WO2020038971A1 - Antisense oligonucleotides targeting vcan - Google Patents

Antisense oligonucleotides targeting vcan Download PDF

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Publication number
WO2020038971A1
WO2020038971A1 PCT/EP2019/072319 EP2019072319W WO2020038971A1 WO 2020038971 A1 WO2020038971 A1 WO 2020038971A1 EP 2019072319 W EP2019072319 W EP 2019072319W WO 2020038971 A1 WO2020038971 A1 WO 2020038971A1
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seq
oligonucleotide
nucleosides
region
vcan
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PCT/EP2019/072319
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French (fr)
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Nanna ALBÆK
Lykke PEDERSEN
Steffen Schmidt
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Roche Innovation Center Copenhagen A/S
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4725—Proteoglycans, e.g. aggreccan
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C12N2310/3341—5-Methylcytosine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===

Definitions

  • the present invention relates to antisense LNA oligonucleotides (oligomers) complementary to VCAN pre-mRNA intron sequences, which are capable of inhibiting the expression of VCAN. Inhibition of VCAN expression is beneficial for a range of medical disorders including inflammatory lung disease (such as chronic obstructive pulmonary disease or asthma), diabetic retinopathy, cancer and fibrotic disorders (such as fibrotic lung disease, cystic fibrosis, or pulmonary fibrosis).
  • inflammatory lung disease such as chronic obstructive pulmonary disease or asthma
  • diabetic retinopathy such as chronic obstructive pulmonary disease or asthma
  • fibrotic disorders such as fibrotic lung disease, cystic fibrosis, or pulmonary fibrosis.
  • VCAN (Versican) is a member of the aggrecan/versican proteoglycan family (other names: CSPG2, ERVR, GHAP, PG-M, WGN, WGN1 ).
  • the VCAN protein is a large chondroitin sulfate proteoglycan and is a major component of the extracellular matrix. This protein is involved in cell adhesion, proliferation, proliferation, migration and angiogenesis and plays a central role in tissue morphogenesis and maintenance. VCAN interacts with many proteins and molecules to facilitate the assembly of the extracellular matrix and ensure its stability.
  • the VCAN gene encodes four extracellular matrix isoforms which differ in the presence or length of a central glycosaminoglycan-binding region (Wight et al., 2002, Curr. Opin. Cell Biol. 14: 617-623).
  • VCAN has been associated with many diseases and disorders.
  • VCAN mutations are the cause of VCAN-related vitreoretinopathy.
  • At least 1 1 mutations in the VCAN gene have been found to cause Wagner syndrome, a condition that leads to progressive vision loss starting in childhood or early adulthood (see e.g. Rothschild et al.,
  • VCAN was suggested as a target in cancer therapy (Keire et al., Matrix Biol. 2016 Mar; 50: 67-81 ).
  • VCAN chronic obstructive pulmonary disease
  • CN103468681 discloses a siRNA which inhibits expression of a VCAN isoform for the treatment of COPD.
  • W02009/061368 discloses that siRNA molecules which target VCAN can be used to treat fibrotic disorders, arterial restenosis, atherosclerosis, cancer, fibrotic lung disease, cystic fibrosis, pulmonary fibrosis, or emphysema.
  • EP 2 078 728 A1 discloses that inhibitors of a VCAN isoform can be used in the treatment of breast cancer.
  • the inhibitor could be e.g. an antibody or an antisense nucleic acid molecule directed against the isoform.
  • W02008/029868 discloses that siRNAs which target VCAN can be used in the treatment of chorioretinal neovascularization diseases such as diabetic retinopathy.
  • the inventors have identified particularly effective regions of the VCAN transcript (VCAN) for antisense inhibition in vitro or in vivo, and provides for antisense oligonucleotides, including LNA gapmer oligonucleotides, which target these regions of the VCAN premRNA.
  • the present invention identifies oligonucleotides which inhibit human VCAN which are useful in the treatment of a range of medical disorders including inflammatory lung disease (such as chronic obstructive pulmonary disease or asthma), diabetic retinopathy, cancer and fibrotic disorders (such as fibrotic lung disease, cystic fibrosis, or pulmonary fibrosis).
  • the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, targeting a human VCAN target nucleic acid, wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN in a cell which is expressing human VCAN.
  • the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, targeting a human VCAN pre-mRNA target nucleic acid, wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN in a cell which is expressing human VCAN, and wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence of at least 10 nucleotides which are fully complementary to the human VCAN pre-mRNA target nucleic acid.
  • the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, targeting a human VCAN target nucleic acid, wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN in a cell which is expressing human VCAN; and wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence of at least 10 nucleotides which are fully complementary to an intron region of VCAN pre-mRNA target nucleic acid.
  • the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, targeting a human VCAN target nucleic acid, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully
  • the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, targeting a human VCAN target nucleic acid, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully
  • the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to SEQ ID NO 13 wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN in a cell which is expressing human VCAN.
  • the invention provides for an LNA antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to SEQ ID NO 13, wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN in a cell which is expressing human VCAN.
  • the invention provides for a gapmer antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to SEQ ID NO 13 wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN in a cell which is expressing human VCAN.
  • the invention provides for an LNA gapmer antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to SEQ ID NO 13 wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN in a cell which is expressing human VCAN.
  • the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to a sequence shown in SEQ ID NO 1 1 or in SEQ ID NO 12, wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN in a cell which is expressing human VCAN.
  • the invention provides for an LNA antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to a sequence shown in SEQ ID NO 1 1 or in SEQ ID NO 12, wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN in a cell which is expressing human VCAN.
  • the invention provides for a gapmer antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to a sequence shown in SEQ ID NO 1 1 or in SEQ ID NO 12, wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN in a cell which is expressing human VCAN.
  • the invention provides for an LNA gapmer antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to a sequence shown in SEQ ID NO 1 1 or in SEQ ID NO 12, wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN in a cell which is expressing human VCAN.
  • the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to SEQ ID NO 1 1 , wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN transcript in a cell which is expressing human VCAN transcript.
  • the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to SEQ ID NO 12, wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN transcript in a cell which is expressing human VCAN transcript.
  • oligonucleotide of the invention as referred to or claimed herein may be in the form of a pharmaceutically acceptable salt.
  • the invention provides for a conjugate comprising the oligonucleotide according to the invention, and at least one conjugate moiety covalently attached to said oligonucleotide.
  • the invention provides for a pharmaceutical composition
  • a pharmaceutical composition comprising the oligonucleotide or conjugate of the invention and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.
  • the invention provides for an in vivo or in vitro method for modulating VCAN expression in a target cell which is expressing VCAN, said method comprising administering an oligonucleotide or conjugate or pharmaceutical composition of the invention in an effective amount to said cell.
  • the invention provides for a method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide, conjugate or the pharmaceutical composition of the invention to a subject suffering from or susceptible to the disease.
  • the disease is selected from the group consisting of inflammatory lung disease (such as chronic obstructive pulmonary disease or asthma), diabetic retinopathy, cancer and fibrotic disorders (such as fibrotic lung disease, cystic fibrosis, or pulmonary fibrosis).
  • inflammatory lung disease such as chronic obstructive pulmonary disease or asthma
  • diabetic retinopathy such as diabetic retinopathy
  • cancer such as fibrotic disorders
  • fibrotic lung disease, cystic fibrosis, or pulmonary fibrosis such as chronic obstructive pulmonary disease or asthma
  • fibrotic disorders such as fibrotic lung disease, cystic fibrosis, or pulmonary fibrosis
  • the invention provides for the oligonucleotide, conjugate or the pharmaceutical composition of the invention for use in medicine.
  • the invention provides for the oligonucleotide, conjugate or the pharmaceutical composition of the invention for use in the treatment or prevention of a disease selected from the group consisting of inflammatory lung disease (such as chronic obstructive pulmonary disease or asthma), diabetic retinopathy, cancer and fibrotic disorders (such as fibrotic lung disease, cystic fibrosis, or pulmonary fibrosis).
  • a disease selected from the group consisting of inflammatory lung disease (such as chronic obstructive pulmonary disease or asthma), diabetic retinopathy, cancer and fibrotic disorders (such as fibrotic lung disease, cystic fibrosis, or pulmonary fibrosis).
  • the invention provides for the use of the oligonucleotide, conjugate or the pharmaceutical composition of the invention, for the preparation of a medicament for treatment or prevention of a disease selected from the group consisting of inflammatory lung disease (such as chronic obstructive pulmonary disease or asthma), diabetic retinopathy, cancer and fibrotic disorders (such as fibrotic lung disease, cystic fibrosis, or pulmonary fibrosis).
  • a disease selected from the group consisting of inflammatory lung disease (such as chronic obstructive pulmonary disease or asthma), diabetic retinopathy, cancer and fibrotic disorders (such as fibrotic lung disease, cystic fibrosis, or pulmonary fibrosis).
  • Figure 1 Testing in vitro efficacy of various antisense oligonucleotides targeting human VCAN mRNA in HEK293 and A549 cell lines at single concentration.
  • Figure 2 Comparison of in vitro efficacy for antisense oligonucleotides targeting human VCAN mRNA in HEK293 and A549 cell lines at single concentration shows good correlation. One motif with very efficient targeting is highlighted.
  • Figure 3 IC50 values for selected oligonucleotides targeting human VCAN mRNA in vitro in HEK293 and A549 cell lines.
  • Figure 4 Testing selected oligonucleotides targeting human VCAN mRNA in vitro for concentration dependent potency and efficacy in A549 cell line.
  • Figure 5 Testing selected oligonucleotides targeting human VCAN mRNA in vitro for concentration dependent potency and efficacy in HEK293 cell line.
  • oligonucleotide as used herein is defined as it is generally understood by the skilled person as a molecule comprising two or more covalently linked nucleosides. Such covalently bound nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are commonly made in the laboratory by solid-phase chemical synthesis followed by purification. When referring to a sequence of the oligonucleotide, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides.
  • the oligonucleotide of the invention is man-made, and is chemically synthesized, and is typically purified or isolated.
  • the oligonucleotide of the invention may comprise one or more modified nucleosides or nucleotides.
  • Antisense oligonucleotide as used herein is defined as oligonucleotides capable of modulating expression of a target gene by hybridizing to a target nucleic acid, in particular to a contiguous sequence on a target nucleic acid.
  • the antisense oligonucleotides are not essentially double stranded and are therefore not siRNAs or shRNAs.
  • the antisense oligonucleotides of the present invention are single stranded.
  • single stranded oligonucleotides of the present invention can form hairpins or intermolecular duplex structures (duplex between two molecules of the same oligonucleotide), as long as the degree of intra or inter self-complementarity is less than 50% across of the full length of the oligonucleotide
  • sequence refers to the region of the oligonucleotide which is complementary to the target nucleic acid.
  • the term is used interchangeably herein with the term“contiguous nucleobase sequence” and the term“oligonucleotide motif sequence”.
  • nucleotides of the oligonucleotide constitute the contiguous nucleotide sequence.
  • the oligonucleotide comprises the contiguous nucleotide sequence, such as a F-G-F’ gapmer region, and may optionally comprise further nucleotide(s), for example a nucleotide linker region which may be used to attach a functional group to the contiguous nucleotide sequence.
  • the nucleotide linker region may or may not be complementary to the target nucleic acid.
  • the contiguous nucleotide sequence is 100% complementary to the target nucleic acid.
  • Nucleotides are the building blocks of oligonucleotides and polynucleotides, and for the purposes of the present invention include both naturally occurring and non-naturally occurring nucleotides.
  • nucleotides such as DNA and RNA nucleotides comprise a ribose sugar moiety, a nucleobase moiety and one or more phosphate groups (which is absent in nucleosides).
  • Nucleosides and nucleotides may also interchangeably be referred to as“units” or“monomers”.
  • modified nucleoside or“nucleoside modification” as used herein refers to nucleosides modified as compared to the equivalent DNA or RNA nucleoside by the introduction of one or more modifications of the sugar moiety or the (nucleo)base moiety.
  • the modified nucleoside comprise a modified sugar moiety.
  • modified nucleoside may also be used herein interchangeably with the term“nucleoside analogue” or modified“units” or modified“monomers”.
  • Nucleosides with an unmodified DNA or RNA sugar moiety are termed DNA or RNA nucleosides herein. Nucleosides with modifications in the base region of the DNA or RNA nucleoside are still generally termed DNA or RNA if they allow Watson Crick base pairing.
  • modified internucleoside linkage is defined as generally understood by the skilled person as linkages other than phosphodiester (PO) linkages, that covalently couples two nucleosides together.
  • the oligonucleotides of the invention may therefore comprise modified internucleoside linkages.
  • the modified internucleoside linkage increases the nuclease resistance of the oligonucleotide compared to a phosphodiester linkage.
  • the internucleoside linkage includes phosphate groups creating a phosphodiester bond between adjacent nucleosides.
  • Modified internucleoside linkages are particularly useful in stabilizing oligonucleotides for in vivo use, and may serve to protect against nuclease cleavage at regions of DNA or RNA nucleosides in the oligonucleotide of the invention, for example within the gap region of a gapmer oligonucleotide, as well as in regions of modified nucleosides, such as region F and F’.
  • the oligonucleotide comprises one or more internucleoside linkages modified from the natural phosphodiester, such one or more modified internucleoside linkages that is for example more resistant to nuclease attack.
  • Nuclease resistance may be determined by incubating the oligonucleotide in blood serum or by using a nuclease resistance assay (e.g. snake venom phosphodiesterase (SVPD)), both are well known in the art.
  • SVPD snake venom phosphodiesterase
  • Internucleoside linkages which are capable of enhancing the nuclease resistance of an oligonucleotide are referred to as nuclease resistant internucleoside linkages.
  • At least 50% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof are modified, such as at least 60%, such as at least 70%, such as at least 80 or such as at least 90% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are nuclease resistant internucleoside linkages.
  • all of the internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof are nuclease resistant internucleoside linkages. It will be recognized that, in some embodiments the nucleosides which link the oligonucleotide of the invention to a non-nucleotide functional group, such as a conjugate, may be phosphodiester.
  • a preferred modified internucleoside linkage is phosphorothioate.
  • Phosphorothioate internucleoside linkages are particularly useful due to nuclease resistance, beneficial pharmacokinetics and ease of manufacture.
  • at least 50% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof are phosphorothioate, such as at least 60%, such as at least 70%, such as at least 80% or such as at least 90% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate.
  • all of the internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof are phosphorothioate.
  • Nuclease resistant linkages such as phosphorothioate linkages, are particularly useful in oligonucleotide regions capable of recruiting nuclease when forming a duplex with the target nucleic acid, such as region G for gapmers.
  • Phosphorothioate linkages may, however, also be useful in non-nuclease recruiting regions and/or affinity enhancing regions such as regions F and F’ for gapmers.
  • Gapmer oligonucleotides may, in some embodiments comprise one or more phosphodiester linkages in region F or F’, or both region F and F’, which the internucleoside linkage in region G may be fully phosphorothioate.
  • all the internucleoside linkages in the contiguous nucleotide sequence of the oligonucleotide are phosphorothioate linkages.
  • antisense oligonucleotide may comprise other internucleoside linkages (other than phosphodiester and phosphorothioate), for example alkyl phosphonate / methyl phosphonate internucleosides, which according to EP2 742 135 may for example be tolerated in an otherwise DNA phosphorothioate gap region.
  • nucleobase includes the purine (e.g. adenine and guanine) and pyrimidine (e.g. uracil, thymine and cytosine) moiety present in nucleosides and nucleotides which form hydrogen bonds in nucleic acid hybridization.
  • pyrimidine e.g. uracil, thymine and cytosine
  • nucleobase also encompasses modified nucleobases which may differ from naturally occurring nucleobases, but are functional during nucleic acid hybridization.
  • nucleobase refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, as well as non-naturally occurring variants. Such variants are for example described in Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid
  • the nucleobase moiety is modified by changing the purine or pyrimidine into a modified purine or pyrimidine, such as substituted purine or substituted pyrimidine, such as a nucleobased selected from isocytosine, pseudoisocytosine, 5-methyl cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil 5- thiazolo-uracil, 2-thio-uracil, 2’thio-thymine, inosine, diaminopurine, 6-aminopurine, 2- aminopurine, 2,6-diaminopurine and 2-chloro-6-aminopurine.
  • a nucleobased selected from isocytosine, pseudoisocytosine, 5-methyl cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromour
  • the nucleobase moieties may be indicated by the letter code for each corresponding nucleobase, e.g. A, T, G, C or U, wherein each letter may optionally include modified nucleobases of equivalent function.
  • the nucleobase moieties are selected from A, T, G, C, and 5-methyl cytosine.
  • 5-methyl cytosine LNA nucleosides may be used.
  • modified oligonucleotide describes an oligonucleotide comprising one or more sugar-modified nucleosides and/or modified internucleoside linkages.
  • chimeric oligonucleotide is a term that has been used in the literature to describe oligonucleotides with modified nucleosides.
  • Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A) - thymine (T)/uracil (U).
  • G guanine
  • A adenine
  • T thymine
  • U uracil
  • oligonucleotides may comprise nucleosides with modified nucleobases, for example 5-methyl cytosine is often used in place of cytosine, and as such the term complementarity encompasses Watson Crick base-paring between non-modified and modified nucleobases (see for example Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009)
  • % complementary refers to the number of nucleotides in percent of a contiguous nucleotide sequence in a nucleic acid molecule (e.g. oligonucleotide) which, at a given position, are complementary to (i.e. form Watson Crick base pairs with) a contiguous sequence of nucleotides, at a given position of a separate nucleic acid molecule (e.g. the target nucleic acid or target sequence).
  • a nucleic acid molecule e.g. oligonucleotide
  • the percentage is calculated by counting the number of aligned bases that form pairs between the two sequences (when aligned with the target sequence 5’-3’ and the oligonucleotide sequence from 3’-5’), dividing by the total number of nucleotides in the oligonucleotide and multiplying by 100. In such a comparison a nucleobase/nucleotide which does not align (form a base pair) is termed a mismatch.
  • insertions and deletions are not allowed in the calculation of % complementarity of a contiguous nucleotide sequence.
  • nucleic acid molecule refers to the proportion of nucleotides (expressed in percent) of a contiguous nucleotide sequence in a nucleic acid molecule (e.g.
  • oligonucleotide which across the contiguous nucleotide sequence, are identical to a reference sequence (e.g. a sequence motif).
  • nucleobases are disregarded as long as the functional capacity of the nucleobase to form Watson Crick base pairing is retained (e.g. 5-methyl cytosine is considered identical to a cytosine for the purpose of calculating % identity).
  • hybridizing or“hybridizes” as used herein is to be understood as two nucleic acid strands (e.g. an oligonucleotide and a target nucleic acid) forming hydrogen bonds between base pairs on opposite strands thereby forming a duplex.
  • the affinity of the binding between two nucleic acid strands is the strength of the hybridization. It is often described in terms of the melting temperature (T m ) defined as the temperature at which half of the oligonucleotides are duplexed with the target nucleic acid. At physiological conditions T m is not strictly proportional to the affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537).
  • AG° is the energy associated with a reaction where aqueous concentrations are 1 M, the pH is 7, and the temperature is 37°C.
  • the hybridization of oligonucleotides to a target nucleic acid is a spontaneous reaction and for spontaneous reactions AG° is less than zero.
  • AG° can be measured experimentally, for example, by use of the isothermal titration calorimetry (ITC) method as described in Hansen et al. , 1965, Chem. Comm. 36-38 and Holdgate et al. , 2005, Drug Discov Today. The skilled person will know that commercial equipment is available for AG° measurements.
  • ITC isothermal titration calorimetry
  • AG° can also be estimated numerically by using the nearest neighbor model as described by SantaLucia, 1998, Proc Natl Acad Sci USA. 95: 1460-1465 using appropriately derived thermodynamic parameters described by Sugimoto et al., 1995, Biochemistry 34:1 121 1-1 1216 and McTigue et al., 2004, Biochemistry 43:5388-5405.
  • oligonucleotides of the present invention hybridize to a target nucleic acid with estimated AG° values below -10 kcal for oligonucleotides that are 10-30 nucleotides in length.
  • the degree or strength of hybridization is measured by the standard state Gibbs free energy AG°.
  • the oligonucleotides may hybridize to a target nucleic acid with estimated AG° values below the range of -10 kcal, such as below -15 kcal, such as below - 20 kcal and such as below -25 kcal for oligonucleotides that are 8-30 nucleotides in length.
  • the oligonucleotides hybridize to a target nucleic acid with an estimated AG° value of -10 to -60 kcal, such as -12 to -40, such as from -15 to -30 kcal or- 16 to -27 kcal such as -18 to -25 kcal.
  • the target nucleic acid is a nucleic acid which encodes mammalian VCAN and may for example be a gene, a VCAN RNA, a mRNA, a pre-mRNA, a mature mRNA or a cDNA sequence.
  • the target may therefore be referred to as an VCAN target nucleic acid.
  • the target nucleic acid encodes an VCAN protein, in particular mammalian VCAN, such as the human VCAN gene encoding pre-mRNA or mRNA sequences provided herein as SEQ ID NO 13, 14, 15, 16 or 17.
  • VCAN protein in particular mammalian VCAN, such as the human VCAN gene encoding pre-mRNA or mRNA sequences provided herein as SEQ ID NO 13, 14, 15, 16 or 17.
  • the target nucleic acid is SEQ ID NO 13 or a naturally occurring variant thereof (e.g. VCAN sequences encoding a mammalian VCAN protein).
  • the target nucleic acid may be a cDNA or a synthetic nucleic acid derived from DNA or RNA.
  • the oligonucleotide of the invention is typically capable of inhibiting the expression of the VCAN target nucleic acid in a cell which is expressing the VCAN target nucleic acid.
  • the contiguous sequence of nucleobases of the oligonucleotide of the invention is typically complementary to the VCAN target nucleic acid, as measured across the length of the oligonucleotide, optionally with the exception of one or two mismatches, and optionally excluding nucleotide based linker regions which may link the oligonucleotide to an optional functional group such as a conjugate, or other noncomplementary terminal nucleotides (e.g. region D’ or D”).
  • the target nucleic acid is a messenger RNA, such as a mature mRNA or a pre-mRNA which encodes mammalian VCAN protein, such as human VCAN, e.g. the human VCAN pre-mRNA sequence, such as that disclosed as SEQ ID NO 13, or VCAN mature mRNA, such as that disclosed as SEQ ID NO 14, 15, 16 or 17.
  • SEQ ID NOs 13 - 17 are DNA sequences - it will be understood that target RNA sequences have uracil (U) bases in place of the thymidine bases (T).
  • the oligonucleotide of the invention targets SEQ ID NO 13.
  • the oligonucleotide of the invention targets SEQ ID NO 14.
  • the oligonucleotide of the invention targets SEQ ID NO 15.
  • the oligonucleotide of the invention targets SEQ ID NO 16.
  • the oligonucleotide of the invention targets SEQ ID NO 17.
  • target sequence refers to a sequence of nucleotides present in the target nucleic acid which comprises the nucleobase sequence which is complementary to the oligonucleotide of the invention.
  • the target sequence consists of a region on the target nucleic acid which is complementary to the contiguous nucleotide sequence of the oligonucleotide of the invention.
  • target sequence regions as defined by regions of the human VCAN pre-mRNA (using SEQ ID NO 13 as a reference) which may be targeted by the oligonucleotides of the invention.
  • the target sequence is longer than the complementary sequence of a single oligonucleotide, and may, for example represent a preferred region of the target nucleic acid which may be targeted by several oligonucleotides of the invention.
  • the oligonucleotide of the invention comprises a contiguous nucleotide sequence which is complementary to or hybridizes to the target nucleic acid, such as a sub-sequence of the target nucleic acid, such as a target sequence described herein.
  • the oligonucleotide comprises a contiguous nucleotide sequence which are complementary to a target sequence present in the target nucleic acid molecule.
  • the contiguous nucleotide sequence (and therefore the target sequence) comprises of at least 10 contiguous nucleotides, such as 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous nucleotides, such as from 12-25, such as from 14-18 contiguous nucleotides.
  • the inventors have identified particularly effective sequences of the VCAN target nucleic acid which may be targeted by the oligonucleotide of the invention.
  • the target sequence is SEQ ID NO 1 1.
  • the target sequence is SEQ ID NO 12.
  • the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to an exon region of SEQ ID NO 13, selected from the group consisting if Ex_1 - Ex_15 (see the following table).
  • the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to a region of SEQ ID NO 13, selected from the group consisting of 1 - 559; 12049 - 12124; 18634 - 19008; 22082 - 22256; 22340 - 22467; 40639 - 40932; 47885 - 50845; 65543 - 70804; 74073 - 74186; 76507 - 76620; 81900 - 82058; 83492 - 83574; 100952 - 101096; 108516 - 108698; and 108843 - 1 10839.
  • the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to an intron region of SEQ ID NO 13, selected from the group consisting of lnt_1 - lnt_14 (see the following table)
  • the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to a region of SEQ ID NO 13, selected from the group consisting of 559 - 12049; 12124 - 18634; 19008 - 22082; 22256 - 22340; 22467 - 40639; 40932 - 47885; 50845 - 65543; 70804 - 74073;
  • the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to a region of SEQ ID NO 13, selected from the group consisting of 1 - 138, 140 - 161 , 163 - 218, 220 - 348, 350 - 447.457 - 555.568 - 598.601 - 619.629 - 643.643 - 672.674 - 730.732 - 750.752 -
  • the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to a region of SEQ ID NO 13, selected from the group consisting of 174 - 200, 191 - 214, 213 - 261 , 265 - 320, 569 - 584, 588 - 607, 675 - 692, 925 - 942, 953 - 973, 1087 - 1 101 , 1261 - 1275, 1316 - 1333, 1596 - 161 1 , 1830 - 1855, 1988 - 2004, 2010 - 2025, 2070 - 2086, 2092 - 21 13, 2161 2178, 3152 - 3177, 4271 - 4293, 4296
  • a“target cell” as used herein refers to a cell which is expressing the target nucleic acid.
  • the target cell may be in vivo or in vitro.
  • the target cell is a mammalian cell such as a rodent cell, such as a mouse cell or a rat cell, or a primate cell such as a monkey cell or a human cell.
  • the target cell expresses VCAN mRNA, such as the VCAN pre- mRNA, e.g. SEQ ID NO 13, or VCAN mature mRNA(e.g. SEQ ID NO 14, 15, 16 or 17).
  • VCAN mRNA such as the VCAN pre- mRNA, e.g. SEQ ID NO 13, or VCAN mature mRNA(e.g. SEQ ID NO 14, 15, 16 or 17).
  • the poly A tail of VCAN mRNA is typically disregarded for antisense oligonucleotide targeting.
  • naturally occurring variant refers to variants of VCAN gene or transcripts which originate from the same genetic loci as the target nucleic acid, but may differ for example, by virtue of degeneracy of the genetic code causing a multiplicity of codons encoding the same amino acid, or due to alternative splicing of pre-mRNA, or the presence of polymorphisms, such as single nucleotide polymorphisms (SNPs), and allelic variants. Based on the presence of the sufficient complementary sequence to the oligonucleotide, the
  • oligonucleotide of the invention may therefore target the target nucleic acid and naturally occurring variants thereof.
  • the homo sapiens VCAN gene is located at chromosome 5, 83471674..83582303, complement (NC_000005.10, Gene ID 1462).
  • the naturally occurring variants have at least 95% such as at least 98% or at least 99% homology to a mammalian VCAN target nucleic acid, such as a target nucleic acid selected form the group consisting of SEQ ID NO 13, 14, 15, 16 or 17. In some embodiments the naturally occurring variants have at least 99% homology to the human VCAN target nucleic acid of SEQ ID NO 13.
  • modulation of expression is to be understood as an overall term for an oligonucleotide’s ability to alter the amount of VCAN protein or VCAN mRNA when compared to the amount of VCAN or VCAN mRNA prior to administration of the
  • oligonucleotide Alternatively modulation of expression may be determined by reference to a control experiment. It is generally understood that the control is an individual or target cell treated with a saline composition or an individual or target cell treated with a non-targeting oligonucleotide (mock).
  • One type of modulation is an oligonucleotide’s ability to inhibit, down-regulate, reduce, suppress, remove, stop, block, prevent, lessen, lower, avoid or terminate expression of VCAN, e.g. by degradation of VCAN mRNA.
  • a high affinity modified nucleoside is a modified nucleotide which, when incorporated into the oligonucleotide enhances the affinity of the oligonucleotide for its complementary target, for example as measured by the melting temperature (T m ).
  • a high affinity modified nucleoside of the present invention preferably result in an increase in melting temperature between +0.5 to + 12°C, more preferably between +1.5 to +10°C and most preferably between+3 to +8°C per modified nucleoside.
  • Numerous high affinity modified nucleosides are known in the art and include for example, many 2’ substituted nucleosides as well as locked nucleic acids (LNA) (see e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr.
  • the oligomer of the invention may comprise one or more nucleosides which have a modified sugar moiety, i.e. a modification of the sugar moiety when compared to the ribose sugar moiety found in DNA and RNA.
  • nucleosides with modification of the ribose sugar moiety have been made, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and/or nuclease resistance.
  • modifications include those where the ribose ring structure is modified, e.g. by replacement with a hexose ring (HNA), or a bicyclic ring, which typically have a biradicle bridge between the C2 and C4 carbons on the ribose ring (LNA), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e.g. UNA).
  • HNA hexose ring
  • LNA bicyclic ring
  • UNA unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons
  • sugar modified nucleosides include, for example, bicyclohexose nucleic acids (WO201 1/017521 ) or tricyclic nucleic acids (WO2013/154798). Modified nucleosides also include nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example in the case of peptide nucleic acids (PNA), or morpholino nucleic acids.
  • PNA peptide nucleic acids
  • Sugar modifications also include modifications made via altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2’-OH group naturally found in DNA and RNA nucleosides. Substituents may, for example be introduced at the 2’, 3’, 4’ or 5’ positions.
  • a 2’ sugar modified nucleoside is a nucleoside which has a substituent other than H or -OH at the 2’ position (2’ substituted nucleoside) or comprises a 2’ linked biradicle capable of forming a bridge between the 2’ carbon and a second carbon in the ribose ring, such as LNA (2’ - 4’ biradicle bridged) nucleosides.
  • the 2’ modified sugar may provide enhanced binding affinity and/or increased nuclease resistance to the oligonucleotide.
  • 2’ substituted modified nucleosides are 2’-0-alkyl-RNA, 2’-0-methyl-RNA, 2’- alkoxy-RNA, 2’-0-methoxyethyl-RNA (MOE), 2’-amino-DNA, 2’-Fluoro-RNA, and 2’-F-ANA nucleoside.
  • MOE methoxyethyl-RNA
  • 2’-amino-DNA 2’-Fluoro-RNA
  • 2’-F-ANA nucleoside please see e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and
  • 2’ substituted does not include 2’ bridged molecules like LNA.
  • LNA Locked Nucleic Acids
  • A“LNA nucleoside” is a 2’- modified nucleoside which comprises a biradical linking the
  • nucleosides are also termed bridged nucleic acid or bicyclic nucleic acid (BNA) in the literature.
  • BNA bicyclic nucleic acid
  • the locking of the conformation of the ribose is associated with an enhanced affinity of hybridization (duplex stabilization) when the LNA is incorporated into an oligonucleotide for a complementary RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the oligonucleotide/complement duplex.
  • Non limiting, exemplary LNA nucleosides are disclosed in WO 99/014226, WO 00/66604, WO 98/039352 , WO 2004/046160, WO 00/047599, WO 2007/134181 , WO 2010/077578, WO 2010/036698, WO 2007/090071 , WO 2009/006478, WO 201 1/156202,
  • LNA nucleosides are beta-D-oxy-LNA, 6’-methyl-beta-D-oxy LNA such as (S)-6’-methyl-beta-D-oxy-LNA (ScET) and ENA.
  • a particularly advantageous LNA is beta-D-oxy-LNA.
  • the RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule.
  • WO01/23613 provides in vitro methods for determining RNaseH activity, which may be used to determine the ability to recruit RNaseH.
  • an oligonucleotide is deemed capable of recruiting RNase H if it, when provided with a complementary target nucleic acid sequence, has an initial rate, as measured in pmol/l/min, of at least 5%, such as at least 10% or more than 20% of the of the initial rate determined when using a oligonucleotide having the same base sequence as the modified oligonucleotide being tested, but containing only DNA monomers with
  • the antisense oligonucleotide of the invention, or contiguous nucleotide sequence thereof may be a gapmer.
  • the antisense gapmers are commonly used to inhibit a target nucleic acid via RNase H mediated degradation.
  • a gapmer oligonucleotide comprises at least three distinct structural regions a 5’-flank, a gap and a 3’-flank, F-G-F’ in the‘5 -> 3’ orientation.
  • The“gap” region (G) comprises a stretch of contiguous DNA nucleotides which enable the oligonucleotide to recruit RNase H.
  • the gap region is flanked by a 5’ flanking region (F) comprising one or more sugar modified nucleosides, advantageously high affinity sugar modified nucleosides, and by a 3’ flanking region (F’) comprising one or more sugar modified nucleosides, advantageously high affinity sugar modified nucleosides.
  • the one or more sugar modified nucleosides in region F and F’ enhance the affinity of the oligonucleotide for the target nucleic acid (i.e. are affinity enhancing sugar modified nucleosides).
  • the one or more sugar modified nucleosides in region F and F’ are 2’ sugar modified nucleosides, such as high affinity 2’ sugar modifications, such as independently selected from LNA and 2’-MOE.
  • the 5’ and 3’ most nucleosides of the gap region are DNA nucleosides, and are positioned adjacent to a sugar modified nucleoside of the 5’ (F) or 3’ (F’) region respectively.
  • the flanks may further defined by having at least one sugar modified nucleoside at the end most distant from the gap region, i.e. at the 5’ end of the 5’ flank and at the 3’ end of the 3’ flank.
  • Regions F-G-F’ form a contiguous nucleotide sequence.
  • Antisense oligonucleotides of the invention, or the contiguous nucleotide sequence thereof, may comprise a gapmer region of formula F-G-F’.
  • the overall length of the gapmer design F-G-F’ may be, for example 12 to 32 nucleosides, such as 13 to 24, such as 14 to 22 nucleosides, Such as from 14 to17, such as 16 to18 nucleosides.
  • the gapmer oligonucleotide of the present invention can be represented by the following formulae:
  • the overall length of the gapmer regions F-G-F’ is at least 12, such as at least 14 nucleotides in length.
  • Regions F, G and F’ are further defined below and can be incorporated into the F-G-F’ formula. Gapmer - Region G
  • Region G is a region of nucleosides which enables the oligonucleotide to recruit RNaseH, such as human RNase H1 , typically DNA nucleosides.
  • RNaseH is a cellular enzyme which recognizes the duplex between DNA and RNA, and enzymatically cleaves the RNA molecule.
  • gapmers may have a gap region (G) of at least 5 or 6 contiguous DNA nucleosides, such as 5 - 16 contiguous DNA nucleosides, such as 6 - 15 contiguous DNA nucleosides, such as 7-14 contiguous DNA nucleosides, such as 8 - 12 contiguous DNA nucleotides, such as 8 - 12 contiguous DNA nucleotides in length.
  • the gap region G may, in some embodiments consist of 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 contiguous DNA nucleosides.
  • One or more cytosine (C) DNA in the gap region may in some instances be methylated (e.g.
  • the gap region G may consist of 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 contiguous phosphorothioate linked DNA nucleosides. In some embodiments, all internucleoside linkages in the gap are phosphorothioate linkages.
  • Modified nucleosides which allow for RNaseH recruitment when they are used within the gap region include, for example, alpha-L-LNA, C4’ alkylated DNA (as described in PCT/EP2009/050349 and Vester et al., Bioorg. Med. Chem. Lett. 18 (2008) 2296 - 2300, both incorporated herein by reference), arabinose derived nucleosides like ANA and 2'F-ANA (Mangos et al. 2003 J. AM. CHEM. SOC.
  • UNA locked nucleic acid
  • the modified nucleosides used in such gapmers may be nucleosides which adopt a 2’ endo (DNA like) structure when introduced into the gap region, i.e. modifications which allow for RNaseH recruitment).
  • the DNA Gap region (G) described herein may optionally contain 1 to 3 sugar modified nucleosides which adopt a 2’ endo (DNA like) structure when introduced into the gap region.
  • gapmers with a gap region comprising one or more 3’endo modified nucleosides are referred to as“gap-breaker” or“gap-disrupted” gapmers, see for example WO2013/022984.
  • Gap-breaker oligonucleotides retain sufficient region of DNA nucleosides within the gap region to allow for RNaseH recruitment. The ability of gapbreaker
  • oligonucleotide design to recruit RNaseH is typically sequence or even compound specific - see Rukov et al. 2015 Nucl. Acids Res. Vol. 43 pp. 8476-8487, which discloses“gapbreaker” oligonucleotides which recruit RNaseH which in some instances provide a more specific cleavage of the target RNA.
  • Modified nucleosides used within the gap region of gap- breaker oligonucleotides may for example be modified nucleosides which confer a 3’endo confirmation, such 2’ -O-methyl (OMe) or 2’-0-MOE (MOE) nucleosides, or beta-D LNA nucleosides (the bridge between C2’ and C4’ of the ribose sugar ring of a nucleoside is in the beta conformation), such as beta-D-oxy LNA or ScET nucleosides.
  • 2’ -O-methyl (OMe) or 2’-0-MOE (MOE) nucleosides or beta-D LNA nucleosides (the bridge between C2’ and C4’ of the ribose sugar ring of a nucleoside is in the beta conformation), such as beta-D-oxy LNA or ScET nucleosides.
  • the gap region of gap-breaker or gap-disrupted gapmers have a DNA nucleosides at the 5’ end of the gap (adjacent to the 3’ nucleoside of region F), and a DNA nucleoside at the 3’ end of the gap (adjacent to the 5’ nucleoside of region F’).
  • Gapmers which comprise a disrupted gap typically retain a region of at least 3 or 4 contiguous DNA nucleosides at either the 5’ end or 3’ end of the gap region.
  • Exemplary designs for gap-breaker oligonucleotides include
  • region G is within the brackets [D n -E r - D m ], D is a contiguous sequence of DNA nucleosides, E is a modified nucleoside (the gap-breaker or gap-disrupting nucleoside), and F and F’ are the flanking regions as defined herein, and with the proviso that the overall length of the gapmer regions F-G-F’ is at least 12, such as at least 14 nucleotides in length.
  • region G of a gap disrupted gapmer comprises at least 6 DNA nucleosides, such as 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 DNA nucleosides.
  • the DNA nucleosides may be contiguous or may optionally be interspersed with one or more modified nucleosides, with the proviso that the gap region G is capable of mediating RNaseH recruitment.
  • Region F is positioned immediately adjacent to the 5’ DNA nucleoside of region G.
  • the 3’ most nucleoside of region F is a sugar modified nucleoside, such as a high affinity sugar modified nucleoside, for example a 2’ substituted nucleoside, such as a MOE nucleoside, or an LNA nucleoside.
  • Region F’ is positioned immediately adjacent to the 3’ DNA nucleoside of region G.
  • the 5’ most nucleoside of region F’ is a sugar modified nucleoside, such as a high affinity sugar modified nucleoside, for example a 2’ substituted nucleoside, such as a MOE nucleoside, or an LNA nucleoside.
  • Region F is 1 - 8 contiguous nucleotides in length, such as 2-6, such as 3-4 contiguous nucleotides in length.
  • the 5’ most nucleoside of region F is a sugar modified nucleoside.
  • the two 5’ most nucleoside of region F are sugar modified nucleoside.
  • the 5’ most nucleoside of region F is an LNA nucleoside.
  • the two 5’ most nucleoside of region F are LNA nucleosides.
  • the two 5’ most nucleoside of region F are 2’ substituted nucleoside nucleosides, such as two 3’ MOE nucleosides.
  • the 5’ most nucleoside of region F is a 2’ substituted nucleoside, such as a MOE nucleoside.
  • Region F’ is 2 - 8 contiguous nucleotides in length, such as 3-6, such as 4-5 contiguous nucleotides in length.
  • the 3’ most nucleoside of region F’ is a sugar modified nucleoside.
  • the two 3’ most nucleoside of region F’ are sugar modified nucleoside.
  • the two 3’ most nucleoside of region F’ are LNA nucleosides.
  • the 3’ most nucleoside of region F’ is an LNA nucleoside.
  • the two 3’ most nucleoside of region F’ are 2’ substituted nucleoside nucleosides, such as two 3’ MOE nucleosides.
  • the 3’ most nucleoside of region F’ is a 2’ substituted nucleoside, such as a MOE nucleoside. It should be noted that when the length of region F or F’ is one, it is advantageously an LNA nucleoside.
  • region F and F’ independently consists of or comprises a contiguous sequence of sugar modified nucleosides.
  • the sugar modified nucleosides of region F may be independently selected from 2’-0-alkyl-RNA units, 2’-0- methyl-RNA, 2’-amino-DNA units, 2’-fluoro-DNA units, 2’-alkoxy-RNA, MOE units, LNA units, arabino nucleic acid (ANA) units and 2’-fluoro-ANA units.
  • region F and F’ independently comprises both LNA and a 2’ substituted modified nucleosides (mixed wing design).
  • region F and F’ consists of only one type of sugar modified nucleosides, such as only MOE or only beta-D-oxy LNA or only ScET. Such designs are also termed uniform flanks or uniform gapmer design.
  • all the nucleosides of region F or F’, or F and F’ are LNA
  • region F consists of 1-5, such as 2-4, such as 3-4 such as 1 , 2, 3, 4 or 5 contiguous LNA nucleosides.
  • all the nucleosides of region F and F’ are beta-D-oxy LNA nucleosides.
  • all the nucleosides of region F or F’, or F and F’ are 2’ substituted nucleosides, such as OMe or MOE nucleosides.
  • region F consists of 1 , 2, 3, 4, 5, 6, 7, or 8 contiguous OMe or MOE nucleosides.
  • flanking regions can consist of 2’ substituted nucleosides, such as OMe or MOE nucleosides. In some embodiments it is the 5’ (F) flanking region that consists 2’ substituted nucleosides, such as OMe or MOE nucleosides whereas the 3’ (F’) flanking region comprises at least one LNA nucleoside, such as beta-D-oxy LNA nucleosides or cET nucleosides.
  • LNA nucleoside such as beta-D-oxy LNA nucleosides or cET nucleosides.
  • the 3’ (F’) flanking region that consists 2’ substituted nucleosides, such as OMe or MOE nucleosides whereas the 5’ (F) flanking region comprises at least one LNA nucleoside, such as beta-D-oxy LNA nucleosides or cET nucleosides.
  • all the modified nucleosides of region F and F’ are LNA nucleosides, such as independently selected from beta-D-oxy LNA, ENA or ScET nucleosides, wherein region F or F’, or F and F’ may optionally comprise DNA nucleosides (an alternating flank, see definition of these for more details).
  • all the modified nucleosides of region F and F’ are beta-D-oxy LNA nucleosides, wherein region F or F’, or F and F’ may optionally comprise DNA nucleosides (an alternating flank, see definition of these for more details).
  • the 5’ most and the 3’ most nucleosides of region F and F’ are LNA nucleosides, such as beta-D-oxy LNA nucleosides or ScET nucleosides.
  • the internucleoside linkage between region F and region G is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage between region F’ and region G is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkages between the nucleosides of region F or F’, F and F’ are phosphorothioate internucleoside linkages.
  • An LNA gapmer is a gapmer wherein either one or both of region F and F’ comprises or consists of LNA nucleosides.
  • a beta-D-oxy gapmer is a gapmer wherein either one or both of region F and F’ comprises or consists of beta-D-oxy LNA nucleosides.
  • the LNA gapmer is of formula: [LNA ⁇ .s-tregion G] -[LNA] ⁇ , wherein region G is as defined in the Gapmer region G definition.
  • a MOE gapmers is a gapmer wherein regions F and F’ consist of MOE nucleosides.
  • the MOE gapmer is of design [MOEJ ⁇ s-lRegion G]-[MOE] 1-8 , such as [MOE] 2.7 -[Region G] 5 -ie-[MOE] 2- 7, such as [MOE] 3.6 -[Region G]-[MOE] 3-6 , wherein region G is as defined in the Gapmer definition.
  • MOE gapmers with a 5-10-5 design have been widely used in the art.
  • a mixed wing gapmer is an LNA gapmer wherein one or both of region F and F’ comprise a 2’ substituted nucleoside, such as a 2’ substituted nucleoside independently selected from the group consisting of 2’-0-alkyl-RNA units, 2’-0-methyl-RNA, 2’-amino-DNA units, 2’- fluoro-DNA units, 2’-alkoxy-RNA, MOE units, arabino nucleic acid (ANA) units and 2’-fluoro- ANA units, such as a MOE nucleosides.
  • a 2’ substituted nucleoside independently selected from the group consisting of 2’-0-alkyl-RNA units, 2’-0-methyl-RNA, 2’-amino-DNA units, 2’- fluoro-DNA units, 2’-alkoxy-RNA, MOE units, arabino nucleic acid (ANA) units and 2’-fluoro- ANA units, such as a MOE nucleosides.
  • region F and F’, or both region F and F’ comprise at least one LNA nucleoside
  • the remaining nucleosides of region F and F’ are independently selected from the group consisting of MOE and LNA.
  • at least one of region F and F’, or both region F and F’ comprise at least two LNA nucleosides
  • the remaining nucleosides of region F and F’ are independently selected from the group consisting of MOE and LNA.
  • one or both of region F and F’ may further comprise one or more DNA nucleosides.
  • Oligonucleotides with alternating flanks are LNA gapmer oligonucleotides where at least one of the flanks (F or F’) comprises DNA in addition to the LNA nucleoside(s).
  • at least one of region F or F’, or both region F and F’ comprise both LNA nucleosides and DNA nucleosides.
  • the flanking region F or F’, or both F and F’ comprise at least three nucleosides, wherein the 5’ and 3’ most nucleosides of the F and/or F’ region are LNA nucleosides.
  • region F or F’, or both region F and F’ comprise both LNA nucleosides and DNA nucleosides.
  • the flanking region F or F’, or both F and F’ comprise at least three nucleosides, wherein the 5’ and 3’ most nucleosides of the F or F’ region are LNA nucleosides, and there is at least one DNA nucleoside positioned between the 5’ and 3’ most LNA nucleosides of region F or F’ (or both region F and F’).
  • the oligonucleotide of the invention may in some embodiments comprise or consist of the contiguous nucleotide sequence of the oligonucleotide which is complementary to the target nucleic acid, such as the gapmer F-G-F’, and further 5’ and/or 3’ nucleosides.
  • the further 5’ and/or 3’ nucleosides may or may not be fully complementary to the target nucleic acid.
  • nucleosides may be referred to as region D’ and D” herein.
  • region D’ or D may be used for the purpose of joining the contiguous nucleotide sequence, such as the gapmer, to a conjugate moiety or another functional group.
  • region D may be used for joining the contiguous nucleotide sequence with a conjugate moiety.
  • a conjugate moiety is can serve as a biocleavable linker. Alternatively it may be used to provide exonucleoase protection or for ease of synthesis or manufacture.
  • Region D’ and D can be attached to the 5’ end of region F or the 3’ end of region F’, respectively to generate designs of the following formulas D’-F-G-F’, F-G-F’-D” or
  • F-G-F’ is the gapmer portion of the oligonucleotide and region D’ or D” constitute a separate part of the oligonucleotide.
  • Region D’ or D may independently comprise or consist of 1 , 2, 3, 4 or 5 additional nucleotides, which may be complementary or non-complementary to the target nucleic acid.
  • the nucleotide adjacent to the F or F’ region is not a sugar-modified nucleotide, such as a DNA or RNA or base modified versions of these.
  • the D’ or D’ region may serve as a nuclease susceptible biocleavable linker (see definition of linkers).
  • the additional 5’ and/or 3’ end nucleotides are linked with phosphodiester linkages, and are DNA or RNA.
  • Nucleotide based biocleavable linkers suitable for use as region D’ or D are disclosed in WO2014/076195, which include by way of example a phosphodiester linked DNA dinucleotide.
  • the use of biocleavable linkers in poly-oligonucleotide constructs is disclosed in WO2015/1 13922, where they are used to link multiple antisense constructs (e.g. gapmer regions) within a single oligonucleotide.
  • the oligonucleotide of the invention comprises a region D’ and/or D” in addition to the contiguous nucleotide sequence which constitutes the gapmer.
  • the oligonucleotide of the present invention can be represented by the following formulae:
  • D’-F-G-F’ in particular D’i- 3 -Fi- 8 -G 5 -i6-F’2-8
  • the internucleoside linkage positioned between region D’ and region F is a phosphodiester linkage. In some embodiments the internucleoside linkage positioned between region F’ and region D” is a phosphodiester linkage.
  • conjugate refers to an oligonucleotide which is covalently linked to a non-nucleotide moiety (conjugate moiety or region C or third region).
  • Conjugation of the oligonucleotide of the invention to one or more non-nucleotide moieties may improve the pharmacology of the oligonucleotide, e.g. by affecting the activity, cellular distribution, cellular uptake or stability of the oligonucleotide.
  • the conjugate moiety modify or enhance the pharmacokinetic properties of the oligonucleotide by improving cellular distribution, bioavailability, metabolism, excretion, permeability, and/or cellular uptake of the oligonucleotide.
  • the conjugate may target the oligonucleotide to a specific organ, tissue or cell type and thereby enhance the effectiveness of the oligonucleotide in that organ, tissue or cell type.
  • the conjugate may serve to reduce activity of the oligonucleotide in non-target cell types, tissues or organs, e.g. off target activity or activity in non-target cell types, tissues or organs.
  • the non-nucleotide moiety is selected from the group consisting of carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g. bacterial toxins), vitamins, viral proteins (e.g. capsids) or combinations thereof.
  • a linkage or linker is a connection between two atoms that links one chemical group or segment of interest to another chemical group or segment of interest via one or more covalent bonds.
  • Conjugate moieties can be attached to the oligonucleotide directly or through a linking moiety (e.g. linker or tether).
  • Linkers serve to covalently connect a third region, e.g. a conjugate moiety (Region C), to a first region, e.g. an oligonucleotide or contiguous nucleotide sequence or gapmer region F-G-F’ (region A).
  • the conjugate or oligonucleotide conjugate of the invention may optionally, comprise a linker region (second region or region B and/or region Y) which is positioned between the oligonucleotide or contiguous nucleotide sequence complementary to the target nucleic acid (region A or first region) and the conjugate moiety (region C or third region).
  • a linker region second region or region B and/or region Y
  • Region B refers to biocleavable linkers comprising or consisting of a physiologically labile bond that is cleavable under conditions normally encountered or analogous to those encountered within a mammalian body.
  • Conditions under which physiologically labile linkers undergo chemical transformation include chemical conditions such as pH, temperature, oxidative or reductive conditions or agents, and salt concentration found in or analogous to those encountered in mammalian cells.
  • Mammalian intracellular conditions also include the presence of enzymatic activity normally present in a mammalian cell such as from proteolytic enzymes or hydrolytic enzymes or nucleases.
  • the biocleavable linker is susceptible to S1 nuclease cleavage.
  • DNA phosphodiester containing biocleavable linkers are described in more detail in WO 2014/076195 (hereby incorporated by reference) - see also region D’ or D” herein.
  • Region Y refers to linkers that are not necessarily biocleavable but primarily serve to covalently connect a conjugate moiety (region C or third region), to an oligonucleotide (region A or first region).
  • the region Y linkers may comprise a chain structure or an oligomer of repeating units such as ethylene glycol, amino acid units or amino alkyl groups.
  • the oligonucleotide conjugates of the present invention can be constructed of the following regional elements A-C, A-B-C, A-B-Y-C, A-Y-B-C or A-Y-C.
  • the linker (region Y) is an amino alkyl, such as a C2 - C36 amino alkyl group, including, for example C6 to C12 amino alkyl groups. In a preferred embodiment the linker (region Y) is a C6 amino alkyl group.
  • treatment refers to both treatment of an existing disease ⁇ e.g. a disease or disorder as herein referred to), or prevention of a disease, i.e. prophylaxis. It will therefore be recognized that treatment as referred to herein may, in some embodiments, be prophylactic.
  • the invention relates to oligonucleotides, such as antisense oligonucleotides, targeting VCAN expression.
  • the oligonucleotides of the invention targeting VCAN are capable of hybridizing to and inhibiting the expression of a VCAN target nucleic acid in a cell which is expressing the VCAN target nucleic acid.
  • the VCAN target nucleic acid may be a mammalian VCAN mRNA or premRNA, such as a human VCAN mRNA or premRNA, for example a premRNA or mRNA originating from the Homo sapiens versican (VCAN), RefSeqGene on chromosome 5, exemplified by NCBI Reference Sequence NG_012682.1 or Ensembl ENSG00000038427 (SEQ ID NO 13).
  • a mature human mRNA target sequence is illustrated herein by the cDNA sequences SEQ ID NO 14, 15, 16 or 17.
  • the oligonucleotides of the invention are capable of inhibiting the expression of VCAN target nucleic acid, such as the VCAN mRNA, in a cell which is expressing the target nucleic acid, such as the VCAN mRNA.
  • the oligonucleotides of the invention are capable of inhibiting the expression of VCAN target nucleic acid in a cell which is expressing the target nucleic acid, so to reduce the level of VCAN target nucleic acid (e.g. the mRNA) by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% inhibition compared to the expression level of the VCAN target nucleic acid (e.g. the mRNA) in the cell.
  • the cell is selected from the group consisting of HEK293 and A549.
  • Example 1 provides a suitable assay for evaluating the ability of the oligonucleotides of the invention to inhibit the expression of the target nucleic acid.
  • the evaluation of a compounds ability to inhibit the expression of the target nucleic acid is performed in vitro, such a gymnotic in vitro assay, for example as according to Example 1.
  • An aspect of the present invention relates to an antisense oligonucleotide, such as an LNA antisense oligonucleotide gapmer which comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length with at least 90% complementarity, such as is fully
  • the oligonucleotide comprises a contiguous sequence of 10 - 30 nucleotides, which is at least 90% complementary, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or 100% complementary with a region of the target nucleic acid or a target sequence.
  • the oligonucleotide of the invention comprises a contiguous nucleotides sequence of 12 - 24, such as 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, or 23, contiguous nucleotides in length, wherein the contiguous nucleotide sequence is fully complementary to SEQ ID NO 1 1.
  • the oligonucleotide of the invention comprises a contiguous nucleotides sequence of 12 - 20, such as 13, 14, 15, 16, 17, 18, 19 or 20, contiguous nucleotides in length, wherein the contiguous nucleotide sequence is fully complementary to SEQ ID NO 12.
  • the antisense oligonucleotide of the invention or the contiguous nucleotide sequence thereof is a gapmer, such as an LNA gapmer, a mixed wing gapmer, or an alternating flank gapmer.
  • the antisense oligonucleotide according to the invention comprises a contiguous nucleotide sequence of at least 10 contiguous nucleotides, such as at least 12 contiguous nucleotides, such as at least 13 contiguous nucleotides, such as at least 14 contiguous nucleotides, such as at least 15 contiguous nucleotides, which is fully complementary to SEQ ID NO 1 1.
  • the antisense oligonucleotide according to the invention comprises a contiguous nucleotide sequence of at least 10 contiguous nucleotides, such as at least 12 contiguous nucleotides, such as at least 13 contiguous nucleotides, such as at least 14 contiguous nucleotides, such as at least 15 contiguous nucleotides, which is fully complementary to SEQ ID NO 12.
  • the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is less than 20 nucleotides in length. In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12 - 24 nucleotides in length. In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12 - 22 nucleotides in length.
  • the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12 - 20 nucleotides in length. In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12 - 18 nucleotides in length. In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12 - 16 nucleotides in length.
  • all of the internucleoside linkages between the nucleosides of the contiguous nucleotide sequence are phosphorothioate internucleoside linkages. In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 1 1.
  • the contiguous nucleotide sequence is fully complementary to SEQ ID NO 12.
  • the antisense oligonucleotide is a gapmer oligonucleotide comprising a contiguous nucleotide sequence of formula 5’-F-G-F’-3’, where region F and F’ independently comprise 1 - 8 sugar modified nucleosides, and G is a region between 5 and 16 nucleosides which are capable of recruiting RNaseH.
  • the sugar modified nucleosides of region F and F’ are independently selected from the group consisting of 2’-0-alkyl-RNA, 2’-0-methyl-RNA, 2’-alkoxy-RNA, 2’- O-methoxyethyl-RNA, 2’-amino-DNA, 2’-fluoro-DNA, arabino nucleic acid (ANA), 2’-fluoro- ANA and LNA nucleosides.
  • region G comprises 5 - 16 contiguous DNA nucleosides.
  • the antisense oligonucleotide is a gapmer oligonucleotide, such as an LNA gapmer oligonucleotide.
  • the LNA nucleosides are beta-D-oxy LNA nucleosides.
  • the internucleoside linkages between the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.
  • the invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides 12 - 24, such as 12 - 18 in length, nucleosides in length wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence comprising at least 12, such as at least 14, such as at least 15 contiguous nucleotides present in SEQ ID NO 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides 12 - 24 nucleosides in length, such as 12 - 18 in length, wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence comprising at least 12, such as at least 13, such as at least 14, such as at least 15 contiguous nucleotides present in SEQ ID NO 5, 6, 7 or 10.
  • the invention provides LNA gapmers according to the invention comprising or consisting of a contiguous nucleotide sequence selected from SEQ ID NO 1 - 10.
  • the invention provides antisense oligonucleotides selected from the group consisting of: TTgtcttataacTGCA (SEQ ID NO 1 ), GGcttattttacCACA (SEQ ID NO 2), TATtattgaggaaGCC (SEQ ID NO 3),ATCAaatctgctatCA (SEQ ID NO 4), GAtttagttggcATGA (SEQ ID NO 5), TAGTtggcatgataAT (SEQ ID NO 6), TTtagttggcatGATA (SEQ ID NO 7), CTTggcattataaCTA (SEQ ID NO 8), GTTCacaaagaaagCC (SEQ ID NO 9), AGAtttagttggcATG (SEQ ID NO 10); wherein a capital letter is a LNA nucleoside, and a lower case letter is a DNA nucleoside.
  • all internucleoside linkages in contiguous nucleoside sequence are phosphorothioate internucleoside linkages.
  • LNA cytosine may be 5-methyl cytosine.
  • DNA cytosine may be 5-methyl cytosine.
  • the invention provides antisense oligonucleotides selected from the group consisting of: TTgtcttataacTGCA (SEQ ID NO 1 ), GGcttattttacCACA (SEQ ID NO 2), TATtattgaggaaGCC (SEQ ID NO 3),ATCAaatctgctatCA (SEQ ID NO 4), GAtttagttggcATGA (SEQ ID NO 5), TAGTtggcatgataAT (SEQ ID NO 6), TTtagttggcatGATA (SEQ ID NO 7), CTTggcattataaCTA (SEQ ID NO 8), GTTCacaaagaaagCC (SEQ ID NO 9), AGAtttagttggcATG (SEQ ID NO 10); wherein a capital letter is a beta-D-oxy-LNA nucleoside, and a lower case letter is a DNA nucleoside.
  • all internucleoside linkages in contiguous nucleoside sequence are phosphorothioate internucleoside linkages.
  • LNA cytosine may be 5-methyl cytosine.
  • DNA cytosine may be 5-methyl cytosine.
  • the invention provides antisense oligonucleotides selected from the group consisting of: TTgtcttataacTGCA (SEQ ID NO 1 ), GGcttattttacCACA (SEQ ID NO 2), TATtattgaggaaGCC (SEQ ID NO 3),ATCAaatctgctatCA (SEQ ID NO 4), GAtttagttggcATGA (SEQ ID NO 5), TAGTtggcatgataAT (SEQ ID NO 6), TTtagttggcatGATA (SEQ ID NO 7), CTTggcattataaCTA (SEQ ID NO 8), GTTCacaaagaaagCC (SEQ ID NO 9), AGAtttagttggcATG (SEQ ID NO 10); wherein a capital letter is a beta-D-oxy-LNA nucleoside, wherein all LNA cytosinese are 5- methyl cytosine, and a lower case letter is a DNA nucleoside, where
  • the invention provides methods for manufacturing the oligonucleotides of the invention comprising reacting nucleotide units and thereby forming covalently linked contiguous nucleotide units comprised in the oligonucleotide.
  • the method uses phophoramidite chemistry (see for example Caruthers et al, 1987, Methods in Enzymology vol. 154, pages 287-313).
  • the method further comprises reacting the contiguous nucleotide sequence with a conjugating moiety (ligand) to covalently attach the conjugate moiety to the oligonucleotide.
  • composition of the invention comprising mixing the oligonucleotide or conjugated oligonucleotide of the invention with a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.
  • the invention provides pharmaceutical compositions comprising any of the aforementioned oligonucleotides and/or oligonucleotide conjugates or salts thereof and a pharmaceutically acceptable diluent, carrier, salt and/or adjuvant.
  • a pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS) and pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
  • the pharmaceutically acceptable diluent is sterile phosphate buffered saline.
  • the oligonucleotide is used in the pharmaceutically acceptable diluent at a concentration of 50 - 300pM solution.
  • the compounds according to the present invention may exist in the form of their
  • pharmaceutically acceptable salts refers to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds of the present invention and are formed from suitable nontoxic organic or inorganic acids or organic or inorganic bases.
  • Acid-addition salts include for example those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like.
  • Base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethyl ammonium hydroxide.
  • the chemical modification of a pharmaceutical compound into a salt is a technique well known to pharmaceutical chemists in order to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of compounds. It is for example described in Bastin, Organic Process Research & Development 2000, 4, 427-435 or in Ansel, In:
  • the pharmaceutically acceptable salt of the compounds provided herein may be a sodium salt.
  • Suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, pro-drug formulations are also provided in W02007/031091.
  • Oligonucleotides or oligonucleotide conjugates of the invention may be mixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations.
  • compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
  • compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered.
  • the resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration.
  • the pH of the preparations typically will be between 3 and 1 1 , more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5.
  • the resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents, such as in a sealed package of tablets or capsules.
  • the composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.
  • the oligonucleotide or oligonucleotide conjugate of the invention is a prodrug.
  • the conjugate moiety is cleaved of the oligonucleotide once the prodrug is delivered to the site of action, e.g. the target cell.
  • oligonucleotides of the invention may be utilized as research reagents for, for example, diagnostics, therapeutics and prophylaxis.
  • such oligonucleotides may be used to specifically modulate the synthesis of VCAN protein in cells (e.g. in vitro cell cultures) and experimental animals thereby facilitating functional analysis of the target or an appraisal of its usefulness as a target for therapeutic intervention.
  • the target modulation is achieved by degrading or inhibiting the mRNA producing the protein, thereby prevent protein formation or by degrading or inhibiting a modulator of the gene or mRNA producing the protein.
  • the target nucleic acid may be a cDNA or a synthetic nucleic acid derived from DNA or RNA.
  • the present invention provides an in vivo or in vitro method for modulating VCAN expression in a target cell which is expressing VCAN, said method comprising administering an oligonucleotide of the invention in an effective amount to said cell.
  • the target cell is a mammalian cell in particular a human cell.
  • the target cell may be an in vitro cell culture or an in vivo cell forming part of a tissue in a mammal.
  • the oligonucleotides may be used to detect and quantitate VCAN expression in cell and tissues by northern blotting, in-situ hybridisation or similar techniques.
  • an animal or a human, suspected of having a disease or disorder which can be treated by modulating the expression of VCAN
  • the invention provides methods for treating or preventing a disease, comprising
  • an oligonucleotide administered a therapeutically or prophylactically effective amount of an oligonucleotide, an oligonucleotide conjugate or a pharmaceutical composition of the invention to a subject suffering from or susceptible to the disease.
  • the invention also relates to an oligonucleotide, a composition or a conjugate as defined herein for use as a medicament.
  • oligonucleotide, oligonucleotide conjugate or a pharmaceutical composition according to the invention is typically administered in an effective amount.
  • the invention also provides for the use of the oligonucleotide or oligonucleotide conjugate of the invention as described for the manufacture of a medicament for the treatment of a disorder as referred to herein, or for a method of the treatment of as a disorder as referred to herein.
  • disease or disorder is associated with expression of VCAN.
  • disease or disorder may be associated with a mutation in the VCAN gene. Therefore, in some embodiments, the target nucleic acid is a mutated form of the VCAN sequence.
  • the methods of the invention are preferably employed for treatment or prophylaxis against diseases caused by abnormal levels and/or activity of VCAN.
  • the invention further relates to use of an oligonucleotide, oligonucleotide conjugate or a pharmaceutical composition as defined herein for the manufacture of a medicament for the treatment of abnormal levels and/or activity of VCAN.
  • the invention relates to oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions for use in the treatment of diseases or disorders selected from inflammatory lung disease (such as chronic obstructive pulmonary disease or asthma), diabetic retinopathy, cancer and fibrotic disorders (such as fibrotic lung disease, cystic fibrosis, or pulmonary fibrosis).
  • diseases or disorders selected from inflammatory lung disease (such as chronic obstructive pulmonary disease or asthma), diabetic retinopathy, cancer and fibrotic disorders (such as fibrotic lung disease, cystic fibrosis, or pulmonary fibrosis).
  • oligonucleotides or pharmaceutical compositions of the present invention may be administered topical or enteral or parenteral (such as, intravenous, subcutaneous, intramuscular, intracerebral, intracerebroventricular or intrathecal).
  • topical or enteral or parenteral such as, intravenous, subcutaneous, intramuscular, intracerebral, intracerebroventricular or intrathecal.
  • the oligonucleotide or pharmaceutical compositions of the present invention are administered by a parenteral route including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intrathecal or intracranial, e.g. intracerebral or intraventricular, intravitreal administration.
  • a parenteral route including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intrathecal or intracranial, e.g. intracerebral or intraventricular, intravitreal administration.
  • the active oligonucleotide or oligonucleotide conjugate is administered intravenously.
  • the active oligonucleotide or oligonucleotide conjugate is administered subcutaneously.
  • the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention is administered at a dose of 0.1 - 15 mg/kg, such as from 0.2 - 10 mg/kg, such as from 0.25 - 5 mg/kg.
  • the administration can be once a week, every 2 nd week, every third week or even once a month.
  • the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention is for use in a combination treatment with another therapeutic agent.
  • the therapeutic agent can for example be the standard of care for the diseases or disorders described above.
  • Example 1 Testing in vitro efficacy of antisense oligonucleotides targeting VCAN mRNA in human HEK293 and A549 cells at single concentration.
  • HEK293 and A549 cell lines were purchased from ATCC and maintained as recommended by the supplier in a humidified incubator at 37°C with 5% C02.
  • 3500 cells/well of HEK293 or 4500 cells/well of A549 were seeded in a 96 multi well plate in culture media. Cells were incubated for 24 hours before addition of oligonucleotides dissolved in PBS. Final concentration of oligonucleotides: 25 pM. 3 days after addition of oligonucleotides, the cells were harvested.
  • RNA was extracted using the PureLink Pro 96 RNA Purification kit (Thermo Fisher Scientific) according to the manufacturer’s instructions and eluated in 50mI water. The RNA was subsequently diluted 10 times with DNase/RNase free Water (Gibco) and heated to 90°C for one minute.
  • One Step RT-qPCR was performed using qScriptTM XLT One-Step RT-qPCR ToughMix®, Low ROXTM (Quantabio) in a duplex set up.
  • the following TaqMan primer assays were used for qPCR: VCAN Hs 00171642 _m1 ) [FAM-MGB] and endogenous control GAPDH, Hs99999905_m1 [VIC-MGB] All primer sets were purchased from Thermo Fisher Scientific.
  • the relative VCAN mRNA expression level in the table is shown as percent of control (PBS-treated cells).
  • VCAN mRNA levels from cells treated with a selection of the compounds are shown in figures 1 and 2, evaluated in human HEK293 and A549 cell lines. From the initial library screen one motif on the VCAN human transcript was identified which provided surprisingly effective and potent compounds in the cell lines tested: Motif A (SEQ ID NO 1 1 ).
  • LNA nucleosides (beta-D-oxy LNA nucleosides were used), all LNA cytosines are 5-methyl cytosine, lower case letters represent DNA nucleosides. All internucleoside linkages are phosphorothioate internucleoside linkages.
  • Example 2 Testing in vitro potency and efficacy of selected oligonucleotides targeting human VCAN mRNA in human HEK293 and A549 cells at different concentrations for a concentration response curve.
  • PBS percent of control
  • the IC50 values for selected oligonucleotides targeting human VCAN mRNA in vitro in the human cell lines A549 and HEK293 are shown in Figure 3.
  • the concentration response curves in human cell lines A549 and HEK293 are provided as Figures 4 and 5, respectively.

Abstract

The present invention relates to antisense LNA oligonucleotides (oligomers) complementary to VCAN pre-mRNA intron sequences, which are capable of inhibiting the expression of VCAN protein. Inhibition of VCAN expression is beneficial for a range of medical disorders including inflammatory lung disease (such as chronic obstructive pulmonary disease or asthma), diabetic retinopathy, cancer and fibrotic disorders (such as fibrotic lung disease, cystic fibrosis, or pulmonary fibrosis).

Description

ANTISENSE OLIGONUCLEOTIDES TARGETING VCAN
FIELD OF INVENTION
The present invention relates to antisense LNA oligonucleotides (oligomers) complementary to VCAN pre-mRNA intron sequences, which are capable of inhibiting the expression of VCAN. Inhibition of VCAN expression is beneficial for a range of medical disorders including inflammatory lung disease (such as chronic obstructive pulmonary disease or asthma), diabetic retinopathy, cancer and fibrotic disorders (such as fibrotic lung disease, cystic fibrosis, or pulmonary fibrosis).
BACKGROUND
VCAN (Versican) is a member of the aggrecan/versican proteoglycan family (other names: CSPG2, ERVR, GHAP, PG-M, WGN, WGN1 ). The VCAN protein is a large chondroitin sulfate proteoglycan and is a major component of the extracellular matrix. This protein is involved in cell adhesion, proliferation, proliferation, migration and angiogenesis and plays a central role in tissue morphogenesis and maintenance. VCAN interacts with many proteins and molecules to facilitate the assembly of the extracellular matrix and ensure its stability. The VCAN gene encodes four extracellular matrix isoforms which differ in the presence or length of a central glycosaminoglycan-binding region (Wight et al., 2002, Curr. Opin. Cell Biol. 14: 617-623).
VCAN has been associated with many diseases and disorders. For example, VCAN mutations are the cause of VCAN-related vitreoretinopathy. At least 1 1 mutations in the VCAN gene have been found to cause Wagner syndrome, a condition that leads to progressive vision loss starting in childhood or early adulthood (see e.g. Rothschild et al.,
Mol Vis. 2013; 19: 2040-2049).
Increased expression of VCAN has been detected in cancer tissue such as breast, brain, ovary, gastrointestinal tract skin and prostate cancer (see e.g. Paulus et al., 1996, J.
Neuropathol. Exp. Neurol. 55 (5): 528-33). Numerous studies have shown that increased VCAN levels are correlated with elevated metastatic potential and poor disease prognosis. Therefore, VCAN was suggested as a target in cancer therapy (Keire et al., Matrix Biol. 2016 Mar; 50: 67-81 ).
Increased VCAN levels were also observed in inflammatory lung diseases such as chronic obstructive pulmonary disease (COPD) and asthma. Therefore, VCAN was suggested as a therapeutic target in these diseases (Andersson-Sjoland et al. , Glycobiology. 2015;
25(3):243-251 ).
CN103468681 discloses a siRNA which inhibits expression of a VCAN isoform for the treatment of COPD.
W02009/061368 discloses that siRNA molecules which target VCAN can be used to treat fibrotic disorders, arterial restenosis, atherosclerosis, cancer, fibrotic lung disease, cystic fibrosis, pulmonary fibrosis, or emphysema.
EP 2 078 728 A1 discloses that inhibitors of a VCAN isoform can be used in the treatment of breast cancer. The inhibitor could be e.g. an antibody or an antisense nucleic acid molecule directed against the isoform.
W02008/029868 discloses that siRNAs which target VCAN can be used in the treatment of chorioretinal neovascularization diseases such as diabetic retinopathy.
There is a need for therapeutic agents which can inhibit VCAN specifically.
We have screened a large number of LNA gapmers targeting mouse and human VCAN and identified sequences and compounds which are particularly potent and effective to specifically target for VCAN antisense in vitro (human and mouse cells).
OBJECTIVE OF THE INVENTION
The inventors have identified particularly effective regions of the VCAN transcript (VCAN) for antisense inhibition in vitro or in vivo, and provides for antisense oligonucleotides, including LNA gapmer oligonucleotides, which target these regions of the VCAN premRNA. The present invention identifies oligonucleotides which inhibit human VCAN which are useful in the treatment of a range of medical disorders including inflammatory lung disease (such as chronic obstructive pulmonary disease or asthma), diabetic retinopathy, cancer and fibrotic disorders (such as fibrotic lung disease, cystic fibrosis, or pulmonary fibrosis).
STATEMENT OF THE INVENTION
The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, targeting a human VCAN target nucleic acid, wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN in a cell which is expressing human VCAN.
The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, targeting a human VCAN pre-mRNA target nucleic acid, wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN in a cell which is expressing human VCAN, and wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence of at least 10 nucleotides which are fully complementary to the human VCAN pre-mRNA target nucleic acid.
The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, targeting a human VCAN target nucleic acid, wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN in a cell which is expressing human VCAN; and wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence of at least 10 nucleotides which are fully complementary to an intron region of VCAN pre-mRNA target nucleic acid.
The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, targeting a human VCAN target nucleic acid, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully
complementary, to a sequence selected from the group consisting of SEQ ID NO 13, 14, 15, 16 and 17.
The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, targeting a human VCAN target nucleic acid, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully
complementary to SEQ ID NO 13.
The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to SEQ ID NO 13 wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN in a cell which is expressing human VCAN.
The invention provides for an LNA antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to SEQ ID NO 13, wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN in a cell which is expressing human VCAN.
The invention provides for a gapmer antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to SEQ ID NO 13 wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN in a cell which is expressing human VCAN.
The invention provides for an LNA gapmer antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to SEQ ID NO 13 wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN in a cell which is expressing human VCAN.
The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to a sequence shown in SEQ ID NO 1 1 or in SEQ ID NO 12, wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN in a cell which is expressing human VCAN.
The invention provides for an LNA antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to a sequence shown in SEQ ID NO 1 1 or in SEQ ID NO 12, wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN in a cell which is expressing human VCAN.
The invention provides for a gapmer antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to a sequence shown in SEQ ID NO 1 1 or in SEQ ID NO 12, wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN in a cell which is expressing human VCAN.
The invention provides for an LNA gapmer antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary, to a sequence shown in SEQ ID NO 1 1 or in SEQ ID NO 12, wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN in a cell which is expressing human VCAN.
The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to SEQ ID NO 1 1 , wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN transcript in a cell which is expressing human VCAN transcript.
The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to SEQ ID NO 12, wherein the antisense oligonucleotide is capable of inhibiting the expression of human VCAN transcript in a cell which is expressing human VCAN transcript.
The oligonucleotide of the invention as referred to or claimed herein may be in the form of a pharmaceutically acceptable salt.
The invention provides for a conjugate comprising the oligonucleotide according to the invention, and at least one conjugate moiety covalently attached to said oligonucleotide.
The invention provides for a pharmaceutical composition comprising the oligonucleotide or conjugate of the invention and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.
The invention provides for an in vivo or in vitro method for modulating VCAN expression in a target cell which is expressing VCAN, said method comprising administering an oligonucleotide or conjugate or pharmaceutical composition of the invention in an effective amount to said cell.
The invention provides for a method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide, conjugate or the pharmaceutical composition of the invention to a subject suffering from or susceptible to the disease.
In some embodiments, the disease is selected from the group consisting of inflammatory lung disease (such as chronic obstructive pulmonary disease or asthma), diabetic retinopathy, cancer and fibrotic disorders (such as fibrotic lung disease, cystic fibrosis, or pulmonary fibrosis).
The invention provides for the oligonucleotide, conjugate or the pharmaceutical composition of the invention for use in medicine.
The invention provides for the oligonucleotide, conjugate or the pharmaceutical composition of the invention for use in the treatment or prevention of a disease selected from the group consisting of inflammatory lung disease (such as chronic obstructive pulmonary disease or asthma), diabetic retinopathy, cancer and fibrotic disorders (such as fibrotic lung disease, cystic fibrosis, or pulmonary fibrosis).
The invention provides for the use of the oligonucleotide, conjugate or the pharmaceutical composition of the invention, for the preparation of a medicament for treatment or prevention of a disease selected from the group consisting of inflammatory lung disease (such as chronic obstructive pulmonary disease or asthma), diabetic retinopathy, cancer and fibrotic disorders (such as fibrotic lung disease, cystic fibrosis, or pulmonary fibrosis).
BRIEF DESCRIPTION OF FIGURES
Figure 1 : Testing in vitro efficacy of various antisense oligonucleotides targeting human VCAN mRNA in HEK293 and A549 cell lines at single concentration.
Figure 2: Comparison of in vitro efficacy for antisense oligonucleotides targeting human VCAN mRNA in HEK293 and A549 cell lines at single concentration shows good correlation. One motif with very efficient targeting is highlighted.
Figure 3: IC50 values for selected oligonucleotides targeting human VCAN mRNA in vitro in HEK293 and A549 cell lines.
Figure 4: Testing selected oligonucleotides targeting human VCAN mRNA in vitro for concentration dependent potency and efficacy in A549 cell line.
Figure 5: Testing selected oligonucleotides targeting human VCAN mRNA in vitro for concentration dependent potency and efficacy in HEK293 cell line. DEFINITIONS
Oligonucleotide
The term“oligonucleotide” as used herein is defined as it is generally understood by the skilled person as a molecule comprising two or more covalently linked nucleosides. Such covalently bound nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are commonly made in the laboratory by solid-phase chemical synthesis followed by purification. When referring to a sequence of the oligonucleotide, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides. The oligonucleotide of the invention is man-made, and is chemically synthesized, and is typically purified or isolated. The oligonucleotide of the invention may comprise one or more modified nucleosides or nucleotides.
Antisense oligonucleotides
The term“Antisense oligonucleotide” as used herein is defined as oligonucleotides capable of modulating expression of a target gene by hybridizing to a target nucleic acid, in particular to a contiguous sequence on a target nucleic acid. The antisense oligonucleotides are not essentially double stranded and are therefore not siRNAs or shRNAs. Preferably, the antisense oligonucleotides of the present invention are single stranded. It is understood that single stranded oligonucleotides of the present invention can form hairpins or intermolecular duplex structures (duplex between two molecules of the same oligonucleotide), as long as the degree of intra or inter self-complementarity is less than 50% across of the full length of the oligonucleotide
Contiguous Nucleotide Sequence
The term“contiguous nucleotide sequence” refers to the region of the oligonucleotide which is complementary to the target nucleic acid. The term is used interchangeably herein with the term“contiguous nucleobase sequence” and the term“oligonucleotide motif sequence”.
In some embodiments all the nucleotides of the oligonucleotide constitute the contiguous nucleotide sequence. In some embodiments the oligonucleotide comprises the contiguous nucleotide sequence, such as a F-G-F’ gapmer region, and may optionally comprise further nucleotide(s), for example a nucleotide linker region which may be used to attach a functional group to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid. Adventurously, the contiguous nucleotide sequence is 100% complementary to the target nucleic acid. Nucleotides
Nucleotides are the building blocks of oligonucleotides and polynucleotides, and for the purposes of the present invention include both naturally occurring and non-naturally occurring nucleotides. In nature, nucleotides, such as DNA and RNA nucleotides comprise a ribose sugar moiety, a nucleobase moiety and one or more phosphate groups (which is absent in nucleosides). Nucleosides and nucleotides may also interchangeably be referred to as“units” or“monomers”.
Modified nucleoside
The term“modified nucleoside” or“nucleoside modification” as used herein refers to nucleosides modified as compared to the equivalent DNA or RNA nucleoside by the introduction of one or more modifications of the sugar moiety or the (nucleo)base moiety. In a preferred embodiment the modified nucleoside comprise a modified sugar moiety. The term modified nucleoside may also be used herein interchangeably with the term“nucleoside analogue” or modified“units” or modified“monomers”. Nucleosides with an unmodified DNA or RNA sugar moiety are termed DNA or RNA nucleosides herein. Nucleosides with modifications in the base region of the DNA or RNA nucleoside are still generally termed DNA or RNA if they allow Watson Crick base pairing.
Modified internucleoside linkages
The term“modified internucleoside linkage” is defined as generally understood by the skilled person as linkages other than phosphodiester (PO) linkages, that covalently couples two nucleosides together. The oligonucleotides of the invention may therefore comprise modified internucleoside linkages. In some embodiments, the modified internucleoside linkage increases the nuclease resistance of the oligonucleotide compared to a phosphodiester linkage. For naturally occurring oligonucleotides, the internucleoside linkage includes phosphate groups creating a phosphodiester bond between adjacent nucleosides. Modified internucleoside linkages are particularly useful in stabilizing oligonucleotides for in vivo use, and may serve to protect against nuclease cleavage at regions of DNA or RNA nucleosides in the oligonucleotide of the invention, for example within the gap region of a gapmer oligonucleotide, as well as in regions of modified nucleosides, such as region F and F’.
In an embodiment, the oligonucleotide comprises one or more internucleoside linkages modified from the natural phosphodiester, such one or more modified internucleoside linkages that is for example more resistant to nuclease attack. Nuclease resistance may be determined by incubating the oligonucleotide in blood serum or by using a nuclease resistance assay (e.g. snake venom phosphodiesterase (SVPD)), both are well known in the art. Internucleoside linkages which are capable of enhancing the nuclease resistance of an oligonucleotide are referred to as nuclease resistant internucleoside linkages. In some embodiments at least 50% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are modified, such as at least 60%, such as at least 70%, such as at least 80 or such as at least 90% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are nuclease resistant internucleoside linkages. In some embodiments all of the internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof, are nuclease resistant internucleoside linkages. It will be recognized that, in some embodiments the nucleosides which link the oligonucleotide of the invention to a non-nucleotide functional group, such as a conjugate, may be phosphodiester.
A preferred modified internucleoside linkage is phosphorothioate.
Phosphorothioate internucleoside linkages are particularly useful due to nuclease resistance, beneficial pharmacokinetics and ease of manufacture. In some embodiments at least 50% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate, such as at least 60%, such as at least 70%, such as at least 80% or such as at least 90% of the internucleoside linkages in the oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate. In some embodiments all of the internucleoside linkages of the oligonucleotide, or contiguous nucleotide sequence thereof, are phosphorothioate.
Nuclease resistant linkages, such as phosphorothioate linkages, are particularly useful in oligonucleotide regions capable of recruiting nuclease when forming a duplex with the target nucleic acid, such as region G for gapmers. Phosphorothioate linkages may, however, also be useful in non-nuclease recruiting regions and/or affinity enhancing regions such as regions F and F’ for gapmers. Gapmer oligonucleotides may, in some embodiments comprise one or more phosphodiester linkages in region F or F’, or both region F and F’, which the internucleoside linkage in region G may be fully phosphorothioate.
Advantageously, all the internucleoside linkages in the contiguous nucleotide sequence of the oligonucleotide are phosphorothioate linkages.
It is recognized that, as disclosed in EP2 742 135, antisense oligonucleotide may comprise other internucleoside linkages (other than phosphodiester and phosphorothioate), for example alkyl phosphonate / methyl phosphonate internucleosides, which according to EP2 742 135 may for example be tolerated in an otherwise DNA phosphorothioate gap region.
Nucleobase
The term nucleobase includes the purine (e.g. adenine and guanine) and pyrimidine (e.g. uracil, thymine and cytosine) moiety present in nucleosides and nucleotides which form hydrogen bonds in nucleic acid hybridization. In the context of the present invention the term nucleobase also encompasses modified nucleobases which may differ from naturally occurring nucleobases, but are functional during nucleic acid hybridization. In this context “nucleobase” refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, as well as non-naturally occurring variants. Such variants are for example described in Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid
Chemistry Suppl. 37 1.4.1.
In a some embodiments the nucleobase moiety is modified by changing the purine or pyrimidine into a modified purine or pyrimidine, such as substituted purine or substituted pyrimidine, such as a nucleobased selected from isocytosine, pseudoisocytosine, 5-methyl cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil 5- thiazolo-uracil, 2-thio-uracil, 2’thio-thymine, inosine, diaminopurine, 6-aminopurine, 2- aminopurine, 2,6-diaminopurine and 2-chloro-6-aminopurine.
The nucleobase moieties may be indicated by the letter code for each corresponding nucleobase, e.g. A, T, G, C or U, wherein each letter may optionally include modified nucleobases of equivalent function. For example, in the exemplified oligonucleotides, the nucleobase moieties are selected from A, T, G, C, and 5-methyl cytosine. Optionally, for LNA gapmers, 5-methyl cytosine LNA nucleosides may be used.
Modified oligonucleotide
The term modified oligonucleotide describes an oligonucleotide comprising one or more sugar-modified nucleosides and/or modified internucleoside linkages. The term chimeric” oligonucleotide is a term that has been used in the literature to describe oligonucleotides with modified nucleosides.
Complementarity
The term“complementarity” describes the capacity for Watson-Crick base-pairing of nucleosides/nucleotides. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A) - thymine (T)/uracil (U). It will be understood that oligonucleotides may comprise nucleosides with modified nucleobases, for example 5-methyl cytosine is often used in place of cytosine, and as such the term complementarity encompasses Watson Crick base-paring between non-modified and modified nucleobases (see for example Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009)
Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1 ).
The term“% complementary” as used herein, refers to the number of nucleotides in percent of a contiguous nucleotide sequence in a nucleic acid molecule (e.g. oligonucleotide) which, at a given position, are complementary to (i.e. form Watson Crick base pairs with) a contiguous sequence of nucleotides, at a given position of a separate nucleic acid molecule (e.g. the target nucleic acid or target sequence). The percentage is calculated by counting the number of aligned bases that form pairs between the two sequences (when aligned with the target sequence 5’-3’ and the oligonucleotide sequence from 3’-5’), dividing by the total number of nucleotides in the oligonucleotide and multiplying by 100. In such a comparison a nucleobase/nucleotide which does not align (form a base pair) is termed a mismatch.
Preferably, insertions and deletions are not allowed in the calculation of % complementarity of a contiguous nucleotide sequence.
The term“fully complementary”, refers to 100% complementarity.
Identity
The term“Identity” as used herein, refers to the proportion of nucleotides (expressed in percent) of a contiguous nucleotide sequence in a nucleic acid molecule (e.g.
oligonucleotide) which across the contiguous nucleotide sequence, are identical to a reference sequence (e.g. a sequence motif). The percentage of identity is thus calculated by counting the number of aligned bases that are identical (a match) between two sequences (e.g. in the contiguous nucleotide sequence of the compound of the invention and in the reference sequence), dividing that number by the total number of nucleotides in the aligned region and multiplying by 100. Therefore, Percentage of Identity = (Matches x 100)/Length of aligned region (e.g. the contiguous nucleotide sequence). Insertions and deletions are not allowed in the calculation the percentage of identity of a contiguous nucleotide sequence. It will be understood that in determining identity, chemical modifications of the nucleobases are disregarded as long as the functional capacity of the nucleobase to form Watson Crick base pairing is retained (e.g. 5-methyl cytosine is considered identical to a cytosine for the purpose of calculating % identity).
Hybridization
The term“hybridizing” or“hybridizes” as used herein is to be understood as two nucleic acid strands (e.g. an oligonucleotide and a target nucleic acid) forming hydrogen bonds between base pairs on opposite strands thereby forming a duplex. The affinity of the binding between two nucleic acid strands is the strength of the hybridization. It is often described in terms of the melting temperature (Tm) defined as the temperature at which half of the oligonucleotides are duplexed with the target nucleic acid. At physiological conditions Tm is not strictly proportional to the affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The standard state Gibbs free energy DQ° is a more accurate representation of binding affinity and is related to the dissociation constant (Kd) of the reaction by AG°=-RTIn(Kd), where R is the gas constant and T is the absolute temperature. Therefore, a very low AG° of the reaction between an oligonucleotide and the target nucleic acid reflects a strong
hybridization between the oligonucleotide and target nucleic acid. AG° is the energy associated with a reaction where aqueous concentrations are 1 M, the pH is 7, and the temperature is 37°C. The hybridization of oligonucleotides to a target nucleic acid is a spontaneous reaction and for spontaneous reactions AG° is less than zero. AG° can be measured experimentally, for example, by use of the isothermal titration calorimetry (ITC) method as described in Hansen et al. , 1965, Chem. Comm. 36-38 and Holdgate et al. , 2005, Drug Discov Today. The skilled person will know that commercial equipment is available for AG° measurements. AG° can also be estimated numerically by using the nearest neighbor model as described by SantaLucia, 1998, Proc Natl Acad Sci USA. 95: 1460-1465 using appropriately derived thermodynamic parameters described by Sugimoto et al., 1995, Biochemistry 34:1 121 1-1 1216 and McTigue et al., 2004, Biochemistry 43:5388-5405. In order to have the possibility of modulating its intended nucleic acid target by hybridization, oligonucleotides of the present invention hybridize to a target nucleic acid with estimated AG° values below -10 kcal for oligonucleotides that are 10-30 nucleotides in length. In some embodiments the degree or strength of hybridization is measured by the standard state Gibbs free energy AG°. The oligonucleotides may hybridize to a target nucleic acid with estimated AG° values below the range of -10 kcal, such as below -15 kcal, such as below - 20 kcal and such as below -25 kcal for oligonucleotides that are 8-30 nucleotides in length.
In some embodiments the oligonucleotides hybridize to a target nucleic acid with an estimated AG° value of -10 to -60 kcal, such as -12 to -40, such as from -15 to -30 kcal or- 16 to -27 kcal such as -18 to -25 kcal.
Target nucleic acid
According to the present invention, the target nucleic acid is a nucleic acid which encodes mammalian VCAN and may for example be a gene, a VCAN RNA, a mRNA, a pre-mRNA, a mature mRNA or a cDNA sequence. The target may therefore be referred to as an VCAN target nucleic acid.
Suitably, the target nucleic acid encodes an VCAN protein, in particular mammalian VCAN, such as the human VCAN gene encoding pre-mRNA or mRNA sequences provided herein as SEQ ID NO 13, 14, 15, 16 or 17. In some embodiments, the target nucleic acid is SEQ ID NO 13 or a naturally occurring variant thereof (e.g. VCAN sequences encoding a mammalian VCAN protein).
If employing the oligonucleotide of the invention in research or diagnostics the target nucleic acid may be a cDNA or a synthetic nucleic acid derived from DNA or RNA.
For in vivo or in vitro application, the oligonucleotide of the invention is typically capable of inhibiting the expression of the VCAN target nucleic acid in a cell which is expressing the VCAN target nucleic acid. The contiguous sequence of nucleobases of the oligonucleotide of the invention is typically complementary to the VCAN target nucleic acid, as measured across the length of the oligonucleotide, optionally with the exception of one or two mismatches, and optionally excluding nucleotide based linker regions which may link the oligonucleotide to an optional functional group such as a conjugate, or other noncomplementary terminal nucleotides (e.g. region D’ or D”). The target nucleic acid is a messenger RNA, such as a mature mRNA or a pre-mRNA which encodes mammalian VCAN protein, such as human VCAN, e.g. the human VCAN pre-mRNA sequence, such as that disclosed as SEQ ID NO 13, or VCAN mature mRNA, such as that disclosed as SEQ ID NO 14, 15, 16 or 17. SEQ ID NOs 13 - 17 are DNA sequences - it will be understood that target RNA sequences have uracil (U) bases in place of the thymidine bases (T).
Figure imgf000014_0001
In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 13.
In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 14.
In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 15.
In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 16.
In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 17. Target Sequence
The term“target sequence” as used herein refers to a sequence of nucleotides present in the target nucleic acid which comprises the nucleobase sequence which is complementary to the oligonucleotide of the invention. In some embodiments, the target sequence consists of a region on the target nucleic acid which is complementary to the contiguous nucleotide sequence of the oligonucleotide of the invention.
Herein are provided numerous target sequence regions, as defined by regions of the human VCAN pre-mRNA (using SEQ ID NO 13 as a reference) which may be targeted by the oligonucleotides of the invention.
In some embodiments the target sequence is longer than the complementary sequence of a single oligonucleotide, and may, for example represent a preferred region of the target nucleic acid which may be targeted by several oligonucleotides of the invention.
The oligonucleotide of the invention comprises a contiguous nucleotide sequence which is complementary to or hybridizes to the target nucleic acid, such as a sub-sequence of the target nucleic acid, such as a target sequence described herein.
The oligonucleotide comprises a contiguous nucleotide sequence which are complementary to a target sequence present in the target nucleic acid molecule. The contiguous nucleotide sequence (and therefore the target sequence) comprises of at least 10 contiguous nucleotides, such as 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous nucleotides, such as from 12-25, such as from 14-18 contiguous nucleotides.
Target Sequence Regions
The inventors have identified particularly effective sequences of the VCAN target nucleic acid which may be targeted by the oligonucleotide of the invention.
In some embodiments the target sequence is SEQ ID NO 1 1.
In some embodiments the target sequence is SEQ ID NO 12.
SEQ ID NO 1 1 : TGCCATTATCATGCCAACTAAATCTGCAGT (13)
SEQ ID NO 12: ATT AT CAT GCCAACTAAAT CT (13)
(numbers in brackets refer to the SEQ ID of VCAN premRNA in which the target sequence is found). In a further aspect, the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to an exon region of SEQ ID NO 13, selected from the group consisting if Ex_1 - Ex_15 (see the following table).
Figure imgf000016_0001
In a further aspect, the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to a region of SEQ ID NO 13, selected from the group consisting of 1 - 559; 12049 - 12124; 18634 - 19008; 22082 - 22256; 22340 - 22467; 40639 - 40932; 47885 - 50845; 65543 - 70804; 74073 - 74186; 76507 - 76620; 81900 - 82058; 83492 - 83574; 100952 - 101096; 108516 - 108698; and 108843 - 1 10839.
In a further aspect, the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to an intron region of SEQ ID NO 13, selected from the group consisting of lnt_1 - lnt_14 (see the following table)
Figure imgf000017_0001
In a further aspect, the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to a region of SEQ ID NO 13, selected from the group consisting of 559 - 12049; 12124 - 18634; 19008 - 22082; 22256 - 22340; 22467 - 40639; 40932 - 47885; 50845 - 65543; 70804 - 74073;
74186 - 76507; 76620 - 81900; 82058 - 83492; 83574 - 100952; 101096 - 108516; and 108698 - 108843.
In a further aspect, the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to a region of SEQ ID NO 13, selected from the group consisting of 1 - 138, 140 - 161 , 163 - 218, 220 - 348, 350 - 447.457 - 555.568 - 598.601 - 619.629 - 643.643 - 672.674 - 730.732 - 750.752 -
796, 810 - 858, 860 - 930, 938- 1133, 1134- 1180, 1182- 1275, 1277- 1299, 1301 - 1335, 1337- 1382, 1384- 1398, 1401 - 1421, 1429 1492, 1505 - 1522, 1527 - 1567, 1578 - 1667, 1669 - 1684, 1686 - 1705, 1707 - 1728, 1739 1754, 1765 - 1805, 1822 - 1894, 1896 - 1928, 1930 - 1948, 1952 - 1977, 1989 - 2055, 2057 2124, 2132-2192, 2206-2265, 2322-2361, 2363 -2411, 2421 - 2442, 2444 - 2460, 2471 2500, 2506 - 2524, 2581 - 2603, 2612 - 2626, 2628 - 2642, 2644 - 2681 , 2683 -2711, 2755 2801 , 2821 - 2844, 2846 - 2905, 2907 - 2931 , 2934 - 2970, 2972 - 2995, 3019 - 3043, 3046 3062, 3066 - 3095, 3097 - 3113, 3115 - 3135, 3137 - 3152, 3151 - 3177, 3179 - 3205, 3207 3261 , 3323 - 3341 , 3359 - 3378, 3406 - 3441 , 3443 - 3461, 3476 - 3497, 3499 - 3513, 3525 3554, 3575 - 3608, 3610 - 3639, 3641 - 3674, 3681 - 3699, 3701 - 3716, 3740 - 3780, 3810 3826, 3828 - 3861 , 3864 - 3879, 3898 - 3922, 3924 - 3974, 3976 - 3990, 3992 - 4012, 4016 4031, 4033-4048, 4050-4140, 4140-4179, 4186 - 4204, 4219 - 4233, 4244 - 4269, 4283 4382, 4396 - 4470, 4472 - 4590, 4605 - 4626, 4628 - 4649, 4673 - 4728, 4742 - 4788, 4812 4888, 4918 - 4936, 4933 - 4960, 4962 - 5009, 5011 -5029, 5031 -5103, 5100-5114, 5120 5148, 5151 - 5209, 5225 - 5240, 5246 - 5320, 5322 - 5352, 5354 - 5382, 5384 - 5418, 5422 5470, 5472 - 5502, 5522 - 5577, 5579 - 5604, 5606 - 5634, 5683 - 5700, 5702 - 5724, 5726 5744, 5763 - 5791 , 5793 - 5820, 5822 - 5853, 5870 - 5891, 5910 - 5934, 5936 - 5965, 5967 6016, 6029 - 6067, 6070 - 6090, 6098 - 6142, 6144 - 6168, 6182 - 6210, 6214 - 6229, 6268 6282, 6284 - 6299, 6301 - 6319, 6322 - 6357, 6359 - 6373, 6384 - 6453, 6456 - 6474, 6470 6491 , 6486 - 6514, 6526 - 6568, 6573 - 6591 , 6597 -6611, 6621 - 6691 , 6693 - 6732, 6735 6766, 6793 - 6853, 6865 - 6884, 6886 - 6906, 6910 - 6929, 6960 - 6976, 6988 - 7009, 7011 7101, 7104-7124, 7124-7156, 7154-7173, 7175 - 7206, 7215 - 7234, 7252 - 7352, 7360 7374, 7377 - 7433, 7435 - 7471 , 7484 - 7509, 7511 - 7532, 7537 - 7567, 7574 - 7632, 7634 7648, 7651 - 7679, 7729 - 7751 , 7763 - 7794, 7799 - 7823, 7833 - 7884, 7886 - 7949, 7951 8037, 8039 - 8075, 8083 -8111,8113- 8232, 8235 - 8251 , 8253 - 8277, 8283 - 8301 , 8339 8368, 8371 - 8397, 8401 - 8429, 8431 - 8448, 8456 - 8498, 8523 - 8581 , 8583 - 8600, 8602 8624, 8626 - 8640, 8642 - 8665, 8667 - 8720, 8722 - 8757, 8791 -8811, 8828 - 8861 , 8865 8898, 8917 - 8938, 8940 - 9018, 9021 - 9047, 9059-9094, 9097-9112, 9114-9133, 9152 9169, 9186 - 9237, 9250 - 9271 , 9285 - 9307, 9309 - 9446, 9449 - 9494, 9520 - 9546, 9552 9567, 9569 - 9585, 9610 - 9646, 9653 - 9727, 9739 - 9765, 9779 - 9802, 9795 - 9810, 9804 9819, 9831 - 9866, 9883 - 9927, 9956 - 9993, 9995 - 10014, 10024 - 10040, 10075 - 10107, 10118- 10134, 10136- 10156, 10172 -
10231, 10233- 10258, 10294- 10320, 10339- 10353, 10370- 10388, 10414- 10447,
10457 - 10496, 10542 - 10558, 10560 - 10654, 10656 - 10681 , 10683 - 10732, 10734 - 10769, 10771 - 10854, 10863- 10931, 10956- 10973, 10975- 10991, 11024- 11073, 11075- 11102, 11109- 11124, 11129 - 11152, 11154 - 11192, 11213- 11238, 11251 - 11368, 11370- 11385, 11388- 11448, 11460- 11500, 11507- 11557, 11559- 11591, 11604- 11625, 11649- 11671, 11686- 11701, 11703- 11717, 11752- 11772, 11765- 11818, 11820- 11845, 11870- 11888, 11890- 11941, 11944- 11981, 12017- 12039, 12041 - 12059, 12073 - 12103, 12105 - 12140, 12142 - 12183, 12193 - 12215, 12217 - 12252, 12254 - 12272, 12274 - 12316, 12330 - 12345, 12347 - 12367, 12389 - 12407, 12409- 12428, 12430- 12475, 12477- 12540, 12546- 12561, 12551 - 12581, 12583- 12623, 12645- 12667, 12662- 12679, 12684- 12721, 12723- 12751, 12773- 12791, 12799- 12815, 12849- 12868, 12870- 12928, 12930- 12947, 12949- 12971, 12975- 13001, 13003- 13022, 13021 - 13044, 13046- 13091, 13085- 13102, 13106- 13123, 13125 - 13183, 13253 - 13338, 13349 - 13379, 13383 - 13403, 13408 - 13437, 13454 - 13473, 13489 - 13541 , 13543 - 13571 , 13573 - 13621 , 13623 - 13641 , 13649 - 13677, 13685 - 13702, 13704 - 13726, 13728 - 13742, 13750 - 13790, 13828 - 13842, 13834 - 13853, 13906 - 13935, 13939 - 13988, 13990 - 14015, 14017 - 14034, 14045 - 14077, 14086- 14119, 14121 - 14135, 14175- 14209, 14220- 14239, 14256- 14271, 14275- 14310, 14333 - 14355, 14359 - 14377, 14370 - 14387, 14389 - 14410, 14439 - 14463, 14567- 14596, 14598- 14615, 14619- 14645, 14677- 14698, 14693- 14721, 14753- 14771, 14778- 14793, 14815- 14832, 14847- 14877, 14879- 14913, 14951 - 14965, 14968- 14988, 14994- 15064, 15111 - 15143, 15160- 15193, 15242- 15276, 15287- 15315, 15317 - 15339, 15341 - 15365, 15410 - 15443, 15443 - 15482, 15478 - 15504,
15506 - 15566, 15568 - 15593, 15593 - 15608, 15625 - 15641 , 15643 - 15677, 15696 - 15711 , 15713 - 15739, 15741 - 15764, 15766 - 15784, 15793 - 15809, 15811 - 15842, 15853 - 15868, 15878 - 15904, 15915 - 15933, 15948 - 15986, 15988 - 16004, 16006 - 16100, 16102 - 16128, 16130 - 16182, 16199 - 16238, 16240 - 16258, 16267 - 16288,
16299 - 16327, 16341 - 16386, 16433 - 16461 , 16463 - 16480, 16473 - 16488, 16489 - 16519, 16521 - 16547, 16555 - 16621 , 16626 - 16661 , 16663 - 16694, 16696 - 16745,
16747 - 16772, 16774 - 16791 , 16793 - 16825, 16827 - 16870, 16872 - 16896, 16899 - 16913, 16916 - 16973, 16975 - 16999, 17001 - 17036, 17056 - 17071 , 17074 - 17089, 17091 - 17140, 17147 - 17162, 17164 - 17193, 17198 - 17215, 17217 - 17254, 17270 - 17292, 17307 - 17336, 17338 - 17409, 17431 - 17457, 17465 - 17479, 17482 - 17550, 17553 - 17567, 17564 - 17635, 17637 - 17654, 17664 - 17727, 17729 - 17782, 17784 - 17837, 17870- 17889, 17891 - 17911, 17921 - 17961, 17963- 17993, 17997- 18012, 18014 - 18085, 18087 - 18104, 18106 - 18139, 18155 - 18180, 18182 - 18198, 18200 - 18221 , 18223 - 18251 , 18262 - 18293, 18295 - 18332, 18334 - 18349, 18364 - 18397, 18397 - 18416, 18432 - 18516, 18518 - 18550, 18563 - 18583, 18585 - 18613, 18615 - 18655, 18658 - 18706, 18708 - 18802, 18804 - 18889, 18891 - 18940, 18957 - 18973, 18981 - 18998, 19000- 19020, 19022- 19068, 19070- 19112, 19144- 19168, 19180 - 19203, 19208 - 19237, 19239 - 19332, 19340 - 19402, 19400 - 19414, 19426 - 19456, 19465 - 19482, 19484 - 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31836, 31854 - 31880, 31892 - 31907, 31912 - 31929, 31931 - 31957, 31959 - 31973, 31975 - 31998, 32000 - 32015, 32038 - 32052, 32041 - 32056, 32121 - 32145, 32147 - 32173, 32175 - 32230, 32242 - 32277, 32299 - 32332, 32334 - 32367, 32379 - 32400, 32402 - 32438, 32439 - 32525, 32529 - 3261 1 , 32616 - 32635, 32637 - 32664, 32668 - 32687, 32686 - 32703, 32723 - 32741 , 32743 - 32766, 32768 - 32816, 32818 - 32846, 32848 - 32863, 32880 - 32897, 32909 - 32942, 32935 - 32950, 32948 - 32969, 32982 - 33024, 33044 - 33071 , 33075 - 33101 , 33105 - 33128, 33133 - 33155, 33155 - 33170, 33166 - 33182, 33184 - 33202, 33216 - 33250, 33252 - 33303, 33305 - 33400, 33402 - 33423, 33452 - 33482, 33479 - 33494, 33496 - 33510, 33499 - 33538, 33545 - 33560, 33562 - 33577, 33584 - 33606, 33629 - 33668, 33681 - 33780, 33781 - 3381 1 , 33835 - 33856, 33877 - 33898, 33936 - 33951 , 33988 - 34022, 34033 - 34048, 34074 - 34103, 34152 - 34190, 34192 - 34214, 34252 - 34300, 34302 - 34325, 34327 - 34341 , 34363 - 34382, 34393 - 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39779, 39771 - 39789, 39780 - 39799, 39830 - 39879, 39925 - 39939, 39959 - 39975, 39979 - 39994, 40007 - 40025, 40032-40056, 40078 - 40111 , 40106 - 40122, 40117-40137, 40148-40169, 40189 - 40236, 40259 - 40278, 40303 - 40342, 40350 - 40374, 40374 - 40396, 40400 - 40414, 40419 - 40433, 40443 - 40492, 40494 - 40550, 40564 - 40623, 40630 - 40659, 40661 - 40687, 40689 - 40711 , 40713 - 40747, 40749 - 40964, 40967 - 41015, 41017 - 41036, 41038-41119, 41124-41165, 41167 - 41181 , 41183 - 41202, 41210-41233, 41280-41308, 41333-41348, 41364 - 41381 , 41383 - 41407, 41413-41433, 41435- 41461, 41463-41477, 41479-41494, 41524-41538, 41540-41559, 41561 -41581, 41597-41611, 41615-41645, 41707-41750, 41757 - 41801 , 41802 - 41817, 41819 - 41847, 41849-41877, 41898-41949, 41951 -41973, 41975 - 41991 , 41996 - 42016, 42019 - 42056, 42058 - 42073, 42076 - 42099, 42102 - 42122, 42176 - 42240, 42242 - 42287, 42289 - 42355, 42369 - 42399, 42401 - 42422, 42427 - 42490, 42492 - 42509, 42511 - 42553, 42555 - 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55037, 55031 - 55054, 55056 - 55072, 55089 - 55106, 55108 - 55130, 55132 - 55155, 55172 - 55212, 55214 - 55310, 55312 - 55381 , 55412 - 55434, 55452 - 55466, 55482 - 55500, 55502 - 55556, 55575 - 55598, 55615 - 55702, 55704 - 55740, 55753 - 55779, 55783 - 55800, 55802 - 55838, 55840 - 55866, 55868 - 55885, 55887 - 55912, 55928 - 55951 , 55960 - 55979, 56009 - 56039, 56041 - 56086, 56093 - 56125, 56127 - 56151 , 56153 - 56184, 56198 - 56241 , 56274 - 56319, 56321 - 56335, 56387 - 56412, 56417 - 56453, 56462 - 56481 , 56483 - 56501 , 56520 - 56534, 56528 - 56549, 56558 - 56581 , 56583 - 56637, 56656 - 56678, 56738 - 56754, 56771 - 56800, 56816 - 56851 , 56851 - 5691 1 , 56913 - 56945, 56947 - 56983, 56989 - 57006, 57031 - 57052, 57072 - 57086, 57075 - 57091 , 57104 - 57127, 57129 - 57179, 57188 - 57261 , 57267 - 57292, 57301 - 57383, 57417 - 57452, 57454 - 57475, 57482 - 57567, 57603 - 57619, 57636 - 57652, 57654 - 57676, 57684 - 57722, 57728 - 57751 , 57758 - 5781 1 , 57813 - 57833, 57836 - 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63855, 63860 - 63918, 63920 - 63981 , 63985 - 64010, 64012 - 64029, 64039 - 64057, 64059 - 64105, 64107 - 64132, 64134 - 64155, 64181 - 64218, 64220 - 64263, 64265 - 64293, 64341 - 64361 , 64371 - 64389, 64391 - 64406, 64409 - 64429, 64438 - 64462, 64473 - 64492, 64509 - 64525, 64536 - 64591 , 64593 - 64636, 64651 - 64666, 64682 - 64709, 64758 - 64778, 64780 - 64795, 64797 - 64841 , 64851 - 64879, 64881 - 64896, 64898 - 64914, 64916 - 64936, 64940 - 64955, 64982 - 65057, 65058 - 651 13, 651 16 - 65152, 65155 - 65234, 65248 - 65288, 65315 - 65377, 65381 - 65421 , 65421 - 65442, 65468 - 65756, 65764 - 65786, 65788 - 65861 , 65863 - 65893, 65895 - 65909, 65912 - 65944, 65946 - 65963, 65975 - 66085, 66091 - 661 15, 661 17 - 66148, 66157 - 66231 , 66233 - 66290, 66296 - 66333, 66335 - 66358, 66360 - 66443, 66452 - 66503, 66505 - 66609, 6661 1 - 66656, 66658 - 66686, 66707 - 66746, 66773 - 66799, 66834 - 66910, 66912 - 66946, 66948 - 67014, 67031 - 67064, 67066 - 67103, 67137 - 67154, 67157 - 67197, 67199 - 67219, 67221 - 67237, 67239 - 67257, 67270 - 67287, 67289 - 67334, 67336 - 67354, 67363 - 67397, 67399 - 67436, 67438 - 67462, 67464 - 67588, 67590 - 67606, 67608 - 67626, 67635 - 67661 , 67663 - 67684, 67686 - 67754, 67762 - 67788, 67790 - 67814, 67816 - 67855, 67857 - 67913, 67927 - 67999, 68001 - 68015, 68017 - 68033, 68035 - 68049, 68073 - 68095, 681 19 - 68168, 68176 - 68223, 68225 - 68249, 68251 - 68278, 68280 - 68343, 68345 - 68372, 68400 - 68431 , 68442 - 68483, 68485 - 68509, 68514 - 68531 , 68533 - 68553, 68555 - 68595, 68618 - 68632, 68632 - 68732, 68734 - 68752, 68761 - 68807, 68814 - 68845, 68847 - 68893, 68895 - 68912, 68917 - 68958, 68960 - 68991 , 6901 1 - 69106, 69108 - 69158, 69172 - 69254, 69256 - 69296, 69307 - 69333, 69335 - 69381 , 69383 - 69403, 69405 - 69424, 69464 - 69534, 69545 - 69585, 69593 - 69614, 69626 - 69655, 69663 - 69779, 69781 - 69809, 69811 - 69844, 69847 - 69863, 69865 - 69900, 69929 - 69948, 69964 - 69984, 69994 - 70013, 70024 - 70092, 70099 - 70118, 70124 - 70212, 70214 - 70247, 70258 - 70276, 70278 - 70296, 70298 - 70399, 70405 - 70428, 70439 - 70487, 70522 - 70565, 70573 - 70613, 70616 - 70638, 70640 - 70662, 70684 - 70730, 70732 - 70797, 70799 - 70833, 70835 - 70863, 70878 - 70916, 70918 - 70949, 70955 - 70999, 71005-71022, 71024-71107, 71109-71148, 71150-71166, 71168-71193, 71195-71212, 71217-71248, 71273-71312, 71341 - 71401 , 71409 - 71426, 71435- 71492, 71541 -71555, 71557 - 71581 , 71583 - 71618, 71620-71637, 71639-71691, 71693 - 71746, 71747 - 71779, 71781 - 71798, 71800 - 71825, 71827 - 71853, 71890 - 71914, 71916 - 71935, 71931 - 71956, 71958 - 71985, 71987 - 72042, 72054 - 72130, 72135 - 72174, 72193 - 72219, 72223 - 72259, 72269 - 72290, 72301 - 72315, 72317 - 72351, 72363 - 72416, 72418 - 72433, 72435 - 72468, 72481 - 72507, 72539 - 72567, 72579-72631, 72633-72655, 72657 - 72711 , 72712 - 72727, 72815-72830, 72842- 72856, 72884 - 72899, 72906 - 72921 , 72923 - 72938, 72931 - 72959, 72961 - 72996, 72998 - 73046, 73050 - 73090, 73099 - 73117, 73119 - 73168, 73189 - 73238, 73240 - 73254, 73258 - 73279, 73289 - 73341, 73344 - 73370, 73372 - 73414, 73416 - 73541, 73543 - 73849, 73851 - 74000, 74002 - 74019, 74044 - 74082, 74084 - 74139, 74141 - 74166, 74168 - 74201, 74210 - 74276, 74292 - 74308, 74311 - 74361, 74364 - 74382, 74387 -74411, 74423 - 74446, 74453 - 74480, 74487 - 74572, 74613 - 74633, 74651 - 74669, 74671 - 74695, 74713 - 74737, 74746 - 74784, 74775 - 74790, 74788 - 74863, 74865-74893, 74910-74933, 74935 - 74951 , 74961 -75002, 74996-75014, 75018- 75032, 75037-75057, 75060-75094, 75085 - 75111 , 75103 - 75117, 75126-75145, 75139 - 75192, 75201 - 75226, 75236 - 75318, 75325 - 75366, 75368 - 75387, 75389 - 75515, 75514 - 75625, 75632 - 75665, 75673 - 75693, 75695 - 75720, 75722 - 75749, 75751 -75839, 75848-75867, 75879-75912, 75938 - 75981 , 75983 - 76013, 76017- 76060, 76068 - 76100, 76111 - 76156, 76219 - 76256, 76258 - 76272, 76274 - 76313, 76327 - 76341 , 76351 - 76371 , 76383 - 76428, 76436 - 76483, 76485 - 76537, 76539 - 76615, 76629 - 76643, 76645 - 76667, 76663 - 76692, 76694 - 76739, 76762 - 76800, 76802 - 76819, 76827 - 76852, 76868 - 76889, 76891 - 76915, 76922 - 76941, 76944 - 76964, 76966 - 77051 , 77053 - 77115, 77119 - 77154, 77156 - 77194, 77219 - 77266, 77268 - 77402, 77404 - 77444, 77455 - 77491 , 77494 - 77539, 77535 - 77560, 77562 - 77577, 77579 - 77623, 77625 - 77683, 77685 - 77747, 77766 - 77782, 77784 - 77808, 77810 - 77824, 77826 - 77862, 77875 - 77889, 77909 - 77923, 77927 - 77941, 77945 - 77975, 77979-78008, 78015 - 78081 , 78083 - 78102, 78113-78127, 78139-78166, 78169 - 78243, 78245 - 78259, 78266 - 78298, 78301 - 78320, 78334 - 78387, 78386 - 78433, 78436 - 78467, 78469 - 78483, 78485 - 78501 , 78517 - 78545, 78553 - 78586, 78599 - 78645, 78686 - 78713, 78731 - 78765, 78780 - 78809, 78815 - 78835, 78878 - 78929, 78931 - 78946, 78948 - 78964, 78966 - 79018, 79020 - 79046, 79048 - 79089, 79094 - 791 15, 791 17 - 79157, 79159 - 79186, 79198 - 79214, 79216 - 79290, 79297 - 79319, 79321 - 79374, 79386 - 79409, 7941 1 - 79446, 79443 - 79470, 79468 - 79483, 79472 - 79487, 79478 - 79495, 79529 - 79565, 79572 - 79595, 79610 - 79627, 79634 - 79655, 79691 - 79765, 79764 - 79788, 79785 - 79871 , 79876 - 79919, 79920 - 79939, 79938 - 79962, 79978 - 80014, 80004 - 80031 , 80066 - 80105, 80134 - 80161 , 80163 - 80177, 80203 - 80228, 80237 - 80252, 80261 - 80276, 80282 - 80307, 80309 - 80340, 80342 - 80387, 80395 - 80529, 80531 - 80616, 80618 - 80707, 80709 - 80734, 80736 - 80763, 80765 - 80781 , 80783 - 80797, 80817 - 80835, 80841 - 80863, 80886 - 80909, 80909 - 80928, 80938 - 80976, 80975 - 80993, 81052 - 81 133, 81 149 - 81 174, 81 187 - 81224, 81232 - 81247, 81257 - 81297, 81314 - 81338, 81374 - 81397, 81401 - 81440, 81442 - 81494, 81515 - 81598, 81610 - 81651 , 81653 - 81690, 81744 - 81761 , 81768 - 81825, 81841 - 81859, 81869 - 81900, 81905 - 82046, 82048 - 82074, 82097 - 82186, 82189 - 82205, 82229 - 82251 , 82284 - 82298, 82300 - 82315, 82319 - 82341 , 82358 - 82384, 82386 - 82444, 82447 - 82494, 8251 1 - 82553, 82555 - 82569, 82571 - 82667, 82671 - 82717, 82756 - 8281 1 , 82822 - 82847, 82849 - 82877, 82894 - 82945, 82947 - 82969, 82975 - 83004, 83021 - 83044, 83065 - 83086, 83088 - 83108, 831 17 - 83140, 83146 - 83161 , 83168 - 83184, 83187 - 83206, 83196 - 83214, 83227 - 83249, 83251 - 83266, 83305 - 83333, 83342 - 83377, 8341 1 - 83434, 83436 - 83462, 83464 - 83552, 83554 - 83582, 83584 - 83639, 83642 - 83668, 83679 - 83708, 83710 - 83734, 83736 - 83752, 83781 - 83795, 83805 - 83837, 83839 - 83891 , 83916 - 83931 , 83925 - 83939, 83986 - 84004, 84006 - 84029, 84031 - 84045, 84091 - 84135, 84160 - 84182, 84185 - 8421 1 , 84235 - 84329, 84331 - 84370, 84383 - 84452, 84454 - 84484, 84506 - 84536, 84538 - 84558, 84580 - 84610, 84634 - 84664, 84667 - 84682, 84690 - 84712, 84727 - 84747, 84755 - 84776, 84779 - 84861 , 84881 - 84895, 84925 - 84979, 84981 - 85002, 85004 - 85020, 85022 - 85051 , 85068 - 85082, 85086 - 85109, 85136 - 85159, 85167 - 85222, 85231 - 85248, 85278 - 85294, 85306 - 85330, 85340 - 85355, 85360 - 85377, 85379 - 85393, 85398 - 85439, 85441 - 85465, 85489 - 85558, 85562 - 85598, 85614 - 85628, 85630 - 85649, 85666 - 85701 , 85744 - 85763, 85765 - 85791 , 85793 - 85873, 85897 - 8591 1 , 85937 - 85979, 85981 - 86001 , 86026 - 86083, 86085 - 86124, 86129 - 86174, 86179 - 86195, 86197 - 86225, 86227 - 86256, 86271 - 86305, 86338 - 86369, 86371 - 86392, 86394 - 86418, 86420 - 86481 , 86476 - 86495, 86497 - 86517, 86546 - 86562, 86567 - 86590, 86600 - 86614, 86616 - 86658, 86660 - 86679, 86681 - 86704, 86706 - 86817, 86819 - 86835, 86873 - 86900, 86902 - 86958, 86961 - 86995, 86997 - 87057, 87059 - 87080, 87096 - 87123, 87139 - 87168, 87170 - 87198, 87195 - 87224, 87226 - 87245, 87259 - 87313, 87358 - 87373, 87366 - 87385, 87387 - 87439, 87461 - 87486, 87484 - 87535, 87543 - 87561 , 87577 - 87592, 87595 - 87755, 87770 - 87820, 87827 - 87876, 87892 - 87922, 88018 - 88041 , 88043 - 88069, 88095 - 88109, 88111 - 88165, 88174 - 88192, 88262 - 88285, 88287 - 88303, 88305 - 88327, 88332 - 88362, 88364 - 88387, 88397 - 88430, 88459 - 88527, 88529 - 88560, 88562 - 88582, 88592 - 88678, 88680 - 88725, 88734 - 88762, 88764 - 88820, 88822 - 88869, 88871 - 88917, 88919 - 88941 , 88943 - 88982, 89003 - 89105, 89111 - 89133, 89132 - 89155, 89157- 89187, 89189-89212, 89237 - 89281 , 89283 - 89333, 89346-89364, 89369-89393, 89395 - 89410, 89412 - 89427, 89429 - 89446, 89452 - 89488, 89498 - 89529, 89559 - 89612, 89628 - 89657, 89660 - 89680, 89687 - 89717, 89722 - 89737, 89753 - 89782, 89804 - 89825, 89827 - 89864, 89866 - 89888, 89896 - 89914, 89966 - 90059, 90061 - 90080, 90082 - 90124, 90129 - 90146, 90166 - 90198, 90200 - 90224, 90238 - 90263, 90267 - 90297, 90299 - 90313, 90315 - 90331 , 90338 - 90377, 90395 - 90418, 90450 - 90467, 90483 - 90506, 90508 - 90527, 90534 - 90550, 90552 - 90571 , 90573 - 90595, 90631 - 90714, 90731 - 90750, 90765 - 90780, 90782 - 90805, 90814 - 90842, 90844 - 90865, 90867-90891, 90914-90929, 90947-90972, 90961 -90975, 90978-91117, 91137-91209, 91224-91253, 91255-91283, 91285-91346, 91348-91374, 91376- 91393, 91395-91420, 91433-91665, 91667-91716, 91718 - 91771 , 91783 - 91891 , 91893-91921, 91923-91968, 91970-92002, 92008 - 92051 , 92054 - 92087, 92089- 92111, 92121 -92155, 92157 - 92181 , 92183 - 92202, 92213-92253, 92263-92285, 92302 - 92318, 92320 - 92339, 92352 - 92371 , 92378 - 92402, 92411 - 92426, 92428 - 92448, 92453 - 92499, 92503 - 92521 , 92521 - 92535, 92542 - 92558, 92596 - 92637, 92651 - 92683, 92693 - 92715, 92713 - 92733, 92722 - 92736, 92747 - 92762, 92786 - 92812, 92841 - 92881 , 92900 - 92915, 92917-92949, 92966-93033, 93066-93092, 93115-93144, 93168-93187, 93232-93250, 93267-93310, 93323-93344, 93362- 93381, 93383 - 93405, 93469 - 93516, 93518 - 93532, 93534 - 93559, 93550 - 93564, 93560 - 93594, 93612 - 93664, 93666 - 93719, 93721 - 93738, 93740 - 93762, 93766 - 93803, 93805 - 93854, 93862 - 93912, 93927 - 93963, 93955 - 93969, 93981 - 94009, 94018 - 94075, 94077 - 94115, 94163 - 94179, 94199 - 94227, 94231 - 94248, 94261 - 94309, 94329 - 94355, 94357 - 94401 , 94414 - 94440, 94460 - 94499, 94522 - 94550, 94540 - 94559, 94561 - 94590, 94582 - 94597, 94598 - 94620, 94626 - 94640, 94667 - 94691, 94692 - 94710, 94707 - 94723, 94725 - 94740, 94742 - 94758, 94750 - 94770, 94788 - 94807, 94797 - 94812, 94809 - 94859, 94861 - 94881 , 94900 - 94943, 94975 - 94998, 95046-95103, 95143 - 95161 , 95161 -95188, 95199-95235, 95242-95257,
95280 - 95299, 95301 - 95355, 95368 - 95405, 95421 - 95455, 95464 - 95491 , 95503 - 95573, 95583 - 95606, 95608 - 95626, 95645 - 95685, 95687 - 95792, 95805 - 95831 ,
95832 - 95849, 95839 - 95855, 95844 - 95866, 95880 - 95897, 95899 - 95929, 95928 - 95943, 95944 - 95961, 95962 - 95982, 95996 - 96026, 96015 - 96036, 96027 - 96051,
96053 - 96084, 96083 - 96097, 96089 - 96108, 96110-96131, 96133 - 96156, 96158 - 96212, 96223-96247, 96277 - 96311 , 96313 - 96335, 96337-96356, 96363-96397,
96410 - 96428, 96439 - 96461, 96486 - 96538, 96590 - 96612, 96614 - 96649, 96651 - 96667, 96695 - 96729, 96741 - 96858, 96860 - 96906, 96936 - 97025, 97027 - 97046,
97058 - 97080, 97082 - 97153, 97166 - 97182, 97185 - 97200, 97222 - 97323, 97328 - 97396, 97401 - 97423, 97473 - 97488, 97500 - 97515, 97517 - 97532, 97556 - 97575,
97591 - 97616, 97626 - 97673, 97675 - 97705, 97707 - 97722, 97724 - 97747, 97760 - 97792, 97794 - 97841 , 97843 - 97869, 97899 - 97931 , 97946 - 97965, 97963 - 98002,
98017 - 98044, 98046 - 98076, 98101 - 98133, 98135 - 98162, 98189 - 98211 , 98213 - 98315, 98317 - 98332, 98334 - 98350, 98400 - 98479, 98497 - 98533, 98615 - 98666,
98676 - 98716, 98732 - 98770, 98768 - 98809, 98820 - 98868, 98874 - 98904, 98915 - 98933, 98967-98983, 98985-99005, 99018-99054, 99070-99102, 99125-99141,
99145 - 99159, 99211 - 99262, 99272 - 99290, 99287 - 99302, 99361 - 99378, 99378 - 99407, 99414 - 99439, 99444 - 99459, 99476 - 99497, 99499 - 99576, 99578 - 99597,
99599 - 99630, 99632 - 99657, 99672 - 99748, 99758 - 99781 , 99797 - 99816, 99818- 99848, 99875 - 99904, 99922 - 99959, 99986 - 100013, 100028 - 100060, 100062 - 100102, 100105 - 100147, 100149 - 100175, 100221 - 100257, 100268 - 100293, 100295 - 100312,
100317 - 100345, 100353 - 100398, 100400 - 100479, 100481 - 100495, 100506 - 100523,
100532 - 100585, 100606 - 100661 , 100681 - 100707, 100722 - 100741 , 100759 - 100799,
100813- 100902, 100911 - 100947, 100948- 100968, 100970- 101104, 101111 - 101140,
101142- 101167, 101170- 101189, 101211 - 101236, 101238- 101253, 101255- 101273,
101275- 101306, 101327- 101371, 101381 - 101411, 101447- 101476, 101514- 101528,
101527- 101541, 101530- 101579, 101590- 101635, 101626- 101655, 101656- 101685,
101681 - 101695, 101687- 101725, 101715- 101732, 101756- 101776, 101825- 101861,
101890 - 101987, 101993 - 102036, 102073 - 102089, 102091 - 102259, 102276 - 102297,
102299- 102326, 102351 - 102371, 102376- 102399, 102427- 102442, 102436- 102459,
102461 - 102491 , 102507 - 102522, 102516- 102534, 102536 - 102610, 102637 - 102678,
102681 - 102707, 102724 - 102769, 102771 - 102790, 102808 - 102823, 102823 - 102837,
102840 - 102879, 102930 - 102955, 102957 - 102971 , 102977 - 103060, 103072 - 103108, 1031 10 - 103125, 103127 - 103177, 103181 - 103199, 103252 - 103279, 103293 - 103329, 103346 - 103360, 103362 - 103397, 103416 - 103552, 103555 - 103577, 103607 - 103627, 103641 - 103664, 103674 - 103688, 103691 - 103746, 103764 - 103785, 103804 - 103823, 103825 - 103866, 103869 - 103894, 103908 - 103927, 103923 - 103938, 103976 - 103994, 104018 - 104046, 104047 - 104073, 104075 - 104138, 104140 - 104189, 104191 - 104210, 104249 - 104279, 104289 - 104305, 104295 - 104310, 10431 1 - 104362, 104370 - 104384, 104386 - 104410, 104431 - 104448, 104450 - 104480, 104508 - 104525, 104527 - 104544, 104546 - 104562, 104583 - 104601 , 104622 - 104644, 104646 - 104668, 104687 - 104710, 104716 - 104736, 104738 - 104817, 104822 - 104847, 104858 - 104885, 104880 - 104930, 104932 - 104949, 104951 - 104966, 104976 - 104999, 105001 - 105016, 105033 - 105051 , 105081 - 105109, 1051 1 1 - 105261 , 105263 - 105387, 105399 - 105452, 105454 - 105535, 105578 - 105596, 105613 - 105643, 105645 - 105707, 105709 - 105759, 105761 - 105838, 105840 - 105860, 105873 - 105920, 105925 - 105948, 105950 - 105992, 105994 - 106019, 106031 - 106055, 106058 - 106086, 106107 - 106209, 106216 - 106241 , 106272 - 106286, 106289 - 106375, 106385 - 106434, 106436 - 106498, 106512 - 106527, 106528 - 106544, 106546 - 106578, 106580 - 106617, 106619 - 106640, 106642 - 106693, 106695 - 106713, 106715 - 106732, 106734 - 106754, 106756 - 106780, 106847 - 106877, 106886 - 106909, 10691 1 - 106931 , 106933 - 106980, 106998 - 107027, 107033 - 107060, 107080 - 1071 14, 107120 - 107137, 107165 - 107183, 107202 - 107220, 107222 - 107245, 107247 - 107293, 107307 - 107343, 107372 - 107386, 107377 - 107394, 107409 - 107483, 107516 - 107572, 107591 - 107617, 107621 - 107636, 107638 - 107653, 107656 - 107685, 107688 - 107720, 107746 - 107771 , 107768 - 107784, 107774 - 107788, 107778 - 107792, 107782 - 107818, 107826 - 107841 , 107843 - 107862, 107869 - 107890, 107891 - 107915, 107926 - 107955, 107957 - 107986, 107991 - 108030, 108048 - 108080, 108095 - 108109, 1081 16 - 108158, 108206 - 108224, 108238 - 108260, 108262 - 108279, 108292 - 108330, 108332 - 108357, 108368 - 108393, 108405 - 108467, 108469 - 108516, 108518 - 108642, 108654 - 108807, 108807 - 108958, 108960 - 109056, 109058 - 109246, 109256 - 109281 , 109283 - 109401 , 109403 - 109540, 109542 - 109588, 109590 - 109610, 109625 - 109699, 109701 - 109766, 109777 - 109792, 109805 - 109848, 109846 - 1 10128, 1 10130 - 1 10155, 1 10157 - 1 10190, 1 10192 - 1 10213, 1 10225 - 1 10355, 1 10357 - 1 10396, 1 10407 - 1 10486, 1 10492 - 1 10517, 1 10513 - 1 10528, 1 10529 - 1 10658, 1 10660 - 1 10675, 1 10684 - 1 10755, and 1 10749 - 1 10836.
In a further aspect, the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to a region of SEQ ID NO 13, selected from the group consisting of 174 - 200, 191 - 214, 213 - 261 , 265 - 320, 569 - 584, 588 - 607, 675 - 692, 925 - 942, 953 - 973, 1087 - 1 101 , 1261 - 1275, 1316 - 1333, 1596 - 161 1 , 1830 - 1855, 1988 - 2004, 2010 - 2025, 2070 - 2086, 2092 - 21 13, 2161 2178, 3152 - 3177, 4271 - 4293, 4296 - 4322, 4697 - 471 1 , 4982 - 4998, 5229 - 5247, 6484 6507. 7844 - 7858. 9777 - 9803. 10049 - 10066. 12074 - 12089. 13606 - 13621. 13633 -
13647, 13940 - 13959, 15787 - 15803, 15815 - 15832, 17085 - 17100, 17277 - 17292, 18176 - 18191 , 18347 - 18361 , 18686 - 18706, 18753 - 18769, 18771 - 18794, 18828 - 18856, 19000 - 19015, 19345 - 19360, 19733 - 19749, 19788 - 19803, 1981 1 - 19841 , 19855 - 19886, 19921 - 19935, 20195 - 20210, 22017 - 22032, 22084 - 22103, 22207 - 22226, 22332 - 22348, 22437 - 22473, 22462 - 22476, 22605 - 22621 , 24058 - 24072, 26596 - 26610, 27288 - 27304, 31762 - 31778, 32057 - 32071 , 32464 - 32478, 32921 - 32943, 33176 - 33192, 34790 - 34804, 39088 - 39102, 40107 - 40123, 40671 - 40687, 40782 - 40804, 40806 - 40840, 40842 - 40886, 40898 - 40946, 41653 - 41667, 42965 - 42989, 42989 - 43006, 43658 - 43672, 43836 - 43850, 44141 - 44155, 4441 1 - 44426, 44825 - 44842, 451 18 - 45134, 45177 - 45196, 45475 - 45505, 45508 - 45527, 45732 - 45748, 45933 - 45957, 46045 - 46075, 46101 - 46133, 47225 - 47250, 47362 - 47377, 47861 - 47876, 47913 - 47928, 47930 - 47957, 48077 - 48091 , 48305 - 48329, 48479 - 48498, 48847 - 48863, 48993 - 49010, 49609 - 49624, 49732 - 49746, 49772 - 49787, 50109 - 50129, 50329 - 50345, 50704 - 50721 , 50809 - 50837, 50839 - 50861 , 50926 - 50940, 50993 - 51008, 51063 - 51088, 51 158 - 51 173, 51 194 - 51210, 51256 - 51270, 51306 - 51325, 51875 - 51893, 51943 - 51961 , 52653 - 52670, 53580 - 53601 , 53606 - 53627, 53747 - 53768, 53780 - 53796, 53866 - 53886, 56606 - 56621 , 57208 - 57222, 57526 - 57563, 57554 - 57572, 57873 - 57888, 59495 - 59509, 59539 - 59553, 60318 - 60338, 60401 - 60415, 60418 - 60445, 60448 - 60468, 60484 - 60500, 60719 - 60733, 60786 - 60800, 61946 - 61960, 63531 - 63547, 65257 - 65271 , 65510 - 65531 , 65531 - 65547, 65568 - 65600, 6561 1 - 65628, 65812 - 65839, 65842 - 65868, 66023 - 66044, 66161 - 66189, 66229 - 66243, 66387 - 66401 , 66474 - 66488, 66501 - 66516, 67529 - 67548, 68295 - 68318, 68436 - 68450, 68833 - 68851 , 69724 - 69740, 69820 - 69835, 70749 - 70765, 71725 - 71741 , 73469 - 73514, 73516 - 73536, 73538 - 73558, 73586 - 73619, 73654 - 73675, 73677 - 73699, 73699 - 73713, 73719 - 73754, 73756 - 73794, 73880 - 73907, 73957 - 73975, 74105 - 74127, 74159 - 74191 , 75126 - 75143, 76501 - 76528, 76590 - 76609, 76617 - 76632, 77125 - 77143, 77955 - 77969, 78175 - 78189, 78389 - 78405, 78407 - 78429, 78452 - 78467, 78987 - 79002, 79317 - 79331 , 79423 - 79446, 79466-79488, 80141 -80155, 80531 -80545, 80814-80828, 81112-81126, 81180-81194, 81184-81203, 81845-81863, 81905-81939, 81962 - 81981 , 82310 - 82331 , 82405 - 82420, 83484 - 83507, 83518 - 83540, 86444 - 86460, 86743 - 86761 , 87206 - 87221, 87398 - 87413, 87790 - 87805, 88538 - 88556, 90189 - 90205, 90660 - 90680, 90687-90709, 91157-91180, 91465 - 91481 , 91522 - 91545, 91658-91679, 91681 -91701, 91750-91764, 92720-92734, 93215-93232, 93590-93604, 93927- 93966, 94507 - 94521 , 95542 - 95557, 95833 - 95853, 96363 - 96379, 98949 - 98963, 99207-99221, 99361 -99380, 100087- 100102, 100964- 100986, 101397- 101440, 101762 - 101776, 102626 - 102640, 105254 - 105268, 107766 - 107796, 107790 - 107819,
107814 - 107828, 108530 - 108573, 108578 - 108592, 108605 - 108627, 108632 - 108652,
108686 - 108720, 108779 - 108803, 108832 - 108869, 108975 - 108992, 108996 - 109017,
109026 - 109044, 109158 - 109174, 109195 - 109217, 109256 - 109275, 109284 - 109354,
109371 - 109392, 109439- 109456, 109465- 109481, 109648- 109662, 109669- 109689, 109703 - 109718, 109728 - 109743, 109850 - 109882, 109891 - 109906, 109919 - 109945, 109961 - 109983, 109985- 110012, 110091 - 110106, 110684- 110703, 110725- 110746, and 110748- 110822.
Target Cell
The term a“target cell” as used herein refers to a cell which is expressing the target nucleic acid. In some embodiments the target cell may be in vivo or in vitro. In some embodiments the target cell is a mammalian cell such as a rodent cell, such as a mouse cell or a rat cell, or a primate cell such as a monkey cell or a human cell.
In preferred embodiments the target cell expresses VCAN mRNA, such as the VCAN pre- mRNA, e.g. SEQ ID NO 13, or VCAN mature mRNA(e.g. SEQ ID NO 14, 15, 16 or 17). The poly A tail of VCAN mRNA is typically disregarded for antisense oligonucleotide targeting.
Naturally occurring variant
The term“naturally occurring variant” refers to variants of VCAN gene or transcripts which originate from the same genetic loci as the target nucleic acid, but may differ for example, by virtue of degeneracy of the genetic code causing a multiplicity of codons encoding the same amino acid, or due to alternative splicing of pre-mRNA, or the presence of polymorphisms, such as single nucleotide polymorphisms (SNPs), and allelic variants. Based on the presence of the sufficient complementary sequence to the oligonucleotide, the
oligonucleotide of the invention may therefore target the target nucleic acid and naturally occurring variants thereof. The homo sapiens VCAN gene is located at chromosome 5, 83471674..83582303, complement (NC_000005.10, Gene ID 1462).
In some embodiments, the naturally occurring variants have at least 95% such as at least 98% or at least 99% homology to a mammalian VCAN target nucleic acid, such as a target nucleic acid selected form the group consisting of SEQ ID NO 13, 14, 15, 16 or 17. In some embodiments the naturally occurring variants have at least 99% homology to the human VCAN target nucleic acid of SEQ ID NO 13.
Modulation of expression
The term“modulation of expression” as used herein is to be understood as an overall term for an oligonucleotide’s ability to alter the amount of VCAN protein or VCAN mRNA when compared to the amount of VCAN or VCAN mRNA prior to administration of the
oligonucleotide. Alternatively modulation of expression may be determined by reference to a control experiment. It is generally understood that the control is an individual or target cell treated with a saline composition or an individual or target cell treated with a non-targeting oligonucleotide (mock).
One type of modulation is an oligonucleotide’s ability to inhibit, down-regulate, reduce, suppress, remove, stop, block, prevent, lessen, lower, avoid or terminate expression of VCAN, e.g. by degradation of VCAN mRNA.
High affinity modified nucleosides
A high affinity modified nucleoside is a modified nucleotide which, when incorporated into the oligonucleotide enhances the affinity of the oligonucleotide for its complementary target, for example as measured by the melting temperature (Tm). A high affinity modified nucleoside of the present invention preferably result in an increase in melting temperature between +0.5 to + 12°C, more preferably between +1.5 to +10°C and most preferably between+3 to +8°C per modified nucleoside. Numerous high affinity modified nucleosides are known in the art and include for example, many 2’ substituted nucleosides as well as locked nucleic acids (LNA) (see e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr.
Opinion in Drug Development, 2000, 3(2), 293-213).
Sugar modifications
The oligomer of the invention may comprise one or more nucleosides which have a modified sugar moiety, i.e. a modification of the sugar moiety when compared to the ribose sugar moiety found in DNA and RNA.
Numerous nucleosides with modification of the ribose sugar moiety have been made, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and/or nuclease resistance. Such modifications include those where the ribose ring structure is modified, e.g. by replacement with a hexose ring (HNA), or a bicyclic ring, which typically have a biradicle bridge between the C2 and C4 carbons on the ribose ring (LNA), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e.g. UNA). Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids (WO201 1/017521 ) or tricyclic nucleic acids (WO2013/154798). Modified nucleosides also include nucleosides where the sugar moiety is replaced with a non-sugar moiety, for example in the case of peptide nucleic acids (PNA), or morpholino nucleic acids.
Sugar modifications also include modifications made via altering the substituent groups on the ribose ring to groups other than hydrogen, or the 2’-OH group naturally found in DNA and RNA nucleosides. Substituents may, for example be introduced at the 2’, 3’, 4’ or 5’ positions.
2’ sugar modified nucleosides.
A 2’ sugar modified nucleoside is a nucleoside which has a substituent other than H or -OH at the 2’ position (2’ substituted nucleoside) or comprises a 2’ linked biradicle capable of forming a bridge between the 2’ carbon and a second carbon in the ribose ring, such as LNA (2’ - 4’ biradicle bridged) nucleosides.
Indeed, much focus has been spent on developing 2’ substituted nucleosides, and numerous 2’ substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides. For example, the 2’ modified sugar may provide enhanced binding affinity and/or increased nuclease resistance to the oligonucleotide. Examples of 2’ substituted modified nucleosides are 2’-0-alkyl-RNA, 2’-0-methyl-RNA, 2’- alkoxy-RNA, 2’-0-methoxyethyl-RNA (MOE), 2’-amino-DNA, 2’-Fluoro-RNA, and 2’-F-ANA nucleoside. For further examples, please see e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and
Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Below are illustrations of some 2’ substituted modified nucleosides.
Figure imgf000034_0001
a'-O-Me 2'F-ftNA 2'F-ANA
Figure imgf000035_0001
2'-0-M0E 2'-0-Aliyl 2'-0-Ethylamine
In relation to the present invention 2’ substituted does not include 2’ bridged molecules like LNA.
Locked Nucleic Acids (LNA)
A“LNA nucleoside” is a 2’- modified nucleoside which comprises a biradical linking the
C2’ and C4’ of the ribose sugar ring of said nucleoside (also referred to as a“2’- 4’ bridge”), which restricts or locks the conformation of the ribose ring. These nucleosides are also termed bridged nucleic acid or bicyclic nucleic acid (BNA) in the literature. The locking of the conformation of the ribose is associated with an enhanced affinity of hybridization (duplex stabilization) when the LNA is incorporated into an oligonucleotide for a complementary RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the oligonucleotide/complement duplex.
Non limiting, exemplary LNA nucleosides are disclosed in WO 99/014226, WO 00/66604, WO 98/039352 , WO 2004/046160, WO 00/047599, WO 2007/134181 , WO 2010/077578, WO 2010/036698, WO 2007/090071 , WO 2009/006478, WO 201 1/156202,
WO 2008/154401 , WO 2009/067647, WO 2008/150729, Morita et al., Bioorganic & Med.Chem. Lett. 12, 73-76, Seth et al. J. Org. Chem. 2010, Vol 75(5) pp. 1569-81 , and Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667.
Further non limiting, exemplary LNA nucleosides are disclosed in Scheme 1.
Scheme 1 :
Figure imgf000036_0001
Particular LNA nucleosides are beta-D-oxy-LNA, 6’-methyl-beta-D-oxy LNA such as (S)-6’-methyl-beta-D-oxy-LNA (ScET) and ENA.
A particularly advantageous LNA is beta-D-oxy-LNA.
RNase H Activity and Recruitment
The RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule. WO01/23613 provides in vitro methods for determining RNaseH activity, which may be used to determine the ability to recruit RNaseH. Typically an oligonucleotide is deemed capable of recruiting RNase H if it, when provided with a complementary target nucleic acid sequence, has an initial rate, as measured in pmol/l/min, of at least 5%, such as at least 10% or more than 20% of the of the initial rate determined when using a oligonucleotide having the same base sequence as the modified oligonucleotide being tested, but containing only DNA monomers with
phosphorothioate linkages between all monomers in the oligonucleotide, and using the methodology provided by Example 91 - 95 of WO01/23613 (hereby incorporated by reference). For use in determining RHase H activity, recombinant human RNase H1 is available from Lubio Science GmbH, Lucerne, Switzerland. Gapmer
The antisense oligonucleotide of the invention, or contiguous nucleotide sequence thereof may be a gapmer. The antisense gapmers are commonly used to inhibit a target nucleic acid via RNase H mediated degradation. A gapmer oligonucleotide comprises at least three distinct structural regions a 5’-flank, a gap and a 3’-flank, F-G-F’ in the‘5 -> 3’ orientation. The“gap” region (G) comprises a stretch of contiguous DNA nucleotides which enable the oligonucleotide to recruit RNase H. The gap region is flanked by a 5’ flanking region (F) comprising one or more sugar modified nucleosides, advantageously high affinity sugar modified nucleosides, and by a 3’ flanking region (F’) comprising one or more sugar modified nucleosides, advantageously high affinity sugar modified nucleosides. The one or more sugar modified nucleosides in region F and F’ enhance the affinity of the oligonucleotide for the target nucleic acid (i.e. are affinity enhancing sugar modified nucleosides). In some embodiments, the one or more sugar modified nucleosides in region F and F’ are 2’ sugar modified nucleosides, such as high affinity 2’ sugar modifications, such as independently selected from LNA and 2’-MOE.
In a gapmer design, the 5’ and 3’ most nucleosides of the gap region are DNA nucleosides, and are positioned adjacent to a sugar modified nucleoside of the 5’ (F) or 3’ (F’) region respectively. The flanks may further defined by having at least one sugar modified nucleoside at the end most distant from the gap region, i.e. at the 5’ end of the 5’ flank and at the 3’ end of the 3’ flank.
Regions F-G-F’ form a contiguous nucleotide sequence. Antisense oligonucleotides of the invention, or the contiguous nucleotide sequence thereof, may comprise a gapmer region of formula F-G-F’.
The overall length of the gapmer design F-G-F’ may be, for example 12 to 32 nucleosides, such as 13 to 24, such as 14 to 22 nucleosides, Such as from 14 to17, such as 16 to18 nucleosides.
By way of example, the gapmer oligonucleotide of the present invention can be represented by the following formulae:
F i-8-G5-i6-F’i-8, such as
F 1-8-G7-i6-F’2-8
with the proviso that the overall length of the gapmer regions F-G-F’ is at least 12, such as at least 14 nucleotides in length.
Regions F, G and F’ are further defined below and can be incorporated into the F-G-F’ formula. Gapmer - Region G
Region G (gap region) of the gapmer is a region of nucleosides which enables the oligonucleotide to recruit RNaseH, such as human RNase H1 , typically DNA nucleosides. RNaseH is a cellular enzyme which recognizes the duplex between DNA and RNA, and enzymatically cleaves the RNA molecule. Suitably gapmers may have a gap region (G) of at least 5 or 6 contiguous DNA nucleosides, such as 5 - 16 contiguous DNA nucleosides, such as 6 - 15 contiguous DNA nucleosides, such as 7-14 contiguous DNA nucleosides, such as 8 - 12 contiguous DNA nucleotides, such as 8 - 12 contiguous DNA nucleotides in length. The gap region G may, in some embodiments consist of 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 contiguous DNA nucleosides. One or more cytosine (C) DNA in the gap region may in some instances be methylated (e.g. when a DNA c is followed by a DNA g) such residues are either annotated as 5-methyl-cytosine (meC). In some embodiments the gap region G may consist of 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 contiguous phosphorothioate linked DNA nucleosides. In some embodiments, all internucleoside linkages in the gap are phosphorothioate linkages.
Whilst traditional gapmers have a DNA gap region, there are numerous examples of modified nucleosides which allow for RNaseH recruitment when they are used within the gap region. Modified nucleosides which have been reported as being capable of recruiting RNaseH when included within a gap region include, for example, alpha-L-LNA, C4’ alkylated DNA (as described in PCT/EP2009/050349 and Vester et al., Bioorg. Med. Chem. Lett. 18 (2008) 2296 - 2300, both incorporated herein by reference), arabinose derived nucleosides like ANA and 2'F-ANA (Mangos et al. 2003 J. AM. CHEM. SOC. 125, 654-661 ), UNA (unlocked nucleic acid) (as described in Fluter ef al., Mol. Biosyst., 2009, 10, 1039 incorporated herein by reference). UNA is unlocked nucleic acid, typically where the bond between C2 and C3 of the ribose has been removed, forming an unlocked“sugar” residue. The modified nucleosides used in such gapmers may be nucleosides which adopt a 2’ endo (DNA like) structure when introduced into the gap region, i.e. modifications which allow for RNaseH recruitment). In some embodiments the DNA Gap region (G) described herein may optionally contain 1 to 3 sugar modified nucleosides which adopt a 2’ endo (DNA like) structure when introduced into the gap region.
Region G -“Gap-breaker”
Alternatively, there are numerous reports of the insertion of a modified nucleoside which confers a 3’ endo conformation into the gap region of gapmers, whilst retaining some RNaseH activity. Such gapmers with a gap region comprising one or more 3’endo modified nucleosides are referred to as“gap-breaker” or“gap-disrupted” gapmers, see for example WO2013/022984. Gap-breaker oligonucleotides retain sufficient region of DNA nucleosides within the gap region to allow for RNaseH recruitment. The ability of gapbreaker
oligonucleotide design to recruit RNaseH is typically sequence or even compound specific - see Rukov et al. 2015 Nucl. Acids Res. Vol. 43 pp. 8476-8487, which discloses“gapbreaker” oligonucleotides which recruit RNaseH which in some instances provide a more specific cleavage of the target RNA. Modified nucleosides used within the gap region of gap- breaker oligonucleotides may for example be modified nucleosides which confer a 3’endo confirmation, such 2’ -O-methyl (OMe) or 2’-0-MOE (MOE) nucleosides, or beta-D LNA nucleosides (the bridge between C2’ and C4’ of the ribose sugar ring of a nucleoside is in the beta conformation), such as beta-D-oxy LNA or ScET nucleosides.
As with gapmers containing region G described above, the gap region of gap-breaker or gap-disrupted gapmers, have a DNA nucleosides at the 5’ end of the gap (adjacent to the 3’ nucleoside of region F), and a DNA nucleoside at the 3’ end of the gap (adjacent to the 5’ nucleoside of region F’). Gapmers which comprise a disrupted gap typically retain a region of at least 3 or 4 contiguous DNA nucleosides at either the 5’ end or 3’ end of the gap region. Exemplary designs for gap-breaker oligonucleotides include
Figure imgf000039_0001
wherein region G is within the brackets [Dn-Er- Dm], D is a contiguous sequence of DNA nucleosides, E is a modified nucleoside (the gap-breaker or gap-disrupting nucleoside), and F and F’ are the flanking regions as defined herein, and with the proviso that the overall length of the gapmer regions F-G-F’ is at least 12, such as at least 14 nucleotides in length. In some embodiments, region G of a gap disrupted gapmer comprises at least 6 DNA nucleosides, such as 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 DNA nucleosides. As described above, the DNA nucleosides may be contiguous or may optionally be interspersed with one or more modified nucleosides, with the proviso that the gap region G is capable of mediating RNaseH recruitment.
Gapmer - flanking regions, F and F’
Region F is positioned immediately adjacent to the 5’ DNA nucleoside of region G. The 3’ most nucleoside of region F is a sugar modified nucleoside, such as a high affinity sugar modified nucleoside, for example a 2’ substituted nucleoside, such as a MOE nucleoside, or an LNA nucleoside.
Region F’ is positioned immediately adjacent to the 3’ DNA nucleoside of region G. The 5’ most nucleoside of region F’ is a sugar modified nucleoside, such as a high affinity sugar modified nucleoside, for example a 2’ substituted nucleoside, such as a MOE nucleoside, or an LNA nucleoside.
Region F is 1 - 8 contiguous nucleotides in length, such as 2-6, such as 3-4 contiguous nucleotides in length. Advantageously the 5’ most nucleoside of region F is a sugar modified nucleoside. In some embodiments the two 5’ most nucleoside of region F are sugar modified nucleoside. In some embodiments the 5’ most nucleoside of region F is an LNA nucleoside. In some embodiments the two 5’ most nucleoside of region F are LNA nucleosides. In some embodiments the two 5’ most nucleoside of region F are 2’ substituted nucleoside nucleosides, such as two 3’ MOE nucleosides. In some embodiments the 5’ most nucleoside of region F is a 2’ substituted nucleoside, such as a MOE nucleoside.
Region F’ is 2 - 8 contiguous nucleotides in length, such as 3-6, such as 4-5 contiguous nucleotides in length. Advantageously, embodiments the 3’ most nucleoside of region F’ is a sugar modified nucleoside. In some embodiments the two 3’ most nucleoside of region F’ are sugar modified nucleoside. In some embodiments the two 3’ most nucleoside of region F’ are LNA nucleosides. In some embodiments the 3’ most nucleoside of region F’ is an LNA nucleoside. In some embodiments the two 3’ most nucleoside of region F’ are 2’ substituted nucleoside nucleosides, such as two 3’ MOE nucleosides. In some embodiments the 3’ most nucleoside of region F’ is a 2’ substituted nucleoside, such as a MOE nucleoside. It should be noted that when the length of region F or F’ is one, it is advantageously an LNA nucleoside.
In some embodiments, region F and F’ independently consists of or comprises a contiguous sequence of sugar modified nucleosides. In some embodiments, the sugar modified nucleosides of region F may be independently selected from 2’-0-alkyl-RNA units, 2’-0- methyl-RNA, 2’-amino-DNA units, 2’-fluoro-DNA units, 2’-alkoxy-RNA, MOE units, LNA units, arabino nucleic acid (ANA) units and 2’-fluoro-ANA units.
In some embodiments, region F and F’ independently comprises both LNA and a 2’ substituted modified nucleosides (mixed wing design).
In some embodiments, region F and F’ consists of only one type of sugar modified nucleosides, such as only MOE or only beta-D-oxy LNA or only ScET. Such designs are also termed uniform flanks or uniform gapmer design.
In some embodiments, all the nucleosides of region F or F’, or F and F’ are LNA
nucleosides, such as independently selected from beta-D-oxy LNA, ENA or ScET nucleosides. In some embodiments region F consists of 1-5, such as 2-4, such as 3-4 such as 1 , 2, 3, 4 or 5 contiguous LNA nucleosides. In some embodiments, all the nucleosides of region F and F’ are beta-D-oxy LNA nucleosides. In some embodiments, all the nucleosides of region F or F’, or F and F’ are 2’ substituted nucleosides, such as OMe or MOE nucleosides. In some embodiments region F consists of 1 , 2, 3, 4, 5, 6, 7, or 8 contiguous OMe or MOE nucleosides. In some embodiments only one of the flanking regions can consist of 2’ substituted nucleosides, such as OMe or MOE nucleosides. In some embodiments it is the 5’ (F) flanking region that consists 2’ substituted nucleosides, such as OMe or MOE nucleosides whereas the 3’ (F’) flanking region comprises at least one LNA nucleoside, such as beta-D-oxy LNA nucleosides or cET nucleosides. In some embodiments it is the 3’ (F’) flanking region that consists 2’ substituted nucleosides, such as OMe or MOE nucleosides whereas the 5’ (F) flanking region comprises at least one LNA nucleoside, such as beta-D-oxy LNA nucleosides or cET nucleosides.
In some embodiments, all the modified nucleosides of region F and F’ are LNA nucleosides, such as independently selected from beta-D-oxy LNA, ENA or ScET nucleosides, wherein region F or F’, or F and F’ may optionally comprise DNA nucleosides (an alternating flank, see definition of these for more details). In some embodiments, all the modified nucleosides of region F and F’ are beta-D-oxy LNA nucleosides, wherein region F or F’, or F and F’ may optionally comprise DNA nucleosides (an alternating flank, see definition of these for more details).
In some embodiments the 5’ most and the 3’ most nucleosides of region F and F’ are LNA nucleosides, such as beta-D-oxy LNA nucleosides or ScET nucleosides.
In some embodiments, the internucleoside linkage between region F and region G is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage between region F’ and region G is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkages between the nucleosides of region F or F’, F and F’ are phosphorothioate internucleoside linkages.
LNA Gapmer
An LNA gapmer is a gapmer wherein either one or both of region F and F’ comprises or consists of LNA nucleosides. A beta-D-oxy gapmer is a gapmer wherein either one or both of region F and F’ comprises or consists of beta-D-oxy LNA nucleosides.
In some embodiments the LNA gapmer is of formula: [LNA^.s-tregion G] -[LNA]^, wherein region G is as defined in the Gapmer region G definition.
MOE Gapmers
A MOE gapmers is a gapmer wherein regions F and F’ consist of MOE nucleosides. In some embodiments the MOE gapmer is of design [MOEJ^s-lRegion G]-[MOE] 1-8, such as [MOE]2.7-[Region G]5-ie-[MOE] 2-7, such as [MOE]3.6-[Region G]-[MOE] 3-6, wherein region G is as defined in the Gapmer definition. MOE gapmers with a 5-10-5 design (MOE-DNA-MOE) have been widely used in the art.
Mixed Wing Gapmer
A mixed wing gapmer is an LNA gapmer wherein one or both of region F and F’ comprise a 2’ substituted nucleoside, such as a 2’ substituted nucleoside independently selected from the group consisting of 2’-0-alkyl-RNA units, 2’-0-methyl-RNA, 2’-amino-DNA units, 2’- fluoro-DNA units, 2’-alkoxy-RNA, MOE units, arabino nucleic acid (ANA) units and 2’-fluoro- ANA units, such as a MOE nucleosides. In some embodiments wherein at least one of region F and F’, or both region F and F’ comprise at least one LNA nucleoside, the remaining nucleosides of region F and F’ are independently selected from the group consisting of MOE and LNA. In some embodiments wherein at least one of region F and F’, or both region F and F’ comprise at least two LNA nucleosides, the remaining nucleosides of region F and F’ are independently selected from the group consisting of MOE and LNA. In some mixed wing embodiments, one or both of region F and F’ may further comprise one or more DNA nucleosides.
Mixed wing gapmer designs are disclosed in W02008/049085 and WO2012/109395, both of which are hereby incorporated by reference.
Alternating Flank Gapmers
Oligonucleotides with alternating flanks are LNA gapmer oligonucleotides where at least one of the flanks (F or F’) comprises DNA in addition to the LNA nucleoside(s). In some embodiments at least one of region F or F’, or both region F and F’, comprise both LNA nucleosides and DNA nucleosides. In such embodiments, the flanking region F or F’, or both F and F’ comprise at least three nucleosides, wherein the 5’ and 3’ most nucleosides of the F and/or F’ region are LNA nucleosides.
In some embodiments at least one of region F or F’, or both region F and F’, comprise both LNA nucleosides and DNA nucleosides. In such embodiments, the flanking region F or F’, or both F and F’ comprise at least three nucleosides, wherein the 5’ and 3’ most nucleosides of the F or F’ region are LNA nucleosides, and there is at least one DNA nucleoside positioned between the 5’ and 3’ most LNA nucleosides of region F or F’ (or both region F and F’).
Region D’ or D” in an oligonucleotide
The oligonucleotide of the invention may in some embodiments comprise or consist of the contiguous nucleotide sequence of the oligonucleotide which is complementary to the target nucleic acid, such as the gapmer F-G-F’, and further 5’ and/or 3’ nucleosides. The further 5’ and/or 3’ nucleosides may or may not be fully complementary to the target nucleic acid.
Such further 5’ and/or 3’ nucleosides may be referred to as region D’ and D” herein.
The addition of region D’ or D” may be used for the purpose of joining the contiguous nucleotide sequence, such as the gapmer, to a conjugate moiety or another functional group. When used for joining the contiguous nucleotide sequence with a conjugate moiety is can serve as a biocleavable linker. Alternatively it may be used to provide exonucleoase protection or for ease of synthesis or manufacture.
Region D’ and D” can be attached to the 5’ end of region F or the 3’ end of region F’, respectively to generate designs of the following formulas D’-F-G-F’, F-G-F’-D” or
D’-F-G-F’-D”. In this instance the F-G-F’ is the gapmer portion of the oligonucleotide and region D’ or D” constitute a separate part of the oligonucleotide.
Region D’ or D” may independently comprise or consist of 1 , 2, 3, 4 or 5 additional nucleotides, which may be complementary or non-complementary to the target nucleic acid. The nucleotide adjacent to the F or F’ region is not a sugar-modified nucleotide, such as a DNA or RNA or base modified versions of these. The D’ or D’ region may serve as a nuclease susceptible biocleavable linker (see definition of linkers). In some embodiments the additional 5’ and/or 3’ end nucleotides are linked with phosphodiester linkages, and are DNA or RNA. Nucleotide based biocleavable linkers suitable for use as region D’ or D” are disclosed in WO2014/076195, which include by way of example a phosphodiester linked DNA dinucleotide. The use of biocleavable linkers in poly-oligonucleotide constructs is disclosed in WO2015/1 13922, where they are used to link multiple antisense constructs (e.g. gapmer regions) within a single oligonucleotide.
In one embodiment the oligonucleotide of the invention comprises a region D’ and/or D” in addition to the contiguous nucleotide sequence which constitutes the gapmer.
In some embodiments, the oligonucleotide of the present invention can be represented by the following formulae:
F-G-F’; in particular Fi-8-G5-i6-F’2-8
D’-F-G-F’, in particular D’i-3-Fi-8-G5-i6-F’2-8
F-G-F’-D”, in particular Fi_8-G5-i6-F’2-8-D”i_3
D’-F-G-F’-D”, in particular DV3- Fi_8-G5-i6-F’2-8-D”i_3
In some embodiments the internucleoside linkage positioned between region D’ and region F is a phosphodiester linkage. In some embodiments the internucleoside linkage positioned between region F’ and region D” is a phosphodiester linkage. Conjugate
The term conjugate as used herein refers to an oligonucleotide which is covalently linked to a non-nucleotide moiety (conjugate moiety or region C or third region).
Conjugation of the oligonucleotide of the invention to one or more non-nucleotide moieties may improve the pharmacology of the oligonucleotide, e.g. by affecting the activity, cellular distribution, cellular uptake or stability of the oligonucleotide. In some embodiments the conjugate moiety modify or enhance the pharmacokinetic properties of the oligonucleotide by improving cellular distribution, bioavailability, metabolism, excretion, permeability, and/or cellular uptake of the oligonucleotide. In particular the conjugate may target the oligonucleotide to a specific organ, tissue or cell type and thereby enhance the effectiveness of the oligonucleotide in that organ, tissue or cell type. A the same time the conjugate may serve to reduce activity of the oligonucleotide in non-target cell types, tissues or organs, e.g. off target activity or activity in non-target cell types, tissues or organs.
In an embodiment, the non-nucleotide moiety (conjugate moiety) is selected from the group consisting of carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g. bacterial toxins), vitamins, viral proteins (e.g. capsids) or combinations thereof.
Linkers
A linkage or linker is a connection between two atoms that links one chemical group or segment of interest to another chemical group or segment of interest via one or more covalent bonds. Conjugate moieties can be attached to the oligonucleotide directly or through a linking moiety (e.g. linker or tether). Linkers serve to covalently connect a third region, e.g. a conjugate moiety (Region C), to a first region, e.g. an oligonucleotide or contiguous nucleotide sequence or gapmer region F-G-F’ (region A).
In some embodiments of the invention the conjugate or oligonucleotide conjugate of the invention may optionally, comprise a linker region (second region or region B and/or region Y) which is positioned between the oligonucleotide or contiguous nucleotide sequence complementary to the target nucleic acid (region A or first region) and the conjugate moiety (region C or third region).
Region B refers to biocleavable linkers comprising or consisting of a physiologically labile bond that is cleavable under conditions normally encountered or analogous to those encountered within a mammalian body. Conditions under which physiologically labile linkers undergo chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidative or reductive conditions or agents, and salt concentration found in or analogous to those encountered in mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activity normally present in a mammalian cell such as from proteolytic enzymes or hydrolytic enzymes or nucleases. In one embodiment the biocleavable linker is susceptible to S1 nuclease cleavage. DNA phosphodiester containing biocleavable linkers are described in more detail in WO 2014/076195 (hereby incorporated by reference) - see also region D’ or D” herein.
Region Y refers to linkers that are not necessarily biocleavable but primarily serve to covalently connect a conjugate moiety (region C or third region), to an oligonucleotide (region A or first region). The region Y linkers may comprise a chain structure or an oligomer of repeating units such as ethylene glycol, amino acid units or amino alkyl groups. The oligonucleotide conjugates of the present invention can be constructed of the following regional elements A-C, A-B-C, A-B-Y-C, A-Y-B-C or A-Y-C. In some embodiments the linker (region Y) is an amino alkyl, such as a C2 - C36 amino alkyl group, including, for example C6 to C12 amino alkyl groups. In a preferred embodiment the linker (region Y) is a C6 amino alkyl group.
T reatment
The term’treatment’ as used herein refers to both treatment of an existing disease {e.g. a disease or disorder as herein referred to), or prevention of a disease, i.e. prophylaxis. It will therefore be recognized that treatment as referred to herein may, in some embodiments, be prophylactic.
DETAILED DESCRIPTION OF THE INVENTION
The invention relates to oligonucleotides, such as antisense oligonucleotides, targeting VCAN expression.
The oligonucleotides of the invention targeting VCAN are capable of hybridizing to and inhibiting the expression of a VCAN target nucleic acid in a cell which is expressing the VCAN target nucleic acid.
The VCAN target nucleic acid may be a mammalian VCAN mRNA or premRNA, such as a human VCAN mRNA or premRNA, for example a premRNA or mRNA originating from the Homo sapiens versican (VCAN), RefSeqGene on chromosome 5, exemplified by NCBI Reference Sequence NG_012682.1 or Ensembl ENSG00000038427 (SEQ ID NO 13). The human VCAN pre-mRNA is encoded on Homo sapiens Chromosome 5, NC_000005.10 (83471674..83582303, complement), GENE ID = 1462 {VCAN).
A mature human mRNA target sequence is illustrated herein by the cDNA sequences SEQ ID NO 14, 15, 16 or 17.
The oligonucleotides of the invention are capable of inhibiting the expression of VCAN target nucleic acid, such as the VCAN mRNA, in a cell which is expressing the target nucleic acid, such as the VCAN mRNA.
In some embodiments, the oligonucleotides of the invention are capable of inhibiting the expression of VCAN target nucleic acid in a cell which is expressing the target nucleic acid, so to reduce the level of VCAN target nucleic acid (e.g. the mRNA) by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% inhibition compared to the expression level of the VCAN target nucleic acid (e.g. the mRNA) in the cell. Suitably the cell is selected from the group consisting of HEK293 and A549. Example 1 provides a suitable assay for evaluating the ability of the oligonucleotides of the invention to inhibit the expression of the target nucleic acid. Suitably the evaluation of a compounds ability to inhibit the expression of the target nucleic acid is performed in vitro, such a gymnotic in vitro assay, for example as according to Example 1.
An aspect of the present invention relates to an antisense oligonucleotide, such as an LNA antisense oligonucleotide gapmer which comprises a contiguous nucleotide sequence of 10 to 30 nucleotides in length with at least 90% complementarity, such as is fully
complementary to SEQ ID NO 13, 14, 15, 16 or 17.
In some embodiments, the oligonucleotide comprises a contiguous sequence of 10 - 30 nucleotides, which is at least 90% complementary, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or 100% complementary with a region of the target nucleic acid or a target sequence.
In some embodiments, the oligonucleotide of the invention comprises a contiguous nucleotides sequence of 12 - 24, such as 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, or 23, contiguous nucleotides in length, wherein the contiguous nucleotide sequence is fully complementary to SEQ ID NO 1 1. In some embodiments, the oligonucleotide of the invention comprises a contiguous nucleotides sequence of 12 - 20, such as 13, 14, 15, 16, 17, 18, 19 or 20, contiguous nucleotides in length, wherein the contiguous nucleotide sequence is fully complementary to SEQ ID NO 12.
In some embodiments, the antisense oligonucleotide of the invention or the contiguous nucleotide sequence thereof is a gapmer, such as an LNA gapmer, a mixed wing gapmer, or an alternating flank gapmer.
In some embodiments, the antisense oligonucleotide according to the invention, comprises a contiguous nucleotide sequence of at least 10 contiguous nucleotides, such as at least 12 contiguous nucleotides, such as at least 13 contiguous nucleotides, such as at least 14 contiguous nucleotides, such as at least 15 contiguous nucleotides, which is fully complementary to SEQ ID NO 1 1.
In some embodiments, the antisense oligonucleotide according to the invention, comprises a contiguous nucleotide sequence of at least 10 contiguous nucleotides, such as at least 12 contiguous nucleotides, such as at least 13 contiguous nucleotides, such as at least 14 contiguous nucleotides, such as at least 15 contiguous nucleotides, which is fully complementary to SEQ ID NO 12.
In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is less than 20 nucleotides in length. In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12 - 24 nucleotides in length. In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12 - 22 nucleotides in length.
In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12 - 20 nucleotides in length. In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12 - 18 nucleotides in length. In some embodiments the contiguous nucleotide sequence of the antisense oligonucleotide according to the invention is 12 - 16 nucleotides in length. Advantageously, in some embodiments all of the internucleoside linkages between the nucleosides of the contiguous nucleotide sequence are phosphorothioate internucleoside linkages. In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 1 1.
In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 12. In some embodiments, the antisense oligonucleotide is a gapmer oligonucleotide comprising a contiguous nucleotide sequence of formula 5’-F-G-F’-3’, where region F and F’ independently comprise 1 - 8 sugar modified nucleosides, and G is a region between 5 and 16 nucleosides which are capable of recruiting RNaseH.
In some embodiments, the sugar modified nucleosides of region F and F’ are independently selected from the group consisting of 2’-0-alkyl-RNA, 2’-0-methyl-RNA, 2’-alkoxy-RNA, 2’- O-methoxyethyl-RNA, 2’-amino-DNA, 2’-fluoro-DNA, arabino nucleic acid (ANA), 2’-fluoro- ANA and LNA nucleosides.
In some embodiments, region G comprises 5 - 16 contiguous DNA nucleosides.
In some embodiments, wherein the antisense oligonucleotide is a gapmer oligonucleotide, such as an LNA gapmer oligonucleotide.
In some embodiments, the LNA nucleosides are beta-D-oxy LNA nucleosides.
In some embodiments, the internucleoside linkages between the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.
Sequence Motifs and Compounds of the Invention
Figure imgf000048_0001
Figure imgf000049_0001
In the compound column, capital letters are beta-D-oxy LNA nucleosides, and LNA C are all 5-methyl C, lower case letters are DNA nucleosides, and optionally a superscript m before a lower case c represent a 5-methyl cytosine DNA nucleoside, and all internucleoside linkages are phosphorothioate internucleoside linkages.
The invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides 12 - 24, such as 12 - 18 in length, nucleosides in length wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence comprising at least 12, such as at least 14, such as at least 15 contiguous nucleotides present in SEQ ID NO 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.
The invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides 12 - 24 nucleosides in length, such as 12 - 18 in length, wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence comprising at least 12, such as at least 13, such as at least 14, such as at least 15 contiguous nucleotides present in SEQ ID NO 5, 6, 7 or 10.
The invention provides LNA gapmers according to the invention comprising or consisting of a contiguous nucleotide sequence selected from SEQ ID NO 1 - 10.
The invention provides antisense oligonucleotides selected from the group consisting of: TTgtcttataacTGCA (SEQ ID NO 1 ), GGcttattttacCACA (SEQ ID NO 2), TATtattgaggaaGCC (SEQ ID NO 3),ATCAaatctgctatCA (SEQ ID NO 4), GAtttagttggcATGA (SEQ ID NO 5), TAGTtggcatgataAT (SEQ ID NO 6), TTtagttggcatGATA (SEQ ID NO 7), CTTggcattataaCTA (SEQ ID NO 8), GTTCacaaagaaagCC (SEQ ID NO 9), AGAtttagttggcATG (SEQ ID NO 10); wherein a capital letter is a LNA nucleoside, and a lower case letter is a DNA nucleoside. In some embodiments all internucleoside linkages in contiguous nucleoside sequence are phosphorothioate internucleoside linkages. Optionally LNA cytosine may be 5-methyl cytosine. Optionally DNA cytosine may be 5-methyl cytosine.
The invention provides antisense oligonucleotides selected from the group consisting of: TTgtcttataacTGCA (SEQ ID NO 1 ), GGcttattttacCACA (SEQ ID NO 2), TATtattgaggaaGCC (SEQ ID NO 3),ATCAaatctgctatCA (SEQ ID NO 4), GAtttagttggcATGA (SEQ ID NO 5), TAGTtggcatgataAT (SEQ ID NO 6), TTtagttggcatGATA (SEQ ID NO 7), CTTggcattataaCTA (SEQ ID NO 8), GTTCacaaagaaagCC (SEQ ID NO 9), AGAtttagttggcATG (SEQ ID NO 10); wherein a capital letter is a beta-D-oxy-LNA nucleoside, and a lower case letter is a DNA nucleoside. In some embodiments all internucleoside linkages in contiguous nucleoside sequence are phosphorothioate internucleoside linkages. Optionally LNA cytosine may be 5-methyl cytosine. Optionally DNA cytosine may be 5-methyl cytosine.
The invention provides antisense oligonucleotides selected from the group consisting of: TTgtcttataacTGCA (SEQ ID NO 1 ), GGcttattttacCACA (SEQ ID NO 2), TATtattgaggaaGCC (SEQ ID NO 3),ATCAaatctgctatCA (SEQ ID NO 4), GAtttagttggcATGA (SEQ ID NO 5), TAGTtggcatgataAT (SEQ ID NO 6), TTtagttggcatGATA (SEQ ID NO 7), CTTggcattataaCTA (SEQ ID NO 8), GTTCacaaagaaagCC (SEQ ID NO 9), AGAtttagttggcATG (SEQ ID NO 10); wherein a capital letter is a beta-D-oxy-LNA nucleoside, wherein all LNA cytosinese are 5- methyl cytosine, and a lower case letter is a DNA nucleoside, wherein all internucleoside linkages in contiguous nucleoside sequence are phosphorothioate internucleoside linkages, and optionally DNA cytosine may be 5-methyl cytosine.
Method of manufacture
In a further aspect, the invention provides methods for manufacturing the oligonucleotides of the invention comprising reacting nucleotide units and thereby forming covalently linked contiguous nucleotide units comprised in the oligonucleotide. Preferably, the method uses phophoramidite chemistry (see for example Caruthers et al, 1987, Methods in Enzymology vol. 154, pages 287-313). In a further embodiment the method further comprises reacting the contiguous nucleotide sequence with a conjugating moiety (ligand) to covalently attach the conjugate moiety to the oligonucleotide. In a further aspect a method is provided for manufacturing the composition of the invention, comprising mixing the oligonucleotide or conjugated oligonucleotide of the invention with a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.
Pharmaceutical Composition
In a further aspect, the invention provides pharmaceutical compositions comprising any of the aforementioned oligonucleotides and/or oligonucleotide conjugates or salts thereof and a pharmaceutically acceptable diluent, carrier, salt and/or adjuvant. A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS) and pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. In some embodiments the pharmaceutically acceptable diluent is sterile phosphate buffered saline. In some embodiments the oligonucleotide is used in the pharmaceutically acceptable diluent at a concentration of 50 - 300pM solution.
The compounds according to the present invention may exist in the form of their
pharmaceutically acceptable salts. The term“pharmaceutically acceptable salt” refers to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds of the present invention and are formed from suitable nontoxic organic or inorganic acids or organic or inorganic bases. Acid-addition salts include for example those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethyl ammonium hydroxide. The chemical modification of a pharmaceutical compound into a salt is a technique well known to pharmaceutical chemists in order to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of compounds. It is for example described in Bastin, Organic Process Research & Development 2000, 4, 427-435 or in Ansel, In:
Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th ed. (1995), pp. 196 and 1456-1457. For example, the pharmaceutically acceptable salt of the compounds provided herein may be a sodium salt.
Suitable formulations for use in the present invention are found in Remington's
Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, e.g., Langer (Science 249:1527-1533,
1990). WO 2007/031091 provides further suitable and preferred examples of
pharmaceutically acceptable diluents, carriers and adjuvants (hereby incorporated by reference). Suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, pro-drug formulations are also provided in W02007/031091.
Oligonucleotides or oligonucleotide conjugates of the invention may be mixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of
pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 1 1 , more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents, such as in a sealed package of tablets or capsules. The composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.
In some embodiments, the oligonucleotide or oligonucleotide conjugate of the invention is a prodrug. In particular with respect to oligonucleotide conjugates the conjugate moiety is cleaved of the oligonucleotide once the prodrug is delivered to the site of action, e.g. the target cell.
Applications
The oligonucleotides of the invention may be utilized as research reagents for, for example, diagnostics, therapeutics and prophylaxis.
In research, such oligonucleotides may be used to specifically modulate the synthesis of VCAN protein in cells (e.g. in vitro cell cultures) and experimental animals thereby facilitating functional analysis of the target or an appraisal of its usefulness as a target for therapeutic intervention. Typically the target modulation is achieved by degrading or inhibiting the mRNA producing the protein, thereby prevent protein formation or by degrading or inhibiting a modulator of the gene or mRNA producing the protein.
If employing the oligonucleotide of the invention in research or diagnostics the target nucleic acid may be a cDNA or a synthetic nucleic acid derived from DNA or RNA.
The present invention provides an in vivo or in vitro method for modulating VCAN expression in a target cell which is expressing VCAN, said method comprising administering an oligonucleotide of the invention in an effective amount to said cell.
In some embodiments, the target cell, is a mammalian cell in particular a human cell. The target cell may be an in vitro cell culture or an in vivo cell forming part of a tissue in a mammal.
In diagnostics the oligonucleotides may be used to detect and quantitate VCAN expression in cell and tissues by northern blotting, in-situ hybridisation or similar techniques.
For therapeutics, an animal or a human, suspected of having a disease or disorder, which can be treated by modulating the expression of VCAN
The invention provides methods for treating or preventing a disease, comprising
administering a therapeutically or prophylactically effective amount of an oligonucleotide, an oligonucleotide conjugate or a pharmaceutical composition of the invention to a subject suffering from or susceptible to the disease.
The invention also relates to an oligonucleotide, a composition or a conjugate as defined herein for use as a medicament.
The oligonucleotide, oligonucleotide conjugate or a pharmaceutical composition according to the invention is typically administered in an effective amount.
The invention also provides for the use of the oligonucleotide or oligonucleotide conjugate of the invention as described for the manufacture of a medicament for the treatment of a disorder as referred to herein, or for a method of the treatment of as a disorder as referred to herein.
The disease or disorder, as referred to herein, is associated with expression of VCAN. In some embodiments disease or disorder may be associated with a mutation in the VCAN gene. Therefore, in some embodiments, the target nucleic acid is a mutated form of the VCAN sequence.
The methods of the invention are preferably employed for treatment or prophylaxis against diseases caused by abnormal levels and/or activity of VCAN.
The invention further relates to use of an oligonucleotide, oligonucleotide conjugate or a pharmaceutical composition as defined herein for the manufacture of a medicament for the treatment of abnormal levels and/or activity of VCAN.
In one embodiment, the invention relates to oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions for use in the treatment of diseases or disorders selected from inflammatory lung disease (such as chronic obstructive pulmonary disease or asthma), diabetic retinopathy, cancer and fibrotic disorders (such as fibrotic lung disease, cystic fibrosis, or pulmonary fibrosis).
Administration
The oligonucleotides or pharmaceutical compositions of the present invention may be administered topical or enteral or parenteral (such as, intravenous, subcutaneous, intramuscular, intracerebral, intracerebroventricular or intrathecal).
In a preferred embodiment the oligonucleotide or pharmaceutical compositions of the present invention are administered by a parenteral route including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intrathecal or intracranial, e.g. intracerebral or intraventricular, intravitreal administration. In one embodiment the active oligonucleotide or oligonucleotide conjugate is administered intravenously. In another embodiment the active oligonucleotide or oligonucleotide conjugate is administered subcutaneously. In some embodiments, the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention is administered at a dose of 0.1 - 15 mg/kg, such as from 0.2 - 10 mg/kg, such as from 0.25 - 5 mg/kg. The administration can be once a week, every 2nd week, every third week or even once a month.
Combination therapies
In some embodiments the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the invention is for use in a combination treatment with another therapeutic agent. The therapeutic agent can for example be the standard of care for the diseases or disorders described above.
The work leading to this invention has received funding from the European Union Seventh Framework Programme [FP7-2007-2013] under grant agreement“HEALTH-F2-2013- 602222" (Athero-Flux)
EXAMPLES
Example 1 : Testing in vitro efficacy of antisense oligonucleotides targeting VCAN mRNA in human HEK293 and A549 cells at single concentration.
HEK293 and A549 cell lines were purchased from ATCC and maintained as recommended by the supplier in a humidified incubator at 37°C with 5% C02. For assays, 3500 cells/well of HEK293 or 4500 cells/well of A549 were seeded in a 96 multi well plate in culture media. Cells were incubated for 24 hours before addition of oligonucleotides dissolved in PBS. Final concentration of oligonucleotides: 25 pM. 3 days after addition of oligonucleotides, the cells were harvested. RNA was extracted using the PureLink Pro 96 RNA Purification kit (Thermo Fisher Scientific) according to the manufacturer’s instructions and eluated in 50mI water. The RNA was subsequently diluted 10 times with DNase/RNase free Water (Gibco) and heated to 90°C for one minute.
For gene expressions analysis, One Step RT-qPCR was performed using qScript™ XLT One-Step RT-qPCR ToughMix®, Low ROX™ (Quantabio) in a duplex set up. The following TaqMan primer assays were used for qPCR: VCAN Hs 00171642 _m1 ) [FAM-MGB] and endogenous control GAPDH, Hs99999905_m1 [VIC-MGB] All primer sets were purchased from Thermo Fisher Scientific. The relative VCAN mRNA expression level in the table is shown as percent of control (PBS-treated cells). The VCAN mRNA levels from cells treated with a selection of the compounds are shown in figures 1 and 2, evaluated in human HEK293 and A549 cell lines. From the initial library screen one motif on the VCAN human transcript was identified which provided surprisingly effective and potent compounds in the cell lines tested: Motif A (SEQ ID NO 1 1 ).
Oligonucleotides used:
Figure imgf000055_0001
For Compounds: Capital letters represent LNA nucleosides (beta-D-oxy LNA nucleosides were used), all LNA cytosines are 5-methyl cytosine, lower case letters represent DNA nucleosides. All internucleoside linkages are phosphorothioate internucleoside linkages. Example 2: Testing in vitro potency and efficacy of selected oligonucleotides targeting human VCAN mRNA in human HEK293 and A549 cells at different concentrations for a concentration response curve.
Human A549 and HEK293 cell line was described in Example 1. The assay was performed as described in Example 1. Concentration of oligonucleotides: from 50 pM, 1 :1 dilutions in 8 steps. 3 days after addition of oligonucleotides, the cells were harvested. RNA extraction and duplex One Step RT-qPCR were performed as described in Example 1. n=2 biological replicates. IC50 determinations were performed in GraphPad Prism6. The relative VCAN mRNA level at treatment with 50 pM oligonucleotide is shown in the table as percent of control (PBS).
Figure imgf000056_0001
The IC50 values for selected oligonucleotides targeting human VCAN mRNA in vitro in the human cell lines A549 and HEK293 are shown in Figure 3. The concentration response curves in human cell lines A549 and HEK293 are provided as Figures 4 and 5, respectively.

Claims

1. An antisense oligonucleotide, 10-30 nucleotides in length, wherein said antisense oligonucleotide comprises a contiguous nucleotide sequence 10 - 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as fully complementary to SEQ ID NO 13, wherein the antisense
oligonucleotide is capable of inhibiting the expression of human VCAN in a cell which is expressing human VCAN ; or a pharmaceutically acceptable salt thereof.
2. The antisense oligonucleotide according to claim 1 , wherein the contiguous
nucleotide sequence is at least 90% complementary to the sequence shown in SEQ ID NO 1 1 or SEQ ID NO 12.
3. The antisense oligonucleotide according to claim 1 , wherein the contiguous
nucleotide sequence is fully complementary to SEQ ID NO 1 1 or SEQ ID NO 12.
4. The antisense oligonucleotide according to any one of claim 1 to 3, wherein the
contiguous nucleotide sequence is fully complementary to a region of SEQ ID NO 13, selected from the group consisting of 1 - 138, 140 - 161 , 163 - 218, 220 - 348, 350 - 447, 457 - 555, 568 - 598, 601 - 619, 629 - 643, 643 - 672, 674 - 730, 732 - 750, 752 - 796, 810 - 858, 860 - 930, 938 - 1 133, 1 134 - 1180, 1 182 - 1275, 1277 - 1299, 1301 - 1335, 1337 - 1382, 1384 - 1398, 1401 - 1421 , 1429 - 1492, 1505 - 1522, 1527 - 1567, 1578 - 1667, 1669 - 1684, 1686 - 1705, 1707 - 1728, 1739 - 1754, 1765 -
1805, 1822 - 1894, 1896 - 1928, 1930 - 1948, 1952 - 1977, 1989 - 2055, 2057 -
2124, 2132 - 2192, 2206 - 2265, 2322 - 2361 , 2363 - 241 1 , 2421 - 2442, 2444 -
2460, 2471 - 2500, 2506 - 2524, 2581 - 2603, 2612 - 2626, 2628 - 2642, 2644 -
2681 , 2683 - 271 1 , 2755 - 2801 , 2821 - 2844, 2846 - 2905, 2907 - 2931 , 2934 -
2970, 2972 - 2995, 3019 - 3043, 3046 - 3062, 3066 - 3095, 3097 - 31 13, 31 15 -
3135, 3137 - 3152, 3151 - 3177, 3179 - 3205, 3207 - 3261 , 3323 - 3341 , 3359 -
3378, 3406 - 3441 , 3443 - 3461 , 3476 - 3497, 3499 - 3513, 3525 - 3554, 3575 -
3608, 3610 - 3639, 3641 - 3674, 3681 - 3699, 3701 - 3716, 3740 - 3780, 3810 -
3826, 3828 - 3861 , 3864 - 3879, 3898 - 3922, 3924 - 3974, 3976 - 3990, 3992 -
4012, 4016 - 4031 , 4033 - 4048, 4050 - 4140, 4140 - 4179, 4186 - 4204, 4219 -
4233, 4244 - 4269, 4283 - 4382, 4396 - 4470, 4472 - 4590, 4605 - 4626, 4628 -
4649, 4673 - 4728, 4742 - 4788, 4812 - 4888, 4918 - 4936, 4933 - 4960, 4962 -
5009, 501 1 - 5029, 5031 - 5103, 5100 - 51 14, 5120 - 5148, 5151 - 5209, 5225 - 5240, 5246 - 5320, 5322 - 5352, 5354 - 5382, 5384 - 5418, 5422 - 5470, 5472 -
5502, 5522 - 5577, 5579 - 5604, 5606 - 5634, 5683 - 5700, 5702 - 5724, 5726 - 5744, 5763 - 5791 , 5793 - 5820, 5822 - 5853, 5870 - 5891 , 5910 - 5934, 5936 - 5965, 5967 - 6016, 6029 - 6067, 6070 - 6090, 6098 - 6142, 6144 - 6168, 6182 - 6210, 6214 - 6229, 6268 - 6282, 6284 - 6299, 6301 - 6319, 6322 - 6357, 6359 - 6373, 6384 - 6453, 6456 - 6474, 6470 - 6491 , 6486 - 6514, 6526 - 6568, 6573 - 6591 , 6597 -6611, 6621 - 6691 , 6693 - 6732, 6735 - 6766, 6793 - 6853, 6865 - 6884, 6886 - 6906, 6910 - 6929, 6960 - 6976, 6988 - 7009, 7011 - 7101, 7104 - 7124, 7124 - 7156, 7154 - 7173, 7175 - 7206, 7215 - 7234, 7252 - 7352, 7360 - 7374, 7377 - 7433, 7435 - 7471 , 7484 - 7509, 7511 - 7532, 7537 - 7567, 7574 - 7632, 7634 - 7648, 7651 - 7679, 7729 - 7751 , 7763 - 7794, 7799 - 7823, 7833 - 7884, 7886 - 7949, 7951 - 8037, 8039 - 8075, 8083 -8111,8113- 8232, 8235 - 8251 , 8253 - 8277, 8283 - 8301 , 8339 - 8368, 8371 - 8397, 8401 - 8429, 8431 - 8448, 8456 - 8498, 8523 - 8581 , 8583 - 8600, 8602 - 8624, 8626 - 8640, 8642 - 8665, 8667 - 8720, 8722 - 8757, 8791 - 8811, 8828 - 8861 , 8865 - 8898, 8917- 8938, 8940 - 9018, 9021 - 9047, 9059 - 9094, 9097-9112, 9114-9133, 9152- 9169, 9186-9237,9250-9271,9285- 9307, 9309 - 9446, 9449 - 9494, 9520 - 9546, 9552 - 9567, 9569 - 9585, 9610 - 9646, 9653 - 9727, 9739 - 9765, 9779 - 9802, 9795 - 9810, 9804 - 9819, 9831 - 9866, 9883 - 9927, 9956 - 9993, 9995 - 10014, 10024 - 10040, 10075 - 10107, 10118- 10134, 10136- 10156, 10172 - 10231, 10233- 10258, 10294- 10320, 10339 - 10353, 10370 - 10388, 10414 - 10447, 10457 - 10496, 10542 - 10558, 10560 - 10654, 10656 - 10681, 10683 - 10732, 10734- 10769, 10771 - 10854, 10863 - 10931 , 10956 - 10973, 10975 - 10991, 11024- 11073, 11075- 11102, 11109- 11124, 11129- 11152, 11154- 11192, 11213- 11238, 11251 - 11368, 11370- 11385, 11388- 11448, 11460- 11500, 11507- 11557, 11559- 11591, 11604- 11625, 11649- 11671, 11686- 11701, 11703- 11717, 11752- 11772, 11765- 11818, 11820- 11845, 11870- 11888, 11890- 11941, 11944- 11981, 12017 - 12039, 12041 - 12059, 12073 - 12103, 12105 - 12140, 12142 - 12183, 12193 - 12215, 12217 - 12252, 12254 - 12272, 12274 - 12316, 12330 - 12345, 12347 - 12367, 12389 - 12407, 12409 - 12428, 12430 - 12475, 12477 - 12540, 12546- 12561, 12551 - 12581, 12583- 12623, 12645 - 12667, 12662 - 12679, 12684- 12721, 12723- 12751, 12773- 12791, 12799- 12815, 12849- 12868, 12870 - 12928, 12930 - 12947, 12949 - 12971, 12975- 13001, 13003- 13022, 13021 - 13044, 13046 - 13091, 13085 - 13102, 13106 - 13123, 13125 - 13183, 13253 - 13338, 13349 - 13379, 13383 - 13403, 13408 - 13437, 13454 - 13473, 13489 - 13541 , 13543 - 13571 , 13573 - 13621, 13623- 13641, 13649- 13677, 13685 - 13702, 13704 - 13726, 13728 - 13742, 13750 - 13790, 13828 - 13842, 13834 - 13853, 13906 - 13935, 13939 13988, 13990 - 14015, 14017 - 14034, 14045- 14077, 14086- 14119, 14121 14135, 14175 - 14209, 14220 - 14239, 14256- 14271, 14275- 14310, 14333 14355, 14359 - 14377, 14370 - 14387, 14389 - 14410, 14439 - 14463, 14567 14596, 14598 - 14615, 14619 - 14645, 14677 - 14698, 14693 - 14721, 14753 14771, 14778- 14793, 14815- 14832, 14847 - 14877, 14879 - 14913, 14951 14965, 14968 - 14988, 14994 - 15064, 15111 - 15143, 15160- 15193, 15242 15276, 15287 - 15315, 15317 - 15339, 15341 - 15365, 15410- 15443, 15443 15482, 15478 - 15504, 15506 - 15566, 15568 - 15593, 15593 - 15608, 15625 15641, 15643- 15677, 15696- 15711, 15713- 15739, 15741 - 15764, 15766 15784, 15793- 15809, 15811 - 15842, 15853 - 15868, 15878 - 15904, 15915 15933, 15948 - 15986, 15988 - 16004, 16006 - 16100, 16102 - 16128, 16130 16182, 16199 - 16238, 16240 - 16258, 16267 - 16288, 16299 - 16327, 16341 16386, 16433- 16461, 16463- 16480, 16473 - 16488, 16489 - 16519, 16521 16547, 16555- 16621, 16626- 16661, 16663 - 16694, 16696 - 16745, 16747 16772, 16774- 16791, 16793- 16825, 16827 - 16870, 16872 - 16896, 16899 16913, 16916 - 16973, 16975 - 16999, 17001 - 17036, 17056 - 17071, 17074 17089, 17091 - 17140, 17147- 17162, 17164 - 17193, 17198 - 17215, 17217 17254, 17270 - 17292, 17307 - 17336, 17338- 17409, 17431 - 17457, 17465 17479, 17482 - 17550, 17553 - 17567, 17564 - 17635, 17637 - 17654, 17664 17727, 17729 - 17782, 17784 - 17837, 17870 - 17889, 17891 - 17911 , 17921 17961, 17963- 17993, 17997- 18012, 18014 - 18085, 18087 - 18104, 18106 18139, 18155 - 18180, 18182 - 18198, 18200 - 18221 , 18223 - 18251 , 18262 18293, 18295 - 18332, 18334 - 18349, 18364 - 18397, 18397 - 18416, 18432 18516, 18518 - 18550, 18563 - 18583, 18585 - 18613, 18615 - 18655, 18658 18706, 18708 - 18802, 18804 - 18889, 18891 - 18940, 18957- 18973, 18981 18998, 19000 - 19020, 19022 - 19068, 19070- 19112, 19144- 19168, 19180 19203, 19208 - 19237, 19239 - 19332, 19340 - 19402, 19400 - 19414, 19426 19456, 19465 - 19482, 19484 - 19502, 19535 - 19584, 19599 - 19639, 19641 19714, 19716- 19763, 19771 - 19969, 19971 - 19992, 19994 - 20074, 20088 20121, 20131 -20173, 20175-20507, 20515 - 20538, 20556 - 20570, 20565 20623, 20625 - 20650, 20652 - 20666, 20668 - 20685, 20687 - 20723, 20801 20831 , 20833 - 20851 , 20869 - 20891 , 20893 - 20909, 20898 - 20912, 20931 20960, 20973 - 21000, 21002-21051, 21053-21086, 21088-21129, 21131 21154, 21171 -21186, 21188-21253, 21255 - 21313, 21320 - 21359, 21361 21384 21386 21455 21457 21502 21504 21554 21564 21592 21610
21662 21678 21695 21697 21715 21717 21775 21789 21833 21869
21886 21888 21909 2191 1 21945 21962 21976 22008 22027 22029
22063 22075 22104 22106 22158 22168 22196 22198 22226 22228
22280 22280 22358 22382 22405 22407 22478 22495 22510 22512
22529 22531 22546 22548 22607 22609 22639 22641 22745 22747
22813 22826 22851 22853 22868 22888 22903 22905 22961 22971
22988 22998 23012 23046 23069 23077 23091 23095 231 10 23125
23156 23166 23200 2321 1 23225 23226 23241 23235 23251 23274
23304 23306 23326 23332 23350 23362 23379 23508 23531 23536
23570 23583 23622 23622 23664 23675 23751 23767 23782 23807
23828 23845 23862 23864 23906 23908 24000 24002 24042 24044
24067 24101 24131 24133 24148 24154 24186 24213 24306 24317
24338 24352 24385 24402 24416 24418 24437 24514 24551 24553
24598 24627 24656 24658 24675 24692 24757 24784 24800 24838
24864 24874 24904 24912 24954 24956 24981 24983 25006 25008
25055 25067 251 10 25120 25134 25136 25151 25153 25180 25187
25219 25221 25301 25304 25325 25327 25366 25379 25393 25384
25430 25452 25543 25567 25601 25605 25620 25637 25678 25686
25727 25729 25754 25756 25773 25775 25798 25800 25816 25812
25844 25846 25874 25882 25903 25905 25928 25939 25979 25983
26012 26014 26055 26068 26109 261 1 1 26128 26130 26153 26168
26221 26223 26255 26257 26332 26334 26361 26363 26378 26380
26394 26393 26419 26425 26457 26459 26552 26566 26603 26605
26619 26621 26647 26649 26664 26686 26750 26754 26771 26773
26858 26860 26881 26906 26962 26969 27001 27004 27038 27062
27126 27136 27160 27162 27190 27197 27238 27251 27322 27324
27346 27353 27380 27390 27416 27424 27441 27454 27474 27481
27509 27512 27526 27528 27550 27559 27620 27628 27771 27773
27847 27848 27864 27880 2791 1 27937 27954 27971 28008 28010
28043 28045 28100 281 10 28139 28135 2821 1 28217 28235 28237
28323 28341 28366 28368 28392 28428 28463 28465 28495 28497
28517 28535 28550 28574 28598 28600 28630 28632 28650 28652
28676 28678 28746 28762 28806 28840 28928 28957 28986 29026
29061 29068 29097 291 10 29130 29145 29161 29183 29212 29222 29245 29247 29290 29292 29356 29359 29396 29403 29418 29420
29447 29449 29469 29471 29501 29503 29517 29506 29520 29519
29538 29540 29563 29579 29652 29654 29692 29709 29771 29777
29874 29876 29907 29917 29946 29948 30143 30146 30200 30202
30247 30249 30263 30265 30297 30309 30323 30325 30393 30395
30420 30442 30459 30483 30580 30578 30596 30598 30613 3061 1
30636 30638 30662 30676 30700 30714 30786 30788 30839 30852
30874 30876 30894 30896 30945 30947 30995 30997 31 103 31 105
31 169 31 171 31222 31224 31239 31241 31264 31266 31285 31292
31315 31317 31333 31349 31363 31379 31405 31407 31434 31436
31471 31473 31519 31530 31580 31583 31603 31605 31628 31640
31657 31659 31703 31705 31721 31755 31773 31762 31777 31779
31817 31820 31836 31854 31880 31892 31907 31912 31929 31931
31957 31959 31973 31975 31998 32000 32015 32038 32052 32041
32056 32121 32145 32147 32173 32175 32230 32242 32277 32299
32332 32334 32367 32379 32400 32402 32438 32439 32525 32529
3261 1 32616 32635 32637 32664 32668 32687 32686 32703 32723
32741 32743 32766 32768 32816 32818 32846 32848 32863 32880
32897 32909 32942 32935 32950 32948 32969 32982 33024 33044
33071 33075 33101 33105 33128 33133 33155 33155 33170 33166
33182 33184 33202 33216 33250 33252 33303 33305 33400 33402
33423 33452 33482 33479 33494 33496 33510 33499 33538 33545
33560 33562 33577 33584 33606 33629 33668 33681 33780 33781
3381 1 33835 33856 33877 33898 33936 33951 33988 34022 34033
34048 34074 34103 34152 34190 34192 34214 34252 34300 34302
34325 34327 34341 34363 34382 34393 34409 3441 1 34436 34439
34484 34528 34545 34566 34604 34632 34659 34661 34676 34678
34699 34701 34729 34747 34761 34784 34855 34857 34875 34914
34929 34928 34957 34959 34973 34975 34997 34999 35014 35038
35062 35063 35083 35085 35128 35130 35151 35164 35183 35203
35220 35229 35247 35263 35277 35286 35320 35335 35358 35360
35387 35389 35406 35397 3541 1 35408 35423 35426 35440 35442
35457 35450 35466 35485 35506 35547 35564 35566 35646 35646
35660 35677 35692 35694 35762 35766 35781 35783 35806 35857
35872 35908 35924 35940 35977 35979 35995 36023 36050 36052 36075 36079 36093 36095 36110 361 12 36154 36183 36198 36200
36225 36227 36262 36267 36291 36293 36356 36358 36373 36375
36407 36436 36452 36475 36508 3651 1 36553 36556 36589 36591
36618 36637 36670 36736 36784 36794 36809 36821 36839 36841
36860 36909 36925 36926 36984 36986 37006 37008 37023 37025
37056 37058 37085 37095 37118 37126 37144 37190 37212 37223
37241 37243 37265 37265 37298 37323 37337 37339 37388 37400
37425 37427 37442 37477 37513 37519 37543 37558 37572 37629
37647 37694 37732 37742 37760 37777 37821 37854 37936 37939
37954 37956 38021 38026 38084 38125 38140 38142 38245 38255
38327 38357 38380 38382 3841 1 38413 38461 38472 38512 38514
38559 38561 38584 38618 38632 38634 38663 38665 38701 38726
38741 38743 38767 38799 38822 38824 38852 38854 38882 38884
38930 38932 38950 38952 39013 39021 39038 39040 39081 39083
39103 391 13 39131 39143 39194 39196 39238 39262 39325 39336
39367 39369 39384 39389 39433 39427 39444 39452 39483 39485
39505 39522 39563 39563 39580 39574 39591 39584 39598 39593
39617 39679 39694 39742 39779 39771 39789 39780 39799 39830
39879 39925 39939 39959 39975 39979 39994 40007 40025 40032
40056 40078 401 1 1 40106 40122 401 17 40137 40148 40169 40189
40236 40259 40278 40303 40342 40350 40374 40374 40396 40400
40414 40419 40433 40443 40492 40494 40550 40564 40623 40630
40659 40661 40687 40689 4071 1 40713 40747 40749 40964 40967
41015 41017 41036 41038 41 119 41 124 41 165 41 167 41 181 41 183
41202 41210 41233 41280 41308 41333 41348 41364 41381 41383
41407 41413 41433 41435 41461 41463 41477 41479 41494 41524
41538 41540 41559 41561 41581 41597 4161 1 41615 41645 41707
41750 41757 41801 41802 41817 41819 41847 41849 41877 41898
41949 41951 41973 41975 41991 41996 42016 42019 42056 42058
42073 42076 42099 42102 42122 42176 42240 42242 42287 42289
42355 42369 42399 42401 42422 42427 42490 42492 42509 4251 1
42553 42555 42582 42584 42608 42606 42665 42665 42730 42740
42759 42780 42825 42827 42848 42872 42886 42882 42947 42963
42988 42988 43006 43018 43037 43027 43047 43051 43067 43077
43102 43102 431 17 43152 43176 43167 43181 43170 43192 43202 43217 43210 43230 43219 43245 43248 43273 43269 4331 1 43313
43331 43332 43382 43402 43432 43430 43548 43569 43585 43587
43618 43634 43675 43677 43722 43724 43740 43745 43759 43758
43858 43902 43955 43957 44051 44053 44081 44083 441 10 441 12
44133 44135 44149 44159 44187 44190 44207 44227 44247 44249
44276 44304 44320 44322 44377 44379 44396 4441 1 44426 44428
44461 44466 44487 44501 44522 44524 44539 44551 44599 44588
44608 44597 44617 44631 44655 44697 4471 1 4471 1 44738 44770
44785 44775 44796 44816 44830 44843 44858 44860 44883 44885
44967 44993 45007 45009 45164 45166 45209 4521 1 45226 45236
45278 45280 45294 45296 45312 45313 45346 45355 45381 45392
45407 45409 45528 45530 45549 45539 45553 45551 45603 45605
45647 45649 45720 45722 45750 45753 45829 45831 45847 45862
45888 45890 45949 45951 46210 46240 46270 46291 46316 46318
46357 46362 46387 46389 46412 46437 46494 46500 46561 46553
46581 46588 46624 46626 46655 46657 46677 46701 46724 46726
46777 46786 46802 46804 46837 46839 46882 46884 46915 46922
46944 46946 46974 46996 47042 47044 47083 47085 47148 47161
47204 47206 47223 47236 47345 47354 47384 47386 47408 47461
47485 47515 47549 47551 47590 47599 47615 47623 47653 47655
47670 47672 47710 47733 47775 4781 1 47835 47837 47852 47854
48032 48034 48048 48051 48084 48086 48130 48154 48190 48237
48251 48256 48283 48285 48353 48355 48409 48419 48445 48447
48528 48530 48568 48586 48610 48645 48661 48664 48680 48682
4871 1 48726 48743 48749 48790 48792 48868 48870 48971 48973
49000 49002 49030 49032 49099 49101 49115 491 17 49148 49150
49171 49173 49189 49214 49231 49241 49270 49272 49289 49291
49367 49369 49443 49445 49465 49467 49507 49509 49543 49545
49585 49587 4961 1 49613 49647 49649 49668 49670 49687 49690
49850 49857 49873 49929 50045 50047 50075 50077 50099 50101
50166 50168 50189 50193 50214 50216 50235 50237 50322 50324
50359 50361 50443 50452 50483 50485 50508 50510 50645 50647
50699 50702 50759 50770 50786 50797 50837 50839 50867 50870
50884 50886 50912 50922 50940 50942 50992 50994 51 1 18 51 120
51 134 51 136 51243 51255 51275 51314 51341 51343 5141 1 51413 51427 51429 51455 51462 51486 51488 51525 51531 51562 51569
51587 51589 51614 51616 51654 51667 51690 5171 1 51734 51739
51758 51760 51822 51850 51865 51879 51893 51906 51933 51942
51969 51978 51997 51999 52088 521 17 52156 52173 5221 1 52256
52360 52362 52377 52380 52403 52402 52428 52459 52480 52482
52500 52511 52545 52564 52582 52592 52624 52626 52645 52649
52709 52711 52737 52746 52782 52808 52839 52841 52862 52860
52878 52881 52895 52897 52913 52939 52959 52961 52989 53006
53030 53032 53055 53055 53070 53073 53139 53141 53157 53159
53268 53270 53325 53327 53405 53407 53433 53435 53533 53535
53557 53559 53581 53580 53594 53583 53682 53686 53725 53727
53822 53824 53907 53909 53931 53936 53965 53977 53998 54000
54077 54079 54095 54097 54154 54180 54214 54229 54253 54258
54297 54312 54335 54337 54364 54366 54392 54394 54432 54434
54449 54451 54481 54479 54493 54499 54513 54507 54526 54516
54535 54552 54600 54601 54623 54631 54674 54693 54710 54725
54758 54767 54789 54814 54829 54871 54890 54892 54909 5491 1
54943 54955 54997 55021 55037 55031 55054 55056 55072 55089
55106 55108 55130 55132 55155 55172 55212 55214 55310 55312
55381 55412 55434 55452 55466 55482 55500 55502 55556 55575
55598 55615 55702 55704 55740 55753 55779 55783 55800 55802
55838 55840 55866 55868 55885 55887 55912 55928 55951 55960
55979 56009 56039 56041 56086 56093 56125 56127 56151 56153
56184 56198 56241 56274 56319 56321 56335 56387 56412 56417
56453 56462 56481 56483 56501 56520 56534 56528 56549 56558
56581 56583 56637 56656 56678 56738 56754 56771 56800 56816
56851 56851 5691 1 56913 56945 56947 56983 56989 57006 57031
57052 57072 57086 57075 57091 57104 57127 57129 57179 57188
57261 57267 57292 57301 57383 57417 57452 57454 57475 57482
57567 57603 57619 57636 57652 57654 57676 57684 57722 57728
57751 57758 5781 1 57813 57833 57836 57861 57871 57899 57915
57938 57984 57999 58023 58048 58051 58068 581 17 58134 58156
58177 58179 58195 58216 58257 58259 58274 58277 58298 58320
58392 58402 58462 58464 58517 58519 58550 58552 58571 58582
58647 58663 58688 58690 58714 58722 58777 58779 58795 58797 58817 58819 58846 58848 58970 58972 59035 59043 59074 59088
59159 59170 59194 59196 59223 59251 59275 59306 59325 59352
59377 59389 59418 59435 59451 59453 59469 59464 59486 59497
59514 59516 59539 59555 59594 59596 59633 59645 59665 59667
59691 59694 59723 59725 59761 59793 59827 59833 59856 59858
59892 59926 59960 59968 59982 59984 60070 60072 60108 601 19
60133 60142 60160 60184 60315 60317 60340 60340 60395 60391
60416 60418 60478 60485 60500 60532 60578 60580 60595 60597
60630 60657 60678 60680 60695 60700 60717 60740 60840 60841
60861 60900 60931 60949 60992 61029 61054 61065 61 121 61 131
61 194 61 196 61214 61253 61283 61288 61308 61310 61349 61351
61391 61415 61439 61443 61467 61493 61564 61567 61679 61681
61721 61735 61784 61798 61828 61846 61868 61872 61891 61903
61945 61947 61963 61974 61989 61996 62025 62027 62050 62061
62076 62108 62124 62138 62155 62157 62180 62216 62238 62240
62254 62256 62276 62305 62343 62345 62372 62414 62452 62454
62507 62516 62552 62569 62610 62612 62633 62649 62685 62687
62744 62746 62765 62767 62788 62791 62808 62821 62862 62864
62894 62922 62993 63037 63073 63078 63098 63104 631 19 63120
63138 63172 63210 63218 63293 63305 63332 63334 63356 63361
63392 63406 63429 63431 63470 63472 63493 63495 63510 63512
63558 63565 63581 63621 63647 63699 63729 63781 63795 63804
63828 63830 63855 63860 63918 63920 63981 63985 64010 64012
64029 64039 64057 64059 64105 64107 64132 64134 64155 64181
64218 64220 64263 64265 64293 64341 64361 64371 64389 64391
64406 64409 64429 64438 64462 64473 64492 64509 64525 64536
64591 64593 64636 64651 64666 64682 64709 64758 64778 64780
64795 64797 64841 64851 64879 64881 64896 64898 64914 64916
64936 64940 64955 64982 65057 65058 65113 651 16 65152 65155
65234 65248 65288 65315 65377 65381 65421 65421 65442 65468
65756 65764 65786 65788 65861 65863 65893 65895 65909 65912
65944 65946 65963 65975 66085 66091 66115 661 17 66148 66157
66231 66233 66290 66296 66333 66335 66358 66360 66443 66452
66503 66505 66609 6661 1 66656 66658 66686 66707 66746 66773
66799 66834 66910 66912 66946 66948 67014 67031 67064 67066 67103 67137 67154 67157 67197 67199 67219 67221 67237 67239
67257 67270 67287 67289 67334 67336 67354 67363 67397 67399
67436 67438 67462 67464 67588 67590 67606 67608 67626 67635
67661 67663 67684 67686 67754 67762 67788 67790 67814 67816
67855 67857 67913 67927 67999 68001 68015 68017 68033 68035
68049 68073 68095 681 19 68168 68176 68223 68225 68249 68251
68278 68280 68343 68345 68372 68400 68431 68442 68483 68485
68509 68514 68531 68533 68553 68555 68595 68618 68632 68632
68732 68734 68752 68761 68807 68814 68845 68847 68893 68895
68912 68917 68958 68960 68991 6901 1 69106 69108 69158 69172
69254 69256 69296 69307 69333 69335 69381 69383 69403 69405
69424 69464 69534 69545 69585 69593 69614 69626 69655 69663
69779 69781 69809 6981 1 69844 69847 69863 69865 69900 69929
69948 69964 69984 69994 70013 70024 70092 70099 701 18 70124
70212 70214 70247 70258 70276 70278 70296 70298 70399 70405
70428 70439 70487 70522 70565 70573 70613 70616 70638 70640
70662 70684 70730 70732 70797 70799 70833 70835 70863 70878
70916 70918 70949 70955 70999 71005 71022 71024 71 107 71 109
71 148 71 150 71 166 71 168 71 193 71 195 71212 71217 71248 71273
71312 71341 71401 71409 71426 71435 71492 71541 71555 71557
71581 71583 71618 71620 71637 71639 71691 71693 71746 71747
71779 71781 71798 71800 71825 71827 71853 71890 71914 71916
71935 71931 71956 71958 71985 71987 72042 72054 72130 72135
72174 72193 72219 72223 72259 72269 72290 72301 72315 72317
72351 72363 72416 72418 72433 72435 72468 72481 72507 72539
72567 72579 72631 72633 72655 72657 7271 1 72712 72727 72815
72830 72842 72856 72884 72899 72906 72921 72923 72938 72931
72959 72961 72996 72998 73046 73050 73090 73099 731 17 731 19
73168 73189 73238 73240 73254 73258 73279 73289 73341 73344
73370 73372 73414 73416 73541 73543 73849 73851 74000 74002
74019 74044 74082 74084 74139 74141 74166 74168 74201 74210
74276 74292 74308 7431 1 74361 74364 74382 74387 7441 1 74423
74446 74453 74480 74487 74572 74613 74633 74651 74669 74671
74695 74713 74737 74746 74784 74775 74790 74788 74863 74865
74893 74910 74933 74935 74951 74961 75002 74996 75014 75018 75032 75037 75057 75060 75094 75085 7511 1 75103 751 17 75126
75145 75139 75192 75201 75226 75236 75318 75325 75366 75368
75387 75389 75515 75514 75625 75632 75665 75673 75693 75695
75720 75722 75749 75751 75839 75848 75867 75879 75912 75938
75981 75983 76013 76017 76060 76068 76100 761 1 1 76156 76219
76256 76258 76272 76274 76313 76327 76341 76351 76371 76383
76428 76436 76483 76485 76537 76539 76615 76629 76643 76645
76667 76663 76692 76694 76739 76762 76800 76802 76819 76827
76852 76868 76889 76891 76915 76922 76941 76944 76964 76966
77051 77053 771 15 771 19 77154 77156 77194 77219 77266 77268
77402 77404 77444 77455 77491 77494 77539 77535 77560 77562
77577 77579 77623 77625 77683 77685 77747 77766 77782 77784
77808 77810 77824 77826 77862 77875 77889 77909 77923 77927
77941 77945 77975 77979 78008 78015 78081 78083 78102 781 13
78127 78139 78166 78169 78243 78245 78259 78266 78298 78301
78320 78334 78387 78386 78433 78436 78467 78469 78483 78485
78501 78517 78545 78553 78586 78599 78645 78686 78713 78731
78765 78780 78809 78815 78835 78878 78929 78931 78946 78948
78964 78966 79018 79020 79046 79048 79089 79094 791 15 791 17
79157 79159 79186 79198 79214 79216 79290 79297 79319 79321
79374 79386 79409 7941 1 79446 79443 79470 79468 79483 79472
79487 79478 79495 79529 79565 79572 79595 79610 79627 79634
79655 79691 79765 79764 79788 79785 79871 79876 79919 79920
79939 79938 79962 79978 80014 80004 80031 80066 80105 80134
80161 80163 80177 80203 80228 80237 80252 80261 80276 80282
80307 80309 80340 80342 80387 80395 80529 80531 80616 80618
80707 80709 80734 80736 80763 80765 80781 80783 80797 80817
80835 80841 80863 80886 80909 80909 80928 80938 80976 80975
80993 81052 81 133 81 149 81 174 81 187 81224 81232 81247 81257
81297 81314 81338 81374 81397 81401 81440 81442 81494 81515
81598 81610 81651 81653 81690 81744 81761 81768 81825 81841
81859 81869 81900 81905 82046 82048 82074 82097 82186 82189
82205 82229 82251 82284 82298 82300 82315 82319 82341 82358
82384 82386 82444 82447 82494 8251 1 82553 82555 82569 82571
82667 82671 82717 82756 8281 1 82822 82847 82849 82877 82894 82945 82947 82969 82975 83004 83021 83044 83065 83086 83088
83108 831 17 83140 83146 83161 83168 83184 83187 83206 83196
83214 83227 83249 83251 83266 83305 83333 83342 83377 8341 1
83434 83436 83462 83464 83552 83554 83582 83584 83639 83642
83668 83679 83708 83710 83734 83736 83752 83781 83795 83805
83837 83839 83891 83916 83931 83925 83939 83986 84004 84006
84029 84031 84045 84091 84135 84160 84182 84185 8421 1 84235
84329 84331 84370 84383 84452 84454 84484 84506 84536 84538
84558 84580 84610 84634 84664 84667 84682 84690 84712 84727
84747 84755 84776 84779 84861 84881 84895 84925 84979 84981
85002 85004 85020 85022 85051 85068 85082 85086 85109 85136
85159 85167 85222 85231 85248 85278 85294 85306 85330 85340
85355 85360 85377 85379 85393 85398 85439 85441 85465 85489
85558 85562 85598 85614 85628 85630 85649 85666 85701 85744
85763 85765 85791 85793 85873 85897 8591 1 85937 85979 85981
86001 86026 86083 86085 86124 86129 86174 86179 86195 86197
86225 86227 86256 86271 86305 86338 86369 86371 86392 86394
86418 86420 86481 86476 86495 86497 86517 86546 86562 86567
86590 86600 86614 86616 86658 86660 86679 86681 86704 86706
86817 86819 86835 86873 86900 86902 86958 86961 86995 86997
87057 87059 87080 87096 87123 87139 87168 87170 87198 87195
87224 87226 87245 87259 87313 87358 87373 87366 87385 87387
87439 87461 87486 87484 87535 87543 87561 87577 87592 87595
87755 87770 87820 87827 87876 87892 87922 88018 88041 88043
88069 88095 88109 881 1 1 88165 88174 88192 88262 88285 88287
88303 88305 88327 88332 88362 88364 88387 88397 88430 88459
88527 88529 88560 88562 88582 88592 88678 88680 88725 88734
88762 88764 88820 88822 88869 88871 88917 88919 88941 88943
88982 89003 89105 891 1 1 89133 89132 89155 89157 89187 89189
89212 89237 89281 89283 89333 89346 89364 89369 89393 89395
89410 89412 89427 89429 89446 89452 89488 89498 89529 89559
89612 89628 89657 89660 89680 89687 89717 89722 89737 89753
89782 89804 89825 89827 89864 89866 89888 89896 89914 89966
90059 90061 90080 90082 90124 90129 90146 90166 90198 90200
90224 90238 90263 90267 90297 90299 90313 90315 90331 90338 90377 90395 90418 90450 90467 90483 90506 90508 90527 90534
90550 90552 90571 90573 90595 90631 90714 90731 90750 90765
90780 90782 90805 90814 90842 90844 90865 90867 90891 90914
90929 90947 90972 90961 90975 90978 91 117 91 137 91209 91224
91253 91255 91283 91285 91346 91348 91374 91376 91393 91395
91420 91433 91665 91667 91716 91718 91771 91783 91891 91893
91921 91923 91968 91970 92002 92008 92051 92054 92087 92089
921 1 1 92121 92155 92157 92181 92183 92202 92213 92253 92263
92285 92302 92318 92320 92339 92352 92371 92378 92402 9241 1
92426 92428 92448 92453 92499 92503 92521 92521 92535 92542
92558 92596 92637 92651 92683 92693 92715 92713 92733 92722
92736 92747 92762 92786 92812 92841 92881 92900 92915 92917
92949 92966 93033 93066 93092 931 15 93144 93168 93187 93232
93250 93267 93310 93323 93344 93362 93381 93383 93405 93469
93516 93518 93532 93534 93559 93550 93564 93560 93594 93612
93664 93666 93719 93721 93738 93740 93762 93766 93803 93805
93854 93862 93912 93927 93963 93955 93969 93981 94009 94018
94075 94077 941 15 94163 94179 94199 94227 94231 94248 94261
94309 94329 94355 94357 94401 94414 94440 94460 94499 94522
94550 94540 94559 94561 94590 94582 94597 94598 94620 94626
94640 94667 94691 94692 94710 94707 94723 94725 94740 94742
94758 94750 94770 94788 94807 94797 94812 94809 94859 94861
94881 94900 94943 94975 94998 95046 95103 95143 95161 95161
95188 95199 95235 95242 95257 95280 95299 95301 95355 95368
95405 95421 95455 95464 95491 95503 95573 95583 95606 95608
95626 95645 95685 95687 95792 95805 95831 95832 95849 95839
95855 95844 95866 95880 95897 95899 95929 95928 95943 95944
95961 95962 95982 95996 96026 96015 96036 96027 96051 96053
96084 96083 96097 96089 96108 961 10 96131 96133 96156 96158
96212 96223 96247 96277 9631 1 96313 96335 96337 96356 96363
96397 96410 96428 96439 96461 96486 96538 96590 96612 96614
96649 96651 96667 96695 96729 96741 96858 96860 96906 96936
97025 97027 97046 97058 97080 97082 97153 97166 97182 97185
97200 97222 97323 97328 97396 97401 97423 97473 97488 97500
97515 97517 97532 97556 97575 97591 97616 97626 97673 97675 97705, 97707 - 97722, 97724 - 97747, 97760 - 97792, 97794-97841, 97843- 97869, 97899 - 97931 , 97946 - 97965, 97963 - 98002, 98017-98044, 98046- 98076, 98101 -98133, 98135-98162, 98189-98211, 98213-98315, 98317- 98332, 98334 - 98350, 98400 - 98479, 98497 - 98533, 98615-98666, 98676- 98716, 98732 - 98770, 98768 - 98809, 98820 - 98868, 98874-98904, 98915- 98933, 98967 - 98983, 98985 - 99005, 99018 - 99054, 99070-99102, 99125- 99141, 99145-99159, 99211 -99262, 99272-99290, 99287 - 99302, 99361 - 99378, 99378 - 99407, 99414 - 99439, 99444 - 99459, 99476 - 99497, 99499 - 99576, 99578 - 99597, 99599 - 99630, 99632 - 99657, 99672 - 99748, 99758 - 99781, 99797 - 99816, 99818 - 99848, 99875 - 99904, 99922 - 99959, 99986 -
100013, 100028- 100060, 100062- 100102, 100105 - 100147, 100149- 100175, 100221 - 100257, 100268- 100293, 100295- 100312, 100317- 100345, 100353- 100398, 100400- 100479, 100481 - 100495, 100506- 100523, 100532- 100585, 100606- 100661, 100681 - 100707, 100722- 100741, 100759- 100799, 100813 - 100902, 100911 - 100947, 100948- 100968, 100970- 101104, 101111 - 101140, 101142- 101167, 101170- 101189, 101211 - 101236, 101238- 101253, 101255- 101273, 101275- 101306, 101327- 101371, 101381 - 101411, 101447- 101476, 101514- 101528, 101527- 101541, 101530- 101579, 101590- 101635, 101626 - 101655, 101656 - 101685, 101681 - 101695, 101687 - 101725, 101715- 101732, 101756- 101776, 101825- 101861, 101890- 101987, 101993- 102036, 102073- 102089, 102091 - 102259, 102276- 102297, 102299- 102326, 102351 - 102371, 102376- 102399, 102427- 102442, 102436- 102459, 102461 - 102491, 102507- 102522, 102516 - 102534, 102536- 102610, 102637- 102678, 102681 - 102707, 102724- 102769, 102771 - 102790, 102808- 102823, 102823- 102837, 102840- 102879, 102930- 102955, 102957- 102971, 102977- 103060, 103072- 103108, 103110- 103125, 103127- 103177, 103181 - 103199, 103252- 103279, 103293- 103329, 103346- 103360, 103362- 103397, 103416 - 103552, 103555- 103577, 103607- 103627, 103641 - 103664, 103674- 103688, 103691 - 103746, 103764- 103785, 103804- 103823, 103825- 103866, 103869- 103894, 103908- 103927, 103923- 103938, 103976- 103994, 104018- 104046, 104047- 104073, 104075- 104138, 104140- 104189, 104191 - 104210, 104249- 104279, 104289- 104305, 104295- 104310, 104311 - 104362, 104370- 104384, 104386- 104410, 104431 - 104448, 104450- 104480, 104508- 104525, 104527- 104544, 104546- 104562, 104583- 104601, 104622- 104644, 104646- 104668, 104687- 104710, 104716- 104736, 104738- 104817, 104822- 104847, 104858- 104885, 104880- 104930, 104932 - 104949, 104951 - 104966, 104976 - 104999, 105001 - 105016, 105033 - 105051 , 105081 - 105109, 1051 1 1 - 105261 , 105263 - 105387, 105399 - 105452, 105454 - 105535, 105578 - 105596, 105613 - 105643, 105645 - 105707, 105709 - 105759, 105761 - 105838, 105840 - 105860, 105873 - 105920, 105925 - 105948, 105950 - 105992, 105994 - 106019, 106031 - 106055, 106058 - 106086, 106107 - 106209, 106216 - 106241 , 106272 - 106286, 106289 - 106375, 106385 - 106434, 106436 - 106498, 106512 - 106527, 106528 - 106544, 106546 - 106578, 106580 - 106617, 106619 - 106640, 106642 - 106693, 106695 - 106713, 106715 - 106732,
106734 - 106754, 106756 - 106780, 106847 - 106877, 106886 - 106909, 10691 1 - 106931 , 106933 - 106980, 106998 - 107027, 107033 - 107060, 107080 - 1071 14, 107120 - 107137, 107165 - 107183, 107202 - 107220, 107222 - 107245, 107247 - 107293, 107307 - 107343, 107372 - 107386, 107377 - 107394, 107409 - 107483, 107516 - 107572, 107591 - 107617, 107621 - 107636, 107638 - 107653, 107656 - 107685, 107688 - 107720, 107746 - 107771 , 107768 - 107784, 107774 - 107788, 107778 - 107792, 107782 - 107818, 107826 - 107841 , 107843 - 107862, 107869 - 107890, 107891 - 107915, 107926 - 107955, 107957 - 107986, 107991 - 108030, 108048 - 108080, 108095 - 108109, 1081 16 - 108158, 108206 - 108224, 108238 - 108260, 108262 - 108279, 108292 - 108330, 108332 - 108357, 108368 - 108393, 108405 - 108467, 108469 - 108516, 108518 - 108642, 108654 - 108807, 108807 - 108958, 108960 - 109056, 109058 - 109246, 109256 - 109281 , 109283 - 109401 , 109403 - 109540, 109542 - 109588, 109590 - 109610, 109625 - 109699, 109701 - 109766, 109777 - 109792, 109805 - 109848, 109846 - 1 10128, 1 10130 - 1 10155, 1 10157 - 1 10190, 1 10192 - 1 10213, 1 10225 - 1 10355, 1 10357 - 110396, 1 10407 - 1 10486, 1 10492 - 1 10517, 1 10513 - 1 10528, 1 10529 - 1 10658, 1 10660 - 1 10675,
1 10684 - 1 10755, and 110749 - 1 10836.
5. The antisense oligonucleotide according to claim 1 or 4, wherein the antisense
oligonucleotide is a gapmer oligonucleotide comprising a contiguous nucleotide sequence of formula 5’-F-G-F’-3’, where region F and F’ independently comprise 1 - 8 sugar modified nucleosides, and G is a region between 5 and 16 nucleosides which are capable of recruiting RNaseH.
6. The antisense oligonucleotide according to claim 5, wherein the sugar modified
nucleosides of region F and F’ are independently selected from the group consisting of 2’-0-alkyl-RNA, 2’-0-methyl-RNA, 2’-alkoxy-RNA, 2’-0-methoxyethyl-RNA, 2’- amino-DNA, 2’-fluoro-DNA, arabino nucleic acid (ANA), 2’-fluoro-ANA and LNA nucleosides.
7. The antisense oligonucleotide according to claim 5 or 6, wherein region G comprises 5 - 16 contiguous DNA nucleosides.
8. The antisense oligonucleotide according to any one of claims 1 - 7, wherein the antisense oligonucleotide is a LNA gapmer oligonucleotide.
9. The antisense oligonucleotide according to any one of claims 5 - 8, wherein the LNA nucleosides are beta-D-oxy LNA nucleosides.
10. The antisense oligonucleotide according to any one of claims 1 - 9, wherein the internucleoside linkages between the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.
1 1. The antisense oligonucleotide according to any one of claims 1 - 10, wherein the oligonucleotide comprises a contiguous nucleotide sequence selected from the group consisting of: SEQ ID NO 1 - 10, such as selected from the group consisting of SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO: 10.
12. The antisense oligonucleotide according to any one of claims 1 - 1 1 , wherein the oligonucleotide comprises or consists of a contiguous nucleotide sequence, selected from the group consisting of:
TT gtcttataacTGCA (SEQ ID NO 1 ), GGcttattttacCACA (SEQ ID NO 2),
TATtattgaggaaGCC (SEQ ID NO 3), ATCAaatctgctatCA (SEQ ID NO 4),
GAtttagttggcATGA (SEQ ID NO 5), TAGTtggcatgataAT (SEQ ID NO 6),
TTtagttggcatGATA (SEQ ID NO 7), CTTggcattataaCTA (SEQ ID NO 8),
GTTCacaaagaaagCC (SEQ ID NO 9), and AGAtttagttggcATG (SEQ ID NO 10), such as selected from the group consisting of
GAtttagttggcATGA (SEQ ID NO 5),
TAGTtggcatgataAT (SEQ ID NO 6),
TTtagttggcatGATA (SEQ ID NO 7), and
AGAtttagttggcATG (SEQ ID NO 10), wherein a capital letter represents a LNA nucleoside, a lower case letter represents a DNA nucleoside.
13. The antisense oligonucleotide according to any one of claims 1 - 12, wherein the oligonucleotide comprises or consists of a contiguous nucleotide sequence, selected from the group consisting of: TTgtcttataacTGCA (SEQ ID NO 1 ), GGcttattttacCACA (SEQ ID NO 2), TATtattgaggaaGCC (SEQ ID NO 3), ATCAaatctgctatCA (SEQ ID NO 4), GAtttagttggcATGA (SEQ ID NO 5), TAGTtggcatgataAT (SEQ ID NO 6),
TTtagttggcatGATA (SEQ ID NO 7), CTTggcattataaCTA (SEQ ID NO 8),
GTTCacaaagaaagCC (SEQ ID NO 9), and AGAtttagttggcATG (SEQ ID NO 10), such as selected from the group consisting of GAtttagttggcATGA (SEQ ID NO 5), TAGTtggcatgataAT (SEQ ID NO 6), TTtagttggcatGATA (SEQ ID NO 7), and
AGAtttagttggcATG (SEQ ID NO 10), wherein a capital letter represents a beta-D-oxy LNA nucleoside, a lower case letter represents a DNA nucleoside, wherein each LNA cytosine is 5-methyl cytosine, and wherein the internucleoside linkages between the nucleosides are phosphorothioate internucleoside linkages.
14. A conjugate comprising the oligonucleotide according to any one of claims 1 - 13, and at least one conjugate moiety covalently attached to said oligonucleotide.
15. A pharmaceutical composition comprising the oligonucleotide of claim 1 -13 or the conjugate of claim 14 and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.
16. An in vivo or in vitro method for modulating VCAN expression in a target cell which is expressing VCAN, said method comprising administering an oligonucleotide of any one of claims 1-13, the conjugate according to claim 14, or the pharmaceutical composition of claim 15 in an effective amount to said cell.
17. A method for treating or preventing a disease comprising administering a
therapeutically or prophylactically effective amount of an oligonucleotide of any one of claims 1 - 13 or the conjugate according to claim 14 or the pharmaceutical composition of claim 15 to a subject suffering from or susceptible to the disease.
18. The method of claim 17, wherein the disease is selected from the group consisting of inflammatory lung disease (such as chronic obstructive pulmonary disease or asthma), diabetic retinopathy, cancer and fibrotic disorders (such as fibrotic lung disease, cystic fibrosis, or pulmonary fibrosis).
19. The oligonucleotide of any one of claims 1 - 13 or the conjugate according to claim 14 or the pharmaceutical composition of claim 15 for use in medicine.
20. The oligonucleotide of any one of claims 1 - 13 or the conjugate according to claim 14 or the pharmaceutical composition of claim 15 for use in the treatment or prevention of a disease selected from the group consisting of inflammatory lung disease (such as chronic obstructive pulmonary disease or asthma), diabetic retinopathy, cancer and fibrotic disorders (such as fibrotic lung disease, cystic fibrosis, or pulmonary fibrosis).
21. Use of the oligonucleotide of claim 1 - 13 or the conjugate according to claim 14 or the pharmaceutical composition of claim 15 for the preparation of a medicament for treatment or prevention of a disease selected from the group consisting of inflammatory lung disease (such as chronic obstructive pulmonary disease or asthma), diabetic retinopathy, cancer and fibrotic disorders (such as fibrotic lung disease, cystic fibrosis, or pulmonary fibrosis).
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