493-20494, 20504, 20506-20508, 20511-20513, 20515-20519, 20521, 20523-20524, 20528-20529, 20532, 20535-20537, 20541, 20543-20545, 20547, 20551-20560, 20564-20565, 20567-20568, 20570, 20572, 20577, 20581-20583, 20585-20586, 20588, 20594-20596, 20599, 20601-20602, 20604-20606, 20608, 20613-20615, 20617-20618, 20625, 20627-20630, 20632-20640, 20642, 20651, 20658- 20659, 20661, 20663-20666, 20668, 20670-20671, 20680-20682, 20685, 20687-20688, 20690-20692, 20694, 20699, 20703, 20707-20710, 20712- 20717, 20721-20722, 20726-20727, 20730-20731, 20734, 20740-20741, 20745, 20747, 20749, 20751, 20756-20762, 20768, 20772-20783, 20785, 20789-20791, 20794-20797, 20799-20801, 20803-20808, 20814, 20816,
20818, 20827, 20829-20830, 20833, 20835-20836, 20842, 20850, 20852- 20854, 20856, 20858, 20860, 20862, 20864, 20868-20872, 20880-20881, 20884-20887, 20891-20893, 20901, 20903, 20905-20906, 20913-20916, 20918, 20920, 20923, 20926-20927, 20931, 20936, 20941-20943, 20945, 20947-20948, 20950-20953, 20956, 20959-20960, 20963-20964, 20968- 20974, 20978-20980, 20984-20985, 20987, 20990-20998, 21000-21002, 21011-21012, 21021-21030, 21032-21033, 21035-21036, 21039, 21043- 21044, 21047-21049, 21053, 21055-21056, 21058, 21060, 21062-21068, 21070-21071, 21073, 21075, 21077, 21080, 21083-21087, 21093, 21102- 21103, 21106-21108, 21110, 21112-21113, 21118-21120, 21123-21127, 21129-21136, 21138, 21141-21149, 21151, 21153, 21156-21158, 21161, 21165-21166, 21168, 21170-21171, 21173, 21175-21182, 21184, 21187, 21191, 21194-21196, 21198, 21200-21204, 21210, 21213, 21216, 21218- 21220, 21223-21224, 21226, 21228, 21230, 21234, 21236-21237, 21239- 21240, 21242, 21246, 21249, 21252, 21256-21260, 21263, 21268, 21272, 21274, 21277-21278, 21281, 21283-21292, 21294-21300, 21302-21308, 21311, 21314-21317, 21319-21325, 21327, 21330, 21338-21342, 21344- 21346, 21352-21353, 21355-21356, 21363-21365, 21369, 21372-21373, 21375, 21378, 21380-21392, 21394-21395, 21397-21400, 21403, 21406, 21409, 21413-21416, 21422-21424, 21427-21428, 21431, 21433, 21442, 21445-21447, 21449, 21451-21452, 21454, 21456, 21459-21460, 21462, 21467, 21469, 21471-21472, 21478-21479, 21484-21486, 21488-21489, 21491-21494, 21496, 21498, 21500, 21502-21504, 21506-21507, 21512, 21514, 21516, 21519, 21527, 21530-21531, 21533, 21535-21536, 21541, 21543-21545, 21547-21553, 21555-21559, 21561, 21565, 21569-21571, 21573-21575, 21577-21578, 21580, 21582, 21584, 21586, 21589-21590, 21592-21593, 21596-21597, 21599-21600, 21603, 21605, 21607, 21611, 21613-21615, 21617, 21619, 21621, 21623-21625, 21627-21634, 21636- 21637, 21643-21646, 21648-21649, 21651, 21655-21656, 21658-21660, 21662, 21665-21666, 21668-21670, 21674, 21676-21682, 21684-21685, 21687, 21690-21693, 21698, 21700-21702, 21704, 21706, 21711-21712, 21714-21718, 21721-21722, 21724-21734, 21739-21740, 21742-21744, 21746, 21748, 21751, 21758-21760, 21762-21766, 21770-21773, 21777- 21779, 21781, 21783-21788, 21790-21792, 21797-21799, 21801-21805, 21807-21808, 21811-21814, 21818, 21820, 21822, 21826, 21828, 21832, 21834, 21836, 21838-21839, 21842, 21844, 21846-21847, 21849, 21851- 21853, 21856-21863, 21869-21871, 21876, 21878, 21880-21882, 21884- 21894, 21896-21897, 21899, 21902-21904, 21908-21914, 21916-21920, 21923, 21925, 21927-21933, 21935, 21937-21942, 21944-21951, 21954, 21957, 21960-21961, 21964, 21968-21969, 21972-21973, 21976-21980, 21982, 21985, 21987, 21990, 21998, 22000, 22003-22004, 22006-22008, 22011, 22014, 22019, 22021-22024, 22028, 22031-22032, 22034, 22036- 22042, 22047-22048, 22050-22051, 22054, 22057-22059, 22062, 22065, 22069-22071, 22082, 22084-22085, 22089-22091, 22097-22101, 22107- 22114, 22116-22117, 22119-22121, 22129, 22137, 22139, 22141, 22143- 22145, 22147-22149, 22151-22153, 22155-22156, 22159-22160, 22162- 22164, 22167, 22169-22171, 22176-22177, 22179, 22181-22182, 22184- 22187, 22189-22192, 22194-22197, 22200-22201, 22203-22210, 22212- 22213, 22220-22221, 22223, 22225-22230, 22232-22233, 22237-22239, 22241-22244, 22246, 22249-22264, 22268, 22270-22272, 22274, 22278- 22279, 22281-22285, 22287-22289, 22291-22295, 22297-22303, 22305, 22308-22311, 22317-22320, 22322-22323, 22325-22331, 22334, 22337- 22338, 22342-22348, 22350-22355, 22357, 22361-22362, 22364-22365, 22369-22370, 22372-22375, 22377-22381, 22385-22388, 22390-22398, 22400-22402, 22404-22409, 22411-22423, 22425-22426, 22428, 22436, 22449, 22452, 22455, 22457-22458, 22463, 22467, 22472, 22478, 22481- 22482, 22485, 22488-22492, 22494-22495, 22499-22500, 22502, 22504- 22506, 22508, 22510-22513, 22515, 22517-22521, 22523-22524, 22527- 22529, 22532, 22535, 22537-22540, 22546-22550, 22552-22555, 22560- 22563, 22565-22567, 22569-22572, 22574, 22576-22577, 22579-22580, 22582, 22585, 22588-22593, 22595, 22601, 22607-22609, 22612-22613, 22616, 22618, 22620-22622, 22624, 22626-22630, 22634, 22636-22643, 22645-22646, 22648-22656, 22658-22659, 22661-22662, 22664, 22668, 22672, 22674-22681, 22684-22685, 22687-22691, 22693-22696, 22698- 22699, 22701-22705, 22709, 22712, 22717-22719, 22722-22727, 22729, 22732, 22740-22742, 22744, 22751-22753, 22761-22765, 22768-22773, 22780-22781, 22783-22786, 22788-22789, 22794, 22797-22798, 22800, 22803, 22805, 22810-22814, 22816, 22819, 22821-22827, 22829, 22832, 22836-22840, 22842, 22850, 22852, 22856, 22858-22862, 22866, 22872, 22874, 22876-22877, 22879-22881, 22886-22887, 22889, 22892-22896, 22898-22906, 22908, 22910, 22913-22918, 22928, 22930-22933, 22935- 22937, 22941, 22944, 22950-22951, 22953-22958, 22963-22965, 22968- 22972, 22974-22976, 22979-22984, 22986, 22988-22989, 22991-22999, 23001, 23003, 23006-23011, 23015-23016, 23024-23028, 23030-23032, 23034-23035, 23037-23039, 23042, 23051, 23054, 23057, 23061-23063, 23065-23068, 23073-23074, 23076-23081, 23083-23086, 23088-23099, 23101-23107, 23110, 23113-23117, 23119-23120, 23122, 23128-23130, 23132, 23137, 23140-23141, 23143, 23146, 23150, 23152, 23156, 23163- 23164, 23166-23167, 23177, 23180-23184, 23186-23187, 23190-23191, 23193, 23196-23199, 23201-23203, 23206, 23208, 23212-23215, 23217, 23220, 23222, 23225-23226, 23229, 23231-23236, 23239, 23243, 23246- 23247, 23249-23251, 23253, 23256, 23258, 23261-23263, 23265, 23272, 23274- 23277, 23282- 23283, 23285, 23288, 23292 - 23296, 23299- 23301,
23303- 23304, 23310- 23315, 23318- 23325, 23331, 23335- 23337, 23339,
23341- 23342, 23344- 23345, 23349- 23354, 23356- 23357, 23359-23360,
23362- 23363, 23365, 23368- 23369, 23371, 23373 f 23375 -23376, 23378-
23380, 23382, 23385 , 23389 -23391 , 23393 , 23397 -23398 , 23400 , 23404
23405, 23409, 23411 -23416, 23418 , 23420 -23423 r 23427 , 23429 -23431,
23436- 23444, 23446- 23447, 23450- 23451, 23454- 2345Ί, 23461, 23463-
23465, 23468- ■23475, 23477- 23482, 23485- 23487, 23490- 23495, 23499-
23500, 23502- ■23507, 23513, 23515 -23520, 23522 - 23527, 23529- 23532,
23534- 23538, 23540, 23543- 23545, 23548- 23549, 23551- 23561, 23563,
23565, 23567- ■23568, 23570- 23572, 23575- 23578, 23580- 23584, 23586,
23592- 23595, 23597- 23599, 23601, 23603, 23606 - 23607, 23610- 23611,
23615- 23616, 23621, 23625- 23634, 23638- 23639, 23642, 23645, 23652-
23654, 23660- ■23663, 23665- 23667, 23670, 23673 - 23674, 23676- 23678,
23681- 23684, 23687, 23689, 23693 -23695, 23701 - 23702, 23705- 23712,
23716- 23717, 23720, 23724- 23725, 23735, 23737 - 23738, 23740- 23741,
23745- 23746, 23748, 23751- 23761, 23764- 23765, 23769, 23775, 23777-
23779, 23781- ■23789, 23791- 23793, 23795, 23799 - 23800, 23802- 23803,
23805- 23810, 23812, 23814, 23817 -23820, 23823 - 23824, 23829- 23830,
23834- 23838, 23841, 23843- 23850, 23852, 23856 r 23860 , 23862 , 23868
23870, 23872- ■23874, 23876, 23879 , 23881 -23883 r 23887 -23890, 23892,
23895, 23899- ■23902, 23904- 23907, 23910, 23914 - 23917, 23920, 23923,
23926- 23929, 23932- 23936, 23938- 23939, 23941- 23942, 23944-23946,
23948- 23949, 23951, 23953- 23954, 23956- 23962, 23965, 23969- 23973,
23976- 23983, 23985, 23987, 23992 , 23994 , 23996 -23998 , 24001 , 24005
24006, 24008- ■24009, 24013- 24018, 24020- 24021, 24027- 24029, 24034-
24035, 24037, 24039 , 24041 , 24045-24052 , 24054 -24055 , 24057 , 24059
24060, 24065, 24067 , 24069 , 24073, 24077-24083 , 24085, 24087-24089
24093, 24095- ■24096, 24099, 24104 -24107, 24109 - 24110, 24112, 24114,
24116, 24122- ■24124, 24127, 24129 -24130, 24132 - 24134, 24136, 24138,
24140, 24144- ■24145, 24148- 24149, 24151, 24153 r 24155 -24164, 24166,
24168- 24169, 24171, 24173- 24177, 24179- 24180, 24182, 24184, 24188-
24190, 24192- ■24194, 24198, 24200 -24204, 24206 r 24208 , 24211 -24215,
24217- 24218, 24221, 24224, 24226 -24232, 24234 r 24237 , 24239 -24247,
24253- 24254, 24256, 24259- 24260, 24262- 24264, 24266, 24268, 24271-
24273, 24278- ■24279, 24281- 24282, 24285- 24287, 24289, 24291- 24294,
24296- 24302, 24304- 24308, 24310, 24312- 24313, 24322, 24324- 24327,
24329- 24333, 24335, 24337, 24341 -24342, 24347 r 24349 -24353, 24355,
24357, 24359, 24361 -24372, 24375 , 24379 , 24381 -24384 , 24387 , 24390
24392, 24395- ■24396, 24399, 24401 -24402, 24407 - 24408, 24411- 24413,
24415- 24416, 24419- 24423, 24426- 24427, 24429- 24430, 24434, 24437-
24439, 24441, 24446 -24449, 24452 -24457, 24459 r 24461 -24469, 24471,
24473, 24476- ■24477, 24479, 24481 -24483, 24486 - 24487, 24490- 24491,
24493, 24498- ■24500, 24502- 24504, 24506- 24511, 24513, 24519- 24522,
24525, 24528, 24530 -24532, 24534 , 24538 -24541 r 24544 -24545, 24547-
24550, 24553- ■24557, 24561- 24563, 24566, 24569 r 24571 -24572, 24574-
24578, 24582, 24584 -24585, 24589 -24590, 24593 - 24605, 24607, 24611,
24613, 24615- ■24624, 24626- 24629, 24634, 24637 - 24638, 24642- 24645,
24647- 24648, 24651, 24655, 24658 -24669, 24673 - 24678, 24680, 24684,
24688- 24689, 24692- 24693, 24696- 24698, 24700, 24703, 24705- 24708,
24711, 24713- ■24715, 24717- 24720, 24722- 24723, 24727- 24729, 24731, 24733, 24735-24737, 24740-24743, 24746-24752, 24754, 24756-24761, 24764-24768, 24771-24772, 24774-24778, 24782, 24786, 24788-24790, 24792, 24796, 24800, 24802-24803, 24805-24806, 24809, 24811, 24820- 24823, 24828, 24830, 24834, 24836, 24838-24845, 24847-24848, 24850- 24852, 24854-24855, 24858-24863, 24871, 24873, 24875, 24879-24881, 24883-24885, 24888-24889, 24891, 24894-24902, 24904-24907, 24912- 24915, 24917-24919, 24921, 24924-24927, 24929, 24932-24933, 24935, 24937-24938, 24942, 24944, 24948-24949, 24951, 24957-24959, 24961- 24962, 24964, 24966-24967, 24969-24970, 24972-24978, 24982, 24985, 24988-24989, 24993-24994, 24998, 25000-25002, 25004, 25007-25008, 25010-25011, 25015, 25017-25018, 25022-25023, 25025-25029, 25031- 25042, 25044-25045, 25051, 25053-25054, 25058, 25060-25063, 25065, 25068, 25072, 25077, 25080, 25082-25083, 25085, 25087-25089, 25091- 25094, 25098-25102, 25105-25107, 25113, 25115, 25120-25123, 25125, 25127-25128, 25133, 25138-25140, 25142, 25146, 25149, 25151-25152, 25154, 25156-25158, 25164, 25166, 25168-25169, 25171-25175, 25181, 25183-25190, 25194-25195, 25197-25202, 25209, 25214-25215, 25217- 25219, 25221-25222, 25227-25230, 25232, 25234-25237, 25240, 25247, 25249-25250, 25252, 25255-25256, 25260, 25264-25278, 25280, 25282- 25294, 25296, 25300-25302, 25305, 25308-25309, 25312-25316, 25319- 25321, 25323, 25325, 25329-25330, 25332-25333, 25338-25339, 25345, 25348, 25352, 25354, 25356-25358, 25360-25361, 25366-25370, 25372, 25375, 25378, 25380, 25384-25385, 25387, 25389-25390, 25394, 25397, 25400-25410, 25412, 25414, 25416-25418, 25420, 25429-25437, 25439, 25441-25442, 25445, 25450-25456, 25459-25460, 25462-25465, 25468- 25469, 25472-25476, 25482-25483, 25488-25490, 25494-25495, 25498, 25501-25504, 25506, 25508, 25510, 25512-25515, 25517-25522, 25525- 25531, 25534, 25537-25541, 25543-25544, 25547, 25549, 25552-25555, 25557-25564, 25566-25568, 25570-25572, 25574-25576, 25579, 25581- 25586, 25588, 25596, 25599, 25605, 25607-25608, 25611, 25614-25615, 25617, 25620-25622, 25625-25626, 25629, 25631-25640, 25642-25644, 25650, 25653-25654, 25656-25661, 25664-25672, 25674-25675, 25677, 25683-25686, 25688, 25691, 25693-25697, 25701, 25704-25707, 25709- 25710, 25712, 25714-25719, 25721-25722, 25730-25731, 25734-25739, 25742-25744, 25747-25748, 25750, 25753, 25757, 25760, 25762, 25765, 25768, 25772, 25774, 25776-25779, 25782, 25784, 25786, 25788-25789, 25791-25794, 25796-25797, 25799-25800, 25803-25805, 25807, 25810, 25812-25814, 25817, 25819-25821, 25823-25824, 25826-25831, 25833- 25835, 25837, 25839-25841, 25843-25845, 25847, 25852-25856, 25858, 25861-25863, 25866, 25870-25874, 25878-25879, 25883-25884, 25886- 25892, 25894-25896, 25900-25904, 25907-25910, 25912, 25915, 25923- 25925, 25927-25932, 25936-25939, 25942-25943, 25945, 25947, 25949- 25951, 25953, 25956-25958, 25961-25963, 25966, 25968-25969, 25971, 25975-25976, 25979, 25982-25983, 25985, 25987, 25991, 25994-26000, 26003, 26006-26011, 26013-26015, 26017, 26020, 26025-26027, 26029, 26031-26032, 26037-26038, 26046-26049, 26051, 26054, 26059-26061, 26066, 26069, 26072-26076, 26078, 26080, 26083-26087, 26090, 26092, 26095-26097, 26100, 26102-26108, 26111-26112, 26117-26118, 26121, 26129, 26131-26132, 26134-26135, 26137-26142, 26146, 26149-26151, 26153-26154, 26157-26158, 26161-26162, 26164-26166, 26168-26170,
26172, 26175, 26183, 26185, 26192-26196, 26198-26199, 26203-26206, 26208- 26223, 26228, 26230- 26232, 26234, 26236 - 26238, 26240- 26242,
26245- 26248, 26251, 26256- 26259, 26261- 26267, 26270, 26272- 26273,
26276- 26277, 26279, 26281, 26284 -26285, 26287 - 26290, 26292- 26293,
26298, 26301- ■26311, 26316, 26318 , 26329 -26330 r 26333 -26334, 26337-
26342, 26344- ■26348, 26350, 26352 -26356, 26358 - 26364, 26366- 26373,
26375- 26376, 26378- 26382, 26384- 26385, 26388, 26391, 26393- 26394,
26396, 26398- ■26400, 26402, 26406 -26408, 26410 r 26412 -26419, 26421-
26423, 26426, 26428 -26429, 26431 -26432, 26437 r 26439 -26445, 26447,
26452- 26454, 26458- 26459, 26461- 26463, 26466, 26469- 26470, 26472-
26473, 26476, 26479 , 26481 -26482 , 26485 -26486 r 26491 , 26493 -26494,
26498- 26499, 26503- 26504, 26506- 26510, 26515, 26518, 26521- 26530,
26532- 26535, 26537- 26538, 26542- 26543, 26545- 26546, 26549-26552,
26555- 26557, 26559, 26561- 26562, 26564- 26565, 26567- 26568, 26571-
26574, 26577, 26580 , 26584 -26586 , 26588 -26589 r 26594 -26596, 26598,
26603, 26605- ■26606, 26608, 26610 -26614, 26617 - 26624, 26626- 26630,
26634, 26636- ■26638, 26642, 26644 -26649, 26651 - 26652, 26656, 26659-
26662, 26664, 26671 , 26676 -26680 , 26682 -26687 r 26689 -26690, 26694-
26697, 26699, 26701 , 26703 , 26705-26708 , 26710 -26715 , 26719 -26721,
26723, 26727- ■26730, 26735, 26738 , 26740 -26747 r 26750 -26752, 26755-
26757, 26759- ■26761, 26763- 26765, 26767- 26769, 26772, 26774- 26778,
26780- 26781, 26783, 26786- 26787, 26789- 26792, 26794- 26798, 26801-
26805, 26808- ■26812, 26814, 26816 -26818, 26820 - 26821, 26823- 26825,
26827- 26829, 26831- 26832, 26835- 26841, 26843- 26844, 26848-26851,
26856- 26861, 26863, 26865- 26866, 26868- 26869, 26871- 26872, 26874-
26876, 26878, 26881 -26887, 26889 -26891, 26893 - 26897, 26899, 26901,
26903- 26904, 26908, 26915- 26917, 26922- 26925, 26927- 26928, 26931,
26933, 26935- ■26940, 26943- 26946, 26948- 26955, 26958, 26960- 26962,
26969- 26971, 26974- 26976, 26978- 26980, 26983, 26985- 26986, 26989,
27000- 27002, 27004- 27005, 27007- 27008, 27010- 27014, 27022, 27024,
27029, 27033- ■27034, 27036- 27037, 27039- 27040, 27042- 27043, 27045-
27052, 27054- ■27055, 27064, 27069 , 27072 -27073 f 27075 , 27078 -27079,
27082, 27096- ■27097, 27106, 27110 , 27112 , 27114 , 27119, 27121-27122,
27124- 27125, 27128- 27129, 27131- 27132, 27134- 27135, 27140, 27149,
27160- 27163, 27165- 27166, 27169- 27170, 27172- 27177, 27180, 27182-
27183, 27186- ■27187, 27191, 27193 -27195, 27197 f 27201 -27203, 27205-
27206, 27208- ■27209, 27216- 27220, 27222- 27226, 27235, 27237- 27238,
27241- 27245, 27247, 27252, 27254 , 27256 -27261 r 27263 , 27265 -27266,
27268- 27269, 27273, 27276- 27280, 27282, 27284 - 27286, 27288- 27295,
27297- 27300, 27302- 27305, 27307, 27310- 27315, 27317- 27319, 27322-
27326, 27328- ■27330, 27338, 27342 , 27345 -27353 r 27355 , 27357 -27358,
27360- 27361, 27364- 27369, 27372- 27378, 27381- 27389, 27391-27393,
27396- 27398, 27400- 27402, 27405- 27411, 27413- 27415, 27417-27420,
27422, 27424- ■27429, 27433- 27437, 27439- 27448, 27451- 27452, 27456,
27458- 27460, 27464, 27466- 27470, 27473- 27474, 27479, 27481- 27488,
27490, 27493, 27495 , 27497 -27498 , 27500 , 27502 -27503 , 27506 -27517,
27519- 27525, 27527, 27531- 27540, 27543, 27545 - 27549, 27551, 27554,
27559, 27563- ■27566, 27568- 27570, 27572- 27579, 27581- 27586, 27588-
27590, 27592- ■27594, 27596- 27598, 27600, 27606 r 27609 -27610, 27613-
27614, 27616- ■27620, 27623- 27624, 27626, 27632 - 27633, 27635, 27638-
27640, 27643, 27650 -27659, 27661 , 27668 -27669 r 27671 -27673, 27676-
27678, 27684, 27686 -27690, 27695 , 27698 -27699 r 27701 , 27703 , 27705- 27707, 27711, 27714, 27718-27719, 27724, 27726, 27728-27729 , 27731, 27733-27735, 27738, 27741, 27747, 27750, 27752-27759, 27762, 27764- 27765, 27767-27773, 27777-27781, 27783, 27785, 27789, 27792-27793, 27795, 27799-27803, 27805, 27807-27808, 27810-27814, 27817-27818, 27820-27822, 27825, 27834, 27844, 27852, 27855, 27860, 27862-27868, 27870-27871, 27876-27877, 27879, 27883-27886, 27888, 27891-27892, 27894, 27896-27904, 27907, 27910, 27914, 27916-27922, 27924-27925, 27928-27929, 27936-27937, 27942-27948, 27954-27961, 27963-27969, 27971-27973, 27975, 27981, 27989, 27991-27992, 27994-27995, 27997, 27999-28000, 28003, 28006-28011, 28013, 28017-28018, 28020, 28022, 28024, 28026-28028, 28032-28033, 28036, 28040, 28043-28044, 28046, 28048, 28050-28051, 28053-28057, 28059-28060, 28062, 28065-28072, 28074, 28076, 28079, 28081, 28086, 28088-28092, 28094, 28096-28098, 28101-28104, 28107-28108, 28110, 28113, 28115-28116, 28118-28119, 28128, 28130-28131, 28133-28134, 28136-28139, 28143-28144, 28146- 28147, 28149-28151, 28154-28159, 28163-28164, 28166-28167, 28169- 28177, 28179, 28182-28185, 28188-28195, 28197, 28201, 28203-28204, 28207, 28210, 28214-28215, 28217, 28222-28225, 28227-28229, 28231- 28234, 28237, 28240, 28243-28250, 28253, 28255-28256, 28258-28262, 28264-28267, 28277, 28281-28282, 28284-28285, 28287-28291, 28293- 28295, 28299-28301, 28303-28304, 28306-28309, 28311, 28313-28316, 28318, 28322, 28324-28328, 28330, 28339-28340, 28342, 28344-28345, 28347-28349, 28351-28352, 28354, 28356-28357, 28360-28364, 28366, 28368-28370, 28372, 28374-28376, 28378-28379, 28384-28388, 28390- 28391, 28393, 28395-28400, 28402, 28404-28405, 28408-28410, 28412- 28423, 28426, 28428-28430, 28432-28435, 28437-28438, 28440-28442, 28446-28449, 28451-28459, 28462, 28466-28472, 28474, 28476-28478, 28480-28483, 28485, 28487, 28489, 28491, 28494, 28496, 28498-28500, 28502, 28505, 28509-28510, 28512-28514, 28516, 28518-28520, 28522- 28529, 28531-28533, 28535-28536, 28538-28539, 28542, 28545-28547, 28549, 28551-28552, 28554, 28556, 28558-28561, 28564-28567, 28569- 28572, 28574, 28576-28577, 28579-28581, 28586, 28589, 28591, 28594- 28602, 28604-28605, 28608-28610, 28612-28613, 28616-28617, 28621, 28623, 28629, 28631, 28633, 28635, 28639-28641, 28643-28646, 28648, 28650-28651, 28655, 28658-28660, 28662-28663, 28665-28666, 28690- 28692, 28694, 28696-28697, 28700, 28706-28707, 28714-28715, 28717- 28723, 28725-28727, 28730, 28732, 28737-28742, 28745-28746, 28748, 28755-28756, 28758-28759, 28762, 28765, 28769-28774, 28785, 28788- 28790, 28801-28806, 28808, 28810, 28817-28819, 28821, 28826-28829, 28831, 28834, 28837, 28839-28844, 28846-28847, 28849, 28853, 28856- 28857, 28859, 28861, 28863, 28865-28868, 28870-28871, 28874-28877, 28880-28881, 28883, 28886, 28888, 28890, 28892-28898, 28904-28906, 28913-28921, 28923-28930, 28933-28934, 28937, 28941-28942, 28944- 28946, 28948-28955, 28957-28964, 28967-28968, 28970-28973, 28979- 28981, 28983-28984, 28986, 28989, 28991, 28993-28994, 28996-28997, 29000, 29002, 29004, 29006-29010, 29012-29016, 29018, 29021, 29025- 29027, 29036-29039, 29042, 29044, 29047, 29049-29056, 29058, 29061, 29063, 29065, 29072, 29075, 29081-29084, 29089-29093, 29102, 29104, 29111, 29114-29119, 29121, 29124-29125, 29128, 29132-29141, 29143- 29146, 29149, 29151, 29153, 29155, 29159, 29161, 29163-29165, 29167- 29168, 29170-29171, 29174, 29176-29182, 29185, 29195, 29203, 29211- 29213, 29218-29223, 29225, 29229-29231, 29234-29236, 29239, 29241, 29243-29244, 29248, 29250-29251, 29254-29260, 29262, 29269, 29271, 29274-29276, 29280-29282, 29285-29288, 29290-29291, 29293, 29297- 29298, 29301-29303, 29306-29308, 29310, 29312-29314, 29316-29320, 29322, 29325-29330, 29332, 29334-29336, 29338-29341, 29345-29348, 29350, 29353-29356, 29358-29364, 29368-29371, 29373-29374, 29377- 29378, 29381-29383, 29385-29391, 29394, 29397-29399, 29404-29405, 29407, 29409-29412, 29414-29417, 29421-29423, 29425, 29429-29431, 29433-29434, 29438-29440, 29442-29443, 29445, 29447-29457, 29459, 29462, 29465-29466, 29468, 29470-29474, 29476-29478, 29480-29483, 29486-29487, 29490, 29492-29493, 29495-29496, 29498, 29500-29502, 29504-29506, 29508-29512, 29514, 29516-29519, 29521-29524, 29527, 29529, 29534-29539, 29544-29548, 29552, 29554, 29559-29563, 29565- 29566, 29571-29573, 29579, 29581-29583, 29587, 29589, 29591-29592, 29594-29595, 29597, 29599-29600, 29603, 29605-29606, 29610-29613, 29617-29621, 29623-29626, 29631, 29635-29647, 29649-29652, 29654- 29655, 29657-29659, 29661-29662, 29664-29665, 29667-29668, 29670- 29672, 29674-29676, 29678, 29681, 29683, 29685, 29689, 29691-29692, 29695, 29698, 29702, 29704-29706, 29708-29711, 29715, 29719-29723, 29725-29733, 29735, 29737-29743, 29745-29756, 29758-29763, 29765- 29767, 29769-29770, 29773-29775, 29777-29780, 29782-29785, 29787- 29789, 29791-29795, 29797, 29800-29803, 29805-29809, 29812-29813, 29816, 29820-29829, 29831-29833, 29835, 29838-29840, 29842, 29844, 29847, 29849-29850, 29852, 29855-29860, 29863-29865, 29867, 29869- 29874, 29879, 29881-29882, 29884-29887, 29889, 29891-29893, 29895- 29896, 29899-29900, 29902-29904, 29907, 29909, 29911, 29914, 29916, 29918, 29925, 29933, 29937-29941, 29943, 29949, 29951, 29953-29955, 29957-29961, 29964-29965, 29974, 29976, 29979, 29981, 29983-29984, 29986-29987, 29992, 29995, 29997-29998, 30000, 30003-30004, 30009, 30011-30012, 30014-30017, 30023, 30027, 30029-30030, 30048-30051, 30053, 30056-30061, 30063, 30072-30075, 30078-30079, 30082, 30084- 30087, 30090, 30092-30093, 30095-30098, 30100-30101, 30103-30104, 30106-30109, 30111, 30115, 30117, 30119, 30123-30124, 30126, 30128- 30134, 30136, 30138-30141, 30144-30148, 30150, 30153, 30155-30159, 30163-30177, 30179, 30181-30183, 30185, 30187-30189, 30192, 30194- 30200, 30206-30207, 30210-30211, 30216-30221, 30223, 30225-30229, 30231-30232, 30235-30236, 30238, 30242-30246, 30248-30250, 30252, 30255-30258, 30263, 30265, 30267, 30269, 30273-30276, 30278-30279, 30281-30289, 30291-30294, 30302-30303, 30305-30306, 30308, 30310, 30312-30322, 30324, 30326-30329, 30331-30333, 30335, 30337-30344,
30346, 30348, 30350, 30352-30358, 30361, 30365, 30370, 30379-30381, 30385-30389, 30392-30393, 30399-30400, 30402-30403, 30414-30418, 30424-30425, 30428, 30431, 30436, 30439, 30441-30443, 30448, 30450- 30452, 30454, 30457, 30460-30462, 30464, 30466, 30473, 30475-30476, 30482-30484, 30489-30493, 30495-30496, 30498, 30500, 30503-30504, 30506-30511, 30513-30514, 30518-30519, 30521, 30527, 30533-30534, 30537, 30539, 30541-30544, 30546-30550, 30562, 30564, 30567, 30574, 30577-30579, 30581, 30583, 30590, 30596-30601, 30603-30604, 30607, 30609-30610, 30612, 30615, 30618-30619, 30621, 30623, 30629-30631, 30633, 30636, 30640-30642, 30644-30646, 30648, 30650, 30653, 30655- 30657, 30659-30660, 30662, 30664, 30667, 30672, 30674-30677, 30680- 30684, 30687-■30689, 30691-30694, 30697-30699, 30701-30705, 30709-
30711, 30713- ■30719, 30721- 30724, 30727, 30730 - 30731, 30733, 30737,
30739- 30740, 30743- 30744, 30747- 30749, 30751- 30752, 30758-30766,
30768, 30771- ■30772, 30776- 30779, 30781- 30785, 30788- 30792, 30794-
30802, 30806, 30811 , 30813 , 30817 , 30824-30826 , 30828-30831, 30835-
30839, 30842- ■30843, 30845- 30847, 30854, 30856 30858 -30862, 30864-
30865, 30869- ■30880, 30892, 30894 30897 -30898 30900 , 30902-30904,
30906, 30913, 30919 , 30921 -30927 30929 -30930 30932 -30933, 30936,
30941- 30949, 30951- 30955, 30957, 30959- 30962, 30965- 30967, 30972-
30976, 30979- ■30983, 30985, 30993 ■30995, 30998 31001 -31002, 31010-
31012, 31015- ■31016, 31018, 31021 ■31025, 31027 - 31028, 31030, 31032-
31038, 31042, 31046 -31048, 31050 31053 , 31055 -31062 , 31065, 31072,
31074- 31075, 31078, 31080- 31084, 31086- 31091, 31093- 31095, 31097-
31101, 31105, 31107 -31108, 31113 31115 -31118 31122 -31126, 31129,
31131- 31132, 31136- 31149, 31151- 31152, 31154- 31158, 31161, 31164-
31165, 31167- ■31168, 31171- 31173, 31175- 31180, 31182- 31184, 31186-
31187, 31193, 31195 , 31197 -31199 31201 -31204 31206 , 31210, 31212-
31214, 31216, 31218 , 31220 -31221 31226 -31227 31229 , 31235, 31237-
31241, 31243- ■31244, 31248- 31250, 31255, 31257 31260 , 31263-31269,
31273, 31275- ■31278, 31282- 31290, 31293- 31297, 31299- 31302, 31304-
31305, 31311- ■31312, 31314, 31316 31318 , 31320 -31328 , 31331-31338,
31340- 31341, 31343, 31345- 31346, 31348- 31352, 31355- 31356, 31358,
31360, 31366- ■31372, 31374- 31379, 31381- 31385, 31387- 31391, 31400,
31402- 31403, 31406- 31409, 31411, 31414- 31418, 31420- 31423, 31426-
31427, 31429- ■31432, 31434, 31437 ■31440, 31445 - 31450, 31452-31455,
31458, 31462, 31464 -31467, 31469 ■31472, 31477 - 31479, 31481-31482,
31485- 31486, 31488- 31489, 31493- 31494, 31496- 31500, 31502-31505,
31510- 31512, 31520- 31522, 31527, 31530- 31531, 31534- 31536, 31538-
31541, 31544- ■31545, 31547- 31549, 31556- 31559, 31561- 31563, 31566-
31569, 31572, 31574 -31575, 31578 ■31579, 31581 - 31582, 31584, 31586-
31587, 31589- ■31591, 31593- 31595, 31597- 31599, 31601- 31603, 31606-
31610, 31613- ■31614, 31616, 31618 31622, 31626 31628 -31635, 31638-
31639, 31641- ■31642, 31644- 31646, 31648, 31650 31652 , 31657, 31659-
31662, 31664, 31666 -31669, 31671 ■31672, 31674 31677 -31680, 31682,
31684, 31689- ■31691, 31693- 31694, 31696- 31698, 31700- 31701, 31708,
31710- 31713, 31715- 31716, 31721, 31723- 31727, 31730, 31733-31735,
31739- 31740, 31742- 31743, 31747- 31748, 31750, 31753, 31766-31767,
31769, 31771- ■31772, 31774, 31777 ■31783, 31787 - 31788, 31791, 31795-
31798, 31800- ■31802, 31805, 31809 ■31810, 31812 - 31813, 31815, 31819,
31823- 31828, 31831- 31832, 31835, 31837- 31839, 31841- 31844, 31847,
31849- 31857, 31861- 31866, 31869- 31872, 31874, 31878- 31895, 31897,
31899, 31913, 31915 -31918, 31921 31925 , 31928 -31934 , 31937-31938,
31940- 31945, 31950- 31952, 31954, 31957- 31959, 31962- 31963, 31965-
31970, 31973, 31975 -31980, 31982 ■31989, 31992 31994 -31995, 32000-
32002, 32005, 32008 -32018, 32021 32024 , 32026 , 32028, 32030-32036,
32041- 32045, 32047, 32051- 32052, 32058, 32060 - 32070, 32072, 32081,
32083, 32085- ■32088, 32095, 32097 ■32098, 32100 32107 , 32111, 32115,
32120- 32121, 32123- 32129, 32131- 32132, 32134- 32135, 32138-32139,
32141- 32143, 32145- 32147, 32149- 32152, 32154, 32158- 32160, 32162,
32164, 32166, 32168 -32172, 32174 32176 -32182 32184 -32185, 32187-
32189, 32191- 32192, 32195- 32199, 32201, 32204 32209 -32210, 32212- 32213, 32216-32218, 32224, 32226, 32229-32230, 32235-32236, 32238- 32244, 32247, 32249, 32256-32262, 32264-32265, 32267, 32269, 32272 32276, 32280, 32286-32289, 32292, 32295, 32300-32301, 32303, 32306 32308-32310, 32312, 32314, 32318, 32325-32326, 32328, 32331, 32339 32341-32344, 32346-32349, 32352-32354, 32356-32357, 32360-32361, 32364-32365, 32368-32369, 32372, 32375-32378, 32393, 32395, 32398, 32403, 32408-32410, 32412, 32416-32418, 32423-32425, 32428, 32430, 32432, 32436-32437, 32441-32443, 32445-32446, 32449, 32452, 32454- 32456, 32458-32459, 32461, 32463, 32467, 32471-32472, 32474-32476, 32478-32479, 32483-32489, 32491-32494, 32499, 32501, 32508, 32512, 32514, 32516, 32519-32521, 32524-32526, 32530-32531, 32535, 32537, 32543, 32545, 32547, 32549, 32553, 32555-32562, 32564-32565, 32567 32568, 32570, 32575-32576, 32580, 32585-32589, 32592-32593, 32597- 32601, 32603-32604, 32607, 32612-32613, 32617-32618, 32622-32623, 32628, 32630, 32632, 32634-32635, 32638-32646, 32648, 32650, 32652 32655, 32662, 32664, 32669-32671, 32676-32677, 32679, 32683, 32685 32687, 32689-32691, 32694, 32697, 32699-32700, 32702, 32706-32713, 32724-32726, 32728, 32730, 32732-32733, 32736, 32741-32744, 32746- 32750, 32752, 32754, 32756-32758, 32761-32765, 32768, 32770, 32773 32775, 32778, 32783, 32785-32786, 32789-32795, 32797-32798, 32802, 32804-32808, 32810, 32815, 32817-32820, 32822, 32825-32832, 32834- 32836, 32838, 32840-32846, 32849-32852, 32854-32856, 32859, 32863- 32871, 32877, 32881, 32883, 32885, 32887-32889, 32891-32892, 32894 32897, 32900-32905, 32907-32914, 32920-32922, 32924-32925, 32927, 32929-32930, 32934, 32936, 32940, 32942-32949, 32951-32952, 32954- 32956, 32959, 32961-32962, 32964, 32966, 32968-32971, 32973-32976, 32978-32979, 32981-32984, 32986-32987, 32991-32993, 32996, 32998, 33000-33002, 33004, 33008, 33013-33045, 33048, 33052, 33056-33060, 33062-33063, 33066-33069, 33073, 33075, 33078-33085, 33089-33090, 33096-33100, 33102, 33104, 33106, 33110-33113, 33117-33120, 33125- 33126, 33129-33130, 33132-33133, 33136-33137, 33141-33144, 33146, 33148, 33152, 33161, 33163-33165, 33167, 33170-33171, 33173, 33175 33178, 33181, 33183-33184, 33187-33188, 33196-33198, 33200-33201, 33203-33204, 33207-33208, 33211-33213, 33215, 33219, 33221-33224, 33229-33231, 33233-33234, 33236, 33238-33249, 33252, 33258-33260, 33262, 33269, 33271, 33273-33275, 33277-33279, 33284-33285, 33291- 33297, 33300-33302, 33305, 33310-33311, 33315, 33320, 33322-33328, 33330-33331, 33333, 33337, 33339, 33343-33344, 33347-33349, 33351- 33352, 33354-33355, 33358, 33364, 33373, 33376-33378, 33380, 33383 33384, 33386-33389, 33391-33392, 33394-33396, 33403, 33405, 33410, 33412-33416, 33420, 33425, 33428-33430, 33432-33435, 33438-33441, 33447, 33451, 33454-33461, 33463, 33466-33468, 33472-33473, 33475, 33477-33486, 33490, 33492, 33497, 33499-33503, 33506, 33508-33510, 33521, 33525, 33527, 33529, 33533-33536, 33538-33539, 33541-33543, 33547, 33549-33557, 33559, 33561-33562, 33567, 33572, 33575-33579, 33581-33584, 33586, 33588, 33590, 33592, 33594, 33596-33598, 33600 33602-33604, 33608-33612, 33614-33615, 33617, 33619-33622, 33624- 33625, 33628-33629, 33631-33635, 33637-33642, 33644-33646, 33649- 33650, 33652-33656, 33658, 33660-33667, 33669, 33672, 33674, 33676 33679, 33681-33684, 33686-33689, 33691, 33694-33702, 33708-33709, 33711-33713, 33715, 33717-33718, 33720-33721, 33723, 33725-33731, 33733, 33738-33741, 33743, 33745, 33750, 33759, 33761-33763, 33765 33768, 33771-33772, 33774-33775, 33779-33780, 33782, 33784, 33786- 33797, 33801-33805, 33808-33809, 33814, 33817-33821, 33823-33825, 33827-33832, 33836, 33838, 33840-33842, 33845-33846, 33848, 33853- 33855, 33857, 33859, 33863, 33865-33867, 33874, 33877-33878, 33880 33881, 33885, 33888, 33890-33895, 33897-33899, 33902-33903, 33905- 33907, 33909-33911, 33914-33918, 33921, 33923-33924, 33926, 33928- 33929, 33931, 33936, 33943, 33945-33946, 33949, 33951, 33954-33957 33959, 33961-33964, 33966, 33969, 33971, 33974-33978, 33980, 33982 33986, 33988-33994, 33996-33997, 33999, 34001-34005, 34008-34012, 34014-34015, 34018-34022, 34024-34028, 34030-34034, 34036, 34038- 34043, 34045-34051, 34055-34057, 34059-34062, 34064, 34067-34069, 34071-34077, 34079-34080, 34088-34089, 34091-34094, 34096-34102, 34108-34109, 34111-34113, 34116-34117, 34119, 34121-34125, 34127- 34130, 34134-34136, 34140, 34143, 34145, 34147-34150, 34152-34155, 34157, 34159, 34162, 34166-34168, 34170, 34181, 34185-34187, 34189 34191-34193, 34198, 34200-34202, 34205, 34207-34208, 34211-34213, 34215, 34219-34220, 34222-34233, 34235-34241, 34246, 34249-34258, 34260, 34262, 34264, 34266-34267, 34270, 34272-34273, 34275, 34277 34278, 34280, 34282-34283, 34285, 34287, 34290-34292, 34294-34296, 34298-34302, 34305, 34308-34309, 34312-34317, 34320-34322, 34326, 34328, 34330-34334, 34338, 34340, 34342, 34344-34352, 34355-34360, 34362, 34366, 34372, 34377, 34380, 34382-34383, 34385, 34387-34392 34394-34396, 34399-34407, 34410-34417, 34419, 34421, 34423-34425, 34428-34430, 34434, 34436, 34438, 34442, 34444-34447, 34450-34457, 34459-34461, 34463, 34465-34466, 34469-34471, 34476-34477, 34479, 34482-34483, 34485-34486, 34488, 34490, 34492-34493, 34495, 34497- 34504, 34507-34509, 34513, 34515-34516, 34520-34523, 34525-34528, 34530-34535, 34543, 34545-34547, 34549-34550, 34552-34554, 34556- 34562, 34565, 34568-34571, 34573, 34575-34579, 34581-34587, 34590- 34593, 34595-34599, 34601-34605, 34611-34619, 34621, 34624-34625, 34627, 34631-34634, 34636-34646, 34648-34656, 34658, 34660, 34662- 34666, 34668, 34670-34671, 34673, 34675, 34677, 34679-34681, 34683 34686, 34688-34695, 34697-34699, 34701-34703, 34705-34706, 34710, 34714-34718, 34721-34722, 34725-34728, 34731-34732, 34734-34738,
34741- 34743, 34750- 34752, 34756, 34760, 34762- 34764, 34767- 34769,
34771- 34776, 34778- 34779, 34781, 34783, 34785, 34787 -34789, 34791,
34794, 34796- ■34797, 34799, 34802- 34803, 34805, 34808 -34809, 34811,
34813, 34816, 34819 , 34822 , 34824 -34827 , 34829 , 34832-34833 , 34837
34839, 34841- ■34845, 34849- 34850, 34854, 34856, 34862 -34865, 34867-
34873, 34876- ■34878, 34881, 34883, 34885 -34886, 34888 -34890, 34892-
34895, 34897- ■34899, 34901- 34908, 34910- 34911, 34915- 34916, 34918-
34926, 34933- ■34938, 34940, 34942, 34944 -34946, 34948 -34949, 34951-
34956, 34958- ■34960, 34962- 34963, 34965, 34968- 34972, 34976- 34978,
34986, 34988, 34990 , 34992 , 34995 -35000 , 35002 -35006 , 35009 , 35012
35017, 35021, 35025 -35026, 35028- 35031, 35034- 35036, 35041- 35043,
35045, 35048- ■35050, 35055, 35057- 35058, 35061- 35062, 35067- 35070,
35072- 35074, 35076- 35078, 35080-35082, 35085, 35089- 35090, 35092-
35094, 35097- ■35099, 35101- 35103, 35106- 35107, 35111- 35113, 35115,
35119, 35122, 35124 -35128, 35130, 35140 , 35146 , 35152-35154 , 35156
35159, 35162- ■35164, 35168- 35169, 35171- 35173, 35180- 35182, 35185, 35190, 35196-35197, 35199-35203, 35206, 35208-35210, 35213, 35215- 35216, 35219-35222, 35228-35229, 35231, 35233, 35235, 35237, 35239- 35246, 35248-35249, 35251-35254, 35256-35264, 35268-35272, 35275, 35280-35281, 35288-35296, 35298, 35302, 35304, 35306, 35308-35310, 35313, 35316-35318, 35320-35321, 35323-35329, 35331-35334, 35338, 35340-35341, 35347-35348, 35350-35351, 35353, 35355-35358, 35365, 35369, 35374, 35376-35377, 35381, 35383-35386, 35388-35389, 35392- 35393, 35395, 35398, 35400, 35402, 35404-35407, 35409-35411, 35414- 35415, 35417-35418, 35421-35422, 35424-35425, 35430-35431, 35433- 35437, 35439-35440, 35442, 35445-35447, 35450, 35456, 35459, 35462- 35465, 35467, 35469-35471, 35473-35474, 35478-35484, 35493, 35499, 35501-35504, 35506-35510, 35512, 35514-35518, 35521-35524, 35526- 35527, 35530-35531, 35533, 35535-35540, 35542, 35546, 35549-35551, 35555, 35557-35558, 35564, 35566-35568, 35570, 35572, 35575, 35577- 35578, 35581-35583, 35586, 35588, 35590, 35592, 35597-35598, 35600- 35601, 35609, 35614-35616, 35618, 35621, 35624-35627, 35633-35635, 35637, 35639-35640, 35642-35644, 35650, 35652-35657, 35659, 35662- 35663, 35667, 35669, 35674, 35677-35679, 35681, 35683, 35685, 35687- 35692, 35694, 35698-35705, 35707-35713, 35715-35724, 35726, 35729- 35734, 35736, 35738, 35740-35742, 35744, 35747, 35751-35752, 35754, 35756-35765, 35768-35769, 35773, 35776-35780, 35782, 35785, 35787- 35788, 35790, 35792, 35800, 35803-35817, 35819, 35821-35833, 35835- 35836, 35838-35839, 35842, 35844, 35848-35851, 35854, 35856, 35859, 35862, 35864-35868, 35871, 35873-35882, 35884-35886, 35890, 35898, 35901, 35903, 35905-35906, 35908, 35910-35911, 35914-35916, 35918- 35921, 35925, 35927-35929, 35931, 35933-35935, 35937, 35939-35944, 35947-35949, 35952-35962, 35965-35966, 35970, 35973-35975, 35979- 35981, 35983-35989, 35991, 35997-36008, 36011-36018, 36020-36029, 36031, 36033-36038, 36040-36041, 36044, 36046, 36048-36049, 36051, 36053, 36056, 36058, 36060-36062, 36065-36068, 36070-36074, 36077, 36079, 36081-36088, 36090, 36092, 36097-36098, 36100, 36104-36107, 36109-36110, 36112-36115, 36117-36119, 36122-36123, 36126-36128, 36131-36136, 36138, 36140, 36143-36147, 36149-36151, 36154-36157, 36160-36172, 36174-36176, 36179, 36181, 36183-36186, 36188, 36191- 36193, 36195-36204, 36206-36209, 36211-36213, 36217, 36220, 36222, 36228, 36230, 36232, 36234-36236, 36239-36242, 36244, 36246-36249, 36254-36255, 36257-36258, 36263-36271, 36273, 36276, 36278-36280, 36283, 36285, 36288, 36290-36294, 36302, 36304-36305, 36308, 36310, 36314-36322, 36327, 36329-36330, 36338-36342, 36346, 36349, 36351- 36352, 36355, 36357, 36363, 36365-36367, 36371, 36373-36375, 36378- 36380, 36383, 36386, 36392, 36399-36400, 36402-36406, 36408-36410, 36412-36414, 36416, 36424-36430, 36432-36433, 36436, 36439-36441, 36443-36444, 36446, 36456-36457, 36459, 36462-36471, 36473, 36475- 36478, 36480, 36483-36489, 36492-36494, 36496, 36499-36500, 36502, 36505, 36507-36512, 36514, 36517, 36523, 36526, 36531-36535, 36537, 36545-36546, 36548, 36552-36558, 36563-36565, 36567, 36570-36573, 36575, 36578, 36581, 36585, 36587, 36591-36599, 36603-36605, 36608, 36612, 36621, 36623-36626, 36628-36629, 36631, 36633-36636, 36639, 36641, 36643, 36645, 36649, 36651-36652, 36657, 36659, 36663-36665, 36667-36671, 36677, 36679, 36683, 36686, 36688, 36692-36698, 36703, 36706, 36708, 36710-36716, 36719-36722, 36724-36728, 36732, 36734, 36736, 36740, 36742 -36743, 36746-■36752, 36754, 36756 -36765, 36768,
36773, 36778- ■36780, 36782, 36784- ■36788, 36796- 36798, 36801, 36803-
36807, 36809- ■36811, 36813, 36818- ■36821, 36823- 36825, 36827- 36831,
36833, 36841- ■36853, 36855- 36857, 36863- 36864, 36867, 36870, 36872-
36873, 36875, 36877 -36879, 36881, 36884 -36886, 36890 -36896, 36898-
36900, 36903- ■36905, 36907- 36908, 36910- 36911, 36916- 36917, 36919,
36922, 36924- ■36927, 36930, 36932- ■36939, 36943, 36945 -36952, 36954-
36958, 36960- ■36962, 36964, 36967- ■36968, 36970- 36972, 36975- 36990,
36992- 36994, 36999- 37003, 37005, 37007, 37009, 37013 , 37018 -37023, 37025, 37028, 37032, 37034, 37038, 37042-37044, 37046, 37050, 37054, 37056-37060, 37065-37069, 37073, 37075-37081, 37083, 37086-37088, 37090-37091, 37095, 37097-37098, 37100-37101, 37103, 37107-37111, 37113-37114, 37120, 37123, 37126-37130, 37132, 37134, 37136-37137, 37139-37140, 37142, 37144, 37147-37148, 37152, 37155, 37157, 37162, 37166, 37168, 37170-37171, 37178, 37185-37192, 37194-37196, 37198- 37200, 37203-37204, 37206, 37208, 37212-37214, 37216-37217, 37219- 37221, 37223, 37229-37230, 37232, 37238, 37240-37242, 37245, 37248, 37250-37257, 37261, 37263, 37265-37273, 37275-37281, 37283, 37290- 37294, 37296-37298, 37301-37306, 37310, 37313, 37315, 37318-37323, 37328-37329, 37333, 37335-37337, 37340-37347, 37353, 37356, 37358- 37359, 37361-37364, 37367, 37369, 37371, 37373, 37375, 37379-37381, 37383, 37385-37386, 37388-37397, 37403-37414, 37417, 37419-37424, 37426-37430, 37432, 37434-37435, 37440, 37443-37445, 37447-37450, 37452-37453, 37455-37456, 37458-37466, 37469-37470, 37475, 37477, 37479-37481, 37483-37484, 37486, 37489-37496, 37500-37501, 37504, 37509, 37511, 37520-37525, 37533, 37535, 37537-37538, 37540-37541, 37544, 37546-37547, 37551, 37554-37560, 37562, 37566, 37568, 37575, 37578-37583, 37586, 37588-37590, 37592-37593, 37595-37596, 37598- 37599, 37602, 37604-37606, 37609, 37611, 37614-37615, 37617, 37622, 37625, 37633, 37636-37638, 37644-37654, 37656-37657, 37659-37664, 37668, 37678, 37680, 37682-37687, 37689-37690, 37692-37695, 37699- 37702, 37706-37711, 37714-37715, 37720-37723, 37726-37728, 37730, 37735, 37739-37741, 37744, 37746-37749, 37755-37756, 37758, 37760- 37764, 37766-37768, 37770-37771, 37773-37777, 37779, 37783-37792, 37794-37795, 37800-37801, 37803-37806, 37809-37810, 37817, 37819, 37824-37835, 37837-37839, 37842-37844, 37846-37851, 37853, 37855- 37858, 37861-37862, 37865-37866, 37868-37872, 37874-37879, 37881- 37883, 37885, 37890, 37892, 37894, 37901, 37903-37904, 37906-37907, 37912-37913, 37918, 37920-37923, 37925, 37929-37931, 37933-37938, 37940-37941, 37943-37945, 37949-37951, 37953-37955, 37957-37958,
37960, 37962-37964, 37967-37968, 37971-37975, 37977, 37979, 37981, 37987-37989, 37996-37997, 38001-38011, 38013, 38015-38016, 38018, 38024-38028, 38031-38040, 38042-38046, 38048-38054, 38059, 38061- 38062, 38064, 38067, 38069-38073, 38077, 38081-38082, 38084-38086, 38088-38093, 38095-38096, 38101, 38103-38105, 38107-38108, 38110- 38111, 38113-38116, 38121-38122, 38132-38133, 38135, 38137-38140, 38143-38147, 38155, 38159, 38161, 38163-38164, 38166, 38170, 38172- 38182, 38184-38185, 38187-38189, 38191-38192, 38194-38208, 38210- 38211, 38213-38215, 38217-38220, 38222, 38224-38226, 38229-38233, 38236-38239, 38244, 38246, 38248-38249, 38251-38258, 38261-38262, 38264-38268, 38270-38271, 38273-38280, 38282, 38288-38291, 38293, 38298, 38305- 38307, 38309- 38312, 38314, 38316- 38317, 38319, 38322-
38324, 38326, 38331, 38334 -38338, 38340 38343 , 38345 , 38347 -38352,
38356, 38358- 38363, 38365- 38366, 38369- 38372, 38377, 38380, 38382-
38384, 38395- 38396, 38398, 38402, 38404 38406 -38407, 38411- 38412,
38414, 38416, 38420- ■38422, 38424, 38426 38427, 38435- 38440, 38442-
38443, 38445, 38450- ■38451, 38454- 38455, 38465- 38467, 38475-38478,
38484, 38486- 38487, 38489- 38490, 38492, 38497- 38499, 38502, 38505-
38506, 38511- 38512, 38517, 38519- 38520, 38525, 38528- 38531, 38534,
38536, 38543, 38548, 38550 , 38557 , 38559 , 38565-38569 , 38571 , 38575-
38576, 38578- 38586, 38588, 38591- 38599, 38601- 38602, 38604, 38606,
38612, 38616- 38617, 38619- 38620, 38622- 38624, 38628-38632, 38634,
38639, 38648, 38651, 38654 , 38657 , 38661 -38664 , 38668 , 38670 -38672,
38675, 38677- 38682, 38685- 38686, 38699, 38702- 38703, 38706, 38714,
38717, 38719, 38721- ■38723, 38727, 38733 - 38734, 38740- 38741, 38746-
38750, 38755, 38757, 38759 , 38762 -38763 38767 , 38776 , 38779 -38780,
38782-38784, 38788-38795, 38797-38798, 38811-38812, 38814-38818, 38820-38822, 38824, 38826, 38830, 38832-38836, 38839-38840, 38842, 38844-38847, 38849-38850, 38852-38854, 38856-38857, 38864-38865, 38868-38869, 38871-38873, 38875, 38877, 38879-38880, 38882-38883, 38886-38887, 38889-38893, 38896-38898, 38900-38901, 38905, 38907- 38908, 38910, 38913, 38915-38918, 38920-38923, 38929, 38932-38933, 38937, 38942, 38944, 38949, 38951-38952, 38954-38955, 38961-38965, 38968, 38971-38972, 38978, 38985-38987, 38989-38993, 38995-38997, 38999, 39005-39006, 39009-39010, 39012, 39015, 39019, 39021-39023, 39033, 39035, 39038, 39041, 39047-39048, 39051-39052, 39055-39059, 39061, 39063-39065, 39068, 39070-39074, 39076, 39078, 39080-39082, 39084-39092, 39094, 39096-39097, 39099-39100, 39102, 39104-39107, 39109-39115, 39117-39126, 39128, 39131, 39134-39140, 39142, 39145, 39147, 39149, 39153-39154, 39156-39157, 39159-39160, 39162, 39164, 39167-39168, 39170, 39172-39173, 39175-39177, 39183, 39185, 39187, 39189, 39191, 39193, 39196-39200, 39202, 39204-39208, 39210, 39214- 39215, 39217, 39219, 39223, 39227, 39230, 39236, 39238-39240, 39250- 39251, 39254, 39258-39260, 39265-39272, 39274-39276, 39278, 39282, 39286, 39288, 39291-39299, 39301-39303, 39307-39308, 39310-39317, 39323, 39325-39327, 39329, 39331-39333, 39335, 39339-39346, 39349, 39351, 39353, 39355-39359, 39362-39364, 39366-39367, 39386, 39388- 39389, 39391-39393, 39396, 39401, 39404, 39406-39417, 39421, 39423- 39424, 39426-39427, 39430, 39432, 39434, 39436, 39438, 39440, 39442, 39446-39453, 39455-39459, 39461, 39464, 39466, 39472, 39474-39475, 39477, 39479, 39481-39490, 39494, 39496, 39501, 39503-39504, 39509, 39512-39514, 39516-39519, 39521, 39523-39526, 39528-39533, 39536, 39541-39548, 39550, 39552-39553, 39563, 39565-39566, 39568-39578, 39581, 39593, 39596, 39601-39603, 39606-39608, 39611-39615, 39617, 39619-39622, 39627, 39629-39640, 39642-39650, 39652-39655, 39658- 39665, 39668, 39675-39676, 39680-39681, 39683-39684, 39687-39692, 39694-39696, 39699, 39706-39709, 39711-39713, 39721-39722, 39725- 39727, 39734, 39736, 39739, 39741-39742, 39744, 39747, 39752, 39756- 39758, 39760, 39762-39763, 39765, 39767, 39769, 39771-39780, 39783, 39787-39792, 39794-39795, 39800, 39802-39804, 39806-39807, 39809, 39813, 39816-39823, 39825, 39827-39829, 39833-39834, 39837-39839, 39842-39843, 39847-39849, 39853-39854, 39856-39857, 39859, 39863, 39866, 39870-39871, 39875, 39878, 39884, 39888, 39896, 39898-39900, 39902-39906, 39908-39909, 39912, 39916-39917, 39922, 39924-39926, 39929-39935, 39938-39942, 39944-39945, 39947-39949, 39951-39952, 39954, 39957-39962, 39964, 39968, 39970, 39973-39974, 39977-39979, 39982, 39984-39987, 39995-39997, 39999, 40002, 40004-40007, 40009- 40011, 40017-40020, 40024, 40027, 40029, 40031, 40033, 40038, 40042- 40045, 40052-40053, 40055-40056, 40058-40063, 40065-40067, 40074- 40076, 40078, 40080-40082, 40085, 40087, 40090, 40094-40095, 40097, 40099-40106, 40108-40109, 40111-40117, 40119, 40121-40122, 40124- 40125, 40127, 40131, 40134-40135, 40137, 40139, 40141-40143, 40145- 40146, 40149-40156, 40161, 40163, 40165, 40169-40170, 40172, 40174- 40175, 40177, 40181, 40183-40185, 40187-40189, 40191-40192, 40194- 40199, 40202-40205, 40208, 40213-40215, 40217-40219, 40223, 40225- 40229, 40231, 40236, 40240, 40243, 40253-40256, 40259-40260, 40262- 40264, 40266, 40268, 40270-40278, 40280-40282, 40284, 40288, 40290, 40292-40296, 40298, 40300-40303, 40305, 40307, 40310-40311, 40313- 40314, 40316, 40319-40322, 40329, 40331-40338, 40343, 40347-40360, 40362-40363, 40372-40373, 40375-40376, 40378-40380, 40382-40384, 40387-40388, 40391-40394, 40396-40398, 40402, 40404, 40409-40411, 40418, 40421, 40423-40424, 40427-40428, 40430, 40432-40433, 40438, 40441-40442, 40444-40445, 40447-40449, 40453-40457, 40462-40463, 40468-40473, 40475-40480, 40482, 40485, 40487-40491, 40493, 40495- 40497, 40499, 40501-40507, 40512-40516, 40521-40526, 40531-40533, 40535, 40537, 40543, 40546-40547, 40550, 40553, 40556, 40559, 40561- 40564, 40566, 40570, 40573, 40576, 40578-40581, 40584-40585, 40592- 40594, 40597-40599, 40603-40604, 40606-40609, 40615, 40618-40621, 40624, 40626, 40628, 40630, 40641-40643, 40648, 40652-40653, 40656- 40658, 40660, 40662, 40664, 40666-40668, 40671, 40673, 40676-40677, 40679, 40681, 40685, 40687-40689, 40691-40692, 40695, 40700, 40703- 40706, 40710-40711, 40713, 40715, 40718-40722, 40724-40733, 40736- 40737, 40739-40741, 40744-40746, 40752, 40755-40757, 40759, 40761, 40765-40774, 40778, 40780, 40782-40783, 40785, 40787-40790, 40793- 40794, 40799-40800, 40806-40807, 40811-40814, 40818-40819, 40823, 40830-40831, 40837-40840, 40843, 40849-40853, 40856-40858, 40861- 40871, 40874, 40876, 40878, 40880, 40883, 40887, 40891-40896, 40902- 40905, 40907, 40909-40916, 40919, 40921-40924, 40926, 40928-40931, 40933-40934, 40936, 40938, 40942-40947, 40950, 40954, 40958, 40960, 40966-40971, 40973-40977, 40980-40981, 40984-40988, 40991-40992, 40994-40995, 40997, 41000, 41002, 41004, 41006-41007, 41010-41011, 41015-41016, 41022, 41024-41025, 41027-41029, 41031-41036, 41039, 41041-41043, 41045, 41059, 41064-41065, 41067-41073, 41075-41076, 41078-41079, 41082-41084, 41087, 41089, 41091-41100, 41103, 41106- 41107, 41109, 41113, 41115-41123, 41125-41127, 41130, 41133-41135, 41139-41140, 41144-41149, 41152-41155, 41157-41158, 41162-41164, 41168, 41170-41175, 41178, 41180-41181, 41186-41187, 41189, 41191, 41194, 41199-41201, 41204-41206, 41209, 41214-41216, 41218, 41222, 41226, 41228-41230, 41232, 41239, 41242-41243, 41246, 41248, 41252- 41254, 41263, 41265-41267, 41269-41271, 41273-41274, 41276, 41285- 41288, 41291, 41295-41296, 41307, 41311-41312, 41314-41315, 41320, 41329-41331, 41335-41337, 41340, 41342, 41344-41345, 41347-41350,
41352, 41354-41359, 41361, 41363, 41365, 41367, 41369-41370, 41372- 41373, 41376, 41378-41380, 41383-41384, 41386, 41388, 41390, 41392, 41398, 41400-41404, 41408, 41410-41411, 41414, 41418-41421, 41428- 41430, 41432-41433, 41435-41437, 41440-41441, 41443-41444, 41448- 41449, 41455, 41457, 41459, 41464-41466, 41473, 41475, 41478-41479, 41485, 41487-41490, 41492, 41494, 41497-41499, 41504-41507, 41510, 41513-41515, 41517-41518, 41520-41521, 41525-41526, 41529, 41531, 41533, 41535-41536, 41540-41541, 41545-41546, 41548, 41550-41556, 41558-41560, 41562, 41566-41568, 41570, 41572-41576, 41578, 41582, 41588, 41590-41594, 41596, 41606-41608, 41611, 41613-41615, 41624, 41629, 41637, 41643-41644, 41647-41648, 41650-41655, 41658-41660, 41662-41664, 41666-41674, 41676-41677, 41679-41680, 41682, 41685, 41689, 41692, 41697, 41700-41702, 41707, 41710-41712, 41714, 41716- 41717, 41719, 41724-41731, 41734-41738, 41740-41741, 41746-41747, 41749-41750, 41754-41755, 41758, 41760, 41763-41765, 41771, 41775- 41780, 41787-41788, 41790, 41792, 41799, 41801, 41803-41804, 41806- 41808, 41811, 41814, 41816-41817, 41819, 41821, 41824-41827, 41835, 41840-41842, 41844-41845, 41847, 41849, 41854, 41858-41861, 41865- 41866, 41868-41869, 41872-41879, 41881-41894, 41898, 41903, 41906, 41908, 41910-41913, 41916-41917, 41924, 41927, 41931, 41936-41937, 41940, 41943-41944, 41946, 41948-41949, 41954, 41957-41958, 41960, 41962-41963, 41967, 41971, 41974-41975, 41980, 41982-41986, 41989- 41992, 41994, 41999, 42004, 42007, 42010-42016, 42018, 42020, 42022- 42023, 42027, 42034-42035, 42038, 42042-42045, 42049, 42051-42054, 42063-42067, 42071, 42079-42086, 42094-42100, 42105-42107, 42111- 42112, 42114-42116, 42118, 42120, 42122-42126, 42128, 42130, 42133, 42136, 42138, 42140, 42142-42143, 42145, 42149-42152, 42154, 42156- 42157, 42159-42160, 42164-42166, 42169, 42172, 42174-42178, 42180, 42182-42187, 42191-42195, 42198-42200, 42202-42204, 42208-42212, 42214, 42216-42217, 42219-42220, 42226-42230, 42235, 42240-42244, 42246-42250, 42255, 42261-42262, 42264, 42273-42274, 42276-42278, 42281, 42287, 42290-42291, 42293, 42298-42300, 42304-42306, 42309, 42311, 42318-42321, 42324, 42326-42335, 42340-42341, 42343, 42345, 42353-42354, 42358, 42361-42362, 42364-42367, 42370, 42372-42377, 42379-42380, 42382, 42385-42387, 42389, 42392-42394, 42399, 42402, 42404-42410, 42414, 42418-42419, 42422, 42424-42429, 42431-42433, 42435-42436, 42438, 42444-42445, 42455, 42459, 42462-42464, 42466- 42470, 42472, 42483, 42485-42486, 42488, 42491, 42494-42495, 42497- 42502, 42504-42505, 42508-42513, 42516-42523, 42526-42529, 42531- 42532, 42538, 42542-42546, 42554, 42556-42558, 42561, 42565, 42568, 42570, 42573, 42577-42580, 42585, 42587-42590, 42593-42594, 42601- 42602, 42605, 42608-42609, 42611, 42613-42615, 42618-42624, 42626, 42628-42634, 42636-42643, 42645-42646, 42648-42651, 42653-42656, 42659-42662, 42664-42666, 42669-42671, 42674-42676, 42679-42680, 42682, 42686-42689, 42691, 42694, 42698-42700, 42703, 42705-42707, 42710, 42719-42721, 42728, 42731, 42734-42736, 42740-42741, 42743, 42749, 42751, 42754-42755, 42757, 42759-42760, 42762, 42765-42766, 42768-42769, 42771, 42775, 42784-42785, 42787-42791, 42793-42794, 42799-42803, 42805-42807, 42817, 42819-42820, 42824-42825, 42830, 42833-42837, 42839-42842, 42844-42848, 42850-42852, 42854-42855, 42857, 42859-42863, 42865-42866, 42869, 42873-42876, 42878, 42882, 42886, 42888-42892, 42895-42897, 42904-42907, 42909-42910, 42912- 42920, 42922-42923, 42925, 42929, 42931, 42936, 42939-42942, 42947- 42948, 42952, 42954-42961, 42965, 42967-42968, 42971-42975, 42984, 42986-42987, 42989, 42991, 42994-42996, 42999-43002, 43004, 43006- 43007, 43014-43019, 43021-43022, 43024-43028, 43030-43037, 43039- 43048, 43055, 43057, 43060-43062, 43064, 43068-43070, 43073, 43076- 43077, 43082, 43084-43086, 43088-43089, 43091, 43093, 43095-43097, 43101, 43104-43106, 43114-43122, 43125-43128, 43133-43134, 43136- 43137, 43139-43145, 43147, 43149-43151, 43153, 43156, 43158-43160, 43162-43165, 43167-43168, 43170-43171, 43173, 43175-43179, 43181- 43182, 43184, 43186-43187, 43189, 43195-43196, 43198, 43200-43202, 43205-43206, 43208, 43210-43214, 43216-43218, 43222-43224, 43226, 43228-43233, 43237-43238, 43240-43247, 43250-43251, 43253-43254, 43257-43258, 43260-43262, 43264-43267, 43269-43272, 43277, 43280- 43281, 43283, 43286, 43292, 43294, 43299, 43301, 43305-43307, 43310, 43312, 43314-43317, 43320, 43323, 43325-43328, 43332-43333, 43335, 43341-43344, 43346, 43348-43349, 43352, 43354-43356, 43358, 43361, 43368, 43370-43371, 43373-43374, 43380, 43382-43383, 43385, 43393, 43396, 43398, 43401-43402, 43404-43405, 43411, 43417, 43422, 43426, 43429, 43439, 43442, 43445-43449, 43452-43458, 43460, 43467-43468, 43470-43474, 43477-43481, 43484, 43487-43488, 43491, 43494-43495, 43497-43498, 43500-43505, 43508, 43510-43512, 43514-43515, 43519, 43521-43524, 43530, 43536, 43540, 43542, 43544, 43546-43547, 43551- 43552, 43554, 43558, 43560, 43565-43569, 43571-43573, 43575, 43579- 43580, 43584, 43588-43591, 43593-43594, 43597-43599, 43601, 43603- 43610, 43612-43616, 43618, 43620, 43622-43625, 43630, 43632, 43634, 43637, 43639-43641, 43643, 43645, 43647, 43649-43650, 43652, 43659, 43662-43668, 43673, 43676-43680, 43682, 43687, 43690-43693, 43695- 43696, 43700-43702, 43704, 43706-43708, 43714-43716, 43723, 43728- 43732, 43734, 43737, 43744-43748, 43750-43751, 43755-43763, 43765, 43767, 43771-43777, 43781, 43783-43785, 43789, 43793-43794, 43797- 43804, 43807-43810, 43813, 43818, 43820-43821, 43823, 43826-43827, 43831-43837, 43839-43840, 43846-43849, 43853, 43856-43862, 43864- 43868, 43870-43871, 43874-43875, 43878, 43882-43883, 43886, 43888- 43891, 43894, 43896-43898, 43900, 43902-43903, 43909, 43913, 43916- 43917, 43919-43924, 43926, 43928-43932, 43934-43937, 43939, 43941, 43944, 43946, 43948-43950, 43952, 43955, 43960-43961, 43963-43967, 43970, 43973-43974, 43976, 43984, 43986, 43989, 43992-44001, 44004, 44006-44007, 44009, 44014-44016, 44018, 44021, 44023-44024, 44026, 44028-44037, 44039, 44046, 44048, 44050-44051, 44053-44054, 44056- 44058, 44061-44063, 44065, 44067, 44069-44070, 44072-44073, 44078, 44080-44081, 44083-44084, 44086-44092, 44096-44099, 44105-44106, 44110, 44113-44115, 44118-44122, 44124-44125, 44127-44128, 44130- 44132, 44134-44136, 44138-44144, 44147, 44150, 44152, 44156-44158, 44171, 44174, 44180, 44182, 44184-44185, 44187-44188, 44190, 44194- 44195, 44199, 44203, 44210, 44219, 44223, 44226-44227, 44230, 44235, 44240-44244, 44246-44247, 44249, 44253-44254, 44257-44261, 44266- 44268, 44271, 44275, 44281-44282, 44284, 44289, 44294, 44299-44304, 44306-44307, 44309, 44311-44314, 44316, 44318-44319, 44321, 44323- 44324, 44326-44327, 44329-44331, 44334, 44342-44344, 44346, 44357- 44358, 44362, 44364, 44366, 44372, 44374, 44377-44380, 44382-44383, 44385, 44387, 44395-44396, 44399-44403, 44405, 44413-44416, 44419- 44422, 44424, 44428-44435, 44437-44438, 44440-44442, 44444, 44446, 44452-44454, 44456-44458, 44460, 44464-44465, 44467-44471, 44473, 44475, 44477-44479, 44482-44483, 44485, 44487-44488, 44492-44494, 44496, 44502, 44504-44506, 44508, 44510, 44513, 44516-44517, 44519- 44520, 44522, 44525-44526, 44528, 44533-44534, 44536, 44538-44540, 44553, 44556, 44559-44561, 44569, 44574-44575, 44577-44578, 44580- 44581, 44583-44584, 44586-44587, 44590, 44593-44595, 44597, 44599, 44601-44604, 44606, 44609, 44611, 44615, 44617, 44624-44625, 44629, 44636, 44641-44645, 44647-44648, 44650, 44652, 44655, 44661, 44676, 44678-44680, 44684-44688, 44690, 44692-44693, 44695, 44701, 44704, 44707-44713, 44715, 44717-44721, 44723-44724, 44733-44741, 44745, 44747-44748, 44750-44752, 44756-44757, 44760, 44762-44763, 44765, 44771-44774, 44776, 44779, 44782-44783, 44785-44790, 44794-44796, 44798-44802, 44804-44806, 44808-44810, 44812-44818, 44821, 44823- 44825, 44827-44828, 44832, 44840-44841, 44843-44845, 44848-44850, 44854, 44857, 44861-44863, 44866-44873, 44878-44881, 44883, 44885, 44887-44890, 44892, 44894-44896, 44898, 44900, 44904, 44906-44909, 44912, 44914-44915, 44922, 44925-44926, 44929, 44933, 44935-44941, 44943-44945, 44947, 44952, 44954-44955, 44960, 44963, 44972-44974, 44978, 44984, 44991, 44996-44997, 44999, 45005-45006, 45008, 45010, 45012-45017, 45020-45021, 45023, 45026, 45028-45029, 45031-45032, 45035, 45037-45038, 45043, 45045, 45047, 45051, 45054, 45056, 45058- 45062, 45064-45075, 45078-45080, 45084, 45086, 45088-45092, 45096- 45097, 45102, 45104-45105, 45107-45112, 45114-45115, 45117, 45119- 45120, 45122, 45128-45130, 45132-45133, 45139, 45141, 45147, 45149, 45151-45153, 45155, 45158, 45161, 45165, 45168, 45175, 45177-45178, 45183-45187, 45189-45190, 45192, 45194, 45199-45200, 45202-45203, 45205, 45207-45208, 45210, 45212, 45216, 45222, 45227-45230, 45232, 45238-45240, 45242, 45245, 45247, 45253, 45256, 45258-45259, 45262, 45273-45274, 45276-45278, 45280, 45284, 45294-45298, 45301, 45304, 45308, 45311-45317, 45319-45320, 45322-45323, 45325-45326, 45329- 45331, 45333, 45335, 45338-45339, 45344-45346, 45348-45349, 45351- 45359, 45361, 45363-45364, 45366, 45368, 45370, 45372-45379, 45385- 45389, 45391-45399, 45403, 45409, 45412-45413, 45415-45420, 45424- 45426, 45430-45431, 45433-45438, 45440, 45442, 45444-45451, 45453- 45454, 45457, 45461, 45463, 45465, 45467, 45469-45473, 45482-45487, 45490-45495, 45502, 45507-45508, 45510, 45516, 45518-45521, 45524- 45527, 45529-45534, 45545, 45549-45552, 45554-45556, 45558-45559, 45563-45564, 45567, 45570-45572, 45574, 45576-45581, 45586, 45588- 45589, 45591-45593, 45595, 45599, 45601, 45604, 45606, 45608-45610, 45612-45616, 45618-45620, 45624, 45627, 45630-45632, 45634, 45636, 45638-45643, 45645, 45647-45648, 45652-45653, 45659, 45661, 45663- 45665, 45667-45668, 45670-45677, 45681-45683, 45685, 45690-45693, 45697-45700, 45703, 45706, 45708-45709, 45716, 45722-45729, 45735- 45736, 45741, 45744-45746, 45749, 45751-45752, 45754-45757, 45759- 45760, 45764-45765, 45767, 45769-45771, 45779-45780, 45784, 45786, 45788, 45791-45796, 45798-45802, 45806, 45808, 45811, 45814-45815, 45818, 45827, 45832, 45834-45835, 45838-45839, 45842-45844, 45847- 45848, 45852-45853, 45856-45858, 45865, 45868-45870, 45872-45875, 45877-45879, 45881-45887, 45889-45893, 45900, 45903, 45905-45906, 45909-45919, 45921, 45923, 45925-45926, 45930, 45934-45936, 45938- 45939, 45942-45943, 45945-45947, 45949-45951, 45953, 45955-45957, 45961, 45965-45966, 45969-45971, 45973, 45975-45976, 45979-45980, 45983-45986, 45990, 45997, 46000, 46002-46003, 46006-46007, 46009, 46012-46013, 46015-46016, 46018-46022, 46024-46025, 46027-46033, 46037, 46040, 46042-46043, 46047-46049, 46051-46053, 46055, 46058- 46063, 46068-46069, 46075, 46078-46079, 46081, 46084-46098, 46100- 46108, 46110-46113, 46115, 46119-46122, 46126-46127, 46131-46136, 46138-46139, 46142-46143, 46153, 46157-46158, 46160-46165, 46168- 46170, 46173, 46176-46177, 46179-46180, 46182-46183, 46188-46196, 46198-46201, 46205, 46207-46210, 46212-46214, 46218-46219, 46221, 46227, 46233-46234, 46236-46240, 46242-46244, 46247, 46249, 46252- 46253, 46255-46256, 46258-46267, 46269-46279, 46283, 46285-46288, 46291, 46293-46294, 46296, 46298-46299, 46301-46302, 46308, 46310, 46312-46314, 46316-46319, 46321-46328, 46331, 46334-46335, 46337- 46338, 46340-46342, 46344, 46346-46350, 46352-46354, 46356-46358, 46362-46367, 46374-46375, 46377-46379, 46383, 46385-46388, 46390- 46393, 46395, 46398, 46403, 46405-46414, 46416-46418, 46420, 46422, 46424, 46428-46429, 46431, 46433-46434, 46436, 46441-46442, 46444, 46446, 46448, 46450, 46452-46453, 46455-46456, 46459-46460, 46462, 46464-46465, 46467-46472, 46475, 46478-46480, 46482-46483, 46485- 46488, 46491, 46493-46494, 46496-46497, 46501-46504, 46507, 46511- 46513, 46515-46516, 46518-46525, 46528-46529, 46532-46533, 46540- 46541, 46543, 46545, 46547-46550, 46552, 46555, 46557, 46560-46561, 46563, 46566, 46569-46570, 46583-46586, 46590-46593, 46595, 46601, 46604, 46608-46609, 46612-46616, 46618, 46621, 46623, 46625-46629,
46631-46633, 46636, 46638, 46641, 46646, 46649, 46653, 46655, 46657, 46660-46661, 46664-46666, 46668-46672, 46674-46679, 46690, 46692, 46696-46697, 46700, 46702-46703, 46710-46713, 46716-46717, 46719- 46721, 46723, 46726-46728, 46730, 46732-46733, 46738-46742, 46744- 46745, 46747-46752, 46754-46755, 46757-46762, 46764, 46768, 46771- 46774, 46776, 46779-46781, 46783, 46785-46786, 46795-46796, 46800, 46806, 46809, 46820, 46823-46825, 46827-46833, 46835-46836, 46838- 46839, 46841, 46843, 46845-46846, 46852-46855, 46857-46859, 46861- 46864, 46866-46869, 46871-46872, 46874-46875, 46882, 46884, 46886- 46887, 46889, 46891-46893, 46895-46905, 46907-46910, 46912-46913, 46915-46916, 46918, 46921-46922, 46927, 46929-46933, 46936, 46939- 46940, 46942, 46944-46947, 46950-46951, 46954-46956, 46959-46960, 46963-46965, 46967-46968, 46970-46973, 46978, 46983-46984, 46986- 46990, 46992-46993, 47000, 47002-47005, 47008-47009, 47011-47012, 47014-47017, 47019, 47021, 47029, 47031-47032, 47036, 47038, 47040, 47043, 47045-47046, 47048, 47051, 47055-47057, 47059-47062, 47065, 47068-47070, 47072-47073, 47075-47086, 47088, 47090-47093, 47095- 47099, 47102-47104, 47108, 47110-47112, 47114, 47117, 47119, 47123, 47128-47131, 47133, 47136-47139, 47141, 47144-47148, 47150-47151, 47153, 47155, 47158, 47162-47164, 47168, 47170-47172, 47174, 47176- 47181, 47184, 47188, 47190-47191, 47193-47197, 47199-47201, 47204, 47206-47211, 47213, 47217, 47221-47227, 47231-47232, 47234, 47237, 47239-47240, 47243-47249, 47255-47259, 47265-47271, 47274, 47281- 47285, 47287-47292, 47295, 47298, 47300, 47302, 47306-47307, 47311, 47317-47318, 47320-47322, 47326-47330, 47333-47335, 47340, 47342, 47344-47345, 47347-47350, 47353-47358, 47360-47361, 47366-47369, 47371, 47373, 47375-47377, 47379, 47381-47385, 47387-47389, 47393- 47398, 47400-47401, 47404-47413, 47418, 47420-47421, 47425-47426, 47428, 47431-47441, 47444, 47446-47447, 47451-47457, 47459, 47461, 47463-47466, 47468-47471, 47473-47482, 47485-47493, 47500, 47508- 47509, 47513, 47515, 47517-47518, 47520-47524, 47528, 47532, 47536, 47538-47540, 47542-47551, 47553, 47557, 47560-47562, 47564-47565, 47567, 47570, 47573-47583, 47588-47590, 47592-47596, 47599, 47604- 47605, 47607, 47609-47611, 47613-47616, 47618, 47620-47621, 47623- 47625, 47627-47628, 47630-47631, 47633-47635, 47639, 47642, 47646, 47648, 47650-47651, 47653
Single Strand Oligonucleotides (Sense Strand of Target Gene)
SeqID range : 47462- 83660
SeqIDs w/o G Runs :
47463, 47468 -47483, 47485- ■47551, 47554 -47559, 47561 -47569, 47577-
47608, 47612 -47623, 47625- ■47631, 47633 -47638, 47643 -47674, 47688-
47851, 47866 -47986, 48000- ■48094, 48108 -48129, 48143 -48269, 48321-
48337, 48353 -48404, 48418- ■48478, 48492 -48556, 48571 -48682, 48696-
48755, 48768 -48818, 48831- ■48973, 48987 -49046, 49060 -49081, 49095-
49191, 49205 -49278, 49292- ■49314, 49329 -49522, 49536 -49641, 49655-
49675, 49689 -49930, 49944- ■49988, 50002 -50012, 50028 -50153, 50176-
50367, 50381 -50395, 50415- ■50545, 50560 -50602, 50616 -50664, 50680-
50746, 50760 -50783, 50799- ■51199, 51225 -51348, 51374 -51408, 51422-
51470, 51495 -51511, 51534- ■51721, 51743 -51840, 51859 -52001, 52016-
52040, 52060 -52100, 52115- ■52133, 52147 -52382, 52406 -52416, 52431-
52473, 52487 -52509, 52524- ■52745, 52759 -52766, 52780 -52823, 52837-
52862, 52889 -52956, 52971- ■53167, 53180 -53285, 53294 -53359, 53373-
53428, 53451 -53505, 53530- ■53571, 53585 -53604, 53618 -53784, 53811-
53935, 53949 -54341, 54363- ■54490, 54504 -54683, 54697 -54730, 54744-
54788, 54794 -55004, 55018- ■55204, 55218 -55435, 55456 -55626, 55640-
55644, 55658 -55702, 55710- ■55744, 55758 -55909, 55923 -55926, 55929-
55967, 55980 -55991, 55996- ■56006, 56027 -56082, 56096 -56108, 56123-
56183, 56196 -56198, 56214- ■56228, 56236 -56309, 56323 -56326, 56340-
56392, 56406 -56411, 56425- ■56570, 56584 -56627, 56641 -56666, 56680-
56772, 56786 -56823, 56837- ■57013, 57028 -57164, 57178 -57215, 57229-
57350, 57366 -57375, 57390- ■57466, 57480 -57622, 57636 -57726, 57740-
57847, 57861 -58139, 58153- 58223, 58239 -58267, 58281 -58522, 58536-
58690, 58704 -58874, 58894- ■58907, 58921 -58981, 58995 -59009, 59023-
59119, 59133 -59208, 59222- ■59264, 59278 -59297, 59312 -59359, 59373-
59460, 59474 -59782, 59802- ■59988, 60002 -60175, 60189 -60193, 60207-
60360, 60374 -60437, 60445- ■60506, 60521 -60525, 60530 -60552, 60556-
60563, 60577 -60617, 60627- ■60844, 60854 -60889, 60894 -61004, 61019-
61074, 61088 -61149, 61158- ■61183, 61197 -61221, 61235 -61261, 61268-
61445, 61459 -61480, 61494- ■61511, 61525 -61559, 61573 -61746, 61760-
61784, 61809 -61858, 61870- ■61884, 61892 -61962, 61970 -62018, 62029-
62113, 62127 -62223, 62237- ■62409, 62423 -62449, 62471 -62621, 62635-
62700, 62722 -62823, 62859- ■62874, 62888 -62901, 62915 -62965, 62987-
63173, 63198 -63219, 63233- ■63278, 63292 -63311, 63338 , 63353 -63569,
63597- 63630, 63661- 63663, 63681- 63708, 63722- 63836, 63858- 63880, 63894- 63931, 63946- 63959, 63973- 63984, 63999- 64067, 64081- 64097,
64111- 64222, 64237- 64280, 64294- 64519, 64534- 64563, 64577- 64598,
64612- 64668, 64682- 64701, 64716- 64737, 64752- 64754, 64770- 64836,
64850- 64867, 64881- 64930, 64944- 65009, 65023- 65082, 65097- 65162,
65176- 65216, 65244- 65272, 65299- 65357, 65371- 65413, 65428- 65455,
65480- 65567, 65581- 65638, 65652- 65686, 65700- 65753, 65771- 65805,
65820- 65950, 65964- 66047, 66067- 66129, 66157- 66181, 66195- 66198,
66212- 66218, 66232- 66257, 66293- 66369, 66397- 66492, 66506- 66563,
66577- 66593, 66607- 66622, 66637- 66926, 66945- 67095, 67114- 67181,
67199- 67203, 67217- 67374, 67388- 67543, 67557- 67613, 67628- 67675,
67689- 67717, 67744- 67814, 67830- 67868, 67883, 67908 -67921, 67935-
68334, 68348 -68522, 68557- ■68693, 68707- ■68770, 68784 -68843, 68866,
68880- 68903, 68917- 68979, 68994- 68997, 69011- 69110, 69125- 69143,
69158- 69168, 69182- 69219, 69234- 69269, 69285- 69330, 69345- 69466,
69480- 69484, 69498- 69590, 69605- 69701, 69715- 69722, 69736- 69763,
69783- 69839, 69853- 69869, 69883- 69945, 69968- 69979, 69993- 70085,
70115- 70138, 70152- 70232, 70246- 70268, 70282- 70290, 70308- 70315,
70329- 70345, 70359- 70456, 70470- 70478, 70493- 70655, 70670- 70707,
70721- 70828, 70842- 70855, 70870- 70983, 70997, 71011 -71024, 71038-
71094, 71109 -71334, 71348- ■71555, 71569- ■71653, 71667 -71747, 71761-
71820, 71834 -71837, 71852- ■71880, 71894- ■71925, 71939 -71965, 71981-
71983, 71997 -72034, 72048- ■72064, 72079- ■72280, 72294 -72366, 72380-
72395, 72439 -72459, 72473- ■72488, 72509- ■72546, 72560 -72645, 72660-
72670, 72685 -72866, 72880- ■72949, 72964, 72978 -73070 , 73084 -73203,
73222- 73229, 73244- 73466, 73493- 73508, 73537- 73545, 73559- 73560,
73574- 73617, 73631- 73646, 73660- 73694, 73708- 73739, 73753- 73760,
73776- 73885, 73899- 73961, 73975- 73988, 74002- 74092, 74106- 74128,
74142- 74143, 74174- 74227, 74241- 74574, 74588- 74677, 74691- 74744,
74758- 74869, 74883- 74956, 74975- 75006, 75021- 75101, 75115- 75165,
75179- 75213, 75227- 75280, 75294- 75310, 75324- 75355, 75388- 75412,
75438- 75452, 75473- 75525, 75540- 75543, 75570- 75597, 75624- 75632,
75646- 75683, 75703- 75723, 75737- 75792, 75833- 75859, 75884- 75924,
75938- 75956, 75970- 75989, 76013- 76030, 76051- 76053, 76068- 76104,
76124- 76134, 76148- 76159, 76174- 76263, 76293- 76330, 76344- 76395,
76445- 76521, 76535- 76621, 76635- 76647, 76669- 76670, 76705, 76719-
76740, 76754 -76879, 76893- ■77017, 77031- ■77116, 77130 -77185, 77208-
77210, 77225 -77229, 77262- ■77314, 77328- ■77361, 77376 -77396, 77438-
77491, 77505 -77585, 77599- ■77610, 77631- ■77703, 77719 -77790, 77806-
77813, 77828 -77851, 77865- ■77875, 77889- ■77897, 77911 -78128, 78156-
78163, 78177 -78235, 78250- ■78282, 78296- ■78459, 78473 -78487, 78510-
78525, 78543 -78562, 78585- ■78605, 78639- ■78718, 78734 -78787, 78801-
78891, 78916 -78967, 78982- ■78986, 79022- ■79030, 79045 -79048, 79062-
79080, 79111 -79142, 79156- ■79164, 79190- ■79211, 79225 -79234, 79248-
79303, 79317 -79340, 79354- ■79389, 79404- ■79429, 79443 -79466, 79481-
79530, 79546 -79551, 79572- ■79595, 79609- ■79665, 79679 -79838, 79852-
79893, 79916 -79946, 79971- ■80016, 80030- ■80077, 80091 -80099, 80143-
80220, 80234 -80260, 80274- ■80321, 80336- ■80343, 80357 -80396, 80410-
80476, 80491 -80497, 80512- ■80540, 80554- ■80617, 80633 -80702, 80717-
80755, 80777 -80901, 80917, 80932 -80934, 80949 -80953 , 80968 , 80983
81032, 81047 -81070, 81092- ■81104, 81118- ■81161, 81175 -81182, 81193-
81198, 81200 -81232, 81255- ■81258, 81272- ■81377, 81389 -81626, 81640- 81679, 81693-81699, 81725-81743, 81757-81789, 81809-81912, 81927- 81935, 81957-82023, 82037-82122, 82144-82217, 82248-82256, 82270- 82278, 82293-82323, 82351-82418, 82442-82509, 82524-82687, 82701- 82706, 82722-82732, 82748-82777, 82799, 82813-82816, 82830-82865, 82879-82981, 83006-83018, 83032-83063, 83085, 83105-83113, 83127- 83239, 83254-83308, 83323-83370, 83399-83504, 83518-83606, 83620- 83653
SeqIDs w/o miR Seeds:
47463-47466, 47468-47471, 47473-47482, 47485-47493, 47500,
47508-47509, 47513, 47515, 47517-47518, 47520-47524, 47528, 47532,
47536, 47538-47540, 47542-47551, 47553, 47557, 47560-47562, 47564-
47565, 47567, 47570, 47573-47583, 47588-47590, 47592-47596, 47599,
47604-47605, 47607, 47609-47611, 47613-47616, 47618, 47620-47621, 47623-47625, 47627-47628, 47630-47631, 47633-47635, 47639, 47642, 47646, 47648, 47650-47651, 47653, 47657, 47660-47662, 47664-47665, 47667-47668, 47670-47678, 47680, 47682, 47687, 47690-47691, 47695- 47696, 47698-47700, 47702, 47704-47706, 47710, 47714-47716, 47718, 47722-47730, 47733, 47735, 47737, 47739, 47741, 47747, 47749, 47751- 47769, 47774, 47776-47777, 47781, 47783-47785, 47788, 47790-47791, 47793-47794, 47797-47801, 47803, 47807, 47809, 47811-47812, 47815, 47817, 47822-47823, 47826-47830, 47832-47837, 47839, 47841-47844, 47848-47850, 47853, 47856-47860, 47863, 47865-47866, 47869-47873, 47875-47876, 47878, 47882, 47886-47888, 47892, 47894-47895, 47897, 47904, 47909-47911, 47913, 47915, 47917-47922, 47926, 47931-47933, 47935-47936, 47938, 47940-47942, 47946, 47951-47952, 47955, 47957- 47959, 47961, 47963-47967, 47969, 47974-47975, 47977-47992, 47994, 47996, 48001, 48003, 48005, 48007, 48009-48016, 48018-48020, 48026- 48027, 48029, 48031, 48036-48039, 48041, 48043-48045, 48047, 48049- 48050, 48052, 48057-48059, 48061, 48063-48066, 48068, 48070-48071, 48073-48076, 48078-48080, 48088-48095, 48097, 48100-48101, 48104- 48105, 48107-48108, 48111-48114, 48117, 48120-48123, 48125-48126, 48128-48130, 48133, 48135-48136, 48138-48139, 48142, 48144-48146, 48149-48151, 48154-48156, 48158, 48160, 48162, 48166-48167, 48169, 48171-48174, 48176, 48178, 48180, 48182, 48184-48188, 48195, 48199- 48204, 48207-48211, 48213-48217, 48219, 48221-48226, 48231-48235, 48237, 48239-48241, 48243-48244, 48246-48248, 48251-48253, 48255- 48262, 48264-48265, 48268, 48270, 48273-48274, 48276, 48281-48287, 48291, 48297-48299, 48301, 48303-48304, 48308, 48313, 48320, 48322- 48323, 48327-48328, 48333-48334, 48338-48340, 48342-48346, 48353- 48356, 48359-48366, 48368, 48370-48373, 48376-48377, 48379, 48384, 48386-48393, 48396, 48399-48402, 48405-48407, 48409-48410, 48412, 48414-48415, 48417-48418, 48423-48428, 48431-48435, 48437, 48439- 48444, 48447-48450, 48454, 48457-48459, 48461, 48463-48465, 48467- 48469, 48473-48476, 48478, 48482-48483, 48485-48486, 48492-48496, 48499, 48501, 48503, 48505, 48507-48508, 48512-48513, 48515-48516, 48519, 48521-48525, 48527-48534, 48537-48538, 48541, 48543, 48545, 48547-48549, 48552-48558, 48560-48561, 48563-48565, 48567, 48571- 48575, 48577, 48579, 48581, 48583, 48586-48599, 48602-48603, 48605- 48610, 48612-48613, 48616-48619, 48621-48624, 48628-48639, 48641- 48643, 48648, 48650, 48652-48658, 48660-48662, 48664, 48667, 48669- 48672, 48675-48683, 48685-48690, 48692-48693, 48696-48700, 48702, 48708, 48710-48712, 48714, 48720, 48722, 48726-48732, 48736-48737, 48739-48740, 48742-48764, 48767-48768, 48770, 48772, 48774-48779, 48781-48786, 48788-48789, 48792-48798, 48800, 48802-48811, 48813- 48816, 48818, 48820, 48829-48832, 48834-48841, 48843-48846, 48848, 48850, 48852, 48854, 48856-48862, 48864-48865, 48868-48871, 48873- 48875, 48877-48882, 48884-48885, 48887-48889, 48891, 48893-48897, 48899, 48903-48904, 48908, 48914-48919, 48921, 48928, 48930-48931, 48933, 48935-48938, 48940-48942, 48944, 48946-48948, 48951-48953, 48958-48959, 48966, 48969-48970, 48972, 48974-48975, 48977-48979, 48981, 48987, 48989, 48991-48992, 48997-49004, 49006-49008, 49011- 49016, 49018, 49020-49025, 49027-49035, 49038-49041, 49043-49050, 49052, 49054, 49058, 49060, 49064, 49067-49068, 49070-49071, 49073- 49081, 49083, 49085-49088, 49090, 49094-49095, 49097-49101, 49105- 49106, 49109, 49112-49113, 49115-49116, 49118-49121, 49124-49125, 49128-49130, 49133-49136, 49138, 49140, 49142, 49144-49148, 49151, 49153, 49159-49160, 49162-49164, 49166-49167, 49170-49173, 49176- 49177, 49179, 49181, 49183-49185, 49187, 49190, 49193, 49197, 49199, 49201, 49205-49209, 49211, 49213-49222, 49225, 49229-49231, 49233, 49236-49239, 49241, 49245-49247, 49252-49253, 49255, 49257-49266, 49269-49271, 49274, 49279, 49281-49286, 49288, 49291, 49295-49301, 49304-49305, 49307-49314, 49316, 49319, 49321-49322, 49329, 49331- 49336, 49338, 49340-49342, 49344-49346, 49349, 49351, 49353-49354, 49356-49360, 49362-49366, 49368-49370, 49372-49374, 49377-49380, 49383-49386, 49389-49395, 49397-49403, 49405-49406, 49408-49409, 49411-49413, 49415-49419, 49421, 49424, 49426-49431, 49433-49434, 49436-49443, 49445-49450, 49452-49461, 49463-49466, 49469-49472, 49476-49478, 49480-49481, 49484-49485, 49488-49489, 49491-49494, 49501, 49503-49504, 49506-49510, 49512-49513, 49515, 49517-49518, 49524-49525, 49527, 49529, 49531-49533, 49535-49536, 49539-49541, 49545-49546, 49549, 49552-49553, 49559-49561, 49563, 49565, 49567- 49573, 49575-49577, 49579-49580, 49582-49585, 49587-49588, 49592- 49597, 49599-49602, 49604-49610, 49615, 49617-49619, 49622-49623, 49625-49629, 49631, 49633-49634, 49637-49638, 49640, 49642-49646, 49648, 49653, 49658-49660, 49662, 49664-49669, 49674-49675, 49679- 49681, 49683, 49686, 49689, 49691, 49693-49700, 49703-49707, 49709- 49710, 49713, 49716-49723, 49725-49726, 49728-49729, 49736-49741, 49743-49744, 49746, 49748-49750, 49752, 49754, 49756-49757, 49760, 49762-49763, 49765-49768, 49771-49773, 49775-49780, 49782-49787, 49789-49794, 49796-49798, 49800, 49802-49808, 49811, 49813-49819, 49821, 49823-49824, 49826-49827, 49830-49833, 49836, 49838-49841, 49843, 49845-49849, 49851-49858, 49860-49866, 49868-49878, 49881- 49882, 49885, 49887-49888, 49890-49895, 49898, 49901-49902, 49904- 49905, 49907, 49910, 49912-49915, 49917, 49919-49931, 49933-49936, 49938, 49941, 49944, 49946, 49948-49949, 49951, 49953, 49955-49961, 49963-49973, 49975, 49978, 49981-49984, 49986, 49988-49989, 49991, 49993, 49999, 50002-50004, 50008, 50015-50020, 50029, 50031-50035, 50037-50040, 50042-50058, 50060-50062, 50064, 50066-50071, 50073- 50074, 50079-50080, 50082, 50085, 50087-50094, 50096, 50099-50100, 50103, 50105-50108, 50110-50120, 50123-50124, 50131-50132, 50135, 50138-50140, 50142-50143, 50145, 50147, 50151, 50154-50155, 50157- 50161, 50167-50169, 50178-50184, 50186-50189, 50191-50209, 50211, 50213, 50217-50219, 50222-50243, 50246-50247, 50249, 50254-50256, 50258-50259, 50261-50270, 50274, 50278-50279, 50282, 50285, 50287- 50290, 50292-■50293, 50296- 50298, 50300, 50303 - 50309, 50315- 50317,
50319, 50325- ■50327, 50331- 50332, 50334- 50338, 50342, 50344- 50347,
50352- 50353, 50364- 50365, 50370, 50372- 50374, 50376- 50377, 50379,
50381- 50383, 50385, 50387- 50402, 50409, 50414 - 50417, 50420- 50422,
50424- 50425, 50427- 50432, 50434, 50436- 50440, 50442, 50444, 50446-
50451, 50454- ■50457, 50459, 50461 50463 -50464 50467 -50471, 50473,
50475- 50476, 50478, 50481- 50483, 50485- 50487, 50489- 50490, 50492-
50496, 50500- ■50503, 50505- 50509, 50511- 50512, 50514- 50515, 50520-
50524, 50526- ■50533, 50536- 50537, 50539- 50547, 50549, 50553- 50554,
50557, 50559- ■50561, 50565- 50568, 50572, 50574 50577 , 50580 -50581,
50585- 50586, 50593- 50595, 50600- 50602, 50607- 50608, 50610, 50612-
50613, 50615- ■50616, 50618, 50620 ■50621, 50623 50625 , 50627 , 50629,
50632, 50635- ■50637, 50639, 50642 50644 -50646 50648 , 50656 , 50659,
50662- 50664, 50666, 50669, 50671 ■50672, 50674 50677 , 50680 -50682,
50684- 50693, 50700- 50701, 50704- 50706, 50708- 50709, 50711, 50713-
50716, 50718- ■50721, 50723, 50725 50727 -50728 50730 , 50733 , 50736,
50738- 50741, 50743- 50746, 50748, 50754, 50759 50764 , 50773 -50775,
50778- 50786, 50789, 50796, 50798 50800 -50802 50804 , 50807 -50813,
50815, 50818, 50820 -50821, 50824 50826 , 50828 -50829 , 50832 -50833,
50836, 50839, 50841 -50842, 50844 ■50847, 50849 50851 , 50853 , 50855,
50857, 50860, 50862 , 50864 , 50871 -50878 , 50880 -50885 , 50889 -50891,
50893, 50896, 50898 -50902, 50904 ■50907, 50909 - 50910, 50912- 50913,
50915- 50917, 50919, 50921- 50922, 50924- 50927, 50930- 50932, 50934-
50953, 50955- ■50958, 50960- 50963, 50965- 50970, 50974, 50976, 50978,
50980- 50991, 50993, 50995- 50996, 50999- 51000, 51005, 51007, 51011,
51013- 51016, 51019- 51020, 51023, 51025- 51027, 51030- 51031, 51033-
51035, 51037- ■51046, 51048, 51050 ■51051, 51053 - 51063, 51066- 51072,
51078- 51079, 51081- 51086, 51089- 51093, 51095- 51099, 51101-51102,
51104, 51106- ■51108, 51110- 51114, 51116, 51120 51122 -51131, 51133-
51141, 51143- ■51144, 51147, 51153 ■51155, 51159 - 51161, 51163- 51164,
51169, 51171, 51173 , 51175 -51176 51178 , 51181 -51184 , 51186 -51192,
51194, 51196, 51198 , 51200 , 51204 -51207 , 51210 -51216 , 51218 , 51223,
51227, 51230- ■51232, 51234- 51236, 51240, 51242 - 51247, 51251, 51259,
51262- 51265, 51268, 51271- 51274, 51276- 51277, 51279- 51280, 51282- 51283, 51285-51293, 51295-51296, 51299, 51301-51302, 51304-51309, 51314, 51318-51319, 51321-51323, 51326, 51328-51329, 51333-51338, 51341-51342, 51346-51347, 51349-51352, 51354-51356, 51363, 51365, 51374-51375, 51378-51379, 51381-51382, 51386-51387, 51389-51398, 51400-51409, 51411, 51413, 51416, 51420, 51423, 51427-51428, 51430, 51432, 51437-51439, 51441, 51444-51451, 51453-51454, 51456-51459, 51467-51472, 51475, 51478, 51480-51481, 51486, 51488, 51491, 51494- 51496, 51498, 51500, 51508-51510, 51512, 51515, 51518, 51520-51522, 51528-51530, 51533-51535, 51537-51541, 51543-51551, 51555-51556, 51558-51559, 51562-51568, 51570, 51577, 51581, 51583, 51585-51586, 51588, 51590-51593, 51595-51598, 51601, 51603, 51605-51606, 51610- 51612, 51616-51617, 51619-51620, 51624, 51628-51630, 51633, 51635- 51637, 51639, 51641, 51644, 51646-51649, 51651-51657, 51659-51662, 51665-51669, 51672-51673, 51676-51678, 51680, 51684-51689, 51691, 51694, 51697, 51700-51701, 51703-51706, 51708-51709, 51711, 51713, 51715-51716, 51719, 51721-51725, 51730-51732, 51734, 51736, 51742- 51745, 51747-51751, 51753-51754, 51757-51761, 51763, 51766-51770, 51772-51776, 51778-51796, 51798-51800, 51803, 51806-51807, 51809- 51816, 51818-51819, 51821, 51823, 51825-51827, 51830, 51832-51834, 51836-51837, 51840-51847, 51849-51850, 51852, 51855, 51859-51862, 51864-51865, 51867-51874, 51879-51882, 51884-51887, 51889-51899, 51901-51907, 51910-51912, 51914-51929, 51932-51935, 51937-51938, 51941, 51943, 51945-51946, 51950, 51954-51959, 51961-51962, 51965, 51967, 51973-51974, 51976, 51979, 51981-51984, 51988, 51991, 51993- 51994, 51998-51999, 52002, 52004, 52007-52009, 52011, 52015-52016, 52018-52019, 52021-52023, 52025, 52029, 52031-52032, 52034, 52036, 52038-52039, 52041-52042, 52045-52046, 52051-52052, 52055, 52057, 52060, 52063-52064, 52066-52070, 52076-52080, 52083-52097, 52099- 52102, 52104-52106, 52109-52111, 52114-52115, 52117, 52121-52123, 52126-52128, 52134, 52137-52139, 52143, 52146-52154, 52156-52157, 52159, 52161, 52166-52170, 52172-52173, 52175-52182, 52184-52188, 52191, 52195-52200, 52202-52207, 52209-52216, 52218, 52221, 52226- 52227, 52229, 52232-52234, 52238-52247, 52251, 52253-52257, 52259- 52264, 52266-52267, 52269, 52272, 52276-52277, 52279, 52282-52285, 52287, 52289-52291, 52293-52294, 52296, 52299, 52301-52304, 52306- 52308, 52310-52312, 52316-52317, 52319-52324, 52328, 52330-52331, 52334-52336, 52339-52341, 52343, 52348-52350, 52352-52353, 52357- 52360, 52362-52364, 52366-52372, 52375-52377, 52379-52380, 52382, 52384, 52388-52391, 52396, 52399, 52407-52408, 52410-52414, 52416- 52423, 52425, 52427, 52430-52434, 52437-52438, 52443-52447, 52451- 52454, 52456, 52458, 52461-52463, 52465, 52467-52468, 52470, 52472- 52482, 52489-52496, 52498-52500, 52502-52503, 52505-52506, 52508, 52511-52512, 52514-52520, 52522, 52524-52525, 52528-52530, 52532, 52535-52538, 52541-52542, 52544, 52549-52550, 52555-52557, 52559- 52560, 52562-52564, 52566, 52570, 52573-52574, 52577-52582, 52584- 52585, 52588, 52590, 52592-52593, 52595-52596, 52598, 52600-52601, 52604-52606, 52611-52613, 52615-52625, 52628, 52630, 52632, 52635, 52639-52640, 52644, 52646-52648, 52650-52653, 52656-52657, 52659- 52660, 52662-52666, 52668, 52670, 52672-52676, 52678-52679, 52681- 52683, 52685, 52687-52689, 52691-52694, 52697, 52699, 52701-52703, 52708, 52711-52712, 52714, 52716, 52722-52727, 52729-52735, 52739, 52741-52743, 52745-52746, 52748, 52750-52753, 52755, 52760-52764, 52767, 52770, 52772, 52776, 52779, 52781-52791, 52793-52794, 52796- 52802, 52805, 52807-52809, 52814, 52817, 52820-52821, 52824, 52828- 52830, 52832, 52838, 52840-52842, 52844, 52850, 52854, 52856-52859, 52861-52866, 52868, 52870-52871, 52878-52883, 52890, 52893, 52895, 52897-52903, 52910-52915, 52918, 52920, 52923-52925, 52930-52931, 52935, 52938-52948, 52950-52951, 52953-52955, 52958-52959, 52961, 52965, 52969, 52971-52972, 52974-52975, 52979-52980, 52983-52989, 52993-52994, 52996, 53000-53001, 53003-53006, 53009, 53011, 53017- 53018, 53021, 53023, 53025-53027, 53030-53032, 53034, 53039-53041, 53044, 53046, 53049-53051, 53056, 53058, 53060, 53062-53067, 53069, 53072-53079, 53081, 53088-53090, 53094, 53097, 53102, 53104-53123, 53125-53127, 53129-53137, 53140, 53145-53146, 53152-53154, 53156, 53159, 53162-53165, 53170-53173, 53175, 53177, 53180-53182, 53184- 53185, 53187, 53189, 53193-53200, 53202-53206, 53210-53212, 53214- 53222, 53225-53226, 53228, 53230-53231, 53234-53236, 53238-53239, 53241, 53243-53247, 53249-53251, 53254, 53257-53260, 53262-53265, 53267, 53269-53271, 53275-53277, 53282-53283, 53285-53287, 53292-
53294, 53296, 53300, 53302-53305, 53312-53320, 53324-53325, 53328-
53329, 53332-53335, 53337, 53339, 53343-53345, 53347-53349, 53351-
53352, 53354-53357, 53359, 53361-53363, 53372, 53374-53377, 53383- 53387, 53389, 53391, 53393, 53396, 53399, 53401-53407, 53409, 53411,
53413- 53418, 53420- 53423, 53426, 53428, 53431- 53432, 53436- 53437,
53441, 53450- ■53451, 53455, 53458 53460 , 53464 -53468 , 53472 -53475,
53477, 53481- ■53482, 53485- 53486, 53491, 53494- 53497, 53500- 53502,
53504, 53508, 53511 , 53517 , 53520 -53527 , 53529 -53531 , 53534 -53535,
53537, 53540, 53542 , 53550 -53551 53553 , 53557 -53558 , 53560 -53565,
53569- 53574, 53578- 53579, 53585, 53587- 53588, 53594- 53597, 53603,
53605, 53614, 53616 , 53619 -53621 53623 -53626, 53629 -53630, 53632,
53635- 53647, 53649- 53654, 53656- 53673, 53675-53680, 53683-53690,
53692- 53697, 53700- 53701, 53706, 53708- 53712, 53714- 53715, 53717-
53718, 53720- ■53722, 53724, 53729 53731 -53733, 53736 , 53738 -53739,
53741- 53745, 53747, 53751, 53753 53754, 53756, 53763 , 53767 , 53771-
53774, 53776, 53779 -53780, 53782 53784, 53786- 53790, 53800, 53805,
53812- 53815, 53817- 53820, 53822, 53824- 53826, 53828- 53830, 53832-
53833, 53836, 53839 -53840, 53842 53844 -53850, 53852 -53853, 53855-
53857, 53859- ■53861, 53863- 53864, 53868- 53872, 53875- 53876, 53878,
53880, 53882- ■53885, 53889- 53891, 53893- 53895, 53897, 53900, 53903-
53906, 53908, 53913 -53914, 53916 53920, 53922, 53924 -53929, 53934-
53945, 53947- ■53950, 53952, 53955 53957, 53959- 53960, 53962- 53963,
53965, 53967, 53970 -53971, 53973 53975, 53979- 53980, 53982, 53984-
53988, 53990- ■53991, 53993, 53995 53997, 54002, 54004 -54005, 54007-
54011, 54013- ■54014, 54016, 54020 54022 -54023, 54026 -54027, 54030-
54031, 54033- ■54034, 54036- 54037, 54040- 54041, 54043- 54046, 54052-
54053, 54055- ■54056, 54058, 54060 54062 , 54064 -54067 , 54074 , 54076,
54079, 54082- ■54084, 54088- 54090, 54095- 54096, 54098- 54099, 54102,
54105- 54109, 54111- 54119, 54121- 54126, 54129, 54131- 54132, 54134-
54137, 54140- ■54141, 54144, 54148 54149, 54151, 54154 -54155, 54160-
54162, 54164, 54168 -54169, 54171 54173 , 54175 -54176 , 54180 -54182,
54188- 54189, 54191- 54193, 54195- 54200, 54204-54208, 54210, 54212,
54214- 54218, 54220- 54222, 54224- 54225, 54228-54230, 54232, 54234, 54236-54242, 54244-54249, 54252, 54254, 54256, 54259-54260, 54262- 54265, 54267, 54273, 54275-54278, 54280-54281, 54283, 54285, 54287, 54289-54293, 54295-54296, 54298-54299, 54301, 54303, 54305, 54309- 54313, 54315, 54317-54321, 54323-54325, 54327-54331, 54333, 54337- 54338, 54341, 54343-54349, 54353, 54359, 54361-54362, 54364, 54366, 54368, 54371, 54373-54374, 54376, 54379, 54381, 54384-54386, 54388, 54392-54395, 54397-54402, 54405-54409, 54411-54415, 54417-54420, 54422-54423, 54427, 54431, 54433-54442, 54446, 54448-54449, 54451- 54455, 54458, 54461, 54463-54464, 54466, 54468-54472, 54474-54475, 54477-54485, 54489-54493, 54495-54496, 54498, 54504, 54506-54510, 54512, 54514-54523, 54526-54530, 54532, 54534-54536, 54538, 54540, 54544-54548, 54554-54556, 54558-54559, 54561, 54563-54566, 54572, 54575-54576, 54581, 54585-54587, 54589-54592, 54595-54596, 54598- 54601, 54603-54607, 54609-54611, 54613-54615, 54617-54622, 54624, 54626-54630, 54633-54637, 54642-54643, 54645-54649, 54651-54652, 54654, 54656-54662, 54664-54673, 54676-54678, 54681, 54683-54688, 54690, 54692-54694, 54698-54701, 54703-54712, 54714-54715, 54718- 54719, 54721-54728, 54732-54736, 54738, 54741, 54745, 54747-54748, 54750-54751, 54753-54755, 54758-54760, 54762-54763, 54765-54766, 54770-54772, 54774-54785, 54787, 54796-54798, 54800, 54806, 54808- 54809, 54811-54812, 54814, 54816, 54818, 54821, 54823-54824, 54828- 54829, 54831, 54834-54836, 54838-54841, 54845, 54849, 54853-54856, 54858-54862, 54864-54865, 54868-54872, 54874-54875, 54877-54878, 54881, 54883-54885, 54887, 54889, 54891, 54893, 54897, 54899-54902, 54905-54912, 54914-54915, 54917, 54920-54921, 54925-54929, 54931- 54932, 54936-54940, 54942, 54944, 54946-54948, 54950, 54953-54960, 54962-54969, 54972, 54974, 54976-54993, 54995-55001, 55003-55005, 55007-55008, 55010-55011, 55014, 55017, 55019, 55022-55025, 55027- 55030, 55032-55034, 55037, 55039, 55045-55048, 55050-55053, 55057- 55060, 55062-55064, 55067-55069, 55072, 55074-55075, 55077-55080, 55083-55084, 55086-55090, 55092-55094, 55096-55098, 55100-55102, 55105, 55107, 55111-55112, 55114, 55116-55121, 55123-55124, 55128, 55130-55134, 55136, 55138, 55140, 55142-55149, 55151-55152, 55156- 55162, 55164, 55168-55174, 55176-55178, 55180-55182, 55186-55195, 55197- 55202, 55204- 55206, 55210- 55211, 55213- 55215, 55218-55223,
55225, 55228- ■55231, 55233- 55236, 55238- 55243, 55247, 55249- 55253,
55256- 55257, 55259- 55266, 55269- 55272, 55274- 55276, 55280, 55283,
55286- 55288, 55291, 55293- 55296, 55299, 55301 f 55303 -55306, 55308-
55312, 55316- ■55320, 55322- 55327, 55329- 55337, 55339, 55341, 55343-
55348, 55350- ■55351, 55354- 55367, 55369, 55371 r 55373 -55374, 55376,
55378, 55381- ■55388, 55390- 55398, 55400- 55402, 55404- 55406, 55409,
55411- 55412, 55415- 55418, 55421, 55425, 55427 55429, 55431- 55433,
55435- 55436, 55438, 55447, 55450 , 55458 -55466 r 55468 -55469, 55473,
55476, 55478, 55480 , 55482 -55487 , 55491 -55492 r 55494 , 55497 , 55499
55502- 55503, 55506- 55507, 55510, 55513- 55515, 55517, 55519- 55520,
55522- 55524, 55526, 55528, 55530 -55533, 55537 r 55541 -55543, 55545-
55546, 55548, 55551 -55558, 55560 -55566, 55570 55571, 55573- 55575,
55578, 55580- ■55582, 55584- 55587, 55590- 55598, 55603- 55605, 55610-
55611, 55613- ■55617, 55619, 55621 , 55623 -55624 r 55626 -55628, 55631,
55633- 55637, 55640- 55641, 55643- 55644, 55646, 55653- 55655, 55658,
55660- 55661, 55664- 55667, 55669, 55671- 55676, 55679- 55685, 55687,
55691, 55693, 55696 , 55699 , 55702-55708 , 55710 -55719 , 55721 , 55727
55729, 55731- ■55732, 55737- 55738, 55740- 55741, 55745, 55747- 55748,
55750- 55751, 55753, 55757- 55759, 55761, 55763 r 55765 -55769, 55773-
55775, 55777- ■55778, 55780- 55784, 55786- 55795, 55798- 55801, 55803,
55806- 55809, 55811- 55815, 55817, 55819- 55828, 55830- 55833, 55835-
55840, 55843- ■55844, 55846- 55848, 55852- 55859, 55861- 55868, 55870-
55871, 55873, 55875 -55877, 55879 -55881, 55883 r 55887 , 55892 -55893,
55895, 55898- ■55903, 55905, 55909 -55915, 55922 r 55924 , 55926 , 55934
55939- 55944, 55952, 55954- 55956, 55958- 55961, 55963- 55964, 55967,
55970- 55975, 55979- 55986, 55988- 55991, 55998, 56000- 56002, 56007-
56009, 56015, 56018 , 56022 -56026 , 56028 -56029 r 56031 -56036, 56038,
56040, 56042- ■56044, 56047, 56050 -56053, 56055 56056, 56058- 56059,
56061- 56066, 56068, 56071, 56073 -56078, 56080 r 56084 , 56089 , 56091
56092, 56096- ■56097, 56104- 56111, 56113, 56116 r 56120 , 56123 -56124,
56126, 56128, 56130, 56132, 56136, 56138-56139, 56144-56148, 56150- 56155, 56158, 56160-56164, 56167, 56170, 56176, 56178-56179, 56186- 56187, 56189, 56192, 56195-56196, 56198-56199, 56201-56203, 56212, 56215- 56218, 56220- 56221, 56223- 56227, 56232, 56234- 56235, 56237,
56239, 56241, 56244 , 56247 -56250 , 56253 -56259, 56262 , 56264 -56268,
56270- 56272, 56274- 56275, 56277- 56280, 56282-56284, 56286, 56288-
56290, 56292- ■56296, 56298, 56306 -56311, 56313- 56314, 56321- 56324,
56326- 56327, 56329, 56332, 56334 -56338, 56340- 56341, 56343- 56348,
56350, 56352- ■56356, 56358- 56359, 56363- 56366, 56372, 56380- 56381,
56384, 56386- ■56393, 56395- 56401, 56403, 56405- 56406, 56413, 56415-
56416, 56425- ■56426, 56428- 56430, 56432, 56435- 56438, 56440- 56445,
56447- 56449, 56451, 56457, 56459 -56461, 56465- 56469, 56471- 56476,
56478, 56481- ■56482, 56484- 56487, 56490- 56495, 56500- 56501, 56504-
56513, 56515- ■56516, 56521, 56526 -56528, 56530- 56532, 56534, 56536-
56544, 56548- ■56550, 56553- 56555, 56557- 56561, 56564- 56568, 56570,
56572, 56574, 56576 , 56578 -56579 , 56582 , 56585 -56588 , 56590 , 56592,
56594, 56597- ■56601, 56603- 56606, 56608- 56609, 56611- 56613, 56616-
56619, 56621, 56623 -56627, 56629 , 56631 -56633, 56635 , 56642 , 56645,
56647, 56650, 56652 -56654, 56657 -56660, 56662, 56664 -56671, 56673-
56675, 56679, 56681 -56688, 56691 , 56693 , 56696 , 56698-56699 , 56701-
56704, 56707, 56710 , 56712 -56715 , 56717 -56718, 56720 , 56722 , 56727-
56729, 56731- ■56732, 56734- 56735, 56737- 56741, 56743- 56744, 56746-
56755, 56757- ■56761, 56766- 56768, 56773- 56780, 56793, 56795- 56799,
56805- 56806, 56808, 56811, 56813 -56814, 56820- 56826, 56836, 56841-
56847, 56849, 56851 , 56853 -56857 , 56859 -56861, 56864 , 56866 -56868,
56870- 56871, 56874- 56879, 56881, 56883- 56890, 56892- 56894, 56896,
56899, 56901, 56903 -56904, 56906 -56907, 56910- 56914, 56917, 56920-
56923, 56925- ■56926, 56929- 56930, 56932- 56933, 56935- 56937, 56939-
56940, 56942- ■56943, 56945- 56946, 56951- 56952, 56955- 56959, 56961,
56963- 56964, 56966, 56970, 56972 -56973, 56975- 56979, 56981- 56982,
56984- 56985, 56987- 56992, 56994, 56997, 56999, 57001 , 57004 -57009,
57011- 57013, 57015- 57020, 57022, 57029- 57032, 57034- 57037, 57039-
57040, 57043- ■57044, 57046- 57049, 57051- 57053, 57055- 57056, 57058-
57060, 57062- ■57069, 57071- 57074, 57076- 57078, 57080- 57081, 57085-
57086, 57090, 57093 , 57095 -57099 , 57101 -57108, 57110 , 57112 -57119,
57121- 57122, 57124- 57125, 57131, 57133- 57137, 57140- 57141, 57143-
57145, 57147- ■57149, 57151- 57158, 57160- 57161, 57164- 57168, 57172, 57177, 57180-57184, 57186-57187, 57190-57192, 57196-57197, 57200, 57202-57203, 57205-57206, 57210-57213, 57219, 57221, 57225, 57227, 57229-57236, 57238, 57240-57242, 57244-57246, 57248, 57251-57253, 57255-57256, 57258, 57260, 57263-57264, 57266-57267, 57269, 57271, 57273-57275, 57277, 57281-57282, 57284-57285, 57292-57296, 57298- 57299, 57301-57305, 57309, 57312-57313, 57316-57324, 57326-57327, 57329-57334, 57338, 57340, 57342, 57344-57349, 57351-57353, 57358, 57366-57370, 57372-57376, 57378-57381, 57383, 57389-57392, 57394- 57396, 57398-57406, 57409-57411, 57413-57414, 57418, 57423-57424, 57426-57433, 57435-57438, 57444, 57447-57449, 57454, 57456, 57458- 57461, 57463, 57465, 57467-57469, 57471, 57474-57475, 57480, 57484- 57489, 57495-57496, 57498-57499, 57503-57504, 57507-57509, 57514, 57518-57519, 57521, 57524-57527, 57529-57534, 57536, 57538, 57540, 57542-57545, 57550-57554, 57556-57559, 57562-57568, 57571-57578, 57580-57587, 57589, 57594-57595, 57597-57599, 57601, 57605, 57608, 57613, 57616, 57618-57619, 57621-57631, 57636, 57638-57641, 57643- 57644, 57648-57649, 57651-57655, 57657, 57659-57661, 57663-57666, 57668-57671, 57673-57676, 57679-57683, 57685-57686, 57689-57694, 57697, 57699-57706, 57708, 57710, 57712, 57715-57720, 57722-57723, 57725, 57727-57731, 57739-57741, 57743-57747, 57749-57761, 57763, 57766-57769, 57771, 57773, 57775-57778, 57780-57781, 57783-57785, 57787-57790, 57792, 57795-57796, 57798-57802, 57805-57806, 57808, 57811, 57813, 57815-57817, 57819-57822, 57824-57826, 57828, 57833, 57835, 57840-57846, 57849, 57854-57855, 57860, 57862-57863, 57865- 57866, 57870-57872, 57875, 57877, 57884-57885, 57887, 57891, 57894, 57898, 57901-57904, 57906-57909, 57911-57919, 57921-57922, 57926, 57929-57931, 57935, 57937, 57939, 57945-57946, 57948, 57951, 57953- 57954, 57958, 57960, 57962-57968, 57970, 57972-57974, 57976-57978, 57980, 57982, 57987-57989, 57991-57994, 57996-58004, 58006, 58009- 58011, 58013-58014, 58016-58017, 58019, 58025, 58029-58031, 58033-
58035, 58037, 58039, 58043, 58046-58047, 58050, 58052, 58054, 58056- 58057, 58060, 58062-58066, 58069, 58071, 58073, 58075, 58080-58084, 58086-58088, 58092-58093, 58096, 58098-58099, 58101-58102, 58106, 58108-58120, 58122-58124, 58126-58132, 58134-58135, 58137, 58140, 58142, 58145, 58149, 58151-58152, 58154, 58159, 58161-58162, 58165, 58167-58171, 58178-58179, 58182, 58188-58190, 58192-58193, 58195, 58197-58200, 58204-58205, 58207, 58209-58210, 58216-58218, 58220, 58223, 58225-58226, 58229-58231, 58239, 58242, 58244, 58246-58250, 58254, 58259-58266, 58270, 58272, 58274-58275, 58280, 58282-58283, 58285, 58288-58289, 58291, 58293-58295, 58297, 58299, 58301, 58303, 58305-58309, 58317-58319, 58321, 58324-58325, 58327, 58331, 58333- 58334, 58340-58342, 58344, 58346-58352, 58355, 58357-58358, 58360, 58365, 58367, 58376-58377, 58379-58380, 58382, 58388, 58392, 58399- 58402, 58404, 58406-58415, 58418-58419, 58422, 58427, 58430-58431, 58433, 58439-58440, 58442, 58444-58448, 58450-58453, 58455, 58457, 58459-58462, 58464-58467, 58469, 58471-58476, 58478-58480, 58484- 58486, 58488, 58490, 58493-58498, 58500, 58503-58505, 58508, 58510, 58512-58514, 58519-58527, 58535-58536, 58539-58542, 58544-58548, 58550, 58553-58554, 58556-58557, 58561, 58563-58564, 58568-58569, 58571-58574, 58577-58596, 58599-58601, 58604, 58606-58613, 58615- 58616, 58619, 58621-58627, 58629, 58632, 58634, 58637-58640, 58643, 58649, 58651, 58654-58659, 58662-58668, 58670-58674, 58676-58677, 58679-58685, 58689-58690, 58692, 58694-58700, 58705, 58708-58710, 58713, 58719-58720, 58722, 58724, 58727-58728, 58731-58733, 58736- 58739, 58742-58746, 58748, 58750-58753, 58755-58757, 58759-58761, 58763-58764, 58767-58769, 58771, 58773-58776, 58778-58779, 58781, 58783, 58785-58788, 58793, 58797-58798, 58800, 58802-58804, 58807- 58809, 58814-58815, 58817-58818, 58820, 58825, 58829, 58831-58832, 58835-58836, 58838, 58840, 58842-58846, 58849-58850, 58852-58853, 58857-58859, 58863, 58865-58866, 58869-58872, 58875-58877, 58879- 58881, 58884, 58888-58889, 58892, 58894-58896, 58898-58899, 58902, 58904-58910, 58912-58917, 58923, 58926-58927, 58929-58933, 58935, 58937, 58939, 58941-58944, 58947, 58951, 58953, 58955, 58957, 58960- 58969, 58971, 58973-58974, 58976, 58978, 58981-58989, 58998-58999, 59001, 59003, 59005-59006, 59008-59013, 59016, 59018-59019, 59025- 59030, 59033, 59036-59038, 59041-59051, 59053-59054, 59056, 59058- 59063, 59067, 59069, 59071-59072, 59074-59075, 59077-59081, 59083- 59086, 59088, 59090-59092, 59094, 59096, 59098-59102, 59106, 59109- - Ill -
59110, 59113, 59115, 59117-59118, 59121, 59127, 59135, 59137, 59139, 59141-59142, 59144-59147, 59149, 59152-59153, 59157-59159, 59164, 59169-59171, 59173-59177, 59179, 59182, 59185-59186, 59188-59193, 59195, 59197-59203, 59206-59207, 59209-59219, 59221-59222, 59224, 59227, 59230-59233, 59238, 59241-59242, 59246-59247, 59249-59251, 59253-59254, 59259-59260, 59263-59264, 59277, 59285, 59287-59288, 59290-59292, 59295-59296, 59304-59306, 59312, 59318-59319, 59321- 59325, 59327, 59329, 59331, 59335-59337, 59339, 59344, 59350-59351, 59355-59357, 59359-59362, 59365-59366, 59369, 59371-59377, 59385- 59386, 59391-59395, 59399, 59402-59405, 59407-59408, 59410, 59416- 59419, 59422-59424, 59426, 59428, 59432-59433, 59435-59438, 59443- 59444, 59449, 59451-59452, 59454, 59458, 59460-59462, 59464-59466, 59468-59469, 59471-59472, 59474-59475, 59478-59480, 59482-59485, 59487, 59490-59492, 59494-59497, 59500-59512, 59515, 59518-59520, 59522-59528, 59532-59535, 59538, 59540, 59542-59556, 59558-59565, 59570-59573, 59577-59579, 59581, 59583-59584, 59586-59587, 59589, 59591-59592, 59594-59595, 59597-59598, 59600-59602, 59604-59607, 59609-59611, 59613, 59615, 59617-59618, 59620-59622, 59624, 59626- 59630, 59632, 59634, 59636-59639, 59641, 59643-59644, 59646, 59648, 59651, 59654-59657, 59661, 59663-59667, 59670, 59673-59675, 59677- 59684, 59687-59690, 59692-59697, 59699, 59701-59702, 59704-59705, 59707-59708, 59711-59715, 59717-59720, 59725-59726, 59729-59730, 59733-59737, 59740-59742, 59745-59746, 59748-59749, 59751-59754, 59758, 59760-59766, 59769-59770, 59772-59775, 59778-59780, 59783- 59785, 59787-59788, 59792, 59797-59798, 59800, 59802-59812, 59814, 59816, 59818, 59821-59824, 59827-59831, 59834-59838, 59843, 59845, 59847-59848, 59850-59854, 59856, 59858-59859, 59866, 59872-59873, 59875, 59877-59881, 59887, 59891-59895, 59900, 59904-59906, 59908- 59914, 59917-59918, 59922-59926, 59929-59933, 59937-59938, 59941, 59945, 59947-59948, 59951-59953, 59957-59958, 59961, 59963-59965, 59968-59972, 59974, 59976-59977, 59979, 59981, 59983-59985, 59987- 59990, 59993, 59995-59996, 59999, 60002, 60004, 60006-60009, 60013, 60016-60018, 60021-60022, 60024, 60026, 60029, 60031-60032, 60034, 60037, 60040-60041, 60043-60044, 60047, 60049-60050, 60052-60055, 60058-60059, 60061-60063, 60065, 60067, 60069-60071, 60073, 60075- 60076, 60078-60079, 60081, 60087-60089, 60092, 60096-60098, 60100, 60102-60104, 60106-60115, 60118, 60120, 60123-60124, 60126-60127, 60129-60131, 60133-60137, 60140-60144, 60146-60151, 60153, 60157, 60162, 60166, 60168-60169, 60173-60174, 60176-60177, 60179, 60181- 60190, 60197-60201, 60206, 60213-60217, 60219, 60222-60224, 60227, 60229-60230, 60233-60238, 60243-60244, 60246, 60248, 60252, 60254- 60261, 60263, 60265-60267, 60269-60270, 60276-60288, 60291, 60296, 60301, 60305-60306, 60310, 60314-60318, 60320, 60322, 60328, 60331, 60335, 60337, 60339-60345, 60347-60352, 60354-60367, 60370, 60375- 60377, 60379-60383, 60385-60387, 60389-60391, 60395, 60397-60401, 60412, 60414, 60416-60417, 60420-60422, 60424, 60426-60430, 60432, 60435-60439, 60442, 60445-60449, 60451, 60453, 60455-60456, 60460- 60463, 60468-60470, 60474, 60476-60478, 60482-60485, 60487, 60489, 60493-60497, 60499-60503, 60505-60506, 60508, 60510-60511, 60514- 60515, 60520-60521, 60524, 60529-60533, 60535-60536, 60540-60544, 60548-60549, 60552-60554, 60556-60558, 60560-60561, 60563, 60566, 60569, 60572, 60579, 60582, 60589-60593, 60595-60602, 60604-60613, 60615, 60617-60621, 60623, 60625-60626, 60628-60629, 60633, 60636, 60638-60645, 60651, 60653, 60655-60656, 60658-60659, 60661-60664, 60666-60668, 60670, 60672-60680, 60683-60684, 60686-60693, 60695, 60699, 60701, 60703, 60705-60706, 60709-60710, 60712-60713, 60715, 60717, 60719, 60721-60722, 60724-60731, 60733-60735, 60738-60739, 60741, 60743-60746, 60750-60751, 60755-60756, 60758-60763, 60765, 60773-60777, 60779, 60781-60782, 60786-60796, 60799, 60801, 60803, 60805, 60807-60809, 60811-60819, 60821, 60826-60828, 60831-60832, 60835-60836, 60840-60844, 60846, 60848-60850, 60853, 60855-60857, 60861-60864, 60868-60876, 60878-60879, 60881, 60883-60886, 60888, 60892-60894, 60896-60898, 60902-60906, 60908-60910, 60912, 60914, 60916-60917, 60921-60924, 60926-60927, 60930-60934, 60936-60938, 60940-60941, 60943-60944, 60946-60950, 60952-60953, 60955-60957, 60959-60967, 60972, 60976, 60978-60979, 60981-60982, 60984, 60986- 60987, 60991-60995, 60998-61002, 61004-61008, 61010-61012, 61015, 61018-61019, 61022-61024, 61026-61043, 61047-61049, 61051-61055, 61057-61058, 61060, 61062-61066, 61068-61075, 61077, 61080-61082, 61084, 61088, 61090-61095, 61097, 61099-61104, 61112, 61114-61116, 61118-61126, 61129, 61131-61139, 61142-61143, 61145-61149, 61154, 61157-61159, 61161, 61163-61166, 61168, 61173-61176, 61178, 61182, 61186, 61188-61191, 61196-61198, 61200, 61203-61211, 61213-61214, 61216-61218, 61220-61222, 61224, 61226-61228, 61231, 61234-61238, 61240, 61244-61249, 61251, 61253-61254, 61256-61259, 61262-61263, 61265, 61270, 61272-61276, 61280, 61284-61285, 61287-61292, 61294, 61296-61298, 61300, 61302-61309, 61312, 61314-61315, 61323, 61326- 61329, 61331-61332, 61334-61337, 61341, 61343, 61345, 61348-61349, 61351-61353, 61355, 61358, 61360-61361, 61364, 61366, 61369-61371, 61373-61375, 61377-61378, 61382-61384, 61386-61390, 61392-61393, 61395, 61399-61400, 61402, 61404-61405, 61408-61409, 61411, 61413, 61415-61420, 61424, 61426, 61430, 61432, 61436-61443, 61445, 61450, 61458-61460, 61462, 61469, 61472-61474, 61476-61477, 61479, 61485- 61490, 61496, 61500-61506, 61509-61512, 61514, 61516-61518, 61521- 61522, 61526-61528, 61530-61532, 61534-61535, 61537-61540, 61542- 61549, 61551-61552, 61555, 61557, 61559-61561, 61563-61564, 61573- 61580, 61582, 61584-61592, 61594, 61599, 61605-61607, 61609-61622, 61624-61627, 61631, 61633, 61637, 61640, 61642, 61648-61649, 61653- 61659, 61663, 61666-61669, 61671, 61673-61682, 61685-61688, 61690- 61694, 61696-61717, 61719-61747, 61749-61751, 61754-61756, 61759, 61761-61763, 61765, 61770-61775, 61777-61780, 61785-61789, 61791, 61793, 61797-61798, 61800, 61803, 61805, 61811-61815, 61817-61819, 61823-61824, 61827-61829, 61836, 61840-61847, 61850, 61853, 61855, 61857-61866, 61869-61874, 61876, 61879-61888, 61890, 61892, 61898, 61903-61906, 61909, 61912, 61914, 61917-61921, 61924-61927, 61929- 61932, 61934-61936, 61940-61941, 61945, 61947-61954, 61956-61967, 61969-61971, 61974-61977, 61979-61980, 61985, 61989-61990, 61993- 61999, 62003-62006, 62008, 62010-62015, 62017-62018, 62022-62023, 62028-62029, 62031-62034, 62036-62038, 62041, 62043-62045, 62053- 62054, 62056, 62058-62060, 62062-62073, 62075, 62077-62081, 62084, 62086-62091, 62093, 62095-62101, 62103-62111, 62113, 62115-62123, 62126-62127, 62132-62134, 62136, 62139-62140, 62142-62146, 62149- 62152, 62154-62160, 62162-62166, 62168, 62170, 62172-62178, 62180- 62181, 62186-62187, 62189, 62191, 62193, 62201-62209, 62211-62215, 62218-62220, 62222-62230, 62236, 62238-62242, 62244-62251, 62255, 62257, 62259, 62263-62264, 62266-62271, 62273-62275, 62277-62279, 62281, 62284-62289, 62291-62293, 62295-62298, 62300-62302, 62306- 62307, 62309-62310, 62312-62313, 62315-62317, 62319-62324, 62327, 62329-62331, 62333, 62335, 62338, 62342-62345, 62351, 62353-62356, 62359, 62361-62365, 62369-62371, 62373-62374, 62376-62385, 62388, 62390, 62393, 62395-62397, 62399, 62401-62402, 62405, 62407-62408, 62415, 62423-62424, 62426-62437, 62439, 62443, 62447, 62451, 62453- 62454, 62456-62457, 62460, 62462-62465, 62467-62468, 62471-62474, 62478, 62480, 62483-62484, 62489-62492, 62494, 62496-62498, 62501, 62504-62505, 62508-62509, 62511-62512, 62514, 62516, 62518-62519, 62522-62526, 62529-62534, 62536-62540, 62542, 62546-62547, 62549, 62553-62555, 62557-62561, 62563-62567, 62570, 62574, 62576-62584, 62586-62593, 62595, 62598-62607, 62609-62610, 62612-62621, 62623, 62627-62628, 62630, 62632, 62635-62636, 62638, 62640, 62643, 62645, 62647-62649, 62651-62654, 62656-62657, 62659, 62661, 62663-62667, 62670-62672, 62674-62677, 62679-62680, 62684-62689, 62696, 62698- 62699, 62702-62708, 62710, 62714-62717, 62724, 62730-62734, 62736, 62745-62747, 62749, 62752-62753, 62755, 62757-62762, 62765-62770, 62773-62779, 62784-62785, 62787-62788, 62790-62792, 62795, 62797, 62800, 62803, 62806-62813, 62815, 62817, 62819, 62821, 62823, 62826- 62831, 62839, 62848-62850, 62852-62853, 62855, 62857-62864, 62867- 62868, 62870, 62872-62875, 62880, 62882, 62885-62886, 62888, 62890, 62894-62896, 62898-62902, 62904, 62907, 62909, 62911, 62917-62921, 62924-62925, 62927-62931, 62933-62936, 62938, 62941-62942, 62945- 62946, 62949, 62951, 62953-62962, 62965, 62967, 62970, 62975-62976, 62979, 62981, 62984-62991, 62994, 62996-62997, 63002-63006, 63008- 63013, 63016-63017, 63019, 63021-63026, 63028-63029, 63031, 63035- 63039, 63043-63047, 63049-63051, 63053-63058, 63063-63064, 63066, 63073, 63075-63082, 63084-63087, 63089-63091, 63093, 63095, 63098, 63101-63108, 63110, 63112-63115, 63119, 63122, 63124, 63126-63129, 63131, 63133, 63136-63140, 63143, 63145-63149, 63151-63152, 63154, 63156, 63158-■63159, 63161, 63163 -63165, 63169- 63172, 63174, 63176-
63177, 63180- ■63181, 63185- 63187, 63190- 63192, 63195, 63198, 63200-
63201, 63204- ■63208, 63213- 63216, 63221, 63225- 63227, 63229, 63231,
63236- 63237, 63239- 63240, 63242- 63243, 63245, 63248, 63251- 63254,
63258- 63259, 63263- 63267, 63269, 63273, 63275, 63277 , 63280 -63283,
63293, 63295- ■63299, 63301, 63303 , 63311 -63313, 63316 -63317, 63319,
63327, 63329, 63333 -63334, 63336 , 63338 , 63341 -63346 , 63351 -63355,
63357- 63358, 63360, 63363- 63365, 63367, 63370- 63372, 63375- 63376,
63378, 63380- ■63381, 63383- 63384, 63386, 63388, 63390 -63397, 63400-
63402, 63404- ■63406, 63408, 63410 -63413, 63415- 63416, 63420- 63421,
63423- 63424, 63428- 63429, 63431, 63434- 63443, 63446- 63447, 63450-
63451, 63458, 63461 -63462, 63465 -63471, 63473- 63474, 63476, 63479-
63487, 63489, 63491 -63493, 63495 , 63498 -63503, 63505 -63507, 63510-
63512, 63514- ■63515, 63517, 63520 -63522, 63525, 63528 , 63532 , 63538
63544, 63547, 63549 -63550, 63553 -63555, 63557, 63559 -63561, 63563,
63565, 63569- ■63575, 63577- 63578, 63580, 63583- 63584, 63586, 63589-
63590, 63594, 63597 , 63600 , 63602-63607 , 63611 -63612 , 63614 -63615,
63617- 63618, 63620, 63622- 63625, 63627- 63628, 63631- 63634, 63636,
63639- 63640, 63642- 63647, 63649, 63652- 63654, 63660, 63666- 63667,
63669- 63674, 63679- 63681, 63683- 63689, 63691, 63693- 63696, 63699-
63704, 63706- ■63709, 63711- 63716, 63719, 63721- 63726, 63729- 63731,
63734, 63736- ■63738, 63742- 63743, 63745- 63749, 63752, 63754, 63758,
63760- 63762, 63765, 63769, 63771 , 63776 , 63779 -63781 , 63783 -63784,
63790- 63791, 63793- 63798, 63801, 63803- 63808, 63810, 63813, 63816-
63817, 63819- ■63820, 63822- 63823, 63825, 63827- 63829, 63831- 63832,
63834- 63837, 63840- 63842, 63844, 63855, 63858- 63865, 63869- 63873,
63876- 63880, 63882- 63884, 63887- 63888, 63890-63891, 63894-63895,
63898, 63900- ■63906, 63908, 63918 -63919, 63921- 63927, 63930- 63935,
63938- 63941, 63947- 63950, 63953- 63955, 63957, 63960- 63964, 63967-
63971, 63973, 63975 , 63977 , 63981-63987 , 63995 , 64000, 64002-64003
64005, 64008, 64010, 64017, 64019, 64021-64029, 64032-64033, 64035, 64037-64038, 64040, 64042-64048, 64052-64054, 64057-64061, 64065- 64068, 64070, 64072-64075, 64080-64083, 64085-64086, 64088-64089, 64092, 64094, 64096-64099, 64101-64103, 64111, 64113, 64115, 64117, 64120-64121, 64123-64126, 64129, 64133, 64136-64138, 64140-64145, 64147-64150, 64152, 64154, 64157-64159, 64162, 64164-64171, 64173- 64176, 64178-64181, 64186-64187, 64189-64190, 64193-64198, 64202, 64205-64209, 64211, 64217-64219, 64222-64223, 64225, 64227-64231, 64235, 64237-64241, 64243-64249, 64252-64255, 64260, 64262, 64264- 64273, 64278-64281, 64284-64288, 64290, 64293, 64295-64297, 64299- 64302, 64304-64305, 64307-64310, 64312-64314, 64318, 64321, 64328- 64329, 64331-64332, 64337-64339, 64341-64342, 64344, 64346, 64348, 64350, 64352, 64355, 64358, 64361-64362, 64364, 64366, 64369-64371, 64374, 64376, 64379-64380, 64382-64384, 64386-64387, 64389-64390, 64392, 64394-64396, 64399-64400, 64404-64406, 64409-64411, 64414- 64418, 64423-64425, 64427, 64431, 64434-64436, 64438, 64441-64442, 64444, 64446-64448, 64450-64453, 64455-64459, 64462-64463, 64465, 64471-64478, 64483-64484, 64486-64488, 64491-64492, 64494-64497, 64499-64502, 64505, 64507, 64510, 64512, 64516-64519, 64521-64525, 64527, 64535-64539, 64543, 64548-64551, 64555-64558, 64561-64562, 64565, 64570, 64572-64573, 64579, 64581-64583, 64587-64588, 64590, 64594, 64596-64597, 64599, 64602-64604, 64606, 64608, 64611, 64614- 64616, 64618, 64624-64628, 64631-64632, 64634-64639, 64645-64653, 64655-64656, 64658-64659, 64661-64662, 64664, 64667, 64669, 64671- 64675, 64677-64680, 64683-64684, 64686, 64688-64690, 64693, 64695- 64696, 64698, 64701-64702, 64704-64705, 64708, 64717, 64719, 64722- 64725, 64727-64728, 64737-64738, 64740-64744, 64749, 64751, 64754- 64755, 64757, 64760-64762, 64766, 64768-64774, 64778-64779, 64781- 64782, 64786, 64788, 64790-64791, 64794, 64796, 64802, 64807-64809, 64814-64815, 64817, 64819-64823, 64825-64836, 64838, 64840-64844, 64851-64853, 64857-64860, 64862, 64865, 64869, 64871-64873, 64875, 64881-64883, 64885-64886, 64888-64890, 64892-64893, 64895-64898, 64900-64908, 64911-64913, 64916-64919, 64922-64923, 64925-64927, 64929, 64931-64932, 64935-64936, 64938-64939, 64942, 64944-64947, 64949, 64951, 64954-64955, 64960, 64962-64965, 64969-64970, 64972- 64973, 64975, 64978, 64983-64984, 64988-64990, 64993, 64997, 64999, 65001, 65004-65005, 65008, 65010, 65013, 65015-65016, 65019, 65021- 65024, 65026-65029, 65031-65033, 65035-65037, 65039, 65042-65045, 65048, 65050, 65055-65060, 65062, 65064-65070, 65072-65076, 65078- 65080, 65082-65083, 65085-65090, 65092, 65097, 65099, 65101-65102, 65104-65105, 65107-65112, 65115, 65118, 65120, 65122, 65124, 65127, 65133, 65136-65137, 65140, 65144, 65146-65147, 65149-65152, 65155- 65157, 65159, 65161-65165, 65167, 65170, 65173, 65175, 65177-65180, 65182-65185, 65187-65195, 65198-65206, 65208-65209, 65211-65214, 65217-65218, 65221-65222, 65225, 65234, 65241-65244, 65246, 65249- 65250, 65252-65253, 65255-65256, 65259, 65267, 65269-65271, 65274, 65277-65279, 65282-65283, 65289-65292, 65298-65299, 65301-65312, 65314-65316, 65321, 65323, 65325, 65327, 65331, 65333, 65335, 65337- 65343, 65346-65347, 65350-65351, 65353-65354, 65356-65357, 65366, 65368, 65370, 65375, 65377, 65382-65383, 65386, 65391-65398, 65400, 65402-65404, 65406, 65408-65410, 65412, 65414-65420, 65431-65435, 65437, 65440, 65443-65445, 65451-65456, 65460, 65462-65464, 65466, 65469-65470, 65472, 65474, 65476, 65478-65480, 65483-65489, 65491- 65492, 65494-65495, 65498, 65501-65509, 65511-65512, 65514, 65516, 65518, 65520-65521, 65523, 65525-65526, 65530, 65532-65533, 65540, 65543-65546, 65548, 65554-65557, 65559, 65562-65567, 65570-65571, 65577-65579, 65581-65583, 65585-65587, 65590-65598, 65600-65602, 65605-65606, 65608, 65610, 65614, 65617-65618, 65622-65624, 65627- 65629, 65632-65634, 65637-65638, 65640, 65643-65645, 65649, 65652, 65654, 65656-65657, 65659-65665, 65667-65668, 65671, 65675, 65682- 65686, 65690, 65692-65693, 65696, 65700-65702, 65704, 65706-65707, 65709, 65711-65712, 65714-65715, 65720-65723, 65728-65730, 65732, 65734, 65736, 65738, 65740-65743, 65745, 65748-65750, 65752-65753, 65756-65757, 65760, 65767, 65769-65776, 65778, 65781, 65783-65787, 65790, 65794-65795, 65797, 65799-65802, 65804-65805, 65807-65809, 65811-65812, 65815, 65820, 65831-65832, 65834, 65836, 65838-65848, 65850-65853, 65855-65860, 65862-65870, 65872-65875, 65877-65879, 65881-65882, 65884, 65891-65892, 65899-65900, 65902-65904, 65906- 65907, 65911-65912, 65914, 65916-65918, 65920-65922, 65924, 65926, 65928, 65934-65937, 65939-65943, 65945-65946, 65949-65950, 65952, 65955, 65957, 65964-65967, 65969-65975, 65977, 65980, 65982, 65984, 65988-65998, 66000-66006, 66010-66013, 66016, 66019-66021, 66030- 66043, 66045, 66047-66054, 66056, 66059, 66061-66062, 66070, 66072- 66076, 66078-66081, 66084, 66086-66088, 66090-66096, 66098-66099, 66101-66103, 66105, 66107, 66109, 66112, 66114, 66118-66119, 66121, 66125-66126, 66130-66131, 66134, 66136, 66138-66139, 66143-66146, 66148, 66150-66152, 66156-66160, 66163-66168, 66170-66174, 66177, 66179-66184, 66186-66188, 66190-66192, 66195-66196, 66201-66208, 66210-66212, 66215, 66219-66220, 66223-66225, 66229, 66232, 66234, 66236, 66238, 66240-66244, 66246-66247, 66249-66252, 66257-66264, 66267, 66271, 66275, 66277, 66284, 66290, 66292-66295, 66297-66300, 66302-66304, 66307-66320, 66322-66323, 66325-66327, 66329-66331,
66337-66343, 66346, 66349, 66351-66354, 66357-66359, 66361, 66365- 66367, 66369-66376, 66378-66379, 66381-66392, 66395, 66400-66403, 66405, 66407-66415, 66417-66421, 66425-66426, 66428-66429, 66431- 66440, 66444, 66447-66450, 66452-66461, 66463, 66468, 66471-66473, 66475-66479, 66481-66490, 66492-66494, 66496, 66498-66499, 66502- 66504, 66506, 66508-66512, 66515-66524, 66526-66527, 66529, 66531, 66533-66536, 66539, 66543, 66546, 66549-66550, 66552-66555, 66557, 66559-66562, 66564, 66568-66571, 66573-66574, 66576, 66578-66579, 66584, 66586, 66588, 66590, 66593-66597, 66605, 66607-66608, 66610- 66611, 66613, 66616, 66618, 66620, 66624, 66628-66631, 66637-66638, 66645-66647, 66650, 66652-66654, 66656-66657, 66659, 66661-66665, 66674-66680, 66682-66684, 66686-66689, 66691-66692, 66695, 66698, 66700, 66702, 66704-66705, 66709, 66711-66713, 66715, 66720-66721, 66723-66724, 66728-66729, 66732, 66735, 66740, 66742-66746, 66748, 66750-66751, 66754, 66756-66759, 66761-66765, 66768, 66770-66771, 66774, 66777, 66781-66782, 66786-66787, 66791, 66796-66801, 66807- 66811, 66813, 66816-66817, 66819, 66822, 66828-66834, 66841, 66843, 66846, 66848, 66851-66853, 66855-66859, 66861, 66863, 66865-66868, 66872, 66877-66879, 66882-66883, 66885, 66891-66895, 66899-66904, 66906-66916, 66918-66920, 66923-66925, 66927-66929, 66931-66932,
66934-66935, 66937, 66940, 66943, 66945-66946, 66948-66952, 66955- 66957, 66959-66965, 66967, 66969, 66972-66974, 66977, 66982-66986, 66989-66990, 66999, 67003, 67005-67008, 67010-67020, 67023-67026, 67028, 67030-67035, 67037-67043, 67045-67054, 67056, 67058, 67061, 67063, 67066-67068, 67070-67072, 67074-67076, 67078, 67085-67086, 67088-67091, 67095, 67099-67103, 67105, 67108-67109, 67118-67120, 67124, 67126-67136, 67140, 67142, 67144-67145, 67147-67162, 67164, 67167, 67169-67171, 67173, 67175-67178, 67180-67181, 67184-67189, 67191-67192, 67197-67199, 67202, 67204-67206, 67208, 67210-67212, 67217-67218, 67220-67221, 67226, 67228, 67231, 67233-67234, 67236, 67239, 67242-67243, 67245, 67248-67250, 67253-67255, 67258, 67264- 67265, 67267, 67272-67274, 67276-67277, 67280-67287, 67289-67291, 67293, 67295-67301, 67304, 67307, 67309-67311, 67313, 67317, 67319, 67326, 67328-67329, 67336, 67339-67340, 67343, 67345, 67347-67348, 67350, 67356, 67360-67362, 67364-67367, 67380, 67384, 67389, 67396- 67401, 67406-67407, 67412-67413, 67416, 67418-67419, 67421, 67425- 67427, 67437-67438, 67440-67442, 67444-67445, 67447, 67449-67450, 67453-67455, 67458-67460, 67463-67464, 67468, 67470-67472, 67474- 67478, 67480, 67483-67484, 67487, 67489-67491, 67493, 67496, 67502- 67509, 67512-67514, 67517-67518, 67520-67521, 67523-67527, 67531- 67532, 67535, 67537, 67542, 67544, 67551, 67557-67561, 67563-67566, 67568-67570, 67575-67576, 67578-67581, 67584, 67588, 67591, 67593, 67596-67597, 67602-67603, 67605-67609, 67611, 67617-67619, 67621- 67623, 67627-67628, 67631-67633, 67636-67642, 67644-67646, 67648- 67650, 67652-67653, 67656-67661, 67664, 67668-67675, 67689, 67691- 67692, 67694-67702, 67704, 67708, 67710-67711, 67713-67716, 67718- 67720, 67723, 67725, 67731-67732, 67734, 67736-67737, 67740-67742, 67744, 67746-67748, 67752, 67754, 67756-67758, 67761-67765, 67767- 67770, 67773, 67775, 67777-67778, 67780, 67784, 67789-67790, 67794- 67797, 67800, 67802-67805, 67807-67817, 67822-67824, 67827, 67829- 67831, 67834, 67836-67837, 67839-67840, 67842-67844, 67847, 67850, 67853-67854, 67857-67858, 67860-67864, 67866-67875, 67877, 67882- 67884, 67887, 67889, 67891-67893, 67896, 67898-67902, 67904, 67907- 67917, 67920, 67922-67923, 67925-67927, 67934-67935, 67937-67948, 67950-67953, 67955-67958, 67960-67973, 67975, 67978-67981, 67983- 67987, 67991-68000, 68002-68006, 68008-68016, 68019, 68024, 68027- 68029, 68031-68034, 68036-68049, 68053, 68055, 68057, 68059-68061, 68063-68065, 68069-68070, 68073, 68076, 68085, 68087, 68089-68090, 68092-68093, 68096, 68099-68101, 68103, 68105-68110, 68113, 68115- 68118, 68120-68121, 68125-68127, 68130, 68132-68133, 68136-68139, 68141-68146, 68151, 68153-68158, 68161, 68164, 68166-68168, 68171- 68172, 68175-68176, 68178, 68180-68181, 68186-68187, 68189-68192, 68194-68197, 68199-68202, 68204-68206, 68209-68210, 68214, 68217- 68219, 68221-68222, 68224, 68227-68230, 68233-68234, 68236-68239, 68241-68246, 68249, 68252-68253, 68255-68259, 68263-68264, 68266- 68267, 68270-68279, 68281, 68283, 68285-68289, 68295-68296, 68300, 68303-68306, 68311-68312, 68314-68319, 68323, 68325, 68331, 68333- 68338, 68340, 68346, 68348-68359, 68361-68365, 68367, 68369, 68372- 68375, 68377-68379, 68381, 68384-68395, 68397-68400, 68403-68413, 68415, 68417, 68420-68423, 68425, 68427-68433, 68435-68444, 68446- 68449, 68453-68458, 68462-68463, 68465, 68469-68470, 68472, 68475, 68477, 68479-68480, 68482, 68487, 68491, 68493-68497, 68499-68500, 68502-68503, 68505-68507, 68509-68512, 68517, 68519, 68521-68522, 68525, 68540, 68542-68546, 68548-68555, 68557, 68559-68560, 68563, 68566, 68568, 68571, 68573-68574, 68577, 68579-68582, 68584-68587, 68590-68592, 68595, 68598, 68601-68605, 68609, 68611-68615, 68617- 68618, 68623-68625, 68627-68634, 68636, 68638-68643, 68645-68651, 68653-68655, 68658, 68660-68665, 68667-68669, 68671-68673, 68676- 68677, 68685, 68687-68694, 68697, 68699-68700, 68703, 68706-68711, 68713-68715, 68717-68721, 68723-68727, 68730-68731, 68734-68736, 68739-68741, 68744, 68748, 68750, 68752, 68754, 68756, 68759, 68761, 68764-68765, 68768, 68770-68771, 68773-68776, 68783-68786, 68788, 68790, 68792-68794, 68796, 68798-68802, 68805, 68808-68811, 68816, 68818, 68824, 68826-68827, 68831-68835, 68837-68839, 68841, 68843, 68847, 68849, 68851, 68855-68860, 68866-68868, 68871, 68873, 68877, 68879-68883, 68887-68888, 68892-68897, 68899-68905, 68907-68908, 68910-68912, 68914, 68916, 68920, 68922-68923, 68927-68929, 68931- 68933, 68935-68936, 68938-68939, 68943-68945, 68947-68953, 68957,
68962, 68964, 68967, 68969-68974, 68977, 68979, 68982, 68985, 68988, 68991, 68993-68994, 68997, 68999-69000, 69002-69005, 69007, 69010- 69013, 69015-69016, 69018-69019, 69021-69022, 69025-69026, 69028- 69031, 69033-69034, 69036, 69038, 69040, 69043, 69045-69050, 69053- 69054, 69056-69057, 69060-69061, 69063-69068, 69070, 69072-69080, 69082, 69086, 69090-69091, 69095-69096, 69098-69100, 69102-69107, 69113, 69115, 69120, 69122, 69126-69127, 69131-69138, 69140-69141, 69143, 69146, 69148-69149, 69156-69161, 69163, 69165-69166, 69172, 69174-69175, 69177, 69181-69184, 69186-69188, 69190, 69193, 69197, 69200-69202, 69210-69212, 69215-69218, 69221-69223, 69225, 69227- 69228, 69234-69236, 69238-69244, 69247-69249, 69251-69254, 69256, 69258-69262, 69264-69266, 69270-69273, 69277-69278, 69282, 69288- 69289, 69291, 69294, 69297-69299, 69303-69306, 69309, 69311-69312, 69314-69317, 69320, 69323-69325, 69328, 69332, 69334, 69337-69339, 69345-69349, 69351-69353, 69355, 69358-69363, 69365-69366, 69368, 69370-69371, 69373-69374, 69378, 69380, 69382-69383, 69385-69386, 69389-69403, 69405, 69407-69408, 69410, 69414, 69416-69417, 69419, 69421, 69427-69435, 69437-69441, 69443, 69445-69447, 69450-69452, 69454, 69456-69460, 69464, 69466, 69471, 69474, 69479-69480, 69482- 69484, 69490, 69493, 69496-69505, 69508, 69510, 69512-69514, 69520- 69521, 69523, 69525, 69528-69532, 69534, 69537-69538, 69540-69541, 69547-69550, 69552, 69554-69560, 69563, 69567, 69570-69573, 69575, 69578, 69580, 69582-69586, 69588-69592, 69595, 69597-69601, 69603- 69607, 69609-69614, 69617-69624, 69626-69634, 69641-69644, 69647, 69649-69652, 69654, 69656, 69658-69659, 69661-69662, 69665-69672, 69674, 69679, 69683, 69686, 69689-69690, 69692-69694, 69696-69699, 69702-69705, 69708-69710, 69712-69714, 69719-69723, 69725, 69728, 69730, 69732, 69735, 69738, 69740, 69743-69744, 69746, 69751-69752, 69754-69757, 69759-69762, 69764, 69767, 69769, 69775-69778, 69785, 69789, 69791-69799, 69801, 69803, 69809, 69812-69814, 69816-69817, 69819, 69821-69823, 69825-69828, 69830, 69832, 69834, 69837-69839, 69841-69845, 69847-69850, 69852-69860, 69862, 69866-69867, 69869- 69870, 69875, 69879, 69883, 69887, 69889, 69891, 69893-69904, 69906- 69911, 69916, 69919, 69921-69925, 69929-69930, 69932-69941, 69943- 69949, 69954, 69960, 69963, 69965-69974, 69976, 69978, 69980-69981, 69983, 69986, 69988-69990, 69995-69996, 70001, 70003-70004, 70008- 70014, 70017, 70019-70020, 70024-70025, 70027, 70029, 70032-70033, 70035, 70039-70041, 70044-70048, 70051-70056, 70058-70062, 70064- 70069, 70072, 70075, 70077-70079, 70082, 70084-70086, 70088, 70090- 70091, 70098-70099, 70102-70103, 70107, 70110-70111, 70118-70119, 70122-70128, 70132-70133, 70135, 70138, 70142-70145, 70147-70149, 70151-70154, 70156-70165, 70169, 70172, 70174, 70176-70178, 70180, 70182, 70185, 70187, 70190, 70192-70196, 70200, 70203-70204, 70207, 70209-70210, 70213, 70215-70220, 70222, 70225, 70227, 70231, 70233, 70236, 70238-70240, 70244, 70247-70249, 70251-70257, 70259-70263, 70267-70271, 70277-70279, 70282, 70284, 70288, 70291, 70294-70295, 70297-70298, 70307, 70310, 70314, 70317-70318, 70320, 70322-70323, 70325-70326, 70328-70331, 70334-70337, 70339, 70342-70343, 70345, 70352-70354, 70356-70357, 70359-70362, 70364-70368, 70370-70371, 70374-70382, 70385-70388, 70392-70395, 70397-70398, 70400, 70402, 70404-70419, 70422-70426, 70428, 70430, 70432-70438, 70440-70446, 70449-70461, 70463, 70466, 70470, 70472-70473, 70475-70476, 70478- 70479, 70483-70484, 70487, 70489, 70493-70494, 70497-70498, 70500- 70501, 70503-70504, 70506, 70508, 70510, 70512, 70516, 70518, 70521- 70523, 70525-70528, 70531, 70536-70539, 70541-70542, 70544-70545, 70547-70548, 70550, 70552-70553, 70555-70557, 70559-70560, 70562- 70571, 70573-70574, 70576-70582, 70584, 70586-70590, 70592-70593, 70595, 70597, 70599-70601, 70605, 70608, 70610-70614, 70616-70617, 70619-70620, 70622-70623, 70625, 70628-70629, 70632-70636, 70639, 70641-70642, 70646, 70648, 70650-70652, 70654-70655, 70657, 70661, 70664, 70666, 70669-70671, 70674, 70679-70685, 70687, 70693-70694, 70696, 70698-70699, 70703, 70705-70706, 70708, 70712-70716, 70719, 70724-70727, 70733, 70735-70743, 70746, 70749-70750, 70762-70763, 70768-70770, 70772, 70775-70776, 70778-70779, 70782, 70784, 70786, 70788-70790, 70792-70793, 70795, 70797-70798, 70801, 70807, 70809- 70810, 70812-70820, 70823-70825, 70830-70831, 70833-70834, 70841- 70842, 70844-70846, 70852-70855, 70859, 70861, 70870-70871, 70875- 70876, 70879-70882, 70884, 70886, 70888, 70893, 70896-70899, 70901- 70902, 70904, 70906, 70909, 70911, 70913-70914, 70922-70923, 70925, 70928, 70932, 70936-70942, 70944-70946, 70950, 70952, 70954, 70956, 70965-70968, 70974, 70976, 70979, 70982-70983, 70987, 70989-70990, 71001, 71003, 71006, 71012-71013, 71015-71017, 71020-71021, 71023- 71032, 71034-71035, 71037-71044, 71046-71051, 71055, 71058, 71060- 71065, 71070, 71076, 71079, 71081-71082, 71084, 71086-71088, 71090, 71094-71095, 71098, 71100-71101, 71103, 71108-71110, 71112-71113, 71116-71117, 71122-71126, 71129, 71131, 71133-71134, 71137-71140, 71143-71144, 71146, 71148-71149, 71151-71152, 71154-71156, 71158- 71164, 71169, 71176-71178, 71181-71182, 71184-71187, 71189-71194, 71200, 71202-71203, 71207-71211, 71214, 71217-71218, 71221-71225, 71227, 71230-71231, 71233, 71235-71240, 71244-71246, 71250, 71253- 71254, 71257, 71260, 71262, 71266-71267, 71272-71273, 71276, 71278- 71281, 71285, 71287-71289, 71291-71293, 71300-71301, 71303, 71305- 71306, 71309, 71315-71318, 71322, 71325-71332, 71334-71341, 71349- 71353, 71355-71358, 71360-71366, 71368-71371, 71373-71382, 71384- 71387, 71389-71399, 71402-71404, 71406-71408, 71411-71412, 71415, 71418, 71421, 71423-71427, 71429, 71431-71434, 71436, 71438, 71440, 71442-71445, 71449-71451, 71454-71458, 71461-71462, 71464-71469,
71471-71475, 71477, 71479, 71481-71488, 71490, 71492, 71495, 71497- 71500, 71503, 71505-71507, 71509-71511, 71513, 71515-71516, 71519- 71521, 71524-71526, 71528-71531, 71533-71534, 71537-71541, 71543- 71544, 71546-71551, 71553-71558, 71561-71562, 71564, 71569-71570, 71572, 71574-71577, 71579-71582, 71584-71590, 71593-71598, 71600- 71603, 71609, 71613, 71615-71617, 71620, 71623-71632, 71634-71637, 71639-71647, 71649-71651, 71653, 71656-71658, 71662-71663, 71669, 71671, 71678, 71680-71682, 71689-71691, 71693, 71701-71702, 71705- 71706, 71708, 71710, 71712, 71714, 71716-71723, 71726-71728, 71730, 71733, 71737, 71739, 71741-71744, 71747-71749, 71751-71752, 71754- 71755, 71757, 71759-71760, 71766, 71768-71771, 71775-71777, 71780, 71784-71785, 71787-71796, 71798-71809, 71812, 71814-71818, 71821, 71823-71824, 71826-71827, 71835, 71837, 71840-71842, 71845-71846, 71851, 71853-71856, 71858-71862, 71864-71866, 71869-71870, 71872, 71874-71880, 71884, 71887-71888, 71890-71892, 71894-71895, 71897, 71899, 71904, 71908-71909, 71911-71918, 71922-71923, 71926, 71930, 71933-71935, 71937-71942, 71945-71952, 71955, 71957-71958, 71961, 71964-71970, 71982-71986, 71990, 71992, 71994, 71999, 72003-72004, 72008-72010, 72012-72015, 72017, 72020-72022, 72025, 72031-72032, 72035, 72037-72038, 72041, 72045, 72048-72054, 72056, 72058, 72062- 72065, 72067, 72069-72073, 72075, 72077, 72079-72081, 72083, 72085, 72088-72090, 72094-72095, 72097, 72099-72100, 72104-72105, 72107- 72112, 72114-72117, 72119, 72123-72124, 72126-72128, 72130, 72132- 72140, 72143, 72145-72150, 72152-72155, 72157-72158, 72160-72166, 72168-72169, 72171, 72173-72175, 72178, 72180-72183, 72185-72192, 72195, 72197, 72199, 72203-72205, 72207-72211, 72214-72232, 72234, 72236-72239, 72241-72242, 72246, 72249-72251, 72253-72256, 72261- 72265, 72268-72269, 72272-72273, 72275, 72277, 72279-72280, 72282- 72283, 72285, 72294-72297, 72300, 72302, 72304, 72306, 72309, 72312, 72314-72315, 72317, 72319-72320, 72325, 72330, 72333-72334, 72337- 72344, 72346-72348, 72350-72353, 72355-72358, 72364-72366, 72368, 72373, 72375-72377, 72379-72382, 72384-72388, 72392-72397, 72400, 72402, 72415-72417, 72419, 72426-72432, 72434, 72436, 72438, 72441- 72452, 72454-72456, 72461-72462, 72468-72473, 72475, 72477-72479,
72481, 72483, 72486, 72489-72490, 72492-72494, 72500, 72502, 72508, 72515-72516, 72523-72527, 72529-72534, 72536-72537, 72539-72551, 72554, 72556, 72559, 72563, 72565-72580, 72582-72586, 72595-72598, 72600-72609, 72611-72612, 72614-72620, 72622-72623, 72626-72629, 72631-72633, 72635-72637, 72640, 72643-72646, 72651-72653, 72657, 72660, 72662-72663, 72668-72670, 72674-72678, 72686, 72690, 72692, 72695, 72703-72704, 72706-72708, 72710-72711, 72715-72718, 72720, 72723, 72728, 72730-72731, 72733, 72738-72739, 72741-72747, 72750, 72752-72754, 72756-72757, 72759, 72761-72769, 72772-72774, 72776, 72778-72779, 72781-72795, 72798, 72802, 72807-72814, 72816, 72819- 72829, 72831-72832, 72840, 72842-72846, 72848-72852, 72854, 72856- 72858, 72860, 72866-72868, 72870-72872, 72874, 72879-72885, 72887- 72889, 72894, 72896, 72900-72906, 72908-72921, 72923, 72925, 72927, 72929, 72931-72933, 72936-72940, 72943-72949, 72951, 72953-72957, 72963-72967, 72969-72974, 72978-72979, 72983-72984, 72987-72995, 72998-73002, 73005, 73007-73018, 73020, 73024-73025, 73027-73031, 73033-73034, 73036, 73038-73043, 73045-73048, 73050, 73052, 73054, 73056-73059, 73061-73064, 73067-73068, 73070, 73073, 73075-73078, 73080, 73082-73084, 73087-73091, 73093-73096, 73098-73105, 73110, 73113-73120, 73122-73123, 73125, 73127, 73129-73130, 73133-73134, 73136-73144, 73146-73150, 73152-73157, 73159-73160, 73163, 73165, 73167-73177, 73180-73181, 73183-73184, 73186-73194, 73196-73198, 73200-73202, 73204, 73206-73211, 73215, 73222-73225, 73229, 73231, 73233-73234, 73236-73238, 73240, 73242, 73244-73245, 73247-73248, 73250-73252, 73255-73259, 73262, 73265-73267, 73269, 73276-73279, 73281, 73283-73286, 73288, 73291, 73296-73297, 73300-73301, 73305, 73307, 73312, 73316-73319, 73325-73337, 73339-73343, 73349-73351, 73353-73355, 73357, 73359, 73362, 73370, 73372-73373, 73375, 73377- 73378, 73380-73381, 73384-73387, 73392, 73394-73395, 73397-73400, 73402-73404, 73407, 73409-73410, 73412-73417, 73419-73420, 73422- 73423, 73425, 73427-73432, 73434-73438, 73440, 73442, 73444-73446, 73448-73449, 73451, 73455, 73458-73465, 73467-73470, 73472-73473, 73477, 73481, 73487, 73489, 73493-73494, 73496, 73498, 73500-73501, 73506, 73508-73510, 73512-73514, 73516, 73521, 73523-73524, 73537, 73539-73545, 73558-73559, 73561, 73563, 73566-73568, 73571-73575, 73577-73581, 73583-73587, 73590-73591, 73593-73595, 73597-73599, 73602-73604, 73606-73607, 73613-73614, 73618, 73620, 73622, 73624- 73625, 73628, 73631-73633, 73635-73638, 73641-73642, 73645, 73647, 73649-73653, 73655, 73657, 73660-73663, 73666-73669, 73673, 73675- 73683, 73685-73687, 73691-73696, 73698, 73700, 73702, 73708-73709, 73711, 73713, 73717, 73721-73722, 73725-73726, 73728, 73734, 73736, 73738-73740, 73742, 73744-73746, 73751-73755, 73757, 73760-73762, 73764-73765, 73769, 73775, 73777-73778, 73780, 73782-73783, 73785- 73788, 73792-73804, 73812-73813, 73815, 73817, 73819, 73821-73822, 73825, 73828-73829, 73832, 73834-73840, 73843-73844, 73846-73849, 73851-73856, 73858-73870, 73872-73877, 73879-73883, 73885, 73887- 73888, 73891, 73893-73895, 73898-73899, 73901, 73903, 73905-73906, 73908, 73910-73911, 73913, 73915, 73919, 73921, 73923, 73926-73928, 73932-73934, 73936, 73945, 73947, 73950, 73953, 73956-73958, 73961, 73964, 73966-73974, 73979-73981, 73987-73989, 73991-73992, 73998- 73999, 74001, 74007, 74009-74010, 74017, 74019-74021, 74023, 74025- 74033, 74042, 74044-74047, 74049, 74051-74057, 74059, 74061-74063, 74065, 74070, 74072, 74075, 74080-74083, 74087-74089, 74095, 74099, 74107, 74110-74112, 74115-74116, 74123-74124, 74126, 74130, 74133, 74135, 74140, 74142-74143, 74145-74147, 74149, 74156-74160, 74162, 74164-74165, 74167, 74169-74173, 74176-74178, 74180-74183, 74185, 74192, 74195, 74197-74201, 74207-74209, 74211-74212, 74214-74219, 74221-74223, 74225-74226, 74228, 74231-74233, 74235-74236, 74239- 74242, 74244-74255, 74257-74259, 74261-74263, 74265-74273, 74275- 74276, 74278, 74282, 74285-74293, 74295-74296, 74298-74299, 74301, 74303, 74306-74307, 74309-74315, 74317-74318, 74321-74323, 74326, 74329-74330, 74333-74335, 74338-74339, 74345, 74347-74354, 74356- 74362, 74364-74365, 74367-74369, 74372-74375, 74377-74379, 74381, 74383-74384, 74387, 74389-74395, 74397-74398, 74401, 74404, 74406- 74407, 74409, 74413-74414, 74416-74423, 74427, 74429-74433, 74436, 74439-74440, 74442-74443, 74445-74449, 74454, 74457-74459, 74465- 74470, 74472-74475, 74477, 74479, 74483, 74485-74490, 74492-74496, 74498-74499, 74503-74507, 74509, 74512-74516, 74518, 74520, 74526- 74532, 74534-74537, 74541-74542, 74545, 74547-74552, 74554-74561, 74564-74566, 74568-74575, 74578-74580, 74582-74585, 74590-74595, 74597-74608, 74610-74623, 74625-74626, 74628, 74632, 74634, 74636, 74638-74639, 74642-74643, 74645-74646, 74648, 74651-74653, 74655- 74659, 74661-74671, 74673, 74675, 74677-74685, 74688, 74690, 74692, 74694, 74698, 74700-74702, 74706-74711, 74713, 74715-74718, 74720- 74726, 74729-74730, 74732-74733, 74736, 74738, 74740-74741, 74743- 74744, 74746, 74750, 74752, 74759-74762, 74764-74767, 74771-74776, 74778-74781, 74783, 74787-74789, 74793-74794, 74796, 74800-74804, 74806-74808, 74810-74814, 74816, 74818-74821, 74823-74825, 74827, 74832-74834, 74840, 74842-74843, 74845, 74847-74849, 74851-74853, 74856-74858, 74861, 74866-74874, 74877-74878, 74884-74886, 74888, 74890, 74892-74896, 74898, 74901, 74903-74905, 74907, 74911, 74913- 74919, 74923-74929, 74931, 74935, 74937-74938, 74941, 74945-74950, 74952-74953, 74957-74965, 74967, 74971, 74974, 74981-74982, 74985, 74987-74989, 74991, 74997-75001, 75006-75008, 75010-75015, 75022, 75026, 75029, 75031-75034, 75037, 75039-75041, 75043, 75045-75046, 75050-75052, 75054, 75057-75058, 75060, 75063-75064, 75067, 75069, 75073-75077, 75079, 75081, 75087, 75091-75092, 75097-75098, 75104- 75106, 75108, 75111, 75113, 75115-75118, 75122, 75124-75127, 75131- 75132, 75135-75136, 75138-75149, 75151, 75154, 75159, 75161-75168, 75170-75171, 75173-75175, 75178-75179, 75181, 75185-75190, 75192- 75193, 75195, 75197-75198, 75203-75204, 75206-75208, 75210-75211, 75216-75217, 75219-75223, 75227-75228, 75230-75231, 75233, 75236, 75241-75249, 75252-75253, 75255-75259, 75261, 75265-75267, 75270- 75274, 75276-75280, 75283, 75285-75290, 75293-75298, 75300-75302, 75305, 75307, 75310-75312, 75314-75316, 75322, 75328-75330, 75332, 75335-75336, 75338-75347, 75350-75351, 75353-75354, 75356-75357,
75360- 75361, 75363- 75365, 75367, 75369, 75373- 75375, 75380, 75384-
75390, 75392- 75402, 75405, 75408- 75414, 75416- 75417, 75421, 75426-
75427, 75429, 75437 -75439, 75442- 75445, 75447, 75449- 75453, 75455-
75458, 75460- 75461, 75464, 75467, 75469, 75472 -75478, 75480, 75482,
75486, 75489, 75491 -75492, 75497, 75499, 75503 , 75505 , 75507 -75509,
75511- 75516, 75518, 75520, 75522- 75529, 75531, 75533, 75539- 75542,
75545- 75548, 75550, 75555, 75561- 75562, 75564, 75566, 75569- 75570,
75572- 75573, 75575, 75578, 75580- 75582, 75584- 75589, 75591-75601,
75606, 75608, 75610 -75612, 75614- 75620, 75623, 75625, 75632, 75634,
75636- 75640, 75643, 75648, 75653, 75658, 75660 , 75662 -75664, 75667-
75668, 75670, 75672 -75673, 75675- 75676, 75678, 75680, 75683, 75686-
75687, 75690- 75691, 75708, 75710, 75712- 75713, 75715- 75719, 75721-
75725, 75727- 75728, 75730, 75734, 75737- 75741, 75743- 75744, 75746,
75748-75749, 75751-75752, 75755-75756, 75759-75764, 75766, 75768, 75775, 75779, 75781-75782, 75785, 75788, 75790-75798, 75802, 75808, 75810-75814, 75818, 75821, 75824, 75832, 75836, 75838-75839, 75841- 75843, 75847-75848, 75851, 75853-75857, 75861, 75864, 75869, 75872- 75873, 75875-75877, 75882-75883, 75885-75887, 75894, 75896-75899, 75906-75908, 75910-75911, 75913-75914, 75916, 75919-75920, 75922- 75924, 75926, 75929, 75932, 75934-75935, 75938-75940, 75942, 75945, 75950-75956, 75958-75961, 75963-75967, 75971, 75973-75981, 75984, 75987, 75989, 75992-75993, 75995-75997, 75999, 76002-76004, 76007, 76009-76010, 76014-76027, 76029-76035, 76037-76041, 76044, 76047- 76048, 76053-76055, 76057-76059, 76062, 76068-76069, 76071-76080, 76082, 76084-76085, 76087, 76089-76092, 76094-76095, 76097, 76100- 76102, 76104, 76106-76107, 76112, 76115, 76118, 76120-76122, 76124- 76128, 76131, 76133, 76136-76139, 76141-76144, 76147-76148, 76150- 76156, 76158, 76160-76167, 76170, 76173, 76175, 76177, 76180, 76184, 76186-76187, 76189-76192, 76194, 76196-76198, 76200-76201, 76204, 76212, 76214-76215, 76218-76219, 76221-76223, 76230-76232, 76235- 76236, 76238-76240, 76243-76244, 76247-76249, 76252-76255, 76258, 76260-76261, 76263, 76265-76270, 76272-76273, 76277-76278, 76280- 76282, 76285, 76287, 76296-76299, 76303, 76305-76307, 76309-76312, 76315-76318, 76321, 76323-76328, 76330-76333, 76335, 76340-76345, 76349-76353, 76355-76357, 76360, 76363, 76365-76366, 76368, 76370- 76372, 76377-76378, 76384-76390, 76392-76393, 76396-76397, 76399- 76403, 76405, 76408-76409, 76413, 76416, 76419-76423, 76428, 76430- 76431, 76435, 76438, 76440, 76442, 76444, 76449-76450, 76453-76458, 76460-76466, 76468-76470, 76472-76481, 76483, 76487-76491, 76493, 76495-76504, 76508, 76511, 76513-76515, 76517, 76520, 76522-76523, 76525-76529, 76531-76532, 76535-76539, 76541-76544, 76547-76552, 76554, 76563-76572, 76574, 76576-76581, 76585-76593, 76596-76597, 76599-76600, 76602-76607, 76609-76610, 76612-76613, 76619, 76621- 76622, 76624, 76626, 76628, 76630-76634, 76637, 76639-76641, 76643- 76645, 76650-76651, 76654, 76656-76658, 76660-76663, 76665-76667, 76669-76671, 76674-76677, 76685, 76687-76689, 76692, 76694, 76698- 76700, 76704-76707, 76711-76712, 76715-76716, 76719-76722, 76724- 76725, 76727-76730, 76732-76734, 76736-76737, 76740-76745, 76747, 76752, 76754, 76756, 76758-76760, 76762-76768, 76770-76775, 76777, 76784, 76787-76788, 76790-76792, 76794-76795, 76799-76800, 76806, 76808, 76811-76812, 76815-76817, 76819-76823, 76825-76829, 76832, 76835-76836, 76841, 76843-76844, 76846, 76849, 76853-76854, 76856- 76859, 76861-76869, 76871, 76873, 76875, 76877, 76879, 76881-76888, 76893, 76896, 76898-76900, 76902-76905, 76907-76909, 76914-76915, 76917-76918, 76920, 76922-76924, 76926-76927, 76929-76930, 76932- 76947, 76951-76952, 76954, 76957, 76960-76961, 76963, 76966-76967, 76970, 76972, 76976-76977, 76980, 76984-76985, 76987-76988, 76991- 76997, 76999, 77001, 77003-77005, 77007, 77010-77012, 77014-77019, 77021-77023, 77025, 77030-77031, 77033-77034, 77036, 77041, 77043, 77045, 77048-77052, 77055, 77057, 77060, 77065, 77068, 77070-77082,
77086, 77091, 77095-77102, 77106-77110, 77112-77114, 77116, 77119,
77121, 77123, 77131-77137, 77139-77140, 77142, 77150-77153, 77156,
77158, 77160, 77162-77165, 77168-77173, 77176, 77180-77182, 77184- 77185, 77188, 77190-77191, 77193, 77195-77200, 77202, 77213-77218,
77223, 77225-77226, 77231-77232, 77239-77240, 77242, 77244, 77251-
77253, 77256-77257, 77259, 77262-77263, 77266, 77268-77270, 77272,
77274, 77277, 77279-77280, 77283-77285, 77287-77290, 77298-77300, 77302-77304, 77306, 77311, 77313-77315, 77319-77320, 77332-77333, 77336-77337, 77339-77340, 77342-77343, 77345-77348, 77352, 77354-
77358, 77360-77362, 77364-77366, 77368-77369, 77371, 77374, 77376-
77380, 77382-77385, 77387-77388, 77390-77391, 77397-77401, 77403-
77406, 77410-77412, 77414-77416, 77418, 77420-77421, 77423, 77425-
77428, 77430-77432, 77434, 77436-77437, 77439-77444, 77448-77450, 77452-77455, 77458-77459, 77461-77462, 77465, 77467-77469, 77472-
77474, 77481, 77483, 77486-77492, 77498, 77502, 77516-77518, 77521, 77523-77525, 77527-77529, 77532, 77534-77536, 77542-77543, 77545-
77546, 77550-77552, 77554-77555, 77557, 77559, 77563-77565, 77568-
77573, 77575-77576, 77581, 77583, 77585, 77587-77595, 77597, 77599, 77605, 77610-77612, 77617-77618, 77624-77628, 77630-77634, 77636-
77640, 77642-77643, 77647-77648, 77650, 77654, 77656-77658, 77660-
77667, 77669-77677, 77679-77680, 77683-77686, 77689, 77692, 77695,
77697, 77699, 77703-77708, 77719, 77724-77729, 77735, 77746, 77749-
77750, 77752-77754, 77757, 77759, 77761, 77764, 77766, 77769-77770, 77772, 77774-77775, 77781, 77783-77784, 77786-77787, 77789, 77792, 77795-77797, 77799, 77806, 77808-77809, 77811, 77814-77816, 77819-
77820, 77822, 77827-77829, 77831-77838, 77840-77841, 77843-77844,
77847, 77853-77855, 77861-77863, 77865, 77867, 77870, 77873, 77877,
77879, 77883, 77885, 77888-77890, 77892, 77895, 77898, 77900-77902, 77911, 77913-77914, 77916-77917, 77919-77920, 77922-77924, 77926-
77929, 77931, 77935-77936, 77940-77944, 77946-77949, 77954-77956,
77958, 77960, 77962-77969, 77971-77973, 77975, 77977-77978, 77982-
77988, 77991-77994, 77999, 78003, 78005-78006, 78008-78010, 78012-
78018, 78021, 78023-78024, 78026-78031, 78033, 78035, 78038-78042, 78044-78047, 78052, 78056-78059, 78061-78062, 78064, 78067-78068, 78072-78073, 78075-78078, 78080, 78082, 78089-78092, 78094-78097, 78099-78101, 78105, 78107-78108, 78110, 78113-78115, 78117, 78120, 78122-78124, 78132-78133, 78136-78138, 78143, 78146, 78148, 78150- 78151, 78157, 78159, 78161, 78165, 78167-78169, 78172-78178, 78186, 78189, 78194, 78196, 78199-78202, 78206-78209, 78211-78212, 78219- 78220, 78222-78224, 78228-78229, 78232, 78234-78235, 78238, 78240- 78243, 78248-78256, 78258-78259, 78261, 78263, 78265, 78267-78272, 78275, 78277, 78279, 78281-78285, 78287-78290, 78292, 78295-78298, 78300, 78302-78305, 78309-78317, 78319-78321, 78323, 78325, 78327, 78329-78332, 78334-78336, 78339-78340, 78342-78345, 78348, 78350- 78351, 78354-78360, 78362, 78365-78367, 78369-78370, 78373, 78375- 78378, 78380, 78382, 78384-78385, 78388, 78390, 78395-78397, 78404- 78406, 78408-78412, 78415-78418, 78420-78422, 78424-78425, 78427- 78428, 78430, 78433-78434, 78442, 78444-78445, 78447, 78449, 78451- 78455, 78457-78464, 78466, 78468-78469, 78472-78477, 78482-78484, 78486, 78488, 78490, 78493-78494, 78496, 78499, 78503, 78509-78512, 78516, 78521-78526, 78528-78529, 78531, 78538, 78542, 78544, 78548, 78550-78551, 78553, 78557-78558, 78561-78562, 78564-78565, 78567- 78568, 78570-78571, 78575, 78577-78579, 78581-78582, 78587-78588, 78591, 78597-78598, 78600, 78602-78611, 78616-78622, 78624, 78627, 78629, 78640-78641, 78643, 78645-78648, 78651-78652, 78654-78656, 78658-78662, 78664-78666, 78669-78670, 78672-78674, 78676-78678, 78680-78682, 78684, 78687-78688, 78695-78701, 78705-78711, 78713- 78721, 78724-78725, 78736-78737, 78739, 78742, 78744-78747, 78751-
78752, 78755, 78757, 78760, 78762-78763, 78765, 78770, 78772, 78774- 78777, 78779-78780, 78782, 78785-78786, 78792, 78794, 78800-78802, 78804-78805, 78807-78808, 78810-78811, 78813-78815, 78820-78821, 78824-78827, 78831-78835, 78837, 78839-78840, 78843-78852, 78858, 78862-78863, 78865, 78867, 78869-78871, 78877, 78879-78883, 78885-
78887, 78891, 78893, 78896, 78899, 78901, 78903-78907, 78910, 78912- 78917, 78920-78921, 78923-78929, 78931-78933, 78936, 78938, 78942- 78947, 78949-78952, 78954, 78960, 78963-78965, 78967-78968, 78970, 78976, 78979, 78984-78986, 78989-78996, 79005, 79007, 79014, 79019, 79021-79023, 79025-79029, 79032-79037, 79044, 79046-79047, 79049, 79051-79055, 79062-79065, 79067, 79069-79071, 79073-79074, 79076- 79083, 79086, 79091, 79093-79097, 79101-79102, 79106-79107, 79113- 79116, 79118-79119, 79121, 79123, 79126, 79129, 79132, 79140, 79142- 79144, 79146-79148, 79163-79165, 79168-79169, 79171-79172, 79175- 79176, 79183-79184, 79186-79187, 79192, 79194-79199, 79201-79203, 79206-79207, 79209-79222, 79227-79229, 79231, 79236-79238, 79240- 79242, 79244-79245, 79247-79251, 79253, 79257, 79260-79261, 79264- 79265, 79267, 79269-79270, 79272, 79274-79284, 79287, 79292, 79294- 79296, 79299-79300, 79302-79309, 79311, 79315, 79318-79320, 79322- 79325, 79331, 79335-79337, 79339-79342, 79344-79353, 79356, 79360- 79362, 79365-79366, 79373-79378, 79382-79383, 79385-79386, 79388- 79390, 79394, 79396-79398, 79402, 79404, 79410-79411, 79413-79416, 79419-79420, 79422, 79425-79426, 79428-79430, 79432-79436, 79438, 79440, 79444, 79451-79452, 79456, 79459-79466, 79468-79473, 79475, 79481-79482, 79484-79486, 79490, 79493-79494, 79496-79499, 79502, 79505, 79509, 79516, 79519, 79521, 79523-79525, 79530, 79533, 79536, 79538, 79546, 79554, 79556-79558, 79560, 79562, 79564, 79567, 79569, 79572-79574, 79580-79581, 79583-79586, 79595, 79599-79601, 79603, 79605, 79607, 79609-79611, 79613, 79615, 79617-79621, 79628, 79631- 79633, 79635, 79638-79639, 79642-79648, 79650, 79652-79658, 79660, 79662-79665, 79667-79668, 79673-79674, 79679-79685, 79687, 79689- 79691, 79694, 79696-79700, 79704-79708, 79710-79712, 79714, 79716, 79719, 79721-79723, 79725-79726, 79728-79731, 79733, 79735, 79737, 79739-79742, 79746-79751, 79754-79759, 79761, 79763-79764, 79766, 79773-79774, 79777, 79782, 79786, 79789-79790, 79793-79796, 79798- 79799, 79802-79803, 79806-79807, 79809-79810, 79812-79814, 79817- 79819, 79821, 79825, 79827-79830, 79832-79833, 79836-79837, 79843- 79845, 79847, 79852, 79855, 79857-79860, 79862, 79864-79865, 79867, 79873, 79875-79876, 79879-79882, 79884, 79886, 79888-79889, 79895- 79896, 79898, 79906, 79911, 79913-79915, 79918-79921, 79924, 79926, 79929, 79931, 79938, 79941-79944, 79948-79949, 79953, 79959-79963, 79965, 79971-79972, 79976-79977, 79979-79980, 79984-79985, 79987, 79989-79990, 79993-79994, 79997-79998, 80000, 80004, 80006, 80008, 80010-80011, 80013-80015, 80017, 80020-80021, 80029-80030, 80032- 80047, 80051-80055, 80059-80061, 80063, 80065, 80069-80071, 80073- 80075, 80080, 80083-80085, 80087, 80090, 80092, 80094, 80096-80102, 80105, 80121, 80128, 80130-80132, 80136, 80141-80145, 80149-80154, 80157, 80159, 80164-80165, 80167, 80170, 80175, 80179, 80181, 80183, 80188, 80192-80193, 80195, 80198, 80201, 80206-80209, 80211, 80214- 80216, 80218, 80222-80223, 80225, 80233, 80235-80236, 80240, 80244, 80249, 80251-80252, 80254-80264, 80274-80275, 80277, 80279-80280, 80283-80293, 80295, 80297-80298, 80300, 80302-80303, 80306, 80308, 80311-80314, 80316-80318, 80320, 80324, 80326-80329, 80333, 80335, 80339, 80342-80343, 80345-80347, 80349, 80353, 80356-80361, 80364- 80367, 80369-80370, 80372, 80374, 80378-80379, 80382, 80388-80389, 80391-80392, 80394, 80397-80400, 80404, 80409, 80411-80415, 80417- 80424, 80428-80431, 80433, 80435, 80437, 80439-80444, 80447, 80450, 80454, 80457-80459, 80461-80462, 80464, 80466, 80468-80470, 80476- 80477, 80479-80485, 80489-80491, 80493, 80496, 80498, 80500, 80502- 80505, 80511-80513, 80515-80520, 80523, 80525, 80529-80533, 80538- 80539, 80548-80551, 80553-80557, 80563-80564, 80566-80569, 80574, 80576, 80578-80579, 80581-80591, 80593-80594, 80596, 80599-80600, 80604-80605, 80608-80610, 80612-80614, 80620-80621, 80623, 80629, 80631-80633, 80639, 80641-80642, 80647, 80649-80654, 80658, 80661- 80662, 80665, 80667-80669, 80674, 80676, 80678-80679, 80683-80684, 80688, 80691, 80693-80694, 80696-80701, 80704, 80708-80710, 80715- 80717, 80723-80725, 80728, 80730-80734, 80736, 80738, 80740-80743, 80745, 80747-80750, 80752-80753, 80755, 80757-80759, 80761, 80768- 80772, 80776-80779, 80782, 80785-80787, 80791, 80793, 80795, 80797- 80799, 80801-80806, 80808, 80811-80819, 80821-80822, 80825-80826, 80828-80832, 80834-80840, 80842-80843, 80846, 80848, 80852, 80859, 80861, 80863-80864, 80867-80868, 80870-80871, 80875, 80877-80878, 80881, 80885-80888, 80890-80894, 80896-80903, 80905-80906, 80921, 80923-80924, 80926, 80936-80940, 80946-80949, 80951, 80954-80957, 80959-80961, 80966, 80968, 80970, 80972, 80974-80976, 80982-80983, 80987-80989, 80992-80993, 80995-80996, 80999, 81004, 81006-81007, 81009, 81011-81013, 81016-81017, 81019, 81021-81022, 81025, 81027- 81033, 81035-81037, 81040-81041, 81047-81048, 81050-81051, 81055, 81061-81062, 81064-81068, 81070-81074, 81078, 81080-81082, 81085, 81088, 81092, 81094-81098, 81100, 81103, 81106, 81108, 81110-81113, 81118-81120, 81126, 81129, 81134-81136, 81140-81143, 81145-81154, 81161-81170, 81172, 81174-81176, 81180-81181, 81183-81184, 81191, 81195-81196, 81200, 81204, 81208, 81210-81211, 81214, 81216-81219, 81223-81224, 81228-81230, 81232-81233, 81237, 81239, 81241, 81243, 81245-81249, 81257-81263, 81273, 81275, 81279, 81282-81294, 81296- 81303, 81305, 81307, 81312-81314, 81316-81322, 81325-81328, 81330, 81333-81337, 81341-81343, 81345-81346, 81348-81350, 81353-81355,
81357, 81359, 81361-81362, 81367, 81369, 81372, 81374, 81376, 81381, 81383, 81385-81386, 81389, 81391, 81393-81396, 81398, 81400, 81402, 81405, 81410, 81412, 81420-81421, 81424, 81427-81430, 81432-81438, 81442-81451, 81453-81455, 81458, 81461-81468, 81470, 81473, 81475- 81476, 81478-81480, 81483, 81497, 81502, 81508-81510, 81513, 81515- 81517, 81519-81524, 81527, 81531-81537, 81540-81546, 81549, 81552, 81554, 81559-81560, 81567, 81578, 81583-81584, 81586-81589, 81592- 81594, 81596-81597, 81599-81600, 81604-81608, 81611-81614, 81617- 81618, 81620-81621, 81625, 81628, 81630-81631, 81640, 81642-81643, 81645-81646, 81648-81655, 81657-81662, 81664-81670, 81672, 81679-
81681, 81687, 81689-81690, 81694, 81697-81698, 81700, 81702, 81704- 81705, 81707, 81712, 81718, 81724, 81728, 81731-81732, 81736-81737, 81739-81741, 81744, 81747-81754, 81757-81761, 81768, 81770, 81772- 81777, 81780-81781, 81783-81784, 81791, 81794, 81796-81798, 81803- 81805, 81808, 81811-81812, 81814-81822, 81825-81830, 81832, 81836, 81839-81840, 81843, 81846, 81849-81857, 81859-81860, 81864, 81866, 81868, 81870-81874, 81876-81877, 81881, 81885-81886, 81888-81891, 81893-81897, 81899-81901, 81904-81905, 81908, 81911, 81915, 81917- 81921, 81927-81931, 81933-81935, 81937, 81940-81942, 81947, 81949- 81950, 81953-81954, 81957, 81959-81961, 81963-81966, 81968, 81971, 81974-81976, 81978-81983, 81985, 81987, 81989, 81992, 81994, 81996, 81998-82000, 82005, 82007-82008, 82010-82011, 82014, 82016-82019, 82021, 82023-82024, 82026, 82028, 82031, 82037, 82039-82040, 82044, 82046-82048, 82051-82053, 82056-82063, 82065-82070, 82072, 82074- 82076, 82078, 82080-82082, 82085, 82087, 82090, 82094-82096, 82098, 82100-82101, 82103, 82105-82111, 82114, 82118-82119, 82122-82126, 82128-82129, 82136-82137, 82147-82148, 82150-82151, 82153, 82155- 82158, 82160-82165, 82168-82172, 82176-82181, 82183, 82185-82186, 82189-82191, 82193-82195, 82197, 82199-82201, 82203-82206, 82209- 82211, 82214-82215, 82217, 82223-82228, 82230-82232, 82235, 82240, 82242, 82247, 82250-82254, 82256-82258, 82262, 82266-82267, 82269, 82271-82273, 82276-82279, 82281-82282, 82294-82301, 82303-82304, 82306, 82309-82310, 82314, 82320-82321, 82325-82329, 82331-82334, 82336-82344, 82351, 82354-82356, 82360, 82366, 82369, 82371-82373, 82376-82379, 82382, 82384-82386, 82390-82391, 82393-82399, 82401, 82403-82405, 82408-82409, 82412, 82415, 82422-82425, 82430, 82432- 82436, 82438-82439, 82442-82447, 82450-82451, 82453-82454, 82459, 82461, 82463-82467, 82469, 82471, 82473-82477, 82480, 82483, 82485- 82487, 82489-82492, 82495, 82500-82502, 82505-82506, 82509, 82511- 82514, 82516, 82518, 82524, 82526-82527, 82530-82531, 82533, 82535, 82543, 82545-82554, 82558, 82560, 82564-82566, 82574, 82583, 82588- 82590, 82593, 82595-82599, 82601, 82604-82605, 82607-82608, 82614- 82621, 82623, 82625, 82627-82628, 82630, 82635, 82637, 82640-82641, 82645, 82648-82650, 82656-82658, 82660-82664, 82667-82669, 82672, 82676-82677, 82683, 82685, 82689-82690, 82692-82694, 82702, 82704- 82706, 82709, 82712-82713, 82716, 82720-82721, 82724, 82726, 82730, 82732-82738, 82740, 82746, 82753, 82756, 82758-82763, 82766-82773, 82775, 82782-82785, 82790, 82792, 82794-82795, 82798, 82800-82801, 82805, 82807-82808, 82810, 82813-82815, 82823, 82825-82826, 82830- 82831, 82833-82834, 82841-82844, 82846, 82850-82856, 82860-82861, 82864, 82866-82867, 82870-82872, 82879-82880, 82882, 82884-82891, 82897-82898, 82900-82903, 82906-82908, 82912, 82915-82916, 82919- 82920, 82922-82923, 82925, 82930-82934, 82937, 82939-82941, 82943, 82945, 82947-82948, 82953-82954, 82956-82957, 82961, 82966-82969,
82973-82980, 82984, 82987, 82992, 82994-82996, 82998, 83001, 83007- 83008, 83011-83012, 83014-83017, 83019, 83022-83024, 83026, 83033, 83043, 83045, 83054-83056, 83059-83060, 83063-83064, 83067, 83070, 83081-83083, 83085, 83087, 83089, 83092, 83094, 83096, 83105, 83107- 83109, 83112, 83114-83116, 83118-83120, 83122, 83125, 83131-83134, 83136-83141, 83147, 83155, 83157, 83160-83161, 83163-83164, 83166, 83168, 83170-83179, 83181-83182, 83184-83190, 83192-83194, 83196, 83198-83202, 83205, 83208-83211, 83213-83215, 83219-83223, 83226- 83229, 83231, 83239-83243, 83248, 83251, 83259, 83261-83262, 83265, 83271, 83273, 83275-83279, 83283-83285, 83287, 83289, 83291, 83293, 83295, 83297-83301, 83303-83305, 83312, 83315, 83321, 83323, 83327, 83329, 83331-83332, 83334, 83336-83337, 83340-83341, 83346, 83348- 83349, 83351, 83354, 83359, 83362-83365, 83367-83370, 83372, 83374- 83376, 83383-83385, 83391-83396, 83399, 83402, 83405, 83408, 83412- 83414, 83416-83420, 83424-83425, 83427-83435, 83437, 83440-83441, 83443-83444, 83446-83448, 83451-83453, 83458, 83460, 83468, 83470, 83472-83474, 83476, 83478, 83480-83482, 83484, 83488, 83490, 83494- 83495, 83498, 83500, 83502-83508, 83510, 83512-83517, 83523, 83527, 83530-83531, 83535, 83537, 83539-83540, 83544, 83546-83548, 83552, 83555-83556, 83562-83564, 83566-83568, 83570-83572, 83574, 83576- 83582, 83584-83587, 83591-83593, 83596-83601, 83603, 83605-83606, 83608-83612, 83614, 83619, 83621, 83623-83624, 83626-83629, 83631- 83634, 83638-83640, 83644-83646, 83648-83651, 83658-83659 The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.

Claims

CLAIMS What is claimed is:
1. A single stranded oligonucleotide having a sequence 5'-X-Y-Z, wherein X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 1-23 nucleotides in length, wherein the single stranded oligonucleotide is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of a MECP2 gene.
2. The single stranded oligonucleotide of claim 1, wherein the oligonucleotide does not comprise three or more consecutive guanosine nucleotides.
3. The single stranded oligonucleotide of claim 1 or 2, wherein the
oligonucleotide does not comprise four or more consecutive guanosine nucleotides.
4. The single stranded oligonucleotide of any one of claims 1 to 3, wherein the oligonucleotide is 8 to 30 nucleotides in length.
5. The single stranded oligonucleotide of any one of claims 1 to 4, wherein the oligonucleotide is 8 to 10 nucleotides in length and all but 1, 2, or 3 of the nucleotides of the complementary sequence of the PRC2-associated region are cytosine or guanosine nucleotides.
6. The single stranded oligonucleotide of any one of claims 1 to 5, wherein at least one nucleotide of the oligonucleotide is a nucleotide analogue.
7. The single stranded oligonucleotide of claim 7, wherein the at least one nucleotide analogue results in an increase in Tm of the oligonucleotide in a range of 1 to 5 °C compared with an oligonucleotide that does not have the at least one nucleotide analogue.
8. The single stranded oligonucleotide of any one of claims 1 to 7, wherein at least one nucleotide of the oligonucleotide comprises a 2' O-methyl.
9. The single stranded oligonucleotide of any one of claims 1 to 8, wherein each nucleotide of the oligonucleotide comprises a 2' O-methyl.
10. The single stranded oligonucleotide of any one of claims 1 to 8, wherein the oligonucleotide comprises at least one ribonucleotide, at least one deoxyribonucleotide, or at least one bridged nucleotide.
11. The single strand oligonucleotide of claim 10, wherein the bridged nucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide.
12. The single stranded oligonucleotide of any one of claims 1 to 6, wherein each nucleotide of the oligonucleotide is a LNA nucleotide.
13. The single stranded oligonucleotide of any one of claims 1 to 6, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2'-fluoro- deoxyribonucleotides.
14. The single stranded oligonucleotide of any one of claims 1 to 6, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and 2'-0- methyl nucleotides.
15. The single stranded oligonucleotide of any one of claims 1 to 6, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and ENA nucleotide analogues.
16. The single stranded oligonucleotide of any one of claims 1 to 6, wherein the nucleotides of the oligonucleotide comprise alternating deoxyribonucleotides and LNA nucleotides.
17. The single stranded oligonucleotide of any one of claims 13 to 16, wherein the 5' nucleotide of the oligonucleotide is a deoxyribonucleotide.
18. The single stranded oligonucleotide of any one of claims 1 to 6, wherein the nucleotides of the oligonucleotide comprise alternating LNA nucleotides and 2' -O-methyl nucleotides.
19. The single stranded oligonucleotide of claim 18, wherein the 5' nucleotide of the oligonucleotide is a LNA nucleotide.
20. The single stranded oligonucleotide of any one of claims 1 to 8, wherein the nucleotides of the oligonucleotide comprise deoxyribonucleotides flanked by at least one LNA nucleotide on each of the 5' and 3' ends of the deoxyribonucleotides.
21. The single stranded oligonucleotide of any one of claims 1 to 20, further comprising phosphorothioate internucleotide linkages between at least two nucleotides.
22. The single stranded oligonucleotide of claim 21, further comprising phosphorothioate internucleotide linkages between all nucleotides.
23. The single stranded oligonucleotide of any one of claims 1 to 22, wherein the nucleotide at the 3' position of the oligonucleotide has a 3' hydroxyl group.
24. The single stranded oligonucleotide of any one of claims 1 to 22, wherein the nucleotide at the 3' position of the oligonucleotide has a 3' thiophosphate.
25. The single stranded oligonucleotide of any one of claims 1 to 24, further comprising a biotin moiety conjugated to the 5' nucleotide.
26. A single stranded oligonucleotide comprising a region of complementarity that is complementary with at least 8 consecutive nucleotides of a PRC2-associated region of a MECP2 gene, wherein the oligonucleotide has at least one of: a) a sequence that is 5'X-Y-Z, wherein X is any nucleotide and wherein X is anchored at the 5' end of the oligonucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a human seed sequence of a microRNA, and Z is a nucleotide sequence of 1 to 23 nucleotides in length; b) a sequence that does not comprise three or more consecutive guanosine
nucleotides; c) a sequence that has less than a threshold level of sequence identity with every sequence of nucleotides, of equivalent length to the second nucleotide sequence, that are between 50 kilobases upstream of a 5 '-end of an off-target gene and 50 kilobases downstream of a 3 '-end of the off-target gene; d) a sequence that is complementary to a PRC2-associated region that encodes an RNA that forms a secondary structure comprising at least two single stranded loops; and/or e) a sequence that has greater than 60% G-C content.
27. The single stranded oligonucleotide of claim 26, wherein the oligonucleotide has the sequence 5'X-Y-Z and wherein the oligonucleotide is 8-50 nucleotides in length.
28. A composition comprising a single stranded oligonucleotide of any one of claims 1 to 27 and a carrier.
29. A composition comprising a single stranded oligonucleotide of any one of claims 1 to 27 in a buffered solution.
30. A composition of claim 28, wherein the oligonucleotide is conjugated to the carrier.
31. The composition of claim 30, wherein the carrier is a peptide.
32. The composition of claim 30, wherein the carrier is a steroid.
33. A pharmaceutical composition comprising a composition of any one of claims 28 to 32 and a pharmaceutically acceptable carrier.
34. A kit comprising a container housing the composition of any one of claims 28 to 33.
35. A method of increasing expression of a MECP2 gene in a cell, the method comprising delivering the single stranded oligonucleotide of any one of claims 1 to 27 into the cell.
36. The method of claim 35, wherein delivery of the single stranded
oligonucleotide into the cell results in a level of expression of the MECP2 gene that is at least 50% greater than a level of expression of the MECP2 gene in a control cell that does not comprise the single stranded oligonucleotide.
37. A method increasing levels of a MECP2 gene in a subject, the method comprising administering the single stranded oligonucleotide of any one of claims 1 to 27 to the subject.
38. A method of treating a condition associated with decreased levels of a MECP2 gene in a subject, the method comprising administering the single stranded oligonucleotide of any one of claims 1 to 27 to the subject.
39. The method of claim 38, wherein the condition is Rett Syndrome, MECP2- related severe neonatal encephalopathy, Angelman syndrome, or PPM-X syndrome.
PCT/US2013/041394 2012-05-16 2013-05-16 Compositions and methods for modulating mecp2 expression WO2013173608A1 (en)

Priority Applications (10)

Application Number Priority Date Filing Date Title
JP2015512847A JP2015518712A (en) 2012-05-16 2013-05-16 Compositions and methods for modulating MECP2 expression
EA201492116A EA201492116A1 (en) 2012-05-16 2013-05-16 COMPOSITIONS AND METHODS FOR MODULATING THE EXPRESSION OF MECP2
CA2873779A CA2873779A1 (en) 2012-05-16 2013-05-16 Compositions and methods for modulating mecp2 expression
US14/401,237 US20150152410A1 (en) 2012-05-16 2013-05-16 Compositions and methods for modulating mecp2 expression
EP13791546.8A EP2850190B1 (en) 2012-05-16 2013-05-16 Compositions and methods for modulating mecp2 expression
AU2013262709A AU2013262709A1 (en) 2012-05-16 2013-05-16 Compositions and methods for modulating MECP2 expression
CN201380037614.0A CN104583402A (en) 2012-05-16 2013-05-16 Compositions and methods for modulating MECP2 expression
US15/787,876 US10655128B2 (en) 2012-05-16 2017-10-19 Compositions and methods for modulating MECP2 expression
US15/872,684 US10837014B2 (en) 2012-05-16 2018-01-16 Compositions and methods for modulating SMN gene family expression
US16/876,271 US11788089B2 (en) 2012-05-16 2020-05-18 Compositions and methods for modulating MECP2 expression

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201261648051P 2012-05-16 2012-05-16
US61/648,051 2012-05-16

Related Parent Applications (2)

Application Number Title Priority Date Filing Date
US14/401,240 Continuation-In-Part US20150232836A1 (en) 2012-05-16 2013-05-16 Compositions and methods for modulating gene expression
PCT/US2013/041434 Continuation-In-Part WO2013173635A1 (en) 2012-05-16 2013-05-16 Compositions and methods for modulating gene expression

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US14/401,237 A-371-Of-International US20150152410A1 (en) 2012-05-16 2013-05-16 Compositions and methods for modulating mecp2 expression
US15/787,876 Continuation US10655128B2 (en) 2012-05-16 2017-10-19 Compositions and methods for modulating MECP2 expression

Publications (1)

Publication Number Publication Date
WO2013173608A1 true WO2013173608A1 (en) 2013-11-21

Family

ID=49584296

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2013/041394 WO2013173608A1 (en) 2012-05-16 2013-05-16 Compositions and methods for modulating mecp2 expression

Country Status (8)

Country Link
US (3) US20150152410A1 (en)
EP (1) EP2850190B1 (en)
JP (1) JP2015518712A (en)
CN (1) CN104583402A (en)
AU (1) AU2013262709A1 (en)
CA (1) CA2873779A1 (en)
EA (1) EA201492116A1 (en)
WO (1) WO2013173608A1 (en)

Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015191780A3 (en) * 2014-06-10 2016-03-17 The General Hospital Corporation Ccctc-binding factor (ctcf) rna interactome
US9328346B2 (en) 2010-11-12 2016-05-03 The General Hospital Corporation Polycomb-associated non-coding RNAs
WO2016141145A1 (en) * 2015-03-03 2016-09-09 Ionis Pharmaceuticals, Inc. Methods for modulating mecp2 expression
US9567581B2 (en) 2012-08-07 2017-02-14 The General Hospital Corporation Selective reactivation of genes on the inactive X chromosome
EP3033422A4 (en) * 2013-08-16 2017-08-02 Rana Therapeutics Inc. Oligonucleotides targeting euchromatin regions of genes
US9920317B2 (en) 2010-11-12 2018-03-20 The General Hospital Corporation Polycomb-associated non-coding RNAs
US10058623B2 (en) 2012-05-16 2018-08-28 Translate Bio Ma, Inc. Compositions and methods for modulating UTRN expression
US10059941B2 (en) 2012-05-16 2018-08-28 Translate Bio Ma, Inc. Compositions and methods for modulating SMN gene family expression
US10174323B2 (en) 2012-05-16 2019-01-08 The General Hospital Corporation Compositions and methods for modulating ATP2A2 expression
US10174315B2 (en) 2012-05-16 2019-01-08 The General Hospital Corporation Compositions and methods for modulating hemoglobin gene family expression
US10221414B2 (en) 2013-10-11 2019-03-05 Ionis Pharmaceuticals, Inc. Compositions for modulating C9ORF72 expression
WO2019048645A1 (en) 2017-09-08 2019-03-14 Mina Therapeutics Limited Stabilized cebpa sarna compositions and methods of use
WO2019048632A1 (en) 2017-09-08 2019-03-14 Mina Therapeutics Limited Stabilized hnf4a sarna compositions and methods of use
WO2019195855A1 (en) * 2018-04-06 2019-10-10 Camp4 Therapeutics Corporation Treating diseases via targeted modulation of gene signaling networks
US10443052B2 (en) 2012-10-15 2019-10-15 Ionis Pharmaceuticals, Inc. Compositions for modulating C9ORF72 expression
US10577604B2 (en) 2012-10-15 2020-03-03 Ionis Pharmaceuticals, Inc. Methods for monitoring C9ORF72 expression
US10655128B2 (en) 2012-05-16 2020-05-19 Translate Bio Ma, Inc. Compositions and methods for modulating MECP2 expression
US10758558B2 (en) 2015-02-13 2020-09-01 Translate Bio Ma, Inc. Hybrid oligonucleotides and uses thereof
US10837014B2 (en) 2012-05-16 2020-11-17 Translate Bio Ma, Inc. Compositions and methods for modulating SMN gene family expression
US10858650B2 (en) 2014-10-30 2020-12-08 The General Hospital Corporation Methods for modulating ATRX-dependent gene repression
US10900036B2 (en) 2015-03-17 2021-01-26 The General Hospital Corporation RNA interactome of polycomb repressive complex 1 (PRC1)
WO2021032777A1 (en) 2019-08-19 2021-02-25 Mina Therapeutics Limited Oligonucleotide conjugate compositions and methods of use
US11129844B2 (en) 2015-03-03 2021-09-28 Ionis Pharmaceuticals, Inc. Compositions and methods for modulating MECP2 expression
US11260073B2 (en) 2015-11-02 2022-03-01 Ionis Pharmaceuticals, Inc. Compounds and methods for modulating C90RF72

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013040429A1 (en) 2011-09-14 2013-03-21 Rana Therapeutics Inc. Multimeric oligonucleotide compounds
US20150247141A1 (en) 2012-09-14 2015-09-03 Rana Therapeutics, Inc. Multimeric oligonucleotide compounds
CA2958524A1 (en) * 2014-08-20 2016-02-25 Lifesplice Pharma Llc Splice modulating oligonucleotides and methods of use thereof
EP4104831A1 (en) 2016-01-26 2022-12-21 Anavex Life Sciences Corp. A2-73 for treating angelman syndrome, williams syndrome smith-magenis syndrome and multiple sclerosis
WO2018022967A1 (en) * 2016-07-28 2018-02-01 Regeneron Pharmaceuticals, Inc. Gpr156 variants and uses thereof
KR102502248B1 (en) 2016-11-01 2023-02-21 더 리서치 파운데이션 포 더 스테이트 유니버시티 오브 뉴욕 5-haluracil-modified microRNA and its use in cancer treatment
WO2018180005A1 (en) * 2017-03-31 2018-10-04 国立研究開発法人日本医療研究開発機構 Antisense nucleic acid for inhibiting biosynthesis of chondroitin sulfate
JP2021527087A (en) * 2018-06-14 2021-10-11 オービッド・セラピューティクス・インコーポレイテッドＯｖｉｄ Ｔｈｅｒａｐｅｕｔｉｃｓ Ｉｎｃ． Use of MIR-92a or MIR-145 in the treatment of Angelman syndrome
CA3110289A1 (en) * 2018-08-30 2020-03-05 The University Of North Carolina At Chapel Hill Feedback enabled synthetic genes, target seed match cassettes, and their uses
US11312963B2 (en) * 2018-10-18 2022-04-26 Synerk Inc. Compositions and methods for inhibiting TIGIT gene expression
US20210395738A1 (en) 2018-11-08 2021-12-23 Greenlight Biosciences, Inc. Control of insect infestation
US20230089497A1 (en) * 2020-03-03 2023-03-23 Arizona Board Of Regents On Behalf Of Arizona State University Rational design of upstream enhancement rna for circuit dynamics regulation and viral diagnostics optimization
WO2022256631A2 (en) * 2021-06-04 2022-12-08 Triplet Therapeutics, Inc. Methods for the treatment of nucleotide repeat expansion disorders associated with msh3 activity

Citations (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3687808A (en) 1969-08-14 1972-08-29 Univ Leland Stanford Junior Synthetic polynucleotides
US4469863A (en) 1980-11-12 1984-09-04 Ts O Paul O P Nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof
US4476301A (en) 1982-04-29 1984-10-09 Centre National De La Recherche Scientifique Oligonucleotides, a process for preparing the same and their application as mediators of the action of interferon
US5023243A (en) 1981-10-23 1991-06-11 Molecular Biosystems, Inc. Oligonucleotide therapeutic agent and method of making same
US5034506A (en) 1985-03-15 1991-07-23 Anti-Gene Development Group Uncharged morpholino-based polymers having achiral intersubunit linkages
US5177196A (en) 1990-08-16 1993-01-05 Microprobe Corporation Oligo (α-arabinofuranosyl nucleotides) and α-arabinofuranosyl precursors thereof
US5188897A (en) 1987-10-22 1993-02-23 Temple University Of The Commonwealth System Of Higher Education Encapsulated 2',5'-phosphorothioate oligoadenylates
US5264423A (en) 1987-03-25 1993-11-23 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5276019A (en) 1987-03-25 1994-01-04 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5278302A (en) 1988-05-26 1994-01-11 University Patents, Inc. Polynucleotide phosphorodithioates
US5321131A (en) 1990-03-08 1994-06-14 Hybridon, Inc. Site-specific functionalization of oligodeoxynucleotides for non-radioactive labelling
US5399676A (en) 1989-10-23 1995-03-21 Gilead Sciences Oligonucleotides with inverted polarity
US5405939A (en) 1987-10-22 1995-04-11 Temple University Of The Commonwealth System Of Higher Education 2',5'-phosphorothioate oligoadenylates and their covalent conjugates with polylysine
US5455233A (en) 1989-11-30 1995-10-03 University Of North Carolina Oligoribonucleoside and oligodeoxyribonucleoside boranophosphates
US5466677A (en) 1993-03-06 1995-11-14 Ciba-Geigy Corporation Dinucleoside phosphinates and their pharmaceutical compositions
US5476925A (en) 1993-02-01 1995-12-19 Northwestern University Oligodeoxyribonucleotides including 3'-aminonucleoside-phosphoramidate linkages and terminal 3'-amino groups
US5519126A (en) 1988-03-25 1996-05-21 University Of Virginia Alumni Patents Foundation Oligonucleotide N-alkylphosphoramidates
US5550111A (en) 1984-07-11 1996-08-27 Temple University-Of The Commonwealth System Of Higher Education Dual action 2',5'-oligoadenylate antiviral derivatives and uses thereof
US5571799A (en) 1991-08-12 1996-11-05 Basco, Ltd. (2'-5') oligoadenylate analogues useful as inhibitors of host-v5.-graft response
US5587361A (en) 1991-10-15 1996-12-24 Isis Pharmaceuticals, Inc. Oligonucleotides having phosphorothioate linkages of high chiral purity
US5625050A (en) 1994-03-31 1997-04-29 Amgen Inc. Modified oligonucleotides and intermediates useful in nucleic acid therapeutics
WO2007115578A1 (en) * 2006-04-07 2007-10-18 Georg-August-Universität Göttingen Stiftung Öffentlichen Rechts Synthetic mecp2 sequence for protein substitution therapy
US20090082297A1 (en) * 2007-06-25 2009-03-26 Lioy Daniel T Compositions and Methods for Regulating Gene Expression

Family Cites Families (303)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2547714B2 (en) 1981-10-23 1996-10-23 モルキユラ− バイオシステムズ インコ−ポレテツド Oligonucleotide therapeutic agent and method for producing the same
DE3329892A1 (en) 1983-08-18 1985-03-07 Köster, Hubert, Prof. Dr., 2000 Hamburg METHOD FOR PRODUCING OLIGONUCLEOTIDES
DE3855864T2 (en) 1987-11-30 1997-09-25 Univ Iowa Res Found DNA MOLECULES STABILIZED BY MODIFICATIONS ON THE 3'-TERMINAL PHOSPHODIESTERBINDING, THEIR USE AS NUCLEIC ACID PROBE AND AS A THERAPEUTIC AGENT FOR INHIBITING THE EXPRESSION OF SPECIFIC TARGET GENES
US5623065A (en) 1990-08-13 1997-04-22 Isis Pharmaceuticals, Inc. Gapped 2' modified oligonucleotides
US6753423B1 (en) 1990-01-11 2004-06-22 Isis Pharmaceuticals, Inc. Compositions and methods for enhanced biostability and altered biodistribution of oligonucleotides in mammals
US5914396A (en) 1990-01-11 1999-06-22 Isis Pharmaceuticals, Inc. 2'-O-modified nucleosides and phosphoramidites
US6005087A (en) 1995-06-06 1999-12-21 Isis Pharmaceuticals, Inc. 2'-modified oligonucleotides
US6395492B1 (en) 1990-01-11 2002-05-28 Isis Pharmaceuticals, Inc. Derivatized oligonucleotides having improved uptake and other properties
AU8196791A (en) 1990-06-27 1992-01-23 Trustees Of Columbia University In The City Of New York, The Methods for detecting spinal muscular atrophy type i and types ii/iii and marker therefor
JP3514779B2 (en) 1991-04-17 2004-03-31 株式会社アズウェル Method for testing apolipoprotein E genotype and primers and probes suitable for the test
US7015315B1 (en) 1991-12-24 2006-03-21 Isis Pharmaceuticals, Inc. Gapped oligonucleotides
WO1994008003A1 (en) 1991-06-14 1994-04-14 Isis Pharmaceuticals, Inc. ANTISENSE OLIGONUCLEOTIDE INHIBITION OF THE ras GENE
US5965722A (en) 1991-05-21 1999-10-12 Isis Pharmaceuticals, Inc. Antisense inhibition of ras gene with chimeric and alternating oligonucleotides
US5661134A (en) 1991-10-15 1997-08-26 Isis Pharmaceuticals, Inc. Oligonucleotides for modulating Ha-ras or Ki-ras having phosphorothioate linkages of high chiral purity
US6831166B2 (en) 1992-10-23 2004-12-14 Isis Pharmaceuticals, Inc. Derivatized oligonucleotides having improved uptake and other properties
US8153602B1 (en) 1991-11-19 2012-04-10 Isis Pharmaceuticals, Inc. Composition and methods for the pulmonary delivery of nucleic acids
ATE204879T1 (en) 1991-12-24 2001-09-15 Isis Pharmaceuticals Inc ANTISENSE OLIGONUCLEOTIDES
US20070032446A1 (en) 1991-12-24 2007-02-08 Isis Pharmaceuticals, Inc. Gapped 2' modified oligonucleotides
US6346614B1 (en) 1992-07-23 2002-02-12 Hybridon, Inc. Hybrid oligonucleotide phosphorothioates
TW244371B (en) 1992-07-23 1995-04-01 Tri Clover Inc
US5652355A (en) 1992-07-23 1997-07-29 Worcester Foundation For Experimental Biology Hybrid oligonucleotide phosphorothioates
CA2140542A1 (en) 1992-07-27 1994-02-03 Abeysingle Padmapriya Oligonucleotide alkylphosphonothioates
WO1994005813A1 (en) 1992-09-10 1994-03-17 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute Compositions and methods for treatment of hepatitis c virus-associated diseases
JPH08508714A (en) 1993-01-25 1996-09-17 ハイブライドン インコーポレイテッド Oligonucleotide alkyl phosphonates and alkyl phosphonothioates
EP1897942A1 (en) 1993-05-11 2008-03-12 The University Of North Carolina At Chapel Hill Antisense oligonucleotides which combat aberrant splicing and methods of using the same
FR2720757B1 (en) 1994-06-03 1996-08-02 Inst Nat Sante Rech Med Method and probes for the detection of markers linked to the locus of infantile spinal muscular atrophies.
US5734039A (en) 1994-09-15 1998-03-31 Thomas Jefferson University Antisense oligonucleotides targeting cooperating oncogenes
EP0708178A1 (en) 1994-10-19 1996-04-24 Institut National De La Sante Et De La Recherche Medicale (Inserm) Survival motor neuron (SMN) gene: a gene for spinal muscular atrophy
US6608035B1 (en) 1994-10-25 2003-08-19 Hybridon, Inc. Method of down-regulating gene expression
US5766903A (en) 1995-08-23 1998-06-16 University Technology Corporation Circular RNA and uses thereof
SK109198A3 (en) 1996-02-14 1999-06-11 Isis Pharmaceuticals Inc Sugar-modified gapped oligonucleotides
US6015710A (en) 1996-04-09 2000-01-18 The University Of Texas System Modulation of mammalian telomerase by peptide nucleic acids
US5898031A (en) 1996-06-06 1999-04-27 Isis Pharmaceuticals, Inc. Oligoribonucleotides for cleaving RNA
US5912332A (en) 1996-07-26 1999-06-15 Hybridon, Inc. Affinity-based purification of oligonucleotides using soluble multimeric oligonucleotides
AU8285998A (en) 1997-07-02 1999-01-25 Sdg, Inc. Targeted liposomal constructs for diagnostic and therapeutic uses
JPH1133863A (en) 1997-07-23 1999-02-09 Howa Mach Ltd Tool replacing device and extruding device for replacing tool
US6383808B1 (en) 2000-09-11 2002-05-07 Isis Pharmaceuticals, Inc. Antisense inhibition of clusterin expression
US6794499B2 (en) 1997-09-12 2004-09-21 Exiqon A/S Oligonucleotide analogues
US7572582B2 (en) 1997-09-12 2009-08-11 Exiqon A/S Oligonucleotide analogues
US7715989B2 (en) 1998-04-03 2010-05-11 Elitech Holding B.V. Systems and methods for predicting oligonucleotide melting temperature (TmS)
US6503754B1 (en) 2000-09-07 2003-01-07 Isis Pharmaceuticals, Inc. Antisense modulation of BH3 interacting domain death agonist expression
US6646113B1 (en) 1998-09-17 2003-11-11 The Trustees Of The University Of Pennsylvania Nucleic acid molecule encoding human survival of motor neuron-interacting protein 1 (SIP1) deletion mutants
US6228642B1 (en) 1998-10-05 2001-05-08 Isis Pharmaceuticals, Inc. Antisense oligonucleotide modulation of tumor necrosis factor-(α) (TNF-α) expression
US6210892B1 (en) 1998-10-07 2001-04-03 Isis Pharmaceuticals, Inc. Alteration of cellular behavior by antisense modulation of mRNA processing
EP0999270A1 (en) 1998-10-09 2000-05-10 Institut National De La Sante Et De La Recherche Medicale (Inserm) Method of regulating SMN gene expression
US6465628B1 (en) 1999-02-04 2002-10-15 Isis Pharmaceuticals, Inc. Process for the synthesis of oligomeric compounds
ES2234563T5 (en) 1999-02-12 2018-01-17 Daiichi Sankyo Company, Limited New nucleoside and oligonucleotide analogs
US9029523B2 (en) 2000-04-26 2015-05-12 Ceres, Inc. Promoter, promoter control elements, and combinations, and uses thereof
US20040002153A1 (en) 1999-07-21 2004-01-01 Monia Brett P. Modulation of PTEN expression via oligomeric compounds
US6284538B1 (en) 1999-07-21 2001-09-04 Isis Pharmaceuticals, Inc. Antisense inhibition of PTEN expression
WO2001007662A1 (en) 1999-07-22 2001-02-01 Genaissance Pharmaceuticals, Inc. Drug target isogenes: polymorphisms in the prostaglandin-endoperoxide synthase 2 gene
US6677445B1 (en) 1999-08-27 2004-01-13 Chiron Corporation Chimeric antisense oligonucleotides and cell transfecting formulations thereof
IL132972A0 (en) 1999-11-16 2001-03-19 Yissum Res Dev Co Pharmaceutical compositions comprising acetylcholinesterase antisense deoxynucleotides for the treatment of muscular and neuromuscular disorders
US6602857B1 (en) 2000-01-18 2003-08-05 Isis Pharmaceuticals, Inc. Antisense modulation of PTP1B expression
US6187545B1 (en) 2000-01-19 2001-02-13 Isis Pharmaceuticals Inc. Antisense modulation of pepck-cytosolic expression
EP1133993A1 (en) 2000-03-10 2001-09-19 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Substances for the treatment of spinal muscular atrophy
EP1268768A2 (en) 2000-03-27 2003-01-02 University Of Delaware Targeted chromosomal genomic alterations with modified single stranded oligonucleotides
AU4036600A (en) 2000-03-28 2001-10-08 Isis Pharmaceuticals Inc Alteration of cellular behavior by antisense modulation of mrna processing
WO2001077384A2 (en) 2000-04-07 2001-10-18 Epigenomics Ag Detection of single nucleotide polymorphisms (snp's) and cytosine-methylations
CA2408011A1 (en) 2000-05-04 2001-11-08 Avi Biopharma, Inc. Splice-region antisense composition and method
EP1289472A4 (en) 2000-05-30 2004-09-08 Advanced Res & Tech Inst Compositions and methods for identifying agents which modulate pten function and pi-3 kinase pathways
US6727355B2 (en) 2000-08-25 2004-04-27 Jcr Pharmaceuticals Co., Ltd. Pharmaceutical composition for treatment of Duchenne muscular dystrophy
US20050054836A1 (en) 2000-11-09 2005-03-10 Cold Spring Harbor Laboratory Chimeric molecules to modulate gene expression
BR0115475A (en) 2000-11-20 2004-02-10 Univ Illinois Membrane structuring proteins
US20040058356A1 (en) 2001-03-01 2004-03-25 Warren Mary E. Methods for global profiling gene regulatory element activity
US6825338B2 (en) * 2001-03-30 2004-11-30 Isis Pharmaceuticals, Inc. Labeled oligonucleotides, methods for making same, and compounds useful therefor
DE60228477D1 (en) 2001-05-08 2008-10-02 Darwin Molecular Corp PROCESS FOR REGULATING THE IMMUNE FUNCTION IN PRIMATES USING THE FOXP3 PROTEIN
US20050176025A1 (en) 2001-05-18 2005-08-11 Sirna Therapeutics, Inc. RNA interference mediated inhibition of B-cell CLL/Lymphoma-2 (BCL-2) gene expression using short interfering nucleic acid (siNA)
AU2002344853A1 (en) 2001-06-14 2003-01-02 Active Pass Pharmaceuticals, Inc. Abca10 transporter
US7407943B2 (en) 2001-08-01 2008-08-05 Isis Pharmaceuticals, Inc. Antisense modulation of apolipoprotein B expression
US7259150B2 (en) 2001-08-07 2007-08-21 Isis Pharmaceuticals, Inc. Modulation of apolipoprotein (a) expression
CA2465129A1 (en) 2001-10-29 2003-05-08 Mcgill University Acyclic linker-containing oligonucleotides and uses thereof
US20030219770A1 (en) 2001-11-08 2003-11-27 Eshleman James R. Methods and systems of nucleic acid sequencing
US20030198983A1 (en) 2002-02-01 2003-10-23 Affymetrix, Inc. Methods of genetic analysis of human genes
US20080207542A1 (en) 2002-03-26 2008-08-28 Sirna Therapeutics, Inc. RNA inteference mediated inhibition of hepatitis C virus (HVC) gene expression using short interfering nucleic acid (siNA)
WO2003085110A2 (en) 2002-04-05 2003-10-16 Santaris Pharma A/S Oligomeric compounds for the modulation hif-1alpha expression
US20030228690A1 (en) * 2002-06-10 2003-12-11 Isis Pharmaceuticals Inc. Antisense modulation of IL-1 receptor-associated kinase-1 expression
US7507808B2 (en) 2002-12-12 2009-03-24 Isis Pharmaceuticals, Inc. Modulation of endothelial lipase expression
US20050130924A1 (en) 2002-06-26 2005-06-16 Monia Brett P. Antisense inhibition via RNAse H-independent reduction in mRNA
AU2003268032A1 (en) 2002-07-29 2004-02-16 Epigenesis Pharmaceuticals, Inc. Composition and methods for treatment and screening
CA2498777C (en) 2002-09-13 2015-01-13 Replicor, Inc. Non-sequence complementary antiviral oligonucleotides
US20050003369A1 (en) 2002-10-10 2005-01-06 Affymetrix, Inc. Method for depleting specific nucleic acids from a mixture
US20040219565A1 (en) 2002-10-21 2004-11-04 Sakari Kauppinen Oligonucleotides useful for detecting and analyzing nucleic acids of interest
AU2003290598A1 (en) * 2002-11-05 2004-06-03 Isis Pharmaceuticals, Inc. Modified oligonucleotides for use in rna interference
EP2314691A3 (en) 2002-11-14 2012-01-18 Dharmacon, Inc. Fuctional and hyperfunctional siRNA
US7250496B2 (en) 2002-11-14 2007-07-31 Rosetta Genomics Ltd. Bioinformatically detectable group of novel regulatory genes and uses thereof
US7655785B1 (en) 2002-11-14 2010-02-02 Rosetta Genomics Ltd. Bioinformatically detectable group of novel regulatory oligonucleotides and uses thereof
EP1569661B1 (en) 2002-11-18 2009-09-09 Santaris Pharma A/S Antisense design
US20040248840A1 (en) 2003-02-10 2004-12-09 Santaris Pharma A/S Oligomeric compounds for the modulation of ras expression
AU2004221760B2 (en) 2003-03-21 2010-03-18 Roche Innovation Center Copenhagen A/S Short interfering RNA (siRNA) analogues
FR2853663B1 (en) 2003-04-14 2007-08-31 Aventis Pharma Sa PROCESS FOR OBTAINING MASTOCYTE LINES FROM PORK TISSUES AND PROCESS FOR PRODUCING HEPARIN TYPE MOLECULES
US8092992B2 (en) 2003-05-29 2012-01-10 Salk Institute For Biological Studies Transcriptional regulation of gene expression by small double-stranded modulatory RNA
WO2004113867A2 (en) 2003-06-16 2004-12-29 University Of Massachusetts Exon analysis
CA2533701A1 (en) 2003-07-31 2005-02-17 Isis Pharmaceuticals, Inc. Oligomeric compounds and compositions for use in modulation of small non-coding rnas
US7888497B2 (en) 2003-08-13 2011-02-15 Rosetta Genomics Ltd. Bioinformatically detectable group of novel regulatory oligonucleotides and uses thereof
WO2005030928A2 (en) 2003-09-23 2005-04-07 Chihiro Koike PORCINE INVARIANT CHAIN PROTEIN, FULL LENGTH cDNA, GENOMIC ORGANIZATION, AND REGULATORY REGION
WO2005045035A2 (en) 2003-10-23 2005-05-19 Sirna Therapeutics, Inc. RNA INTERFERENCE MEDIATED INHIBITION OF NOGO AND NOGO RECEPTOR GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
CN101454451A (en) 2003-10-30 2009-06-10 科勒制药有限公司 C-class oligonucleotide analogs with enhanced immunostimulatory potency
EP1756137A4 (en) 2003-11-05 2007-10-31 Univ Texas Diagnostic and therapeutic methods and compositions involving pten and breast cancer
CA2550258A1 (en) 2003-12-23 2005-07-07 Santaris Pharma A/S Oligomeric compounds for the modulation of bcl-2
US20050244851A1 (en) 2004-01-13 2005-11-03 Affymetrix, Inc. Methods of analysis of alternative splicing in human
US20050164209A1 (en) 2004-01-23 2005-07-28 Bennett C. F. Hepatocyte free uptake assays
CA2553104A1 (en) 2004-01-23 2005-08-11 Avi Biopharma, Inc. Antisense oligomers and methods for inducing immune tolerance and immunosuppression
EP1713912B1 (en) 2004-01-30 2013-09-18 Santaris Pharma A/S Modified short interfering rna (modified sirna)
WO2005089169A2 (en) 2004-03-12 2005-09-29 Exelixis, Inc. Mptens as modifiers of the pten pathway and methods of use
US8314226B2 (en) 2004-03-29 2012-11-20 The General Hospital Corporation Oligonucleotide complex compositions and methods of use as gene alteration tools
US7374927B2 (en) 2004-05-03 2008-05-20 Affymetrix, Inc. Methods of analysis of degraded nucleic acid samples
EP1752536A4 (en) 2004-05-11 2008-04-16 Alphagen Co Ltd Polynucleotide causing rna interfere and method of regulating gene expression with the use of the same
US7687616B1 (en) 2004-05-14 2010-03-30 Rosetta Genomics Ltd Small molecules modulating activity of micro RNA oligonucleotides and micro RNA targets and uses thereof
US20050265927A1 (en) 2004-05-17 2005-12-01 Yale University Intranasal delivery of nucleic acid molecules
JP2008501694A (en) 2004-06-03 2008-01-24 アイシス ファーマシューティカルズ、インク． Double-stranded composition having individually modified strands for use in gene regulation
US7807816B2 (en) 2004-06-28 2010-10-05 University Of Western Australia Antisense oligonucleotides for inducing exon skipping and methods of use thereof
US8029985B2 (en) 2004-09-01 2011-10-04 Vybion, Inc. Amplified bioassay
WO2007011372A2 (en) 2004-09-09 2007-01-25 The Regents Of The University Of California Therapeutic and prognostic factor yy1 in human cancer
US7838657B2 (en) 2004-12-03 2010-11-23 University Of Massachusetts Spinal muscular atrophy (SMA) treatment via targeting of SMN2 splice site inhibitory sequences
US9550990B2 (en) 2004-12-10 2017-01-24 Ionis Pharmaceuticals, Inc. Regulation of epigenetic control of gene expression
US20070099196A1 (en) 2004-12-29 2007-05-03 Sakari Kauppinen Novel oligonucleotide compositions and probe sequences useful for detection and analysis of micrornas and their target mRNAs
US7879992B2 (en) 2005-01-31 2011-02-01 Isis Pharmaceuticals, Inc. Modification of MyD88 splicing using modified oligonucleotides
ES2852549T3 (en) 2005-02-09 2021-09-13 Sarepta Therapeutics Inc Antisense composition for treatment of muscle atrophy
US8999943B2 (en) 2005-03-14 2015-04-07 Board Of Regents, The University Of Texas System Antigene oligomers inhibit transcription
US7449297B2 (en) 2005-04-14 2008-11-11 Euclid Diagnostics Llc Methods of copying the methylation pattern of DNA during isothermal amplification and microarrays
CA2604532C (en) 2005-04-15 2017-03-07 The Regents Of The University Of California Small activating rna molecules and methods of use
US8586719B2 (en) 2005-04-27 2013-11-19 Pacific Arrow Limited Triterpenes for modulating gene expression and cell membrane, and as antiprotozoal agents
DK1910395T3 (en) 2005-06-23 2013-02-04 Isis Pharmaceuticals Inc Compositions and Methods for Modulating SMN2 Splicing
JP5074392B2 (en) 2005-07-01 2012-11-14 インデックス・ファーマシューティカルズ・アクチエボラーグ Immune promotion method
US8067571B2 (en) 2005-07-13 2011-11-29 Avi Biopharma, Inc. Antibacterial antisense oligonucleotide and method
WO2007031091A2 (en) * 2005-09-15 2007-03-22 Santaris Pharma A/S Rna antagonist compounds for the modulation of p21 ras expression
JP2009515887A (en) 2005-11-11 2009-04-16 ザ スクリプス リサーチ インスティテュート Histone deacetylase inhibitors as therapeutic agents for nervous system diseases
EP2431467A3 (en) 2005-11-17 2012-05-02 Board Of Regents, The University Of Texas Modulation of gene expression by oligomers targeted to chromosomal DNA
EP1951263A4 (en) 2005-11-21 2009-11-18 Johnson & Johnson Res Pty Ltd Multitargeting interfering rnas having two active strands and methods for their design and use
CN103301475B (en) 2005-12-28 2016-08-03 斯克里普斯研究所 Method that pharmaceutical composition and expression vector and regulator gene are expressed and the application of nucleic acid molecules
WO2007133812A2 (en) 2005-12-30 2007-11-22 Philadelphia Health & Education Corporation, D/B/A Drexel University College Of Medicine Improved carriers for delivery of nucleic acid agents to cells and tissues
EP2388328A1 (en) 2006-01-27 2011-11-23 Isis Pharmaceuticals, Inc. Oligomeric compounds and compositions for the use in modulation of micrornas
US8362229B2 (en) 2006-02-08 2013-01-29 Quark Pharmaceuticals, Inc. Tandem siRNAS
JP5814505B2 (en) 2006-04-03 2015-11-17 ロシュ・イノベーション・センター・コペンハーゲン・アクティーゼルスカブＲｏｃｈｅ Ｉｎｎｏｖａｔｉｏｎ Ｃｅｎｔｅｒ Ｃｏｐｅｎｈａｇｅｎ Ａ／Ｓ Pharmaceutical composition comprising antimiRNA antisense oligonucleotide
EP3431602A1 (en) 2006-04-03 2019-01-23 Roche Innovation Center Copenhagen A/S Pharmaceutical composition comprising anti-mirna antisense oligonucleotides
WO2007134014A2 (en) 2006-05-05 2007-11-22 Isis Pharmaceuticals, Inc. Compounds and methods for modulating expression of gcgr
WO2008002996A2 (en) 2006-06-27 2008-01-03 Shanghai Institutes For Biological Sciences Rheumatoid arthritis t cell vaccine
EP2054529B1 (en) 2006-08-25 2014-03-26 Duke University Methods for in vivo identification of endogenous mrna targets of micrornas
WO2008025069A1 (en) 2006-08-28 2008-03-06 The Walter And Eliza Hall Institute Of Medical Research Methods of modulating cellular activity and compositions therefor
WO2008029619A1 (en) 2006-09-07 2008-03-13 Daiichi Sankyo Company, Limited Ena antisense oligonucleotide having sequence-specific action
WO2008036282A1 (en) 2006-09-18 2008-03-27 The Regents Of The University Of Californina Upregulating activity or expression of bdnf to mitigate cognitive impairment in asymptomatic huntington's subjects
US20080139472A1 (en) 2006-10-06 2008-06-12 The Regents Of The University Of California Upregulating bdnf levels to mitigate mental retardation
ES2620472T5 (en) 2006-10-18 2020-07-09 Ionis Pharmaceuticals Inc Antisense compounds
WO2008061537A2 (en) 2006-11-23 2008-05-29 Querdenker Aps Oligonucleotides for modulating target rna activity
WO2008066784A2 (en) 2006-11-27 2008-06-05 Ludwig Institute For Cancer Research Expression of foxp3 by cancer cells
AU2008206168B2 (en) 2007-01-19 2012-01-19 The Regents Of The University Of Michigan ADRB2 cancer markers
ITMI20070127A1 (en) 2007-01-29 2008-07-30 Fond I R C C S E-O PEPTIDES PROTEINS FOR THE PREVENTION AND-OR CARE OF NEURODEGENERATIVE DISEASES
WO2008103763A2 (en) * 2007-02-20 2008-08-28 Sequenom, Inc. Methods and compositions for cancer diagnosis and treatment based on nucleic acid methylation
US7858592B2 (en) 2007-02-26 2010-12-28 The Board Of Regents Of The University Of Texas System Interfering RNAs against the promoter region of P53
DK2149605T3 (en) 2007-03-22 2013-09-30 Santaris Pharma As Short RNA antagonist compounds to modulate the desired mRNA
CA2685444A1 (en) 2007-05-01 2008-11-06 Jesper Worm Rna antagonist compounds for the modulation of beta-catenin
WO2008141282A2 (en) 2007-05-11 2008-11-20 The Regents Of The University Of Michigan Materials and methods for foxp3 tumor suppression
WO2008138904A2 (en) 2007-05-11 2008-11-20 Santaris Pharma A/S Rna antagonist compounds for the modulation of her3
US8691965B2 (en) 2007-06-14 2014-04-08 Mirx Therapeutics Aps Oligonucleotides for modulating target RNA activity
ES2694726T3 (en) 2007-06-29 2018-12-26 Sarepta Therapeutics, Inc. Tissue specific peptide conjugates and methods
KR20100063048A (en) 2007-07-31 2010-06-10 더 존스 홉킨스 유니버시티 Polypeptide-nucleic acid conjugate for immunoprophylaxis or immunotherapy for neoplastic or infectious disorders
WO2009027349A2 (en) * 2007-08-24 2009-03-05 Oryzon Genomics Sa Treatment and prevention of neurodegenerative diseases
AU2008306327B2 (en) 2007-10-04 2014-05-15 Roche Innovation Center Copenhagen A/S Micromirs
WO2009046397A2 (en) 2007-10-04 2009-04-09 Board Of Regents, The University Of Texas System Modulating gene expression with agrna and gapmers targeting antisense transcripts
ITRM20070523A1 (en) 2007-10-05 2009-04-06 Consiglio Nazionale Ricerche CODIFYING NUCLEIC ACID FOR A REGULATING PROTEIN SPECIFIC TO THE TRANSCRIPTION OF THE UROPHINE PROTEIN BY IT CODIFIED AND ITS APPLICATIONS
EP2227545A2 (en) 2007-11-09 2010-09-15 Isis Pharmaceuticals, Inc. Modulation of factor 7 expression
US8637478B2 (en) 2007-11-13 2014-01-28 Isis Pharmaceuticals, Inc. Compounds and methods for modulating protein expression
US8937217B2 (en) 2007-12-18 2015-01-20 E. I. Du Pont De Nemours And Company Down-regulation of gene expression using artificial microRNAs
WO2009090182A1 (en) 2008-01-14 2009-07-23 Santaris Pharma A/S C4'-substituted - dna nucleotide gapmer oligonucleotides
US8404659B2 (en) 2008-03-07 2013-03-26 Santaris Pharma A/S Pharmaceutical compositions for treatment of MicroRNA related diseases
EP2265717A4 (en) 2008-03-07 2012-09-12 Senesco Technologies Inc Use of sirna to achieve down regulation of an endogenous gene in combination with the use of a sense construct to achieve expression of a desired polynucleotide
US8846639B2 (en) 2008-04-04 2014-09-30 Isis Pharmaceutical, Inc. Oligomeric compounds comprising bicyclic nucleosides and having reduced toxicity
US20110263687A1 (en) 2008-04-07 2011-10-27 Riken Rna molecules and uses thereof
US20110224280A1 (en) 2008-04-16 2011-09-15 Niels Fisker Nielsen Pharmaceutical Composition Comprising Anti PCSK9 Oligomers
US8916532B2 (en) 2008-04-28 2014-12-23 The Trustees Of The University Of Pennsylvania Methods for enhancing utrophin production via inhibition of microRNA
WO2009151546A2 (en) 2008-05-27 2009-12-17 Ptc Therapeutics, Inc. Methods for treating spinal muscular atrophy
US8222221B2 (en) 2008-06-04 2012-07-17 The Board Of Regents Of The University Of Texas System Modulation of gene expression through endogenous small RNA targeting of gene promoters
CA2635187A1 (en) 2008-06-05 2009-12-05 The Royal Institution For The Advancement Of Learning/Mcgill University Oligonucleotide duplexes and uses thereof
WO2010000665A1 (en) 2008-06-30 2010-01-07 Santaris Pharma A/S Antidote oligomers
CA2730641A1 (en) 2008-07-15 2010-01-21 Santaris Pharma A/S Rna antagonists targeting gli2
EP2317847B1 (en) 2008-07-29 2019-04-17 The Board of Regents of The University of Texas System Selective inhibition of polyglutamine protein expression
EP2331543A4 (en) 2008-09-26 2013-06-19 Agency Science Tech & Res 3-deazaneplanocin derivatives
CA2739464C (en) 2008-10-03 2020-03-31 Opko Curna, Llc Treatment of apolipoprotein-a1 related diseases by inhibition of natural antisense transcript to apolipoprotein-a1
CA2739046A1 (en) 2008-10-16 2010-04-22 Marina Biotech, Inc. Process and compositions for liposomal and effective delivery of nucleic acids
RU2011124146A (en) 2008-11-17 2012-12-27 Ф.Хоффманн-Ля Рош Аг COMPOSITIONS AND METHODS OF SUPPRESSING EXPRESSION OF FACTOR VII GENES
GB0821457D0 (en) 2008-11-24 2008-12-31 Trillion Genomics Ltd Oligonucleotides
US20110294870A1 (en) 2008-12-04 2011-12-01 Opko Curna, Llc Treatment of tumor suppressor gene related diseases by inhibition of natural antisense transcript to the gene
CN108042560A (en) 2008-12-04 2018-05-18 库尔纳公司 By inhibiting to treat 1 relevant disease of Sirtuin for the natural antisense transcript of Sirtuin 1
MX2011005910A (en) 2008-12-04 2011-06-17 Opko Curna Llc Treatment of erythropoietin (epo) related diseases by inhibition of natural antisense transcript to epo.
CA2746003C (en) 2008-12-04 2020-03-31 Opko Curna, Llc Treatment of vascular endothelial growth factor (vegf) related diseases by inhibition of natural antisense transcript to vegf
WO2010076248A1 (en) 2008-12-31 2010-07-08 Santaris Pharma A/S Use of lna apob antisense oligomers for the treatment of acute coronary syndromes
CA2750561C (en) 2009-01-26 2017-10-10 Protiva Biotherapeutics, Inc. Compositions and methods for silencing apolipoprotein c-iii expression
EP2396408B1 (en) 2009-02-12 2017-09-20 CuRNA, Inc. Treatment of glial cell derived neurotrophic factor (gdnf) related diseases by inhibition of natural antisense transcript to gdnf
KR101682735B1 (en) 2009-02-12 2016-12-06 큐알엔에이, 인크. Treatment of brain derived neurotrophic factor (bdnf) related diseases by inhibition of natural antisense transcript to bdnf
WO2010093860A2 (en) 2009-02-13 2010-08-19 The General Hospital Corporation Isolation of factors that associate directly or indirectly with chromatin
CA2754749C (en) 2009-03-04 2019-04-30 Opko Curna, Llc Treatment of sirtuin 1 (sirt1) related diseases by inhibition of natural antisense transcript to sirt1
WO2010107733A2 (en) 2009-03-16 2010-09-23 Curna, Inc. Treatment of nuclear factor (erythroid-derived 2)-like 2 (nrf2) related diseases by inhibition of natural antisense transcript to nrf2
CN102549159B (en) 2009-03-17 2016-08-10 库尔纳公司 By suppressing to treat the disease that DLK1 is correlated with for the natural antisense transcript of δ sample 1 congener (DLK1)
WO2010115993A1 (en) 2009-04-10 2010-10-14 Association Institut De Myologie Tricyclo-dna antisense oligonucleotides, compositions, and methods for the treatment of disease
WO2010122538A1 (en) 2009-04-24 2010-10-28 Santaris Pharma A/S Pharmaceutical compositions for treatment of hcv patients that are non-responders to interferon
US8815586B2 (en) 2009-04-24 2014-08-26 The Board Of Regents Of The University Of Texas System Modulation of gene expression using oligomers that target gene regions downstream of 3′ untranslated regions
US8318690B2 (en) 2009-05-01 2012-11-27 Curna, Inc. Treatment of hemoglobin (HBF/HBG) related diseases by inhibition of natural antisense transcript to HBF/HBG
KR101835889B1 (en) 2009-05-06 2018-03-08 큐알엔에이, 인크. Treatment of lipid transport and metabolism gene related diseases by inhibition of natural antisense transcript to a lipid transport and metabolism gene
CA2761142C (en) 2009-05-06 2021-06-08 Opko Curna, Llc Treatment of tristetraproline (ttp) related diseases by inhibition of natural antisense transcript to ttp
US9012139B2 (en) 2009-05-08 2015-04-21 Curna, Inc. Treatment of dystrophin family related diseases by inhibition of natural antisense transcript to DMD family
DK2432881T3 (en) 2009-05-18 2018-02-26 Curna Inc TREATMENT OF REPROGRAMMING FACTOR-RELATED DISEASES BY INHIBITING NATURAL ANTISENSE TRANSCRIPTS TO A REPROGRAMMING FACTOR
WO2010135695A2 (en) 2009-05-22 2010-11-25 Curna, Inc. TREATMENT OF TRANSCRIPTION FACTOR E3 (TFE3) and INSULIN RECEPTOR SUBSTRATE 2 (IRS2) RELATED DISEASES BY INHIBITION OF NATURAL ANTISENSE TRANSCRIPT TO TFE3
CN103221541B (en) 2009-05-28 2017-03-01 库尔纳公司 Antiviral gene relevant disease is treated by the natural antisense transcript suppressing antiviral gene
ES2629339T3 (en) 2009-06-16 2017-08-08 Curna, Inc. Treatment of diseases related to paraoxonase 1 (pon1) by inhibition of natural antisense transcript to pon1
JP5944311B2 (en) 2009-06-16 2016-07-05 クルナ・インコーポレーテッド Treatment of collagen gene-related diseases by suppression of natural antisense transcripts against collagen genes
CA2765889A1 (en) 2009-06-24 2010-12-29 Opko Curna, Llc Treatment of tumor necrosis factor receptor 2 (tnfr2) related diseases by inhibition of natural antisense transcript to tnfr2
KR101807324B1 (en) 2009-06-26 2017-12-08 큐알엔에이, 인크. Treatment of down syndrome gene related diseases by inhibition of natural antisense transcript to a down syndrome gene
BR112012000421A2 (en) 2009-07-06 2019-09-24 Alnylam Pharmaceuticals Inc compositions and methods for enhancing the production of a biological product.
US20120258104A1 (en) 2009-07-22 2012-10-11 Cenix Bioscience Gmbh Delivery System and Conjugates For Compound Delivery Via Naturally Occurring Intracellular Transport Routes
WO2011010706A1 (en) 2009-07-23 2011-01-27 武田薬品工業株式会社 Fgf21 cis-element binding substance
CA2768947C (en) 2009-07-24 2018-06-19 Opko Curna, Llc Treatment of sirtuin (sirt) related diseases by inhibition of natural antisense transcript to a sirtuin (sirt)
WO2011017516A2 (en) 2009-08-05 2011-02-10 Curna, Inc. Treatment of insulin gene (ins) related diseases by inhibition of natural antisense transcript to an insulin gene (ins)
EP2464731B1 (en) 2009-08-11 2016-10-05 CuRNA, Inc. Treatment of adiponectin (adipoq) related diseases by inhibition of natural antisense transcript to an adiponectin (adipoq)
EP2982755B1 (en) 2009-08-21 2020-10-07 CuRNA, Inc. Treatment of 'c terminus of hsp70-interacting protein' (chip) related diseases by inhibition of natural antisense transcript to chip
EP2470657B1 (en) 2009-08-25 2019-10-23 CuRNA, Inc. Treatment of 'iq motif containing gtpase activating protein' (iqgap) related diseases by inhibition of natural antisense transcript to iqgap
WO2011025862A2 (en) 2009-08-28 2011-03-03 Curna, Inc. Treatment of atp-binding cassette sub-family b member 1 (abcb1) related diseases by inhibition of natural antisense transcript to abcb1
WO2011032109A1 (en) 2009-09-11 2011-03-17 Sma Foundation Biomarkers for spinal muscular atrophy
WO2011038205A2 (en) 2009-09-25 2011-03-31 Curna, Inc. Treatment of growth hormone (gh) related diseases by inhibition of natural antisense transcript to gh
DK2480669T3 (en) 2009-09-25 2018-02-12 Curna Inc TREATMENT OF FILAGGRIN- (FLG) RELATED DISEASES BY MODULATING FLG EXPRESSION AND ACTIVITY
EP2490699A1 (en) 2009-10-20 2012-08-29 Santaris Pharma A/S Oral delivery of therapeutically effective lna oligonucleotides
KR101138048B1 (en) 2009-11-06 2012-04-23 성균관대학교산학협력단 Novel peptides upregulating BDNF expression and pharmaceutical composition for protection and therapy against Alzheimer's disease and Parkinson's disease comprising the same
CA2782366A1 (en) 2009-12-16 2011-07-14 Opko Curna, Llc Treatment of membrane bound transcription factor peptidase, site 1 (mbtps1) related diseases by inhibition of natural antisense transcript to mbtps1
WO2011079261A2 (en) 2009-12-23 2011-06-30 Curna, Inc. Treatment of hepatocyte growth factor (hgf) related diseases by inhibition of natural antisense transcript to hgf
US9068183B2 (en) 2009-12-23 2015-06-30 Curna, Inc. Treatment of uncoupling protein 2 (UCP2) related diseases by inhibition of natural antisense transcript to UCP2
RU2611186C2 (en) 2009-12-29 2017-02-21 Курна, Инк. TREATMENT OF TUMOR PROTEIN 63 (p63) RELATED DISEASES BY INHIBITION OF NATURAL ANTISENSE TRANSCRIPT TO p63
EP2519633B1 (en) 2009-12-29 2017-10-25 CuRNA, Inc. Treatment of nuclear respiratory factor 1 (nrf1) related diseases by inhibition of natural antisense transcript to nrf1
RU2016115782A (en) 2009-12-31 2018-11-28 Курна, Инк. TREATMENT OF DISEASES ASSOCIATED WITH THE SUBSTRATE OF THE INSULIN 2 RECEPTOR (IRS2) BY INHIBITING THE NATURAL ANTISENSE TRANSCRIPT TO IRS2 AND THE TRANSCRIPTION FACTOR E3 (TFE3)
US8946181B2 (en) 2010-01-04 2015-02-03 Curna, Inc. Treatment of interferon regulatory factor 8 (IRF8) related diseases by inhibition of natural antisense transcript to IRF8
WO2011085066A2 (en) 2010-01-06 2011-07-14 Curna, Inc. Treatment of pancreatic developmental gene related diseases by inhibition of natural antisense transcript to a pancreatic developmental gene
EP2524039B1 (en) 2010-01-11 2017-11-29 CuRNA, Inc. Treatment of sex hormone binding globulin (shbg) related diseases by inhibition of natural antisense transcript to shbg
RU2611192C2 (en) 2010-01-25 2017-02-21 Курна, Инк. TREATMENT OF RNase H1 RELATED DISEASES BY INHIBITION OF NATURAL ANTISENSE TRANSCRIPT TO RNase H1
US9574191B2 (en) 2010-02-03 2017-02-21 The Board Of Regents Of The University Of Texas System Selective inhibition of polyglutamine protein expression
WO2011097582A2 (en) 2010-02-08 2011-08-11 Opko Curna Llc Treatment of arachidonate 12-lipoxygenase, 12r type (alox12b) related diseases by inhibition of natural antisense transcript to alox12b
US20130059902A1 (en) 2010-02-08 2013-03-07 Isis Pharmaceuticals, Inc. Methods and compositions useful in treatment of diseases or conditions related to repeat expansion
KR101838308B1 (en) 2010-02-22 2018-03-13 큐알엔에이, 인크. Treatment of pyrroline-5-carboxylate reductase 1 (pycr1) related diseases by inhibition of natural antisense transcript to pycr1
WO2011117353A1 (en) 2010-03-24 2011-09-29 Mirrx Therapeutics A/S Bivalent antisense oligonucleotides
US8980856B2 (en) 2010-04-02 2015-03-17 Curna, Inc. Treatment of colony-stimulating factor 3 (CSF3) related diseases by inhibition of natural antisense transcript to CSF3
RU2610661C2 (en) 2010-04-09 2017-02-14 Курна, Инк. Treatment of fibroblast growth factor 21 (fgf21) related diseases by inhibition of natural antisense transcript to fgf21
DK2558578T3 (en) 2010-04-13 2016-01-25 Life Technologies Corp CONFIGURATIONS AND METHODS FOR INHIBITION OF nucleic acid function
RS56011B1 (en) 2010-04-29 2017-09-29 Ionis Pharmaceuticals Inc Modulation of transthyretin expression
RU2693462C2 (en) 2010-05-03 2019-07-03 Курна, Инк. Treatment of sirtuin (sirt) related diseases by inhibition of natural antisense transcript to sirtuin (sirt)
TWI586356B (en) 2010-05-14 2017-06-11 可娜公司 Treatment of par4 related diseases by inhibition of natural antisense transcript to par4
TW201201819A (en) 2010-05-19 2012-01-16 Opko Curna Llc Treatment of BCL2-like 11 (BCL2L11) related diseases by inhibition of natural antisense transcript to BCL2L11
TW201143782A (en) 2010-05-19 2011-12-16 Opko Curna Llc Treatment of LIM homeobox 2 (LHX2) related diseases by inhibition of natural antisense transcript to LHX2
EP2576783B1 (en) 2010-05-26 2017-11-29 CuRNA, Inc. Treatment of atonal homolog 1 (atoh1) related diseases by inhibition of natural antisense transcript to atoh1
JP5917497B2 (en) 2010-05-26 2016-05-18 カッパーアールエヌエー，インコーポレイテッド Treatment of MSRA-related diseases by inhibition of natural antisense transcripts against methionine sulfoxide reductase A (MSRA)
EP2582397A4 (en) 2010-06-15 2014-10-29 Isis Pharmaceuticals Inc Compounds and methods for modulating interaction between proteins and target nucleic acids
US20120004278A1 (en) 2010-06-18 2012-01-05 The Board Of Trustees Of The Leland Stanford Junior University Linc rnas in cancer diagnosis and treatment
GB201010557D0 (en) 2010-06-23 2010-08-11 Mina Therapeutics Ltd RNA molecules and uses thereof
JP6023705B2 (en) 2010-06-23 2016-11-09 カッパーアールエヌエー，インコーポレイテッド Treatment of SCNA-related diseases by inhibition of natural antisense transcripts on sodium channels, voltage-gated, alpha subunit (SCNA)
US20130122046A1 (en) 2010-07-09 2013-05-16 Institut Pasteur Of Shanghai, Cas Regulatory factor of foxp3 and regulatory t cells and use thereof
WO2012009347A2 (en) 2010-07-12 2012-01-19 Opko Curna Llc Treatment of bcl2 binding component 3 (bbc3) related diseases by inhibition of natural antisense transcript to bbc3
JP5998131B2 (en) 2010-07-14 2016-09-28 カッパーアールエヌエー，インコーポレイテッド DISCSLARGEHOMOLOG (DLG) Treatment of DLG-related diseases by inhibition of natural antisense transcripts on DLG1
US20140011860A1 (en) 2010-07-19 2014-01-09 Isis Pharmaceuticals, Inc. Compounds and methods for modulating target nuclear and sub-nuclear nucleic acid molecules in cells and animals
EP3633038A3 (en) 2010-07-19 2020-07-29 Ionis Pharmaceuticals, Inc. Modulation of dystrophia myotonica-protein kinase (dmpk) expression
WO2012018881A2 (en) 2010-08-03 2012-02-09 Alnylam Pharmaceuticals, Inc. Methods and compositions for the regulation of rna
WO2012024478A2 (en) 2010-08-19 2012-02-23 Opko Curna Llc Treatment of nicotinamide phosphoribosyltransferase (nampt) related diseases by inhibition of natural antisense transcript to nampt
KR102061353B1 (en) 2010-09-10 2020-01-02 에피자임, 인코포레이티드 Inhibitors of human ezh2, and methods of use thereof
KR101235256B1 (en) 2010-09-13 2013-02-21 서울대학교산학협력단 Treatment of Neurodegenerative Diseases by Targeting a miRNA
ES2640755T3 (en) 2010-10-06 2017-11-06 Curna, Inc. Treatment of diseases related to Sialidase 4 (neu4) by inhibition of the natural antisense transcript to the neu4 gene
EP2630241B1 (en) 2010-10-22 2018-10-17 CuRNA, Inc. Treatment of alpha-l-iduronidase (idua) related diseases by inhibition of natural antisense transcript to idua
DK2633052T3 (en) 2010-10-27 2018-07-16 Curna Inc TREATMENT OF INTERFERON-RELATED DEVELOPMENT REGULATOR 1 (IFRD1) -RELATED DISEASES BY INHIBITION OF NATURAL ANTISENCE TRANSCRIPT TO IFRD1
WO2012064806A2 (en) 2010-11-11 2012-05-18 The University Of North Carolina At Chapel Hill Methods and compositions for unsilencing imprinted genes
JP6336755B2 (en) 2010-11-12 2018-06-06 ザ ジェネラル ホスピタル コーポレイション Non-coding RNA related to polycomb
US9920317B2 (en) 2010-11-12 2018-03-20 The General Hospital Corporation Polycomb-associated non-coding RNAs
WO2012068340A2 (en) 2010-11-18 2012-05-24 Opko Curna Llc Antagonat compositions and methods of use
US8987225B2 (en) 2010-11-23 2015-03-24 Curna, Inc. Treatment of NANOG related diseases by inhibition of natural antisense transcript to NANOG
DK2655621T3 (en) 2010-12-20 2018-08-13 Massachusetts Gen Hospital Polycomb-associated non-coding RNAS
US9206479B2 (en) 2011-02-09 2015-12-08 University Of Rochester Methods and compositions related to Staufen 1 binding sites formed by duplexing Alu elements
WO2012144220A1 (en) 2011-04-22 2012-10-26 Oncotherapy Science, Inc. Ezh2 as target gene for cancer therapy and diagnosis
US8557787B2 (en) 2011-05-13 2013-10-15 The Board Of Trustees Of The Leland Stanford Junior University Diagnostic, prognostic and therapeutic uses of long non-coding RNAs for cancer and regenerative medicine
ES2653247T3 (en) 2011-06-09 2018-02-06 Curna, Inc. Treatment of frataxin-related diseases (FXN) by inhibiting the natural antisense transcript to the FXN gene
CA2844144A1 (en) 2011-07-05 2013-01-10 The General Hospital Corporation Rna-yy1 interactions
CN108272782B (en) 2011-09-06 2021-04-23 库尔纳公司 Use of small molecules in preparation of medicine for treating Dravet syndrome or generalized epilepsy with febrile convulsion adjunctive disease
WO2013040429A1 (en) 2011-09-14 2013-03-21 Rana Therapeutics Inc. Multimeric oligonucleotide compounds
EP2816114A3 (en) 2011-12-15 2015-02-25 Oncostamen S.r.l. MicroRNAs and uses thereof
ITMI20120275A1 (en) 2012-02-24 2013-08-25 Biogenera Societa A Responsabilita Limitata OLIGONUCLEOTIDS FOR THE MODULATION OF GENE EXPRESSION AND THEIR USES
US20150031750A1 (en) 2012-03-15 2015-01-29 The Scripps Research Institute Treatment of brain derived neurotrophic factor (bdnf) related diseases by inhibition of natural antisense transcript to bdnf
JP2015523854A (en) 2012-05-16 2015-08-20 ラナ セラピューティクス インコーポレイテッド Compositions and methods for modulating SMN gene family expression
WO2013173635A1 (en) 2012-05-16 2013-11-21 Rana Therapeutics, Inc. Compositions and methods for modulating gene expression
US10837014B2 (en) 2012-05-16 2020-11-17 Translate Bio Ma, Inc. Compositions and methods for modulating SMN gene family expression
US10174323B2 (en) 2012-05-16 2019-01-08 The General Hospital Corporation Compositions and methods for modulating ATP2A2 expression
CA2873801A1 (en) 2012-05-16 2013-11-21 Rana Therapeutics Inc. Compositions and methods for modulating apoa1 and abca1 expression
US20150141320A1 (en) 2012-05-16 2015-05-21 Rana Therapeutics, Inc. Compositions and methods for modulating gene expression
AU2013262656A1 (en) 2012-05-16 2015-01-22 Rana Therapeutics, Inc. Compositions and methods for modulating UTRN expression
WO2013173601A1 (en) 2012-05-16 2013-11-21 Rana Therapeutics, Inc. Compositions and methods for modulating bdnf expression
DK2850189T3 (en) 2012-05-16 2019-02-25 Translate Bio Ma Inc COMPOSITIONS AND PROCEDURES FOR MODULATING GENEPRESSION
WO2013173608A1 (en) 2012-05-16 2013-11-21 Rana Therapeutics, Inc. Compositions and methods for modulating mecp2 expression
WO2013173605A1 (en) 2012-05-16 2013-11-21 Rana Therapeutics, Inc. Compositions and methods for modulating pten expression
AP2014008100A0 (en) 2012-05-16 2014-12-31 Gen Hospital Corp Compositions and methods for modulating hemoglobingene family expression
US9567581B2 (en) 2012-08-07 2017-02-14 The General Hospital Corporation Selective reactivation of genes on the inactive X chromosome
US20150247141A1 (en) 2012-09-14 2015-09-03 Rana Therapeutics, Inc. Multimeric oligonucleotide compounds
MY173826A (en) 2012-11-15 2020-02-24 Roche Innovation Ct Copenhagen As Oligonucleotide conjugates
WO2014172698A1 (en) 2013-04-19 2014-10-23 Isis Pharmaceuticals, Inc. Compositions and methods for modulation nucleic acids through nonsense mediated decay
AU2014274730A1 (en) 2013-06-07 2016-01-21 Rana Therapeutics, Inc. Compositions and methods for modulating FOXP3 expression
US10190117B2 (en) 2013-06-16 2019-01-29 National University Corporation Tokyo Medical And Dental University Double-stranded antisense nucleic acid with exon-skipping effect
CN105555279B (en) 2013-06-28 2019-12-06 伦敦健康科学中心研究公司 MicroRNA inhibition for treatment of sepsis
EP3033423A4 (en) 2013-08-16 2017-04-26 Rana Therapeutics Inc. Epigenetic regulators of frataxin
US20160201064A1 (en) 2013-08-16 2016-07-14 Rana Therapeutics, Inc. Compositions and methods for modulating expression of frataxin
WO2015035476A1 (en) 2013-09-16 2015-03-19 University Of Western Sydney Modulation of gene expression
US10174328B2 (en) 2013-10-04 2019-01-08 Translate Bio Ma, Inc. Compositions and methods for treating amyotrophic lateral sclerosis
US10822369B2 (en) 2014-11-14 2020-11-03 Ionis Pharmaceuticals, Inc. Compounds and methods for the modulation of proteins

Patent Citations (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3687808A (en) 1969-08-14 1972-08-29 Univ Leland Stanford Junior Synthetic polynucleotides
US4469863A (en) 1980-11-12 1984-09-04 Ts O Paul O P Nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof
US5023243A (en) 1981-10-23 1991-06-11 Molecular Biosystems, Inc. Oligonucleotide therapeutic agent and method of making same
US4476301A (en) 1982-04-29 1984-10-09 Centre National De La Recherche Scientifique Oligonucleotides, a process for preparing the same and their application as mediators of the action of interferon
US5550111A (en) 1984-07-11 1996-08-27 Temple University-Of The Commonwealth System Of Higher Education Dual action 2',5'-oligoadenylate antiviral derivatives and uses thereof
US5034506A (en) 1985-03-15 1991-07-23 Anti-Gene Development Group Uncharged morpholino-based polymers having achiral intersubunit linkages
US5264423A (en) 1987-03-25 1993-11-23 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5276019A (en) 1987-03-25 1994-01-04 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5286717A (en) 1987-03-25 1994-02-15 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5188897A (en) 1987-10-22 1993-02-23 Temple University Of The Commonwealth System Of Higher Education Encapsulated 2',5'-phosphorothioate oligoadenylates
US5405939A (en) 1987-10-22 1995-04-11 Temple University Of The Commonwealth System Of Higher Education 2',5'-phosphorothioate oligoadenylates and their covalent conjugates with polylysine
US5519126A (en) 1988-03-25 1996-05-21 University Of Virginia Alumni Patents Foundation Oligonucleotide N-alkylphosphoramidates
US5278302A (en) 1988-05-26 1994-01-11 University Patents, Inc. Polynucleotide phosphorodithioates
US5453496A (en) 1988-05-26 1995-09-26 University Patents, Inc. Polynucleotide phosphorodithioate
US5399676A (en) 1989-10-23 1995-03-21 Gilead Sciences Oligonucleotides with inverted polarity
US5455233A (en) 1989-11-30 1995-10-03 University Of North Carolina Oligoribonucleoside and oligodeoxyribonucleoside boranophosphates
US5563253A (en) 1990-03-08 1996-10-08 Worcester Foundation For Biomedical Research Linear aminoalkylphosphoramidate oligonucleotide derivatives
US5536821A (en) 1990-03-08 1996-07-16 Worcester Foundation For Biomedical Research Aminoalkylphosphorothioamidate oligonucleotide deratives
US5541306A (en) 1990-03-08 1996-07-30 Worcester Foundation For Biomedical Research Aminoalkylphosphotriester oligonucleotide derivatives
US5321131A (en) 1990-03-08 1994-06-14 Hybridon, Inc. Site-specific functionalization of oligodeoxynucleotides for non-radioactive labelling
US5177196A (en) 1990-08-16 1993-01-05 Microprobe Corporation Oligo (α-arabinofuranosyl nucleotides) and α-arabinofuranosyl precursors thereof
US5571799A (en) 1991-08-12 1996-11-05 Basco, Ltd. (2'-5') oligoadenylate analogues useful as inhibitors of host-v5.-graft response
US5587361A (en) 1991-10-15 1996-12-24 Isis Pharmaceuticals, Inc. Oligonucleotides having phosphorothioate linkages of high chiral purity
US5476925A (en) 1993-02-01 1995-12-19 Northwestern University Oligodeoxyribonucleotides including 3'-aminonucleoside-phosphoramidate linkages and terminal 3'-amino groups
US5466677A (en) 1993-03-06 1995-11-14 Ciba-Geigy Corporation Dinucleoside phosphinates and their pharmaceutical compositions
US5625050A (en) 1994-03-31 1997-04-29 Amgen Inc. Modified oligonucleotides and intermediates useful in nucleic acid therapeutics
WO2007115578A1 (en) * 2006-04-07 2007-10-18 Georg-August-Universität Göttingen Stiftung Öffentlichen Rechts Synthetic mecp2 sequence for protein substitution therapy
US20090082297A1 (en) * 2007-06-25 2009-03-26 Lioy Daniel T Compositions and Methods for Regulating Gene Expression

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
"Antisense Research and Applications", 1993, CRC PRESS, pages: 276 - 278
CHAHROUR, MARIA. ET AL.: "MeCP2, a key contributor to neurological disease, activates and represses transcription", SCIENCE, vol. 320, no. 5880, 30 May 2008 (2008-05-30), pages 1224 - 1229, XP055177820 *
DE MESMAEKER ET AL., ACE. CHEM. RES., vol. 28, 1995, pages 366 - 374
GEBEYEHU, G. ET AL., NUCL. ACIDS RES., vol. 15, 1987, pages 4513
KLEIN ET AL., NAT NEUROSCI., vol. 10, no. 12, 2007, pages 1513 - 4
KLEIN, MATTHEW E. ET AL.: "Homeostatic regulation of MeCP2 expression by a CREB-induced microRNA", NATURE NEUROSCIENCE, vol. 10, no. 12, 11 November 2007 (2007-11-11), pages 1513 - 1514, XP003028045 *
KORNBERG: "DNA Replication", 1980, W. H. FREEMAN & CO., pages: 75 - 77
NIELSEN ET AL., SCIENCE, vol. 254, 1991, pages 1497
WANG ET AL., J. AM. CHEM. SOC., vol. 122, 2000, pages 8595 - 8602

Cited By (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10358644B2 (en) 2010-11-12 2019-07-23 The General Hospital Corporation Polycomb-associated non-coding RNAs
US11066673B2 (en) 2010-11-12 2021-07-20 The General Hospital Corporation Polycomb-associated non-coding RNAs
US9328346B2 (en) 2010-11-12 2016-05-03 The General Hospital Corporation Polycomb-associated non-coding RNAs
US10119144B2 (en) 2010-11-12 2018-11-06 The General Hospital Corporation Polycomb-associated non-coding RNAs
US10053694B2 (en) 2010-11-12 2018-08-21 The General Hospital Corporation Polycomb-associated non-coding RNAS
US9816094B2 (en) 2010-11-12 2017-11-14 The General Hospital Corporation Polycomb-associated non-coding RNAs
US9856479B2 (en) 2010-11-12 2018-01-02 The General Hospital Corporation Polycomb-associated non-coding RNAs
US9920317B2 (en) 2010-11-12 2018-03-20 The General Hospital Corporation Polycomb-associated non-coding RNAs
US10174315B2 (en) 2012-05-16 2019-01-08 The General Hospital Corporation Compositions and methods for modulating hemoglobin gene family expression
US10655128B2 (en) 2012-05-16 2020-05-19 Translate Bio Ma, Inc. Compositions and methods for modulating MECP2 expression
US10058623B2 (en) 2012-05-16 2018-08-28 Translate Bio Ma, Inc. Compositions and methods for modulating UTRN expression
US10059941B2 (en) 2012-05-16 2018-08-28 Translate Bio Ma, Inc. Compositions and methods for modulating SMN gene family expression
US10174323B2 (en) 2012-05-16 2019-01-08 The General Hospital Corporation Compositions and methods for modulating ATP2A2 expression
US10837014B2 (en) 2012-05-16 2020-11-17 Translate Bio Ma, Inc. Compositions and methods for modulating SMN gene family expression
US11788089B2 (en) 2012-05-16 2023-10-17 The General Hospital Corporation Compositions and methods for modulating MECP2 expression
US9567581B2 (en) 2012-08-07 2017-02-14 The General Hospital Corporation Selective reactivation of genes on the inactive X chromosome
US10443052B2 (en) 2012-10-15 2019-10-15 Ionis Pharmaceuticals, Inc. Compositions for modulating C9ORF72 expression
US10577604B2 (en) 2012-10-15 2020-03-03 Ionis Pharmaceuticals, Inc. Methods for monitoring C9ORF72 expression
US10041074B2 (en) 2013-08-16 2018-08-07 Translate Bio Ma, Inc. Euchromatic region targeting methods for modulating gene expression
EP3033422A4 (en) * 2013-08-16 2017-08-02 Rana Therapeutics Inc. Oligonucleotides targeting euchromatin regions of genes
US10221414B2 (en) 2013-10-11 2019-03-05 Ionis Pharmaceuticals, Inc. Compositions for modulating C9ORF72 expression
US11339393B2 (en) 2013-10-11 2022-05-24 Ionis Pharmaceuticals, Inc. Compositions for modulating C9ORF72 expression
US11001841B2 (en) 2014-06-10 2021-05-11 The General Hospital Corporation CCCTC-binding factor (CTCF) RNA interactome
WO2015191780A3 (en) * 2014-06-10 2016-03-17 The General Hospital Corporation Ccctc-binding factor (ctcf) rna interactome
US10858650B2 (en) 2014-10-30 2020-12-08 The General Hospital Corporation Methods for modulating ATRX-dependent gene repression
US10758558B2 (en) 2015-02-13 2020-09-01 Translate Bio Ma, Inc. Hybrid oligonucleotides and uses thereof
US11129844B2 (en) 2015-03-03 2021-09-28 Ionis Pharmaceuticals, Inc. Compositions and methods for modulating MECP2 expression
WO2016141145A1 (en) * 2015-03-03 2016-09-09 Ionis Pharmaceuticals, Inc. Methods for modulating mecp2 expression
EP4056703A1 (en) * 2015-03-03 2022-09-14 Ionis Pharmaceuticals, Inc. Methods for modulating mecp2 expression
US10900036B2 (en) 2015-03-17 2021-01-26 The General Hospital Corporation RNA interactome of polycomb repressive complex 1 (PRC1)
US11260073B2 (en) 2015-11-02 2022-03-01 Ionis Pharmaceuticals, Inc. Compounds and methods for modulating C90RF72
WO2019048645A1 (en) 2017-09-08 2019-03-14 Mina Therapeutics Limited Stabilized cebpa sarna compositions and methods of use
WO2019048632A1 (en) 2017-09-08 2019-03-14 Mina Therapeutics Limited Stabilized hnf4a sarna compositions and methods of use
EP4183882A1 (en) 2017-09-08 2023-05-24 MiNA Therapeutics Limited Stabilized hnf4a sarna compositions and methods of use
EP4219715A2 (en) 2017-09-08 2023-08-02 MiNA Therapeutics Limited Stabilized cebpa sarna compositions and methods of use
WO2019195855A1 (en) * 2018-04-06 2019-10-10 Camp4 Therapeutics Corporation Treating diseases via targeted modulation of gene signaling networks
WO2021032777A1 (en) 2019-08-19 2021-02-25 Mina Therapeutics Limited Oligonucleotide conjugate compositions and methods of use

Also Published As

Publication number Publication date
AU2013262709A1 (en) 2015-01-22
US11788089B2 (en) 2023-10-17
CN104583402A (en) 2015-04-29
US20210102203A1 (en) 2021-04-08
EP2850190B1 (en) 2020-07-08
EP2850190A4 (en) 2016-02-10
EP2850190A1 (en) 2015-03-25
CA2873779A1 (en) 2013-11-21
US20180223282A1 (en) 2018-08-09
US20150152410A1 (en) 2015-06-04
EA201492116A1 (en) 2015-05-29
JP2015518712A (en) 2015-07-06
US10655128B2 (en) 2020-05-19

Similar Documents

Publication Publication Date Title
US11788089B2 (en) Compositions and methods for modulating MECP2 expression
US10058623B2 (en) Compositions and methods for modulating UTRN expression
US10174323B2 (en) Compositions and methods for modulating ATP2A2 expression
US10174315B2 (en) Compositions and methods for modulating hemoglobin gene family expression
WO2013173647A1 (en) Compositions and methods for modulating apoa1 and abca1 expression
CA2873794A1 (en) Compositions and methods for modulating smn gene family expression
WO2013173601A1 (en) Compositions and methods for modulating bdnf expression
WO2014197826A1 (en) Compositions and methods for modulating foxp3 expression
EP2850187A1 (en) Compositions and methods for modulating pten expression
EP2850183A1 (en) Compositions and methods for modulating gene expression
EP2850184A1 (en) Compositions and methods for modulating gene expression
WO2018031871A1 (en) Ex vivo modulation of foxp3 expression

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13791546

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2015512847

Country of ref document: JP

Kind code of ref document: A

Ref document number: 2873779

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 14401237

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2013791546

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 201492116

Country of ref document: EA

ENP Entry into the national phase

Ref document number: 2013262709

Country of ref document: AU

Date of ref document: 20130516

Kind code of ref document: A

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112014028641

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 112014028641

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20141117