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WO2020038973A1 - Antisense oligonucleotides targeting sptlc1 - Google Patents

Antisense oligonucleotides targeting sptlc1 Download PDF

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Publication number
WO2020038973A1
WO2020038973A1 PCT/EP2019/072322 EP2019072322W WO2020038973A1 WO 2020038973 A1 WO2020038973 A1 WO 2020038973A1 EP 2019072322 W EP2019072322 W EP 2019072322W WO 2020038973 A1 WO2020038973 A1 WO 2020038973A1
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seq
oligonucleotide
nucleosides
region
nucleotide sequence
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Nanna ALBÆK
Lykke PEDERSEN
Steffen Schmidt
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Roche Innovation Center Copenhagen AS
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Roche Innovation Center Copenhagen AS
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    • 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
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
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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
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    • 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
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    • 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
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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 ===---===
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/11—Applications; Uses in screening processes for the determination of target sites, i.e. of active nucleic acids

Definitions

  • the present invention relates to antisense LNA oligonucleotides (oligomers) complementary to SPTLC1 pre-mRNA intron sequences, which are capable of inhibiting the expression of SPTLC1.
  • Inhibition of SPTLC1 expression is beneficial for a range of medical disorders including hypertension such as cancer, hepatitis C, diabetes (such as type 2 diabetes), opiate/opioid-antinociceptive tolerance, Niemann-Pick type C disease, HIV infection and amphetamine/methamphetamine-induced toxicity.
  • Serine palmitoyltransferase a member of the class-ll pyridoxal-phosphate-dependent aminotransferase family, is the key enzyme in sphingolipid biosynthesis. It converts L-serine and palmitoyl-CoA to 3-oxosphinganine with pyridoxal 5'-phosphate as a cofactor.
  • the enzyme contains two main subunits. SPTLC1 (serine palmitoyltransferase long chain base subunit 1) encodes the long chain base subunit 1 of this enzyme.
  • Serine palmitoyltransferase has been associated with many diseases and disorders.
  • Various mutations in the SPTLC1 gene are the most common cause of hereditary sensory neuropathy type 1.
  • the mutations in the SPTLC1 gene have been shown to alter active site specificity by enhancing the ability of the enzyme to condense L-alanine with the palmitoyl- CoA substrate (see Auer-Grumbach et al., Eur J Med Genet. 2013 May; 56(5): 266-269).
  • increased de novo ceramide synthesis due to an increase in serine palmitoyltransferase activity causes apoptosis (Perry et al., J. Biol. Chem. 2002 275: 9078-9084). Therefore, inhibition of serine palmitoyltransferase activity prevents the proliferation of many cancer cells (Yagushi, Biochem Biophys Res Commun. 2017,
  • WO02/48325 refers to reagents which regulate human serine palmitoyltransferase and reagents which bind to human serine palmitoyltransferase gene products can play a role in preventing, ameliorating, or correcting dysfunctions or diseases including, but not limited to HIV, cancer, obesity, and diabetes.
  • W02008/122037 refers to serine palmitoyltransferase inhibitors as adjuncts to opiates, and methods which provide improved pain management of chronic pain disorders associated with long terms administration of opiates.
  • W02009/001097 refers to an inhibitor compound of sphingolipid biosynthesis for use in the treatment of diseases which have a secondary Niemann-Pick type C disease cellular phenotype.
  • WO2013/149250 refers to a method of treating an amphetamine-type drug-induced toxicity, said method comprising administering to the subject in need thereof an effective amount of e.g. a serine palmitoyltransferase inhibitor.
  • the inhibitor could be, e.g., an antisense nucleic acid, a ribozyme, a triplex-forming oligonucleotide, a siRNA or an antibody.
  • the inventors have identified particularly effective regions of the SPTLC1 transcript ( SPTLC1 ) for antisense inhibition in vitro or in vivo, and provides for antisense
  • oligonucleotides including LNA gapmer oligonucleotides, which target these regions of the SPTLC1 premRNA.
  • the present invention identifies oligonucleotides which inhibit human SPTLC1 which are useful in the treatment of a range of medical disorders including hypertension such as cancer, hepatitis C, diabetes (such as type 2 diabetes), opiate/opioid- antinociceptive tolerance, Niemann-Pick type C disease, HIV infection and
  • amphetamine/methamphetamine-induced toxicity amphetamine/methamphetamine-induced toxicity
  • the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, targeting a human SPTLC1 target nucleic acid, wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 in a cell which is expressing human SPTLC1.
  • the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, targeting a human SPTLC1 pre-mRNA target nucleic acid, wherein the antisense
  • oligonucleotide is capable of inhibiting the expression of human SPTLC1 in a cell which is expressing human SPTLC1 , and wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence of at least 10 nucleotides which are fully complementary to the human SPTLC1 pre-mRNA target nucleic acid.
  • the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, targeting a human SPTLC1 target nucleic acid, wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 in a cell which is expressing human SPTLC1 ; and wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence of at least 10 nucleotides which are fully complementary to an intron region of SPTLC1 pre-mRNA target nucleic acid.
  • the invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, targeting a human SPTLC1 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 SPTLC1 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 27 wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 in a cell which is expressing human SPTLC1.
  • 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 27, wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 in a cell which is expressing human SPTLC1.
  • 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 27 wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 in a cell which is expressing human SPTLC1.
  • 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 27 wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 in a cell which is expressing human SPTLC1.
  • 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 selected from the group consisting of SEQ ID NO 21 , SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26 and SEQ ID NO 31 , wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 in a cell which is expressing human SPTLC1.
  • 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 selected from the group consisting of SEQ ID NO 21 , SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26 and SEQ ID NO 31 , wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 in a cell which is expressing human SPTLC1.
  • 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 selected from the group consisting of SEQ ID NO 21 , SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26 and SEQ ID NO 31 , wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 in a cell which is expressing human SPTLC1.
  • 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 selected from the group consisting of SEQ ID NO 21 , SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26 and SEQ ID NO 31 , wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 in a cell which is expressing human SPTLC1.
  • 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 21 , wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 transcript in a cell which is expressing human SPTLC1 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 22, wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 transcript in a cell which is expressing human SPTLC1 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 23, wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 transcript in a cell which is expressing human SPTLC1 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 24, wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 transcript in a cell which is expressing human SPTLC1 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 25, wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 transcript in a cell which is expressing human SPTLC1 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 26, wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 transcript in a cell which is expressing human SPTLC1 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 31 , wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 transcript in a cell which is expressing human SPTLC1 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
  • 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 SPTLC1 expression in a target cell which is expressing SPTLC1, 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 hypertension such as cancer, hepatitis C, diabetes (such as type 2 diabetes), opiate/opioid-antinociceptive tolerance, Niemann-Pick type C disease, HIV infection and
  • amphetamine/methamphetamine-induced toxicity amphetamine/methamphetamine-induced toxicity
  • 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 hypertension such as cancer, hepatitis C, diabetes (such as type 2 diabetes), opiate/opioid-antinociceptive tolerance, Niemann-Pick type C disease, HIV infection and amphetamine/methamphetamine-induced toxicity.
  • a disease selected from the group consisting of hypertension such as cancer, hepatitis C, diabetes (such as type 2 diabetes), opiate/opioid-antinociceptive tolerance, Niemann-Pick type C disease, HIV infection and amphetamine/methamphetamine-induced toxicity.
  • 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 hypertension such as cancer, hepatitis C, diabetes (such as type 2 diabetes), opiate/opioid-antinociceptive tolerance, Niemann-Pick type C disease, HIV infection and amphetamine/methamphetamine-induced toxicity.
  • a disease selected from the group consisting of hypertension such as cancer, hepatitis C, diabetes (such as type 2 diabetes), opiate/opioid-antinociceptive tolerance, Niemann-Pick type C disease, HIV infection and amphetamine/methamphetamine-induced toxicity.
  • Figure 1 Testing in vitro efficacy of various antisense oligonucleotides targeting human SPTLC1 mRNA in A549 and MDA-MB-231 cell lines at single concentration.
  • Figure 2 Testing in vitro efficacy of various antisense oligonucleotides targeting mouse Sptld mRNA in J774A.1 cells at single concentration.
  • Figure 3 Comparison of in vitro efficacy for antisense oligonucleotides targeting human SPTLC1 mRNA in A549 and MDA-MB-231 cell lines at single concentration shows good correlation. Three motifs with very efficient targeting are highlighted.
  • Figure 4 IC50 values for selected oligonucleotides targeting human SPTLC1 mRNA in vitro in A549 and MDA-MB-231 cell lines.
  • Figure 5 Testing selected oligonucleotides targeting human SPTLC1 mRNA in vitro for concentration dependent potency and efficacy in A549 cell line.
  • Figure 6 Testing selected oligonucleotides targeting human SPTLC1 mRNA in vitro for concentration dependent potency and efficacy in MDA-MB-231 cell line.
  • Figure 7 Testing selected oligonucleotides targeting mouse Sptld mRNA in vitro for concentration dependent potency and efficacy in J774A.1 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 Contiguous Nucleotide Sequence
  • 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”.
  • the 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. Complementarity
  • 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 a!., 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 SPTLC1 and may for example be a gene, a SPTLC1 RNA, a mRNA, a pre- mRNA, a mature mRNA or a cDNA sequence.
  • the target may therefore be referred to as an SPTLC1 target nucleic acid.
  • the target nucleic acid encodes an SPTLC1 protein, in particular mammalian SPTLC1 , such as the human SPTLC1 gene encoding pre-mRNA or mRNA sequences provided herein as SEQ ID NO 27, 28, 29 or 30.
  • SPTLC1 protein in particular mammalian SPTLC1 , such as the human SPTLC1 gene encoding pre-mRNA or mRNA sequences provided herein as SEQ ID NO 27, 28, 29 or 30.
  • the target nucleic acid is selected from the group consisting of SEQ ID NO 27 or a naturally occurring variant thereof (e.g. SPTLC1 sequences encoding a mammalian SPTLC1 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 SPTLC1 target nucleic acid in a cell which is expressing the SPTLC1 target nucleic acid.
  • oligonucleotide of the invention is typically complementary to the SPTLC1 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 non- complementary 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 SPTLC1 protein, such as human SPTLC1 , e.g.
  • SPTLC1 pre-mRNA sequence such as that disclosed as SEQ ID NO 27, or SPTLC1 mature mRNA, such as that disclosed as SEQ ID NO 28, 29 or 30.
  • SEQ ID NOs 27 - 30 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 27.
  • the oligonucleotide of the invention targets SEQ ID NO 28.
  • the oligonucleotide of the invention targets SEQ ID NO 29.
  • the oligonucleotide of the invention targets SEQ ID NO 30.
  • 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 SPTLC1 pre-mRNA (using SEQ ID NO 27 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, 11 , 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 SPTLC1 target nucleic acid which may be targeted by the oligonucleotide of the invention.
  • the target sequence is SEQ ID NO 21.
  • the target sequence is SEQ ID NO 22.
  • the target sequence is SEQ ID NO 23.
  • the target sequence is SEQ ID NO 24.
  • the target sequence is SEQ ID NO 25.
  • the target sequence is SEQ ID NO 26.
  • the target sequence is SEQ ID NO 31.
  • SEQ ID NO 21 AAGAT ACTT GAT AT AT GCGTTT AACATGGT CAG (27)
  • SEQ ID NO 23 GT AAAATT G C CTT AC ATT AT C G AC AT C C C G
  • SEQ ID NO 24 TG C CTT AC ATT ATC G A
  • SEQ ID NO 25 AAG GTGTTCCTGCTT ATT AAAT GTACTTG G (27)
  • the invention provides for an antisense oligonucleotide, 10-30
  • 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 27, selected from the group consisting if Ex_1 - Ex_15 (see the following table).
  • the invention provides for an antisense oligonucleotide, 10-30
  • 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 27, selected from the group consisting of 9 - 71 ; 2823 - 2930; 6551 - 6645; 34422 - 34515; 35297 - 35369; 47287 - 47419; 56077 - 56206; 59891 - 59980; 65318 - 65425;
  • the invention provides for an antisense oligonucleotide, 10-30
  • 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 27 , selected from the group consisting of lnt_1 - lnt_14.
  • the invention provides for an antisense oligonucleotide, 10-30
  • 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 27, selected from the group consisting of 71 - 2823; 2930 - 6551 ; 6645 - 34422;
  • the invention provides for an antisense oligonucleotide, 10-30
  • 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 27, selected from the group consisting of 10 - 40, 66 - 85, 89 - 113, 131 - 151, 153 - 190, 194 - 224, 226 - 255, 276 - 291, 293 - 316, 346 - 394, 396 - 415, 466 - 523, 551 - 573,
  • 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 27, selected from the group consisting of 253-267,1261 - 1275,2832-2851,6557 - 6579, 6617 - 6639, 8893 - 8912, 13803 - 13823, 14533 - 14553, 21090 - 21106, 21901 - 21916, 32671 - 32685, 34030 - 34052, 34441 - 34457, 35357 - 35375, 36893 - 36907,
  • 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 SPTLC1 mRNA, such as the SPTLC1 pre-mRNA, e.g. SEQ ID NO 27, or SPTLC1 mature mRNA (e.g. SEQ ID NO 28, 29 or 30).
  • SPTLC1 mRNA such as the SPTLC1 pre-mRNA, e.g. SEQ ID NO 27, or SPTLC1 mature mRNA (e.g. SEQ ID NO 28, 29 or 30).
  • the poly A tail of SPTLC1 mRNA is typically disregarded for antisense oligonucleotide targeting.
  • naturally occurring variant refers to variants of SPTLC1 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 SPTLC1 gene is located at chromosome 9, 92031 134..921 15474, complement (NC_000009.12, Gene ID 10558).
  • the naturally occurring variants have at least 95% such as at least 98% or at least 99% homology to a mammalian SPTLC1 target nucleic acid, such as a target nucleic acid selected form the group consisting of SEQ ID NO 27, 28, 29 or 30. In some embodiments the naturally occurring variants have at least 99% homology to the human SPTLC1 target nucleic acid of SEQ ID NO 27.
  • modulation of expression is to be understood as an overall term for an oligonucleotide’s ability to alter the amount of SPTLC1 protein or SPTLC1 mRNA when compared to the amount of SPTLC1 or SPTLC1 mRNA prior to administration of the oligonucleotide.
  • 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 SPTLC1 , e.g. by degradation of SPTLC1 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. Opinion in Drug Development, 2000, 3(2), 293-213).
  • 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.
  • 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).
  • HNA hexose ring
  • LNA ribose ring
  • UNA unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons
  • 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
  • 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.
  • 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 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.
  • 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
  • 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, 11 , 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 a!., 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
  • UNA unlocked 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
  • nucleosides such as independently selected from beta-D-oxy LNA, ENA or ScET
  • 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]i_ 5 -[region G] -[LNA] I-5 , 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 [MOE]i-e-[Region G]-[MOE] 1-8, such as [MOE]2-7-[Region G]s-i 6 -[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.
  • 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:
  • F-G-F in particular F1-8-G5-16-F 2-8
  • D’-F-G-F’-D in particular D’ I-3 - Fi-8-G5-i6-F’2-8-D”i -3
  • 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 SPTLC1 expression.
  • the oligonucleotides of the invention targeting SPTLC1 are capable of hybridizing to and inhibiting the expression of a SPTLC1 target nucleic acid in a cell which is expressing the SPTLC1 target nucleic acid.
  • the SPTLC1 target nucleic acid may be a mammalian SPTLC1 mRNA or premRNA, such as a human SPTLC1 mRNA or premRNA, for example a premRNA or mRNA originating from the Homo sapiens serine palmitoyltransferase long chain base subunit 1 (SPTLC1 ), RefSeqGene on chromosome 9, exemplified by NCBI Reference Sequence NG_007950.1 or Ensembl ENSG00000090054 (SEQ ID NO 27).
  • the human SPTLC1 pre-mRNA is encoded on Homo sapiens Chromosome 9, NC_000009.12 (92031 134..921 15474, complement).
  • GENE ID 10558 ( SPTLC1 ).
  • a mature human mRNA target sequence is illustrated herein by the cDNA sequences SEQ ID NO 28, 29 or 30.
  • the oligonucleotides of the invention are capable of inhibiting the expression of SPTLC1 target nucleic acid, such as the SPTLC1 mRNA, in a cell which is expressing the target nucleic acid, such as the SPTLC1 mRNA.
  • the oligonucleotides of the invention are capable of inhibiting the expression of SPTLC1 target nucleic acid in a cell which is expressing the target nucleic acid, so to reduce the level of SPTLC1 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 SPTLC1 target nucleic acid (e.g. the mRNA) in the cell.
  • the cell is selected from the group consisting of A549 cells, MDA-MB-231 cells, J774A.1 cells and MPC-1 1 cells.
  • 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 21.
  • 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 22.
  • the antisense 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 23.
  • the antisense oligonucleotide of the invention comprises a contiguous nucleotides sequence of 12 - 16, such as 13, 14 or 15 contiguous nucleotides in length, wherein the contiguous nucleotide sequence is fully complementary to SEQ ID NO 24.
  • 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 25.
  • the antisense oligonucleotide of the invention comprises a contiguous nucleotides sequence of 12 - 17, such as 13, 14, 15 or 16 contiguous nucleotides in length, wherein the contiguous nucleotide sequence is fully complementary to SEQ ID NO 26.
  • the antisense oligonucleotide of the invention comprises a contiguous nucleotides sequence of 12 - 22, such as 13, 14, 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides in length, wherein the contiguous nucleotide sequence is fully complementary to SEQ ID NO 31.
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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 26.
  • 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 31.
  • 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.
  • the contiguous nucleotide sequence is fully complementary to SEQ ID NO 21.
  • the contiguous nucleotide sequence is fully complementary to SEQ ID NO 22.
  • the contiguous nucleotide sequence is fully complementary to SEQ ID NO 23.
  • the contiguous nucleotide sequence is fully complementary to SEQ ID NO 24. In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 25.
  • the contiguous nucleotide sequence is fully complementary to SEQ ID NO 26.
  • the contiguous nucleotide sequence is fully complementary to SEQ ID NO 31.
  • 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 - 20.
  • 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 3 or 13.
  • 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 8 or 15.
  • 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 17, 18 or 20.
  • 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: TTtaagtgttcGACC (SEQ ID NO 1 ), TTGtgataaagcCGT (SEQ ID NO 2), CGCatatatcaaGTA (SEQ ID NO 3), CGtatgagaggCAAG (SEQ ID NO 4), GGATatacagtcgCG (SEQ ID NO 5), TAatgtcactaCGGG (SEQ ID NO 6), CCGAttactgattTA (SEQ ID NO 7), TCgataatgtaAGGC (SEQ ID NO 8), TTAgtagtcgcTCG (SEQ ID NO 9), GGctattgacgATTC (SEQ ID NO 10), ATGAggcgaagttTA (SEQ ID NO 1 1 ), ATCaacgcataCGC (SEQ ID NO 12), CCAtgttaaacgCAT (SEQ ID NO 13), AT
  • CATttaataagcaGGA SEQ ID NO 20; 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: TTtaagtgttcGACC (SEQ ID NO 1 ), TTGtgataaagcCGT (SEQ ID NO 2), CGCatatatcaaGTA (SEQ ID NO 3), CGtatgagaggCAAG (SEQ ID NO 4), GGATatacagtcgCG (SEQ ID NO 5), TAatgtcactaCGGG (SEQ ID NO 6), CCGAttactgattTA (SEQ ID NO 7), TCgataatgtaAGGC (SEQ ID NO 8), TTAgtagtcgcTCG (SEQ ID NO 9), GGctattgacgATTC (SEQ ID NO 10), ATGAggcgaagttTA (SEQ ID NO 1 1 ), ATCaacgcataCGC (SEQ ID NO 12), CCAtgttaaacgCAT (SEQ ID NO 13), AT
  • CATttaataagcaGGA (SEQ ID NO 20); 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: TTtaagtgttcGACC (SEQ ID NO 1 ), TTGtgataaagcCGT (SEQ ID NO 2), CGCatatatcaaGTA (SEQ ID NO 3), CGtatgagaggCAAG (SEQ ID NO 4), GGATatacagtcgCG (SEQ ID NO 5), TAatgtcactaCGGG (SEQ ID NO 6), CCGAttactgattTA (SEQ ID NO 7), TCgataatgtaAGGC (SEQ ID NO 8), TTAgtagtcgcTCG (SEQ ID NO 9), GGctattgacgATTC (SEQ ID NO 10), ATGAggcgaagttTA (SEQ ID NO 1 1 ), ATCaacgcataCGC (SEQ ID NO 12), CCAtgttaaacgCAT (SEQ ID NO 13), AT
  • CATttaataagcaGGA (SEQ ID NO 20); 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.
  • 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 - 300mM 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 non- toxic 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. 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.
  • 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.
  • oligonucleotides may be used to specifically modulate the synthesis of SPTLC1 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 SPTLC1 expression in a target cell which is expressing SPTLC1, 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 SPTLC1 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 SPTLC1
  • 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.
  • 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 SPTLd.
  • disease or disorder may be associated with a mutation in the SPTLd gene. Therefore, in some embodiments, the target nucleic acid is a mutated form of the SPTLd sequence.
  • the methods of the invention are preferably employed for treatment or prophylaxis against diseases caused by abnormal levels and/or activity of SPTLd.
  • 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 SPTLd.
  • the invention relates to oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions for use in the treatment of diseases or disorders selected from cancer, hepatitis C, diabetes (such as type 2 diabetes), opiate/opioid-antinociceptive tolerance, Niemann-Pick type C disease, HIV infection and
  • amphetamine/methamphetamine-induced toxicity amphetamine/methamphetamine-induced toxicity
  • oligonucleotides or pharmaceutical compositions of the present invention may be administered topical or enteral or parenteral (such as, intravenous, subcutaneous, intra- muscular, 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.
  • Examplel Testing in vitro efficacy of antisense oligonucleotides targeting SPTLC1 mRNA in human A549 and MDA-MB-231 , as well as mouse J774A.1 cell lines at single concentration.
  • A549, MDA-MB-231 and J774A.1 cell lines were purchased from ATCC and maintained as recommended by the supplier in a humidified incubator at 37°C with 5% C02.
  • 4500 cells/well of MDA-MB-231 and A549; 2500 cells/well of J774A.1 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 mM. 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
  • 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: SPTLC1 Hs0027231 1_m1 (Mm00447343_m1 ) [FAM-MGB] and endogenous control GAPDH, Hs99999905_m1 (Mm99999915_g1 ) [VIC- MGB] All primer sets were purchased from Thermo Fisher Scientific.
  • the relative SPTLC1 mRNA expression level in the table is shown as percent of control (PBS-treated cells).
  • SPTLC1 mRNA levels from cells treated with a selection of the compounds are shown in figures 1 to 3, evaluated human A549 and MDA-MB-231 cell lines as well as mouse J77A.1 cell lines. From the initial library screen three motifs on the SPTLC1 human transcript were identified which provided surprisingly effective and potent compounds in the cell lines tested: Motif A (SEQ ID NO 21 ), Motif B (SEQ ID NO 23) and Motif C (SEQ ID NO 25).
  • LNA nucleosides (beta-D-oxy LNA nucleosides were used), all LNA cytosines are 5-methyl cytosine, lower case letters represent DNA nucleosides, DNA cytosines preceded with a superscript m represents a 5-methyl C-DNA nucleoside. All internucleoside linkages are phosphorothioate internucleoside linkages.
  • Example 2 Testing in vitro potency and efficacy of selected oligonucleotides targeting SPTLC1 mRNA in human A549; MDA-MB-231 and mouse J774A.1 cells for a concentration response curve.
  • PBS percent of control
  • the IC50 values for selected oligonucleotides targeting human SPTLC1 mRNA in vitro in the human cell lines MDA-MB-231 and A549 are shown in Figure 4.
  • the concentration response curves in human cell lines A549 and MDA-MB-231 as well as in mouse cell line J77A.1 are provided as Figures 5, 6 and 7, respectively.

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Abstract

The present invention relates to antisense LNA oligonucleotides (oligomers) complementary to SPTLC1 pre-mRNA intron sequences, which are capable of inhibiting the expression of SPTLC1 protein. Inhibition of SPTLC1 expression is beneficial for a range of medical disorders including hypertension such as cancer, hepatitis C, diabetes (such as type 2 diabetes), opiate/opioid-antinociceptive tolerance, Niemann-Pick type C disease, HIV infection and amphetamine/methamphetamine-induced toxicity.

Description

ANTISENSE OLIGONUCLEOTIDES TARGETING SPTLC1
FIELD OF INVENTION
The present invention relates to antisense LNA oligonucleotides (oligomers) complementary to SPTLC1 pre-mRNA intron sequences, which are capable of inhibiting the expression of SPTLC1. Inhibition of SPTLC1 expression is beneficial for a range of medical disorders including hypertension such as cancer, hepatitis C, diabetes (such as type 2 diabetes), opiate/opioid-antinociceptive tolerance, Niemann-Pick type C disease, HIV infection and amphetamine/methamphetamine-induced toxicity.
BACKGROUND
Serine palmitoyltransferase, a member of the class-ll pyridoxal-phosphate-dependent aminotransferase family, is the key enzyme in sphingolipid biosynthesis. It converts L-serine and palmitoyl-CoA to 3-oxosphinganine with pyridoxal 5'-phosphate as a cofactor. The enzyme contains two main subunits. SPTLC1 (serine palmitoyltransferase long chain base subunit 1) encodes the long chain base subunit 1 of this enzyme.
Serine palmitoyltransferase has been associated with many diseases and disorders. Various mutations in the SPTLC1 gene are the most common cause of hereditary sensory neuropathy type 1. The mutations in the SPTLC1 gene have been shown to alter active site specificity by enhancing the ability of the enzyme to condense L-alanine with the palmitoyl- CoA substrate (see Auer-Grumbach et al., Eur J Med Genet. 2013 May; 56(5): 266-269). Further, it has been demonstrated that increased de novo ceramide synthesis due to an increase in serine palmitoyltransferase activity causes apoptosis (Perry et al., J. Biol. Chem. 2002 275: 9078-9084). Therefore, inhibition of serine palmitoyltransferase activity prevents the proliferation of many cancer cells (Yagushi, Biochem Biophys Res Commun. 2017,
1 1 ;484(3):493-500). For example, administration of myriocin, a small molecule inhibitor of serine palmitoyltransferase isolated from Myriococcum albomyces, resulted in suppression of melanoma cells (Lee et al., Cancer Biology & Therapy, 2002, 13:2, 92-100).
Katsume et al. showed that administration of an inhibitor of serine palmitoyltransferase disrupts the HCV replication complex and thereby inhibits HCV (hepatitis C virus) replication. Therefore, serine palmitoyltransferase is considered as a target for the treatment of hepatitis C (Gastroenterology. 2013, 145(4):865-73).
WO02/48325 refers to reagents which regulate human serine palmitoyltransferase and reagents which bind to human serine palmitoyltransferase gene products can play a role in preventing, ameliorating, or correcting dysfunctions or diseases including, but not limited to HIV, cancer, obesity, and diabetes.
W02008/122037 refers to serine palmitoyltransferase inhibitors as adjuncts to opiates, and methods which provide improved pain management of chronic pain disorders associated with long terms administration of opiates.
W02009/001097 refers to an inhibitor compound of sphingolipid biosynthesis for use in the treatment of diseases which have a secondary Niemann-Pick type C disease cellular phenotype.
WO2013/149250 refers to a method of treating an amphetamine-type drug-induced toxicity, said method comprising administering to the subject in need thereof an effective amount of e.g. a serine palmitoyltransferase inhibitor. The inhibitor could be, e.g., an antisense nucleic acid, a ribozyme, a triplex-forming oligonucleotide, a siRNA or an antibody.
There is a need for therapeutic agents which can inhibit SPTLC1 specifically.
We have screened a large number of LNA gapmers targeting mouse and human SPTLC1 and identified sequences and compounds which are particularly potent and effective to specifically target for SPTLC1 antisense in vitro (human and mouse cells).
OBJECTIVE OF THE INVENTION
The inventors have identified particularly effective regions of the SPTLC1 transcript ( SPTLC1 ) for antisense inhibition in vitro or in vivo, and provides for antisense
oligonucleotides, including LNA gapmer oligonucleotides, which target these regions of the SPTLC1 premRNA. The present invention identifies oligonucleotides which inhibit human SPTLC1 which are useful in the treatment of a range of medical disorders including hypertension such as cancer, hepatitis C, diabetes (such as type 2 diabetes), opiate/opioid- antinociceptive tolerance, Niemann-Pick type C disease, HIV infection and
amphetamine/methamphetamine-induced toxicity.
STATEMENT OF THE INVENTION
The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, targeting a human SPTLC1 target nucleic acid, wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 in a cell which is expressing human SPTLC1. The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, targeting a human SPTLC1 pre-mRNA target nucleic acid, wherein the antisense
oligonucleotide is capable of inhibiting the expression of human SPTLC1 in a cell which is expressing human SPTLC1 , and wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence of at least 10 nucleotides which are fully complementary to the human SPTLC1 pre-mRNA target nucleic acid.
The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, targeting a human SPTLC1 target nucleic acid, wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 in a cell which is expressing human SPTLC1 ; and wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence of at least 10 nucleotides which are fully complementary to an intron region of SPTLC1 pre-mRNA target nucleic acid.
The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, targeting a human SPTLC1 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 27, 28, 29 and 30.
The invention provides for an antisense oligonucleotide, 10-30 nucleotides in length, targeting a human SPTLC1 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 27.
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 27 wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 in a cell which is expressing human SPTLC1. 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 27, wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 in a cell which is expressing human SPTLC1.
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 27 wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 in a cell which is expressing human SPTLC1.
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 27 wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 in a cell which is expressing human SPTLC1.
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 selected from the group consisting of SEQ ID NO 21 , SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26 and SEQ ID NO 31 , wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 in a cell which is expressing human SPTLC1.
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 selected from the group consisting of SEQ ID NO 21 , SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26 and SEQ ID NO 31 , wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 in a cell which is expressing human SPTLC1.
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 selected from the group consisting of SEQ ID NO 21 , SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26 and SEQ ID NO 31 , wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 in a cell which is expressing human SPTLC1.
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 selected from the group consisting of SEQ ID NO 21 , SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26 and SEQ ID NO 31 , wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 in a cell which is expressing human SPTLC1.
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 21 , wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 transcript in a cell which is expressing human SPTLC1 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 22, wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 transcript in a cell which is expressing human SPTLC1 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 23, wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 transcript in a cell which is expressing human SPTLC1 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 24, wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 transcript in a cell which is expressing human SPTLC1 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 25, wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 transcript in a cell which is expressing human SPTLC1 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 26, wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 transcript in a cell which is expressing human SPTLC1 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 31 , wherein the antisense oligonucleotide is capable of inhibiting the expression of human SPTLC1 transcript in a cell which is expressing human SPTLC1 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 SPTLC1 expression in a target cell which is expressing SPTLC1, 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 hypertension such as cancer, hepatitis C, diabetes (such as type 2 diabetes), opiate/opioid-antinociceptive tolerance, Niemann-Pick type C disease, HIV infection and
amphetamine/methamphetamine-induced toxicity.
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 hypertension such as cancer, hepatitis C, diabetes (such as type 2 diabetes), opiate/opioid-antinociceptive tolerance, Niemann-Pick type C disease, HIV infection and amphetamine/methamphetamine-induced toxicity.
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 hypertension such as cancer, hepatitis C, diabetes (such as type 2 diabetes), opiate/opioid-antinociceptive tolerance, Niemann-Pick type C disease, HIV infection and amphetamine/methamphetamine-induced toxicity.
BRIEF DESCRIPTION OF FIGURES
Figure 1 : Testing in vitro efficacy of various antisense oligonucleotides targeting human SPTLC1 mRNA in A549 and MDA-MB-231 cell lines at single concentration.
Figure 2: Testing in vitro efficacy of various antisense oligonucleotides targeting mouse Sptld mRNA in J774A.1 cells at single concentration. Figure 3: Comparison of in vitro efficacy for antisense oligonucleotides targeting human SPTLC1 mRNA in A549 and MDA-MB-231 cell lines at single concentration shows good correlation. Three motifs with very efficient targeting are highlighted.
Figure 4: IC50 values for selected oligonucleotides targeting human SPTLC1 mRNA in vitro in A549 and MDA-MB-231 cell lines.
Figure 5: Testing selected oligonucleotides targeting human SPTLC1 mRNA in vitro for concentration dependent potency and efficacy in A549 cell line.
Figure 6: Testing selected oligonucleotides targeting human SPTLC1 mRNA in vitro for concentration dependent potency and efficacy in MDA-MB-231 cell line.
Figure 7: Testing selected oligonucleotides targeting mouse Sptld mRNA in vitro for concentration dependent potency and efficacy in J774A.1 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 AG° 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 a!., 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 SPTLC1 and may for example be a gene, a SPTLC1 RNA, a mRNA, a pre- mRNA, a mature mRNA or a cDNA sequence. The target may therefore be referred to as an SPTLC1 target nucleic acid.
Suitably, the target nucleic acid encodes an SPTLC1 protein, in particular mammalian SPTLC1 , such as the human SPTLC1 gene encoding pre-mRNA or mRNA sequences provided herein as SEQ ID NO 27, 28, 29 or 30.
In some embodiments, the target nucleic acid is selected from the group consisting of SEQ ID NO 27 or a naturally occurring variant thereof (e.g. SPTLC1 sequences encoding a mammalian SPTLC1 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 SPTLC1 target nucleic acid in a cell which is expressing the SPTLC1 target nucleic acid. The contiguous sequence of nucleobases of the
oligonucleotide of the invention is typically complementary to the SPTLC1 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 non- complementary 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 SPTLC1 protein, such as human SPTLC1 , e.g. the human SPTLC1 pre-mRNA sequence, such as that disclosed as SEQ ID NO 27, or SPTLC1 mature mRNA, such as that disclosed as SEQ ID NO 28, 29 or 30. SEQ ID NOs 27 - 30 are DNA sequences - it will be understood that target RNA sequences have uracil (U) bases in place of the thymidine bases (T).
Figure imgf000015_0001
Figure imgf000016_0001
In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 27.
In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 28.
In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 29.
In some embodiments, the oligonucleotide of the invention targets SEQ ID NO 30.
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 SPTLC1 pre-mRNA (using SEQ ID NO 27 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, 11 , 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 SPTLC1 target nucleic acid which may be targeted by the oligonucleotide of the invention.
In some embodiments the target sequence is SEQ ID NO 21.
In some embodiments the target sequence is SEQ ID NO 22.
In some embodiments the target sequence is SEQ ID NO 23.
In some embodiments the target sequence is SEQ ID NO 24.
In some embodiments the target sequence is SEQ ID NO 25.
In some embodiments the target sequence is SEQ ID NO 26.
In some embodiments the target sequence is SEQ ID NO 31.
SEQ ID NO 21 : AAGAT ACTT GAT AT AT GCGTTT AACATGGT CAG (27)
SEQ ID NO 22: T ACTT GAT AT AT GCGTTT AACAT G G (27)
SEQ ID NO 23: GT AAAATT G C CTT AC ATT AT C G AC AT C C C G (27) SEQ ID NO 24: TG C CTT AC ATT ATC G A (27)
SEQ ID NO 25: AAG GTGTTCCTGCTT ATT AAAT GTACTTG G (27)
SEQ ID NO 26: TCCTGCTTATTAAATGT (27)
SEQ ID NO 31 : GGT GTT CCT GCTT ATT AAAT GT (27)
(numbers in brackets refer to the SEQ ID of SPTLC1 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 27, selected from the group consisting if Ex_1 - Ex_15 (see the following table).
Figure imgf000017_0001
Figure imgf000018_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 27, selected from the group consisting of 9 - 71 ; 2823 - 2930; 6551 - 6645; 34422 - 34515; 35297 - 35369; 47287 - 47419; 56077 - 56206; 59891 - 59980; 65318 - 65425;
67677 - 67772; 68117 - 68213; 69332 - 69386; 77020 - 77137; 80502 - 80575; and 82827 - 83386.
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 27 , selected from the group consisting of lnt_1 - lnt_14.
Figure imgf000018_0002
Figure imgf000019_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 27, selected from the group consisting of 71 - 2823; 2930 - 6551 ; 6645 - 34422;
34515-35297; 35369-47287; 47419-56077; 56206-59891; 59980-65318; 65425- 67677; 67772 - 68117; 68213 - 69332; 69386 - 77020; 77137 - 80502; and 80575 - 82827. 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 27, selected from the group consisting of 10 - 40, 66 - 85, 89 - 113, 131 - 151, 153 - 190, 194 - 224, 226 - 255, 276 - 291, 293 - 316, 346 - 394, 396 - 415, 466 - 523, 551 - 573,
589 - 614, 619 - 634, 638 - 656, 667 - 689, 691 - 730, 732 - 752, 763 - 777, 781 - 797, 798 - 822,814 -828, 831 -874, 875-910, 908-923, 1105- 1119, 1132- 1147, 1139- 1159,
1149 - 1166, 1192 - 1213, 1218 - 1235, 1241 - 1259, 1257 - 1276, 1306- 1330, 1334- 1351,
1342 - 1379, 1381 - 1406, 1429 - 1462, 1465- 1501, 1519- 1549, 1591 - 1611, 1614- 1639, 1641 - 1657, 1657 - 1672, 1674 - 1693, 1695 - 1710, 1726 - 1742, 1759 - 1818, 1872 - 1894,
1896 - 1925, 1930 - 1945, 1982 -2001,2004 -2019, 2025-2078,2081 -2126,2131 -2149,
2183 - 2208, 2207 - 2227, 2236 - 2281, 2275 - 2289, 2283 - 2325, 2322 - 2405, 2418 - 2440,
2442 - 2486, 2490 - 2510, 2504 - 2519, 2508 - 2536, 2557 - 2574, 2582 - 2638, 2649 - 2698,
2715 - 2731 , 2737 - 2788, 2796 - 2860, 2862 - 2933, 2946 - 2961 , 2963 - 2981 , 2988 - 3002, 3017 - 3065, 3071 - 3134, 3135 - 3157, 3159 - 3189, 3241 - 3259, 3302 - 3329, 3381 - 3446,
3448 - 3477, 3487 - 3510, 3510 - 3528, 3532 - 3558, 3580 - 3594, 3609 - 3659, 3662 - 3684, 3722 - 3761 , 3763 - 3777, 3779 - 3793, 3795 - 3809, 3820 - 3837, 3863 - 3899, 3909 - 3923,
3940 - 3965, 3983 - 4033, 4035 - 4058, 4060 - 4075, 4086 - 4124, 4135 - 4151 , 4181 - 4216,
4219 - 4243, 4262 - 4276, 4278 - 4295, 4298 - 4331 , 4333 - 4350, 4352 - 4393, 4395 - 4410,
4420 - 4439, 4441 - 4457, 4459 - 4477, 4507 - 4526, 4528 - 4555, 4581 - 4601 , 4603 - 4618,
4640 - 4665, 4678 - 4696, 4698 - 4721 , 4732 - 4770, 4787 - 4872, 4877 - 4896, 4892 - 4947,
4965 - 5002, 5019 - 5049, 5051 - 5074, 5077 - 5099, 51 12 - 5127, 5129 - 5144, 5147 - 5171 ,
5170 - 5184, 5186 - 5298, 5300 - 5317, 5319 - 5341 , 5375 - 5421 , 5425 - 5462, 5464 - 5479,
5476 - 5497, 5489 - 5516, 5517 - 5535, 5558 - 5572, 5577 - 5591 , 5584 - 5635, 5637 - 5673,
5675 - 5694, 5691 - 5750, 5752 - 5777, 5782 - 5847, 5861 - 5886, 5903 - 5921 , 5923 - 5944,
5972 - 5990, 5998 - 6017, 6033 - 6074, 6076 - 6090, 61 11 - 6128, 6136 - 6152, 6154 - 6244,
6247 - 6266, 6269 - 6295, 6297 - 6321 , 6323 - 6343, 6360 - 6382, 6405 - 6474, 6499 - 6546,
6548 - 6579, 6581 - 6606, 6620 - 6668, 6670 - 6696, 6705 - 6735, 6731 - 6747, 6756 - 6775,
6777 - 6816, 6821 - 6840, 6855 - 6875, 6895 - 6929, 6935 - 6968, 6985 - 7001 , 7003 - 7020, 7036 - 7062, 7068 - 7085, 7109 - 7135, 7137 - 7185, 7233 - 7254, 7283 - 7315, 7317 - 7336,
7338 - 7355, 7367 - 7385, 7397 - 7416, 7429 - 7471 , 7498 - 7514, 7516 - 7540, 7592 - 7633,
7623 - 7660, 7680 - 7699, 7701 - 7722, 7724 - 7743, 7745 - 7762, 7769 - 7788, 7777 - 7807,
7798 - 7816, 7806 - 7847, 7862 - 7901 , 7981 - 8005, 8019 - 8040, 8042 - 8105, 8107 - 8133,
8153 - 8170, 8172 - 8204, 8243 - 8278, 8291 - 8396, 8402 - 8432, 8892 - 8924, 8930 - 8945,
8934 - 8963, 8955 - 8974, 8963 - 8986, 8987 - 9026, 9057 - 9073, 9074 - 9092, 9086 - 9105,
9121 - 9136, 9230 - 9245, 9238 - 9255, 9247 - 9270, 9269 - 9288, 9277 - 9296, 9297 - 9330,
9334 - 9349, 9348 - 9368, 9396 - 9414, 9412 - 9429, 9426 - 9440, 9447 - 9462, 9479 - 9495,
9508 - 9542, 9559 - 9575, 9588 - 9603, 9605 - 9622, 9704 - 9737, 9744 - 9767, 9756 - 9774,
9784 - 9807, 9796 - 9814, 9828 - 9847, 9836 - 9854, 9860 - 9874, 9886 - 9908, 9915 - 9944,
9969 - 9983, 10008 - 10028, 10056 - 10071 , 10073 - 10091 , 10094 - 10113, 10151 - 10176,
10208 - 10223, 10225 - 10251 , 10253 - 10267, 10304 - 10356, 10358 - 10405, 10414 - 10447, 10479 - 10519, 10529 - 10552, 10562 - 10576, 10581 - 10629, 10631 - 10680,
10682 - 10738, 10740 - 10782, 10848 - 10891 , 10886 - 10907, 10981 - 10997, 11057 - 1 1071 , 1 1084 - 11 106, 11 146 - 11 192, 11242 - 11263, 11303 - 11330, 11335 - 11357,
1 1359 - 11379, 11410 - 11431 , 11433 - 11448, 11478 - 11514, 11520 - 11562, 11610 - 1 1625, 1 1658 - 11674, 11676 - 11706, 11751 - 11774, 11793 - 11807, 11814 - 11828,
1 1842 - 11880, 11912 - 11946, 11985 - 12010, 12022 - 12040, 12064 - 12078, 12080 - 12100, 12102 - 12157, 12159 - 12174, 12176 - 12194, 12197 - 12231 , 12233 - 12267,
12269 - 12312, 12314 - 12329, 12350 - 12366, 12370 - 12402, 12404 - 12423, 12467 - 12516, 12519 - 12533, 12559 - 12581 , 12583 - 12648, 12659 - 12691 , 12702 - 12740,
12778 - 12804, 12805 - 12832, 12860 - 12902, 12937 - 12956, 12958 - 12992, 12995 - 13017, 13062 - 13077, 13105 - 13122, 13124 - 13166, 13168 - 13207, 13209 - 13229, 13231 - 13250, 13247 - 13263, 13270 - 13288, 13346 - 13362, 13364 - 13404, 13414 - 13437, 13472 - 13502, 13504 - 13535, 13542 - 13561 , 13583 - 13602, 13604 - 13621 , 13623 - 13649, 13665 - 13694, 13754 - 13791 , 13801 - 13835, 13829 - 13847, 13837 - 13874, 13868 - 13929, 13931 - 13954, 13955 - 13970, 13972 - 14004, 14008 - 14025, 14027 - 14052, 14053 - 14091 , 14106 - 14122, 14124 - 14155, 14207 - 14241 , 14243 - 14259, 14269 - 14283, 14272 - 14297, 14326 - 14345, 14350 - 14374, 14416 - 14430, 14460 - 14489, 1451 1 - 14532, 14533 - 14556, 14586 - 14602, 14604 - 14643, 14665 - 14683, 14693 - 14707, 14720 - 14740, 14742 - 14758, 14760 - 14788, 14865 - 14879, 14881 - 14904, 14906 - 14933, 14939 - 14962, 14964 - 14986, 14998 - 15024, 15052 - 15066, 15068 - 15105, 15120 - 15194, 15189 - 15203, 15192 - 15225, 15251 - 15271 , 15274 - 1531 1 , 15321 - 15337, 15346 - 1541 1 , 15419 - 15463, 15503 - 15533, 15535 - 15550, 15558 - 15584, 15630 - 15651 , 15642 - 15659, 15653 - 15742, 15766 - 15797, 15813 - 15833, 15845 - 15862, 15885 - 15905, 15896 - 15925, 15942 - 15979, 15992 - 16015, 16020 - 16034, 16036 - 16058, 16085 - 16106, 16108 - 16123, 16125 - 16163, 16184 - 16203, 16218 - 16244, 16261 - 16300, 16302 - 16326, 16368 - 16385, 16387 - 16401 , 16431 - 16447, 16449 - 16471 , 16476 - 16499, 16509 - 16525, 16534 - 16553, 16562 - 16584, 16586 - 16613, 16615 - 16641 , 16668 - 16682, 16672 - 16687, 16677 - 16750, 16747 - 16808, 16810 - 16866, 16864 - 16878, 16886 - 16900, 17369 - 17386, 17388 - 17405, 17413 - 17435, 17469 - 17491 , 17493 - 17523, 17559 - 17578, 17582 - 17655, 17657 - 17671 , 17662 - 17702, 17704 - 17724, 17726 - 17753, 17750 - 17769, 17771 - 17810, 1781 1 - 17828, 17836 - 17874, 17876 - 17890, 17918 - 17947, 17949 - 17994, 17998 - 18035, 18037 - 18061 , 18072 - 18089, 18097 - 18126, 18128 - 18162, 18164 - 18191 , 18210 - 18270, 18272 - 18314, 18316 - 18344, 18346 - 18360, 18362 - 18378, 18391 - 18405, 18407 - 18443, 18448 - 18484, 18477 - 18552, 18568 - 18587, 18590 - 18646, 18660 - 18680, 18682 - 18747, 18749 - 18776, 18778 - 18837, 18848 - 18868, 18876 - 18896, 18910 - 18930, 18941 - 18964, 19001 - 19023, 19070 - 19147, 19149 - 19183, 19185 - 19232, 19238 - 19325, 19331 - 19348, 19351 - 19368, 19376 - 19400, 19428 - 19479, 19518 - 19543, 19545 - 19571 , 19581 - 19612, 19656 - 19677, 19679 - 19701 , 19703 - 19721 , 19723 - 19737, 19787 - 19823, 19838 - 19863, 19883 - 19898, 19900 - 19980, 19982 - 20022, 20024 - 20043, 20087 - 20106, 20108 - 20164, 20172 - 20190, 20192 - 20209, 20219 - 20235, 20238 - 20255, 20264 - 20279, 20298 - 20319, 20353 - 20383, 20379 - 20394, 20401 - 20439, 20441 - 20463, 20518 - 20536, 20577 - 20594, 20596 - 20610, 20622 - 20677, 20694 - 2071 1 , 20732 - 20802, 20804 - 20823, 20837 - 20851 , 20857 - 20910, 20912 - 20929, 20942 - 20984, 20987 - 21010, 21012 - 21035, 21057 - 21074, 21076 - 21 102, 21 104 - 21 149, 21157 - 21171 , 21190 - 21215, 21222 - 21237, 21258 - 21277, 21285 - 2131 1 , 21313 - 21342, 21376 - 21393, 21395 - 21409, 2141 1 - 21457, 21481 - 21514, 21557 - 21574, 21576 - 21622, 21652 - 21679, 21692 - 21716, 21735 - 21789, 21808 - 21838, 21840 - 21881 , 21884 - 2191 1 , 21901 - 21915, 21915 - 21962, 21964 - 21985, 21987 - 22003, 22035 - 22063, 22065 - 22082, 22123 - 22138, 22143 - 22157, 22161 - 22217, 22226 - 22241 , 22257 - 22282, 22284 - 22305, 22327 - 22343, 22345 - 22362, 22364 - 22403, 22405 - 22432, 22449 - 22473, 22485 - 22500, 22502 - 22526, 22528 - 22556, 22567 - 22602, 22604 - 22619, 22621 - 22640, 22675 - 22700, 22697 - 22713, 22702 - 22717, 22718 - 22735, 22727 - 22776, 22783 - 22802, 22821 - 22850, 22840 - 22864, 22874 - 22889, 22893 - 22918, 22920 - 22934, 22938 - 22952, 22954 - 22983, 22993 - 23040, 23042 - 23092, 23128 - 23143, 23149 - 23199, 23201 - 23229, 23250 - 23279, 23281 - 23299, 23327 - 23342, 23349 - 23372, 23405 - 23425, 23439 - 23461 , 23479 - 23512, 23531 - 23547, 23567 - 23662, 23690 - 23724, 23789 - 23818, 23830 - 23878, 23889 - 23907, 23914 - 23928, 23945 - 23964, 23966 - 23990, 24014 - 24035, 24101 - 24130, 24174 - 2421 1 , 24213 - 24232, 24252 - 24303, 24302 - 24323, 24336 - 24363, 24382 - 24404, 24412 - 24434, 24571 - 24605, 24607 - 24639, 24641 - 24659, 24660 - 24705, 24707 - 24731 , 24755 - 24770, 24764 - 24783, 24782 - 24824, 24821 - 24835, 24857 - 24912, 24906 - 24923, 24924 - 24979, 25015 - 25032, 25036 - 25079, 25080 - 25157, 25184 - 25202, 25206 - 25233, 25300 - 25336, 25338 - 25360, 25362 - 25412, 25420 - 25447, 25467 - 25481 , 25493 - 2551 1 , 25513 - 25547, 25560 - 25591 , 25593 - 25633, 25635 - 25651 , 25675 - 25689, 25688 - 25734, 25751 - 25775, 25780 - 25847, 25849 - 25878, 25880 - 25896, 25898 - 25914, 25916 - 25945, 25954 - 25972, 25997 - 26018, 26019 - 26039, 26041 - 26076, 26072 - 26089, 26100 - 26115, 26104 - 26119, 26121 - 26137, 26152 - 26194, 26183 - 26201 , 26203 - 26243, 26354 - 26372, 26398 - 26433, 26453 - 26484, 26506 - 26536, 26555 - 26602, 26604 - 26620, 26622 - 26641 , 26656 - 26673, 26675 - 26690, 26712 - 26753, 26837 - 26857, 26870 - 26885, 26887 - 26902, 26921 - 26954, 26956 - 26977, 32089 - 32104, 33156 - 33183, 33185 - 33202, 33204 - 33225, 33261 - 33290, 33292 - 33322, 33380 - 33408, 33419 - 33438, 33454 - 33515, 33517 - 33537, 33539 - 33555, 33564 - 33581 , 33583 - 33631 , 33645 - 33758, 33812 - 33882, 33900 - 33969, 33985 - 34000, 34036 - 34067, 34069 - 34108, 34119 - 34160, 34162 - 34199, 34218 - 34233, 34235 - 34276, 34284 - 34333, 34381 - 34396, 34409 - 34438, 34440 - 34466, 34471 - 34529, 34531 - 34569, 34613 - 34672, 34666 - 34684, 34687 - 34710, 34701 - 34742, 34732 - 34748, 34751 - 34782, 34779 - 34807, 34797 - 34812, 34829 - 34846, 34864 - 34899, 34912 - 34927, 34939 - 34957, 34964 - 34991 , 35013 - 35045, 35047 - 35068, 35070 - 35136, 35174 - 35188, 35256 - 35279, 35284 - 35393, 35404 - 35422, 35434 - 35448, 35441 - 35456, 35458 - 35472, 35471 - 35492, 35494 - 35512, 35514 - 35548, 35550 - 35613, 35649 - 35697, 35722 - 35794, 35810 - 35859, 35866 - 35890, 35892 - 35927, 35941 - 36007, 36010 - 36049, 36068 - 36095, 36093 - 36110, 36112 - 36159, 36165 - 36182, 36184 - 36205, 36207 - 36252, 36248 - 36270, 36260 - 36277, 36342 - 36358, 36362 - 36377, 36383 - 36405, 36414 - 36436, 36439 - 36481 , 36485 - 36500, 36503 - 36551 , 36561 - 36584, 36586 - 36628, 36642 - 36661 , 36651 - 36681 , 36689 - 36708, 36710 - 36732, 36782 - 36799, 36813 - 36832, 36834 - 36849, 36876 - 36894, 36896 - 36913, 36915 - 36976, 36984 - 36999, 37022 - 37041 , 37043 - 37089, 371 12 - 37126, 37128 - 37144, 37146 - 37165, 39975 - 39989, 43270 - 43324, 43348 - 43373, 43375 - 43394, 43410 - 43425, 43442 - 43463, 43523 - 43554, 43562 - 43585, 43587 - 43603, 43605 - 43626, 43628 - 43642, 43661 - 43684, 43707 - 43754, 43772 - 43791 , 43796 - 43814, 43816 - 43836, 43855 - 43872, 43918 - 43958, 43964 - 43980, 43982 - 4401 1 , 44031 - 44070, 44069 - 44093, 44094 - 44116, 44113 - 44127, 44118 - 44160, 44150 - 44176, 44181 - 44195, 44197 - 44220, 44222 - 44237, 44239 - 44294, 44285 - 44300, 44299 - 44359, 44360 - 44384, 44415 - 44447, 44447 - 44461 , 44475 - 44490, 44500 - 44516, 44515 - 44532, 44534 - 44567, 44569 - 44600, 44602 - 44631 , 44633 - 44653, 44669 - 44692, 44706 - 44721 , 44723 - 44739, 44741 - 44758, 44760 - 44777, 44797 - 44827, 44836 - 44893, 44900 - 44916, 44927 - 44956, 44958 - 44973, 44975 - 44995, 45009 - 45027, 45046 - 45077, 45088 - 45128, 45139 - 45183, 45185 - 45207, 45209 - 45254, 45263 - 45283, 45285 - 45303, 45302 - 45344, 45346 - 45410, 45412 - 45426, 45450 - 45478, 45480 - 45513, 45515 - 45571 , 45584 - 45621 , 45649 - 45664, 45677 - 45693, 45700 - 45739, 45741 - 45760, 45772 - 45793, 45795 - 45830, 45895 - 45915, 45917 - 45935, 45972 - 45986, 46003 - 46038, 46041 - 46084, 46101 - 461 16, 46133 - 46154, 46157 - 46222, 46230 - 46251 , 46267 - 46298, 46300 - 46320, 46322 - 46355, 46377 - 46410, 46412 - 46440, 46452 - 46476, 46532 - 46565, 46571 - 46594, 46596 - 46621 , 46641 - 46668, 46672 - 46686, 46694 - 46708, 46710 - 46742, 46758 - 46785, 46787 - 46836, 46843 - 46887, 46893 - 46910, 46912 - 46933, 46935 - 46951 , 46953 - 46967, 46977 - 4701 1 , 47013 - 47029, 47035 - 47075, 47077 - 47092, 47094 - 47118, 47120 - 47139, 47141 - 47161 , 47163 - 47190, 47207 - 47222, 47224 - 47251 , 47253 - 47268, 47270 - 47427, 47436 - 47451 , 47477 - 47518, 47527 - 47541 , 47543 - 47599, 47608 - 47626, 47648 - 47694, 47707 - 47723, 47731 - 47769, 47796 - 47819, 47849 - 47864, 47866 - 47880, 47931 - 47953, 47967 - 47988, 48017 - 48035, 48066 - 48081 , 48092 - 48107, 481 13 - 48136, 48126 - 48162, 48164 - 48187, 48189 - 48217, 48214 - 48248, 48250 - 48269, 48267 - 48281 , 48271 - 48289, 48278 - 48315, 48305 - 48321 , 48314 - 48337, 48339 - 48361 , 48353 - 48369, 48372 - 48392, 48381 - 48397, 48393 - 48408, 48401 - 48425, 48465 - 48490, 48481 - 48497, 48512 - 48527, 48527 - 48542, 48546 - 48560, 48550 - 48607, 48597 - 48617, 48615 - 48639, 48641 - 48668, 48673 - 48707, 48716 - 48734, 48758 - 48795, 48828 - 48883, 48905 - 48930, 48936 - 48957, 48983 - 48997, 48995 - 49030, 49042 - 49067, 49083 - 49098, 49100 - 49150, 49166 - 49192, 49222 - 49320, 49322 - 49355, 49357 - 49376, 49395 - 49420, 49443 - 49461 , 49472 - 49536, 49544 - 49597, 49599 - 49631 , 49633 - 49652, 49654 - 49671 , 49673 - 49699, 49701 - 49723, 49725 - 49785, 49787 - 49809, 49826 - 49843, 49853 - 49878, 49892 - 49907, 49929 - 49962, 49981 - 50001 , 50003 - 50036, 50038 - 50069, 50071 - 50126, 50135 - 50153, 50155 - 50173, 50182 - 50240, 50242 - 50270, 50327 - 50351 , 50353 - 50386, 50394 - 50413, 50430 - 50445, 50435 - 50449, 50447 - 50484, 50498 - 50515, 50529 - 50552, 50554 - 50626, 50628 - 50642, 50651 - 50665, 50670 - 50700, 50706 - 50745, 50747 - 50786, 50788 - 50816, 50818 - 50833, 50854 - 50883, 50898 - 50917, 50932 - 50946, 50948 - 50962, 50970 - 50984, 50978 - 51004, 51016 - 51054, 51053 - 51088, 51091 - 51 123, 51 128 - 51 173, 51238 - 51254, 51256 - 51282, 51284 - 51309, 5131 1 - 51331 , 51346 - 51384, 51387 - 51408, 51414 - 51434, 51474 - 51490, 51493 - 51507, 51546 - 51568, 51579 - 51594, 51596 - 51616, 51618 - 51644, 51662 - 51706, 51715 - 51731 , 51739 - 51762, 51773 - 51812, 51814 - 51856, 51858 - 51873, 51898 - 51931 , 51933 - 51948, 51950 - 51976, 51988 - 52014, 52016 - 52048, 52050 - 52091 , 52093 - 52115, 52136 - 52158, 52164 - 52187, 52191 - 52242, 52276 - 52304, 52314 - 52328, 52339 - 52358, 52368 - 52384, 52396 - 52420, 52429 - 52484, 52487 - 52503, 52508 - 52567, 52574 - 52594, 52616 - 52637, 52639 - 52666, 52700 - 52716, 52718 - 52737, 52741 - 52797, 52799 - 52817, 52823 - 52837, 52834 - 52851 , 52856 - 52881 , 52883 - 52904, 52918 - 52934, 52936 - 52957, 52956 - 52972, 52974 - 52996, 52999 - 53013, 53032 - 53050, 53066 - 53094, 53099 - 53133, 53136 - 53182, 53191 - 53206, 53233 - 53247, 53256 - 53273, 53285 - 53337, 53339 - 53390, 53402 - 53419, 53421 - 53435, 53476 - 53508, 53510 - 53529, 53531 - 53569, 53588 - 53609, 53606 - 53638, 53640 - 53659, 53662 - 53676, 53678 - 53696, 53727 - 53761 , 53763 - 53781 , 53775 - 53789, 53791 - 53819, 53833 - 53847, 53849 - 53876, 53895 - 53924, 53926 - 53942, 53944 - 53969, 53971 - 54024, 54034 - 54055, 54049 - 54086, 54090 - 54112, 54114 - 54134, 54136 - 54161 , 54175 - 54210, 54226 - 54270, 54275 - 54292, 54321 - 54343, 54383 - 54410, 54424 - 54441 , 54458 - 54505, 54495 - 54515, 54526 - 54545, 54547 - 54584, 54589 - 54609, 54616 - 54643, 54642 - 54667, 54662 - 54707, 54697 - 5471 1 , 54705 - 54743, 54745 - 54759, 54761 - 54800, 54826 - 54861 , 54886 - 54983, 54985 - 55012, 55014 - 55040, 55047 - 55102, 55104 - 55122, 55128 - 55180, 55189 - 55203, 55205 - 55236, 55289 - 55305, 55307 - 55336, 55338 - 55379, 55383 - 55424, 55426 - 55456, 55459 - 55484, 55486 - 55520, 55569 - 55586, 55588 - 55617, 55623 - 55643, 55649 - 55663, 55665 - 55706, 55708 - 55725, 55748 - 55775, 55777 - 55797, 55807 - 55830, 55851 - 55874, 55884 - 55909, 55924 - 55971, 55986 - 56011 , 56013 - 56066, 56081 - 56273, 56275 - 56323, 56323 - 56348, 56353 - 56392, 56394 - 56413, 56416 - 56439, 56441 - 56470, 56485 - 56504, 56511 - 56546, 56548 - 56577, 56593 - 56632, 56634 - 56698, 56706 - 56723, 56725 - 56754, 56791 - 56834, 56836 - 56865, 56880 - 56930, 56943 - 56979, 56981 - 57000, 57005 - 57021, 57036 - 57053, 57063 - 57122, 57137 - 57201, 57296 - 57321, 57367 - 57392, 57401 - 57426, 57428 - 57457, 57459 - 57480, 57498 - 57568, 57570 - 57593, 57595 - 57616, 57632 - 57654, 57661 - 57686, 57724 - 57738, 57743 - 57813, 57817 - 57834, 57836 - 57884, 57979 -58014, 58016-58035, 58055-58070, 58077-58110, 58112-58142, 58174- 58220, 58222 - 58236, 58238 - 58294, 58296 - 58318, 58320 - 58335, 58347 - 58374, 58385 - 58409, 58406 - 58466, 58479 - 58557, 58564 - 58582, 58583 - 58619, 58623 - 58683, 58701 - 58744, 58762 - 58794, 58796 - 58822, 58824 - 58855, 58889 - 58907, 58918 -58990, 59011 -59061, 59120-59167, 59203-59223, 59225-59242, 59263- 59283, 59297 - 59327, 59332 - 59361 , 59364 - 59421 , 59423 - 59441 , 59443 - 59458, 59460 - 59486, 59500 - 59535, 59549 - 59580, 59586 - 59612, 59618 - 59649, 59651 - 59672, 59708 - 59723, 59725 - 59749, 59751 - 59770, 59780 - 59805, 59807 - 59829, 59831 - 59846, 59848 - 59998, 60002 - 60028, 60032 - 60050, 60067 - 60082, 60084 - 60103, 60105 - 60122, 60135 - 60156, 60172 - 60208, 60218 - 60293, 60292 - 60309, 60309 - 60330, 60374 - 60388, 60385 - 60433, 60423 - 60441, 60436 - 60455, 60498 - 60518, 60518 - 60541 , 60560 - 60589, 60609 - 60674, 60664 - 60680, 60682 - 60698, 60717 - 60742, 60757 - 60796, 60798 - 60817, 60825 - 60856, 60914 - 60957, 60972 - 61012, 61014 -61031, 61050-61077, 61071 -61087, 61089-61103, 61101 -61119, 61120 -61154, 61149-61171, 61180-61199, 61190-61212, 61213-61238, 61235- 61257, 61247 - 61275, 61285 - 61312, 61315 - 61332, 61322 - 61337, 61332 - 61354, 61350 - 61378, 61381 - 61400, 61415 - 61445, 61453 - 61482, 61484 - 61540, 61551 - 61575, 61579 - 61642, 61644 - 61658, 61660 - 61684, 61686 - 61704, 61720 - 61743, 61745-61806, 61839-61900, 61902-61928, 61930-61963, 61965-61981, 61983- 62011, 62025-62045, 62047-62073, 62093-62117, 62132-62148, 62176-62199, 62228 - 62251, 62247 - 62277, 62307 - 62324, 62319 - 62336, 62338 - 62361, 62363 - 62409, 62411 - 62437, 62439 - 62458, 62474 - 62492, 62500 - 62562, 62565 - 62604, 62613 - 62656, 62658 - 62687, 62683 - 62697, 62702 - 62751, 62740 - 62793, 62796 - 62816, 62806 - 62823, 62826 - 62851, 62846 - 62863, 62861 - 62893, 62900 - 62927, 62930 - 62955, 63008 - 63042, 63044 - 63078, 63080 - 63098, 63100 - 63119, 63121 - 63138, 63139 - 63153, 63165 - 63179, 63181 - 63195, 63197 - 63228, 63230 - 63244, 63246 - 63309, 63315 - 63359, 63361 - 63399, 63426 - 63457, 63474 - 63501 , 63503 - 63526, 63528 - 63576, 63577 - 63596, 63598 - 63638, 63653 - 63669, 63689 - 63707, 63709 - 63738, 63754 - 63793, 63807 - 63835, 63837 - 63854, 63856 - 63873, 63876 - 63979, 63997 - 64020, 64016 - 64033, 64035 - 64058, 64060 - 64075, 64090 - 64145, 64186 - 64207, 64223 - 64240, 64242 - 64260, 64280 - 64299, 64325 - 64342, 64359 - 64396, 64407 - 64422, 64412 - 64426, 64424 - 64444, 64467 - 64484, 64477 - 6451 1 , 64517 - 64532, 64525 - 64590, 64600 - 64623, 64625 - 64646, 64662 - 64709, 64720 - 64782, 64796 - 64821 , 64823 - 64844, 64846 - 64860, 64862 - 64890, 64917 - 64947, 64949 - 64963, 64979 - 65001 , 65029 - 65053, 65059 - 65083, 65086 - 65125, 65156 - 65171 , 65173 - 65200, 65202 - 65216, 65223 - 65258, 65279 - 65294, 65302 - 65370, 65372 - 65441 , 65464 - 65486, 65493 - 65516, 65527 - 65580, 65582 - 65596, 65599 - 65620, 65688 - 65718, 65720 - 65747, 65765 - 65787, 65815 - 65839, 65841 - 65865, 65889 - 65910, 65912 - 65954, 65968 - 66002, 66012 - 66060, 66062 - 66084, 66086 - 66142, 66144 - 66158, 66207 - 66226, 66228 - 66243, 66259 - 66310, 66312 - 66329, 66332 - 66355, 66372 - 66396, 66417 - 66437, 66476 - 6651 1 , 66549 - 66588, 66591 - 66629, 66633 - 66653, 66655 - 66669, 66671 - 66690, 66725 - 66758, 66760 - 66779, 66781 - 66797, 66830 - 66890, 66893 - 66907, 66925 - 66944, 66955 - 66971 , 66973 - 66998, 67027 - 67042, 67044 - 67067, 67096 - 67148, 67149 - 67180, 67182 - 67201 , 67203 - 67220, 67245 - 67274, 67282 - 67297, 67310 - 67337, 67339 - 67368, 67381 - 67399, 67401 - 67425, 67427 - 67463, 67465 - 67486, 67498 - 67573, 67590 - 67613, 67616 - 67638, 67627 - 67642, 67641 - 67663, 67672 - 67785, 67792 - 67840, 67853 - 67909, 67919 - 67936, 67977 - 68005, 68007 - 68021 , 68023 - 68038, 68059 - 68077, 68101 - 68224, 68238 - 68259, 68275 - 68320, 68350 - 68365, 68379 - 68410, 68414 - 68439, 68441 - 68461 , 68473 - 68488, 68490 - 68529, 68531 - 68573, 68590 - 68614, 68616 - 68644, 68673 - 68703, 68704 - 68738, 68775 - 68791 , 68793 - 68820, 68845 - 68890, 68912 - 68975, 68983 - 69024, 69036 - 69058, 69057 - 69072, 69074 - 69089, 69091 - 69129, 69131 - 69155, 69174 - 69189, 6921 1 - 69227, 69229 - 69288, 69315 - 69360, 69362 - 69438, 69464 - 69485, 69494 - 69529, 69541 - 69566, 69568 - 69616, 69618 - 69655, 69688 - 69706, 69714 - 69735, 69738 - 69778, 69780 - 69808, 69832 - 69862, 69861 - 69877, 69876 - 69890, 69992 - 70029, 70021 - 70038, 70080 - 70099, 70106 - 70128, 70130 - 70156, 70176 - 70190, 70196 - 70223, 70225 - 70260, 70260 - 70319, 70321 - 70407, 70433 - 70454, 70493 - 70519, 70522 - 70546, 70557 - 70614, 70616 - 70650, 70652 - 70667, 70669 - 70685, 70681 - 70734, 70750 - 70765, 70767 - 70796, 70802 - 70816, 70830 - 70862, 70867 - 70900, 70909 - 70940, 70942 - 70975, 70975 - 71043, 71058 - 71088, 71094 - 71 115, 71 145 - 71 163, 71 180 - 71200, 71221 - 71237, 71244 - 71264, 71266 - 71287, 71289 - 71325, 71357 - 71391 , 71443 - 71461 , 71482 - 71501 , 71532 - 71559, 71572 - 71624, 71626 - 71667, 71680 - 71694, 71726 - 71766, 71774 - 71808, 71858 - 71872, 71867 - 71881 , 71888 - 71920, 71943 - 71961 , 71965 - 71982, 71984 - 72003, 72008 - 72024, 72048 - 72066, 72116 - 72136, 72138 - 72182, 72190 - 72230, 72287 - 72312, 72325 - 72340, 72385 - 72433, 72435 - 72450, 72456 - 72489, 72499 - 72525, 72541 - 72577, 72609 - 72652, 72652 - 72667, 72683 - 72698, 72708 - 72739, 72768 - 72785, 72788 - 72820, 72822 - 72838, 72840 - 72872, 72895 - 72909, 72921 - 72936, 72940 - 72985, 73067 - 73081 , 73100 - 73120, 73132 - 73164, 73169 - 73201 , 73199 - 73213, 73203 - 73238, 73267 - 73283, 73298 - 73328, 73317 - 73354, 73356 - 73377, 73400 - 73428, 73430 - 73445, 73447 - 73462, 73471 - 73485, 73504 - 73605, 73633 - 73680, 73680 - 73706, 73755 - 73785, 73813 - 73830, 73832 - 73857, 73891 - 73917, 73919 - 73940, 73948 - 73976, 73984 - 74008, 74010 - 74036, 74038 - 74060, 74070 - 74098, 74100 - 74116, 74118 - 74158, 74209 - 74239, 74260 - 74290, 74292 - 74343, 74355 - 74372, 74453 - 74502, 74504 - 74532, 74545 - 74577, 74589 - 74606, 74603 - 74631 , 74624 - 74645, 74634 - 74648, 74650 - 74670, 74672 - 74701 , 74718 - 74756, 74759 - 74774, 74764 - 74810, 74813 - 74843, 74833 - 74850, 74865 - 74888, 74914 - 74963, 74974 - 74992, 74994 - 75015, 7501 1 - 75030, 75028 - 75072, 75079 - 75100, 75108 - 75122, 7511 1 - 75137, 75128 - 75142, 75143 - 75157, 75148 - 75162, 75152 - 75169, 75171 - 75217, 75206 - 75240, 75242 - 75282, 75296 - 75317, 75332 - 75347, 75337 - 75357, 75364 - 75389, 75403 - 75439, 75470 - 75497, 75499 - 75525, 75565 - 75624, 75627 - 75651 , 75653 - 75672, 75704 - 75729, 75731 - 75759, 75760 - 75775, 75777 - 75793, 75795 - 75827, 75829 - 75844, 75848 - 75874, 75898 - 75916, 75915 - 75937, 75940 - 75968, 76002 - 76016, 76018 - 76062, 76073 - 76097, 76117 - 76148, 76150 - 76167, 76169 - 76191 , 76193 - 76207, 76242 - 76256, 76253 - 76273, 76275 - 76347, 76349 - 76367, 76390 - 76404, 76393 - 76417, 76419 - 76433, 76435 - 76470, 76489 - 76505, 76515 - 76530, 76532 - 76597, 76599 - 76646, 76663 - 76681 , 76701 - 76716, 76749 - 76786, 76797 - 7681 1 , 76864 - 76885, 76897 - 76916, 76918 - 76965, 76994 - 77019, 77021 - 77070, 77072 - 77147, 77161 - 77208, 77212 - 77227, 77250 - 77274, 77293 - 77331 , 77333 - 77359, 77361 - 77377, 77384 - 77459, 77465 - 77520, 77522 - 77537, 77553 - 77578, 77580 - 77609, 77672 - 77690, 77692 - 77727, 77759 - 77804, 77812 - 77843, 77861 - 77877, 77913 - 77936, 77951 - 77980, 77994 - 78025, 78027 - 78073, 78083 - 78115, 78132 - 78148, 78172 - 78196, 78198 - 78221 , 78228 - 78245, 78247 - 78263, 78265 - 78285, 78287 - 78331 , 78367 - 78391 , 78399 - 78428, 78430 - 78459, 78478 - 78500, 78510 - 78534, 78550 - 78570, 78572 - 78588, 78598 - 78630, 78658 - 78694, 78696 - 78723, 78734 - 78768, 78770 - 78791 , 78799 - 78813, 78815 - 78856, 78858 - 78883, 78897 - 78941 , 78943 - 78980, 78982 - 79002, 78998 - 79012, 79020 - 79046, 79086 - 79112, 79117 - 79147, 79149 - 79169, 79171 - 79191 , 79193 - 79231 , 79259 - 79274, 79347 - 79361 , 79391 - 79411 , 79427 - 79462, 79464 - 79492, 79494 - 79520, 79540 - 79566, 79586 - 79609, 79615 - 79637, 79665 - 79682, 79684 - 79700, 79701 - 79717, 79719 - 79786, 79792 - 79825, 79827 - 79848, 79852 - 79880, 79882 - 79944, 79958 - 79975, 80000 - 80051 , 80053 - 80067, 80074 - 80091 , 80093 - 80124, 80137 - 80162, 80187 - 80208, 80218 - 80246, 80276 - 80301 , 80322 - 80340, 80352 - 80366, 80419 - 80433, 80439 - 80453, 80458 - 80476, 80499 - 80518, 80520 - 80548, 80550 - 80578, 80616 - 80635, 80637 - 80674, 80687 - 80714, 80716 - 80736, 80742 - 80763, 80765 - 80817, 80828 - 80879, 80958 - 80979, 81029 - 81054, 81 144 - 81 173, 81 175 - 81203, 81207 - 81224, 81245 - 81264, 81270 - 81296, 81298 - 81322, 81324 - 81359, 81374 - 81389, 81391 - 81440, 81442 - 81461 , 81481 - 81498, 81500 - 81523, 81548 - 81567, 81590 - 81639, 81633 - 81668, 81676 - 81736, 81735 - 81793, 81828 - 81862, 81864 - 81886, 81888 - 81905, 81907 - 81949, 81984 - 82044, 82058 - 82072, 82074 - 82101 , 82119 - 82175, 82177 - 82192, 82204 - 82222, 82235 - 82252, 82302 - 82323, 82327 - 82358, 82377 - 82400, 82432 - 82446, 82460 - 82476, 82477 - 82492, 82493 - 82570, 82572 - 82640, 82650 - 82664, 82676 - 82691 , 82685 - 82736, 82727 - 82741 , 82735 - 82749, 82747 - 82789, 82783 - 82858, 82861 - 82898, 82900 - 82921 , 82938 - 82955, 82965 - 82988, 83004 - 83047, 83070 - 83097, 83092 - 83171 , 83173 - 83275, 83291 - 83314, 83340 - 83366, 83368 - 83383, 83427 - 83483, 83501 - 83545, 83571 - 83609, 8361 1 - 83626, 83634 - 83684, 83687 - 83707, 83709 - 83733, 83735 - 83759, 83851 - 83903, 83917 - 83933, 83967 - 83983, 83986 - 84025, 84047 - 84079, 84090 - 84229, 85639 - 85654, 85643 - 85674, 85809 - 85824, 85873 - 85892, 85882 - 85899, 86600 - 86622, 86643 - 86665, 86671 - 86686, 86710 - 86735, 86758 - 86775, 86802 - 86819, 87013 - 87052, 87060 - 87094, 87107 - 87121 , 87123 - 87150, 88108 - 88253, 88273 - 88291 , 88284 - 88316, 88310 - 88324, 88326 - 88358, 88358 - 88394, 88396 - 88415, 88424 - 88438, 89475 - 89493, 89484 - 8951 1 , 89502 - 89519, 89518 - 89548, 89548 - 89572, 89567 - 89587, 89589 - 89605, 89616 - 89633, 89693 - 89714, 89704 - 89719, 90241 - 90261 , 90261 - 90275, 90291 - 9031 1 , 90313 - 90330, 90343 - 90364, 90401 - 90415, 90419 - 90436, 90425 - 90446, 90457 - 90471 , 90461 - 90475, 90480 - 90500, 90504 - 90541 , 90544 - 90558, 90552 - 90573, 90563 - 90593, 90602 - 90624, 90634 - 90649, 90651 - 90668, 90712 - 90732, 90721 - 90741 , 90730 - 90749, 90760 - 90783, 90785 - 90806, 90816 - 90835, 90824 - 90849, 93450 - 93475, 93502 - 93516, 93580 - 93599, 93603 - 93621, 93630 - 93655, 93645 - 93661 , 93666 - 93686 , 93691 - 93705, 93714 - 93740, 93960 - 93974, 94410 - 94428, 94480 - 94495, 94555 - 94569, 94561 - 94581, 94586 - 94601, 94620 - 94640, 96242 - 96256, 96246 - 96262 , 96270 - 96286, 96292 - 96338, 96355 - 96376, 96379 - 96396, 96387 - 96408, 96398 - 96435, 96440 - 96455, 96448 - 96469, 96736 - 96750, 96766 - 96796, 96797 - 96829 , 96831 - 96850, 96839 - 96882, 96892 - 96912, 96927 - 96941, 96933 - 96953, 96943 - 96961, 96964 - 96980, 96983 - 97006, 97147 - 97190, 97185 - 97199, 97204 - 97231 , 97242 - 97257, 97265 - 97302, 97304 - 97326, 97332 - 97351, 97352 - 97388, 97390 - 97417, 97407 - 97438, 97440 - 97461, 98217 - 98258, 98259 - 98277, 98279 - 98295 , 98310 - 98348, 98341 - 98356, 98345 - 98366, 98360 - 98377, 98383 - 98401, 98403 - 98418, 98425 - 98442, 98445 - 98464, 98470 -98494, 98483-98503, 99116-99186 , 99188 - 99213, 99208 - 99223, 99215 - 99268, 99259 - 99276, 99283 - 99312, 99302 - 99345, 99347 - 99373, 99367 - 99422, 100868 - 100882, 102411 - 102425, 102783 - 102797, 103436 - 103450, 104811 - 104825, 104853 - 104872, 104874 - 104891, 104919 - 104961, 104972 - 104986, 104975 - 104992, 104988 - 105007, 105001 - 105041, 105051 - 105065, 105068 - 105104, 105106 - 105124, 105126 - 105156, 105158 - 105188, 105192 - 105221, 105219 - 105261, 105276 - 105292, 105312 - 105330, 105362 - 105390, 105388 - 105404, 105649 - 105688, 105686 - 105700, 105690 - 105707, 105702 - 105723, 105737 - 105762, 105793 - 105812, 105817 - 105836, 105843 - 105877, 105909 - 105928, 106311 - 106331, 106324 - 106340, 106329 - 106363, 106385 - 106404, 106399 - 106437, 106464 - 106497, 106486 - 106506, 106509 - 106531, 106525 - 106540, 106533 - 106549, 106550 - 106569, 106580 - 106620, 107575 - 107606, 107609 - 107664, 107666 - 107683, 107690 - 107705, 107694 - 107708, 107699 - 107716, 107709 - 107733, 107769 - 107791, 107795 - 107823, 109491 - 109523, 109532 - 109557, 109576 - 109613, 109615 - 109637, 109639 - 109654, 109657 - 109671, 109667 - 109732, 109736 - 109763, 109764 - 109779, 109845 - 109859, 109853 - 109875, 109887 - 109901, 109901 - 109921, 109924 - 109940, 109929 - 109962, 109953 - 109972, 110010 - 110072, 110065- 110103, 110105- 110120, 110208 - 110222, 110237 - 110254, 110297 - 110313, 110358 - 110374, 110379 - 110398, 110418 - 110437, 110466 - 110490, 110518 - 110535, 110526 - 110545, 110548 - 110565, 110567 - 110581, 110583 - 110601, 110616 - 110630, 110639 - 110661, 110730 - 110745, 110780 - 110809, 110811 - 110840, 112148 - 112163, 112158 - 112172, 112178 - 112199, 112201 - 112249, 112240 - 112258, 112267 - 112294, 112299 - 112369, 112383 - 112443, 112632 - 112649, 112689 - 112721, 112726 - 112741, 112762 - 112789, 112790 - 112813, 112822 - 112855, 112846 - 112867, 112873 - 112889, 112916 - 112930, 112919 - 112950, 112969 - 112986, 113000 - 113027, 113027 - 113046, 113036 - 113050, 113062 - 113078, 113069 - 113089, 113091 - 113114, 113112 - 113126, 113116 - 113130, 113123 - 113139, 113134 - 113195, 113190 - 113215, 113211 - 113228, 113217 - 113247, and 113708- 113725.
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 27, selected from the group consisting of 253-267,1261 - 1275,2832-2851,6557 - 6579, 6617 - 6639, 8893 - 8912, 13803 - 13823, 14533 - 14553, 21090 - 21106, 21901 - 21916, 32671 - 32685, 34030 - 34052, 34441 - 34457, 35357 - 35375, 36893 - 36907,
37181 - 37195, 38864 - 38878, 44081 - 44099, 44135 - 44158, 44214 - 44228, 44286 - 44300, 47365 - 47380, 48066 - 48080, 48374 - 48389, 52564 - 52579, 54458 - 54472, 54622-54643, 56021 -56036, 56102-56121, 56126-56154, 56177-56193, 56195- 56209, 56269 - 56288, 56323 - 56345, 57083 - 57099, 58208 - 58223, 59433 - 59447, 59889 - 59904, 61087 - 61102, 61200 - 61218, 62252 - 62272, 64562 - 64576, 65246 - 65261, 67675-67690, 67704-67720, 67929-67944, 68147-68164, 68180-68216, 68926 - 68940, 69832 - 69861, 73258 - 73272, 74602 - 74616, 74608 - 74628, 75108 - 75122, 80744 - 80759, 81916 - 81931, 82194 - 82208, 82493 - 82509, 83240 - 83257, 83890 - 83904, 84137 - 84158, 84195 - 84214, 84210 - 84228, 86601 - 86620, 87104 - 87118, 88109 - 88133, 88352 - 88366, 89724 - 89740, 90544 - 90558, 92765 - 92779, 96843 - 96860, 97381 - 97397, 99118 - 99138, 99288 - 99310, 99810 - 99825, 100794 - 100809, 104792 - 104808, 104810 - 104824, 104975 - 104992, 107840 - 107864, 108098 - 108112, 109492 - 109507, and 112144- 112163.
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 SPTLC1 mRNA, such as the SPTLC1 pre-mRNA, e.g. SEQ ID NO 27, or SPTLC1 mature mRNA (e.g. SEQ ID NO 28, 29 or 30). The poly A tail of SPTLC1 mRNA is typically disregarded for antisense oligonucleotide targeting. Naturally occurring variant
The term“naturally occurring variant” refers to variants of SPTLC1 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 SPTLC1 gene is located at chromosome 9, 92031 134..921 15474, complement (NC_000009.12, Gene ID 10558).
In some embodiments, the naturally occurring variants have at least 95% such as at least 98% or at least 99% homology to a mammalian SPTLC1 target nucleic acid, such as a target nucleic acid selected form the group consisting of SEQ ID NO 27, 28, 29 or 30. In some embodiments the naturally occurring variants have at least 99% homology to the human SPTLC1 target nucleic acid of SEQ ID NO 27.
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 SPTLC1 protein or SPTLC1 mRNA when compared to the amount of SPTLC1 or SPTLC1 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 SPTLC1 , e.g. by degradation of SPTLC1 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 imgf000033_0001
2'-0-MOE 2'-0-AI!yl 2'-Oithylamine
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 imgf000034_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:
FI-8-G5-I6-F’I-8, such as
F1-8-G7-16-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, 11 , 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 a!., 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 Fluiter et 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
Fl-8-[D3-4-El- D 3-4]-F 1 1 -8
Figure imgf000037_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]i_5-[region G] -[LNA]I-5, 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 [MOE]i-e-[Region G]-[MOE] 1-8, such as [MOE]2-7-[Region G]s-i6-[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 F1-8-G5-16-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 D’I-3- 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.
Treatment
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 SPTLC1 expression.
The oligonucleotides of the invention targeting SPTLC1 are capable of hybridizing to and inhibiting the expression of a SPTLC1 target nucleic acid in a cell which is expressing the SPTLC1 target nucleic acid.
The SPTLC1 target nucleic acid may be a mammalian SPTLC1 mRNA or premRNA, such as a human SPTLC1 mRNA or premRNA, for example a premRNA or mRNA originating from the Homo sapiens serine palmitoyltransferase long chain base subunit 1 (SPTLC1 ), RefSeqGene on chromosome 9, exemplified by NCBI Reference Sequence NG_007950.1 or Ensembl ENSG00000090054 (SEQ ID NO 27). The human SPTLC1 pre-mRNA is encoded on Homo sapiens Chromosome 9, NC_000009.12 (92031 134..921 15474, complement). GENE ID = 10558 ( SPTLC1 ).
A mature human mRNA target sequence is illustrated herein by the cDNA sequences SEQ ID NO 28, 29 or 30.
The oligonucleotides of the invention are capable of inhibiting the expression of SPTLC1 target nucleic acid, such as the SPTLC1 mRNA, in a cell which is expressing the target nucleic acid, such as the SPTLC1 mRNA.
In some embodiments, the oligonucleotides of the invention are capable of inhibiting the expression of SPTLC1 target nucleic acid in a cell which is expressing the target nucleic acid, so to reduce the level of SPTLC1 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 SPTLC1 target nucleic acid (e.g. the mRNA) in the cell. Suitably the cell is selected from the group consisting of A549 cells, MDA-MB-231 cells, J774A.1 cells and MPC-1 1 cells. 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 27, 28, 29 or 30.
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 21.
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 22.
In some embodiments, the antisense 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 23.
In some embodiments, the antisense oligonucleotide of the invention comprises a contiguous nucleotides sequence of 12 - 16, such as 13, 14 or 15 contiguous nucleotides in length, wherein the contiguous nucleotide sequence is fully complementary to SEQ ID NO 24.
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 25.
In some embodiments, the antisense oligonucleotide of the invention comprises a contiguous nucleotides sequence of 12 - 17, such as 13, 14, 15 or 16 contiguous nucleotides in length, wherein the contiguous nucleotide sequence is fully complementary to SEQ ID NO 26.
In some embodiments, the antisense oligonucleotide of the invention comprises a contiguous nucleotides sequence of 12 - 22, such as 13, 14, 15, 16, 17, 18, 19, 20 or 21 contiguous nucleotides in length, wherein the contiguous nucleotide sequence is fully complementary to SEQ ID NO 31. 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 21.
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 22.
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 23.
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 24.
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 25. 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 26.
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 31.
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 21.
In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 22.
In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 23.
In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 24. In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 25.
In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 26.
In some embodiments, the contiguous nucleotide sequence is fully complementary to SEQ ID NO 31.
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 - 20.
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 3 or 13.
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 8 or 15. 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 17, 18 or 20.
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: TTtaagtgttcGACC (SEQ ID NO 1 ), TTGtgataaagcCGT (SEQ ID NO 2), CGCatatatcaaGTA (SEQ ID NO 3), CGtatgagaggCAAG (SEQ ID NO 4), GGATatacagtcgCG (SEQ ID NO 5), TAatgtcactaCGGG (SEQ ID NO 6), CCGAttactgattTA (SEQ ID NO 7), TCgataatgtaAGGC (SEQ ID NO 8), TTAgtagtcgcTCG (SEQ ID NO 9), GGctattgacgATTC (SEQ ID NO 10), ATGAggcgaagttTA (SEQ ID NO 1 1 ), ATCaacgcataCGC (SEQ ID NO 12), CCAtgttaaacgCAT (SEQ ID NO 13), ATCAgaaggtaggCG (SEQ ID NO 14), CGAtaatgtaagGCA (SEQ ID NO 15), GACaaattaccattTCA (SEQ ID NO 16), ACatttaataagcAGGA (SEQ ID NO 17),
TAataagcaggaaCACC (SEQ ID NO 18), GATgttgtagttGAG (SEQ ID NO 19) and
CATttaataagcaGGA (SEQ ID NO 20); 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: TTtaagtgttcGACC (SEQ ID NO 1 ), TTGtgataaagcCGT (SEQ ID NO 2), CGCatatatcaaGTA (SEQ ID NO 3), CGtatgagaggCAAG (SEQ ID NO 4), GGATatacagtcgCG (SEQ ID NO 5), TAatgtcactaCGGG (SEQ ID NO 6), CCGAttactgattTA (SEQ ID NO 7), TCgataatgtaAGGC (SEQ ID NO 8), TTAgtagtcgcTCG (SEQ ID NO 9), GGctattgacgATTC (SEQ ID NO 10), ATGAggcgaagttTA (SEQ ID NO 1 1 ), ATCaacgcataCGC (SEQ ID NO 12), CCAtgttaaacgCAT (SEQ ID NO 13), ATCAgaaggtaggCG (SEQ ID NO 14), CGAtaatgtaagGCA (SEQ ID NO 15), GACaaattaccattTCA (SEQ ID NO 16), ACatttaataagcAGGA (SEQ ID NO 17),
TAataagcaggaaCACC (SEQ ID NO 18), GATgttgtagttGAG (SEQ ID NO 19) and
CATttaataagcaGGA (SEQ ID NO 20); 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: TTtaagtgttcGACC (SEQ ID NO 1 ), TTGtgataaagcCGT (SEQ ID NO 2), CGCatatatcaaGTA (SEQ ID NO 3), CGtatgagaggCAAG (SEQ ID NO 4), GGATatacagtcgCG (SEQ ID NO 5), TAatgtcactaCGGG (SEQ ID NO 6), CCGAttactgattTA (SEQ ID NO 7), TCgataatgtaAGGC (SEQ ID NO 8), TTAgtagtcgcTCG (SEQ ID NO 9), GGctattgacgATTC (SEQ ID NO 10), ATGAggcgaagttTA (SEQ ID NO 1 1 ), ATCaacgcataCGC (SEQ ID NO 12), CCAtgttaaacgCAT (SEQ ID NO 13), ATCAgaaggtaggCG (SEQ ID NO 14), CGAtaatgtaagGCA (SEQ ID NO 15), GACaaattaccattTCA (SEQ ID NO 16), ACatttaataagcAGGA (SEQ ID NO 17),
TAataagcaggaaCACC (SEQ ID NO 18), GATgttgtagttGAG (SEQ ID NO 19) and
CATttaataagcaGGA (SEQ ID NO 20); 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 - 300mM 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 non- toxic 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 SPTLC1 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 SPTLC1 expression in a target cell which is expressing SPTLC1, 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 SPTLC1 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 SPTLC1 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 SPTLd. In some embodiments disease or disorder may be associated with a mutation in the SPTLd gene. Therefore, in some embodiments, the target nucleic acid is a mutated form of the SPTLd sequence.
The methods of the invention are preferably employed for treatment or prophylaxis against diseases caused by abnormal levels and/or activity of SPTLd.
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 SPTLd.
In one embodiment, the invention relates to oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions for use in the treatment of diseases or disorders selected from cancer, hepatitis C, diabetes (such as type 2 diabetes), opiate/opioid-antinociceptive tolerance, Niemann-Pick type C disease, HIV infection and
amphetamine/methamphetamine-induced toxicity.
Administration
The oligonucleotides or pharmaceutical compositions of the present invention may be administered topical or enteral or parenteral (such as, intravenous, subcutaneous, intra- muscular, 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
Examplel : Testing in vitro efficacy of antisense oligonucleotides targeting SPTLC1 mRNA in human A549 and MDA-MB-231 , as well as mouse J774A.1 cell lines at single concentration.
A549, MDA-MB-231 and J774A.1 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, 4500 cells/well of MDA-MB-231 and A549; 2500 cells/well of J774A.1 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 mM. 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: SPTLC1 Hs0027231 1_m1 (Mm00447343_m1 ) [FAM-MGB] and endogenous control GAPDH, Hs99999905_m1 (Mm99999915_g1 ) [VIC- MGB] All primer sets were purchased from Thermo Fisher Scientific. The relative SPTLC1 mRNA expression level in the table is shown as percent of control (PBS-treated cells). The SPTLC1 mRNA levels from cells treated with a selection of the compounds are shown in figures 1 to 3, evaluated human A549 and MDA-MB-231 cell lines as well as mouse J77A.1 cell lines. From the initial library screen three motifs on the SPTLC1 human transcript were identified which provided surprisingly effective and potent compounds in the cell lines tested: Motif A (SEQ ID NO 21 ), Motif B (SEQ ID NO 23) and Motif C (SEQ ID NO 25).
Oligonucleotides used:
Figure imgf000056_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, DNA cytosines preceded with a superscript m represents a 5-methyl C-DNA nucleoside. All internucleoside linkages are phosphorothioate internucleoside linkages.
Example 2: Testing in vitro potency and efficacy of selected oligonucleotides targeting SPTLC1 mRNA in human A549; MDA-MB-231 and mouse J774A.1 cells for a concentration response curve.
Human MDA-MB-231 , A549 and mouse J774A.1 cell line was described in Example 1. The assay was performed as described in Example 1. Concentration of oligonucleotides: from 50 mM, 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 X. n=2 biological replicates. IC50 determinations were performed in GraphPad Prism6. The relative SPTLC1 mRNA level at treatment with 50 mM oligonucleotide is shown in the table as percent of control (PBS).
Figure imgf000057_0001
Figure imgf000058_0001
The IC50 values for selected oligonucleotides targeting human SPTLC1 mRNA in vitro in the human cell lines MDA-MB-231 and A549 are shown in Figure 4. The concentration response curves in human cell lines A549 and MDA-MB-231 as well as in mouse cell line J77A.1 are provided as Figures 5, 6 and 7, 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 27, wherein the antisense
oligonucleotide is capable of inhibiting the expression of human SPTLC1 in a cell which is expressing human SPTLCV, or a pharmaceutically acceptable salt thereof.
2. The antisense oligonucleotide according to claim 1 , 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 21 , SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26 and SEQ ID NO 31.
3. The antisense oligonucleotide according to claim 1 , wherein the contiguous
nucleotide sequence is fully complementary to SEQ ID NO 21.
4. The antisense oligonucleotide according to claim 1 , wherein the contiguous
nucleotide sequence is fully complementary to SEQ ID NO 23 or SEQ ID NO 25.
5. The antisense oligonucleotide according to any one of claim 1 to 4, wherein the
contiguous nucleotide sequence is fully complementary to a region of SEQ ID NO 17, selected from the group consisting of 10 - 40, 66 - 85, 89 - 113, 131 - 151 , 153 - 190, 194 - 224, 226 - 255, 276 - 291 , 293 - 316, 346 - 394, 396 - 415, 466 - 523, 551 - 573, 589 - 614, 619 - 634, 638 - 656, 667 - 689, 691 - 730, 732 - 752, 763 - 777, 781 - 797, 798 - 822, 814 - 828, 831 - 874, 875 - 910, 908 - 923, 1105 - 1 119, 1132 - 1 147, 1139 - 1159, 1 149 - 1166, 1 192 - 1213, 1218 - 1235, 1241 - 1259, 1257 - 1276, 1306 - 1330, 1334 - 1351 , 1342 - 1379, 1381 - 1406, 1429 - 1462, 1465 - 1501 , 1519 - 1549, 1591 - 1611 , 1614 - 1639, 1641 - 1657, 1657 - 1672, 1674 -
1693, 1695 - 1710, 1726 - 1742, 1759 - 1818, 1872 - 1894, 1896 - 1925, 1930 -
1945, 1982 - 2001 , 2004 - 2019, 2025 - 2078, 2081 - 2126, 2131 - 2149, 2183 - 2208, 2207 - 2227, 2236 - 2281 , 2275 - 2289, 2283 - 2325, 2322 - 2405, 2418 -
2440, 2442 - 2486, 2490 - 2510, 2504 - 2519, 2508 - 2536, 2557 - 2574, 2582 -
2638, 2649 - 2698, 2715 - 2731 , 2737 - 2788, 2796 - 2860, 2862 - 2933, 2946 -
2961 , 2963 - 2981 , 2988 - 3002, 3017 - 3065, 3071 - 3134, 3135 - 3157, 3159 -
3189, 3241 - 3259, 3302 - 3329, 3381 - 3446, 3448 - 3477, 3487 - 3510, 3510 -
3528, 3532 - 3558, 3580 - 3594, 3609 - 3659, 3662 - 3684, 3722 - 3761 , 3763 -
3777, 3779 - 3793, 3795 - 3809, 3820 - 3837, 3863 - 3899, 3909 - 3923, 3940 -
3965, 3983 - 4033, 4035 - 4058, 4060 - 4075, 4086 - 4124, 4135 - 4151 , 4181 - 4216, 4219 - 4243, 4262 - 4276, 4278 - 4295, 4298 - 4331 , 4333 - 4350, 4352 - 4393, 4395 - 4410, 4420 - 4439, 4441 - 4457, 4459 - 4477, 4507 - 4526, 4528 - 4555, 4581 - 4601 , 4603 - 4618, 4640 - 4665, 4678 - 4696, 4698 - 4721 , 4732 - 4770, 4787 - 4872, 4877 - 4896, 4892 - 4947, 4965 - 5002, 5019 - 5049, 5051 - 5074, 5077 - 5099, 5112 - 5127, 5129 - 5144, 5147 - 5171 , 5170 - 5184, 5186 - 5298, 5300 - 5317, 5319 - 5341 , 5375 - 5421 , 5425 - 5462, 5464 - 5479, 5476 - 5497, 5489 - 5516, 5517 - 5535, 5558 - 5572, 5577 - 5591 , 5584 - 5635, 5637 - 5673, 5675 - 5694, 5691 - 5750, 5752 - 5777, 5782 - 5847, 5861 - 5886, 5903 - 5921 , 5923 - 5944, 5972 - 5990, 5998 - 6017, 6033 - 6074, 6076 - 6090, 611 1 - 6128, 6136 - 6152, 6154 - 6244, 6247 - 6266, 6269 - 6295, 6297 - 6321 , 6323 - 6343, 6360 - 6382, 6405 - 6474, 6499 - 6546, 6548 - 6579, 6581 - 6606, 6620 - 6668, 6670 - 6696, 6705 - 6735, 6731 - 6747, 6756 - 6775, 6777 - 6816, 6821 - 6840, 6855 - 6875, 6895 - 6929, 6935 - 6968, 6985 - 7001 , 7003 - 7020, 7036 - 7062, 7068 - 7085, 7109 - 7135, 7137 - 7185, 7233 - 7254, 7283 - 7315, 7317 - 7336, 7338 - 7355, 7367 - 7385, 7397 - 7416, 7429 - 7471 , 7498 - 7514, 7516 - 7540, 7592 - 7633, 7623 - 7660, 7680 - 7699, 7701 - 7722, 7724 - 7743, 7745 - 7762, 7769 - 7788, 7777 - 7807, 7798 - 7816, 7806 - 7847, 7862 - 7901 , 7981 - 8005, 8019 - 8040, 8042 - 8105, 8107 - 8133, 8153 - 8170, 8172 - 8204, 8243 - 8278, 8291 - 8396, 8402 - 8432, 8892 - 8924, 8930 - 8945, 8934 - 8963, 8955 - 8974, 8963 - 8986, 8987 - 9026, 9057 - 9073, 9074 - 9092, 9086 - 9105, 9121 - 9136, 9230 - 9245, 9238 - 9255, 9247 - 9270, 9269 - 9288, 9277 - 9296, 9297 - 9330, 9334 - 9349, 9348 - 9368, 9396 - 9414, 9412 - 9429, 9426 - 9440, 9447 - 9462, 9479 - 9495, 9508 - 9542, 9559 - 9575, 9588 - 9603, 9605 - 9622, 9704 - 9737, 9744 - 9767, 9756 - 9774, 9784 - 9807, 9796 - 9814, 9828 - 9847, 9836 - 9854, 9860 - 9874, 9886 - 9908, 9915 - 9944, 9969 - 9983, 10008 - 10028, 10056 - 10071 , 10073 - 10091 , 10094 - 10113, 10151 - 10176, 10208 - 10223, 10225 - 10251 , 10253 - 10267, 10304 - 10356, 10358 - 10405, 10414 - 10447, 10479 - 10519, 10529 - 10552, 10562 - 10576, 10581 - 10629, 10631 - 10680, 10682 - 10738, 10740 - 10782, 10848 - 10891 , 10886 - 10907, 10981 - 10997, 11057 - 1 1071 , 1 1084 - 11 106, 11 146 - 11 192, 11242 - 11263, 11303 - 11330, 11335 - 1 1357, 1 1359 - 11379, 11410 - 11431 , 11433 - 11448, 11478 - 11514, 11520 - 1 1562, 1 1610 - 11625, 11658 - 11674, 11676 - 11706, 11751 - 11774, 11793 - 1 1807, 1 1814 - 11828, 11842 - 11880, 11912 - 11946, 11985 - 12010, 12022 - 12040, 12064 - 12078, 12080 - 12100, 12102 - 12157, 12159 - 12174, 12176 - 12194, 12197 - 12231 , 12233 - 12267, 12269 - 12312, 12314 - 12329, 12350 - 12366 12370 12402 12404 12423 12467 12516 12519 12533 12559
12581 12583 12648 12659 12691 12702 12740 12778 12804 12805
12832 12860 12902 12937 12956 12958 12992 12995 13017 13062
13077 13105 13122 13124 13166 13168 13207 13209 13229 13231
13250 13247 13263 13270 13288 13346 13362 13364 13404 13414
13437 13472 13502 13504 13535 13542 13561 13583 13602 13604
13621 13623 13649 13665 13694 13754 13791 13801 13835 13829
13847 13837 13874 13868 13929 13931 13954 13955 13970 13972
14004 14008 14025 14027 14052 14053 14091 14106 14122 14124
14155 14207 14241 14243 14259 14269 14283 14272 14297 14326
14345 14350 14374 14416 14430 14460 14489 1451 1 14532 14533
14556 14586 14602 14604 14643 14665 14683 14693 14707 14720
14740 14742 14758 14760 14788 14865 14879 14881 14904 14906
14933 14939 14962 14964 14986 14998 15024 15052 15066 15068
15105 15120 15194 15189 15203 15192 15225 15251 15271 15274
15311 15321 15337 15346 1541 1 15419 15463 15503 15533 15535
15550 15558 15584 15630 15651 15642 15659 15653 15742 15766
15797 15813 15833 15845 15862 15885 15905 15896 15925 15942
15979 15992 16015 16020 16034 16036 16058 16085 16106 16108
16123 16125 16163 16184 16203 16218 16244 16261 16300 16302
16326 16368 16385 16387 16401 16431 16447 16449 16471 16476
16499 16509 16525 16534 16553 16562 16584 16586 16613 16615
16641 16668 16682 16672 16687 16677 16750 16747 16808 16810
16866 16864 16878 16886 16900 17369 17386 17388 17405 17413
17435 17469 17491 17493 17523 17559 17578 17582 17655 17657
17671 17662 17702 17704 17724 17726 17753 17750 17769 17771
17810 17811 17828 17836 17874 17876 17890 17918 17947 17949
17994 17998 18035 18037 18061 18072 18089 18097 18126 18128
18162 18164 18191 18210 18270 18272 18314 18316 18344 18346
18360 18362 18378 18391 18405 18407 18443 18448 18484 18477
18552 18568 18587 18590 18646 18660 18680 18682 18747 18749
18776 18778 18837 18848 18868 18876 18896 18910 18930 18941
18964 19001 19023 19070 19147 19149 19183 19185 19232 19238
19325 19331 19348 19351 19368 19376 19400 19428 19479 19518
19543 19545 19571 19581 19612 19656 19677 19679 19701 19703 19721 , 19723 - 19737, 19787 - 19823, 19838 - 19863, 19883 - 19898, 19900 - 19980, 19982 - 20022, 20024 - 20043, 20087 - 20106, 20108 - 20164, 20172 - 20190, 20192 - 20209, 20219 - 20235, 20238 - 20255, 20264 - 20279, 20298 - 20319, 20353 - 20383, 20379 - 20394, 20401 - 20439, 20441 - 20463, 20518 - 20536, 20577 - 20594, 20596 - 20610, 20622 - 20677, 20694 - 2071 1 , 20732 - 20802, 20804 - 20823, 20837 - 20851 , 20857 - 20910, 20912 - 20929, 20942 - 20984, 20987 - 21010, 21012 - 21035, 21057 - 21074, 21076 - 21 102, 21 104 - 21149, 21157 - 21 171 , 21 190 - 21215, 21222 - 21237, 21258 - 21277, 21285 - 21311 , 21313 - 21342, 21376 - 21393, 21395 - 21409, 2141 1 - 21457, 21481 - 21514, 21557 - 21574, 21576 - 21622, 21652 - 21679, 21692 - 21716, 21735 - 21789, 21808 - 21838, 21840 - 21881 , 21884 - 2191 1 , 21901 - 21915, 21915 - 21962, 21964 - 21985, 21987 - 22003, 22035 - 22063, 22065 - 22082, 22123 - 22138, 22143 - 22157, 22161 - 22217, 22226 - 22241 , 22257 - 22282, 22284 - 22305, 22327 - 22343, 22345 - 22362, 22364 - 22403, 22405 - 22432, 22449 - 22473, 22485 - 22500, 22502 - 22526, 22528 - 22556, 22567 - 22602, 22604 - 22619, 22621 - 22640, 22675 - 22700, 22697 - 22713, 22702 - 22717, 22718 - 22735, 22727 - 22776, 22783 - 22802, 22821 - 22850, 22840 - 22864, 22874 - 22889, 22893 - 22918, 22920 - 22934, 22938 - 22952, 22954 - 22983, 22993 - 23040, 23042 - 23092, 23128 - 23143, 23149 - 23199, 23201 - 23229, 23250 - 23279, 23281 - 23299, 23327 - 23342, 23349 - 23372, 23405 - 23425, 23439 - 23461 , 23479 - 23512, 23531 - 23547, 23567 - 23662, 23690 - 23724, 23789 - 23818, 23830 - 23878, 23889 - 23907, 23914 - 23928, 23945 - 23964, 23966 - 23990, 24014 - 24035, 24101 - 24130, 24174 - 2421 1 , 24213 - 24232, 24252 - 24303, 24302 - 24323, 24336 - 24363, 24382 - 24404, 24412 - 24434, 24571 - 24605, 24607 - 24639, 24641 - 24659, 24660 - 24705, 24707 - 24731 , 24755 - 24770, 24764 - 24783, 24782 - 24824, 24821 - 24835, 24857 - 24912, 24906 - 24923, 24924 - 24979, 25015 - 25032, 25036 - 25079, 25080 - 25157, 25184 - 25202, 25206 - 25233, 25300 - 25336, 25338 - 25360, 25362 - 25412, 25420 - 25447, 25467 - 25481 , 25493 - 2551 1 , 25513 - 25547, 25560 - 25591 , 25593 - 25633, 25635 - 25651 , 25675 - 25689, 25688 - 25734, 25751 - 25775, 25780 - 25847, 25849 - 25878, 25880 - 25896, 25898 - 25914, 25916 - 25945, 25954 - 25972, 25997 - 26018, 26019 - 26039, 26041 - 26076, 26072 - 26089, 26100 - 261 15, 26104 - 26119, 26121 - 26137, 26152 - 26194, 26183 - 26201 , 26203 - 26243, 26354 - 26372, 26398 - 26433, 26453 - 26484, 26506 - 26536, 26555 - 26602, 26604 - 26620, 26622 - 26641 , 26656 - 26673, 26675 - 26690, 26712 - 26753, 26837 - 26857, 26870 - 26885, 26887 - 26902, 26921 - 26954, 26956 - 26977, 32089 - 32104, 33156 - 33183, 33185 - 33202, 33204 - 33225, 33261 - 33290, 33292 - 33322, 33380 - 33408, 33419 - 33438, 33454 - 33515, 33517 - 33537, 33539 - 33555, 33564 - 33581 , 33583 - 33631 , 33645 - 33758, 33812 - 33882, 33900 - 33969, 33985 - 34000, 34036 - 34067, 34069 - 34108, 34119 - 34160, 34162 - 34199, 34218 - 34233, 34235 - 34276, 34284 - 34333, 34381 - 34396, 34409 - 34438, 34440 - 34466, 34471 - 34529, 34531 - 34569, 34613 - 34672, 34666 - 34684, 34687 - 34710, 34701 - 34742, 34732 - 34748, 34751 - 34782, 34779 - 34807, 34797 - 34812, 34829 - 34846, 34864 - 34899, 34912 - 34927, 34939 - 34957, 34964 - 34991 , 35013 - 35045, 35047 - 35068, 35070 - 35136, 35174 - 35188, 35256 - 35279, 35284 - 35393, 35404 - 35422, 35434 - 35448, 35441 - 35456, 35458 - 35472, 35471 - 35492, 35494 - 35512, 35514 - 35548, 35550 - 35613, 35649 - 35697, 35722 - 35794, 35810 - 35859, 35866 - 35890, 35892 - 35927, 35941 - 36007, 36010 - 36049, 36068 - 36095, 36093 - 361 10, 361 12 - 36159, 36165 - 36182, 36184 - 36205, 36207 - 36252, 36248 - 36270, 36260 - 36277, 36342 - 36358, 36362 - 36377, 36383 - 36405, 36414 - 36436, 36439 - 36481 , 36485 - 36500, 36503 - 36551 , 36561 - 36584, 36586 - 36628, 36642 - 36661 , 36651 - 36681 , 36689 - 36708, 36710 - 36732, 36782 - 36799, 36813 - 36832, 36834 - 36849, 36876 - 36894, 36896 - 36913, 36915 - 36976, 36984 - 36999, 37022 - 37041 , 37043 - 37089, 37112 - 37126, 37128 - 37144, 37146 - 37165, 39975 - 39989, 43270 - 43324, 43348 - 43373, 43375 - 43394, 43410 - 43425, 43442 - 43463, 43523 - 43554, 43562 - 43585, 43587 - 43603, 43605 - 43626, 43628 - 43642, 43661 - 43684, 43707 - 43754, 43772 - 43791 , 43796 - 43814, 43816 - 43836, 43855 - 43872, 43918 - 43958, 43964 - 43980, 43982 - 4401 1 , 44031 - 44070, 44069 - 44093, 44094 - 44116, 44113 - 44127, 441 18 - 44160, 44150 - 44176, 44181 - 44195, 44197 - 44220, 44222 - 44237, 44239 - 44294, 44285 - 44300, 44299 - 44359, 44360 - 44384, 44415 - 44447, 44447 - 44461 , 44475 - 44490, 44500 - 44516, 44515 - 44532, 44534 - 44567, 44569 - 44600, 44602 - 44631 , 44633 - 44653, 44669 - 44692, 44706 - 44721 , 44723 - 44739, 44741 - 44758, 44760 - 44777, 44797 - 44827, 44836 - 44893, 44900 - 44916, 44927 - 44956, 44958 - 44973, 44975 - 44995, 45009 - 45027, 45046 - 45077, 45088 - 45128, 45139 - 45183, 45185 - 45207, 45209 - 45254, 45263 - 45283, 45285 - 45303, 45302 - 45344, 45346 - 45410, 45412 - 45426, 45450 - 45478, 45480 - 45513, 45515 - 45571 , 45584 - 45621 , 45649 - 45664, 45677 - 45693, 45700 - 45739, 45741 - 45760, 45772 - 45793, 45795 - 45830, 45895 - 45915, 45917 - 45935, 45972 - 45986, 46003 - 46038, 46041 - 46084, 46101 - 46116, 46133 - 46154, 46157 - 46222, 46230 - 46251 , 46267 - 46298, 46300 - 46320, 46322 - 46355, 46377 - 46410, 46412 - 46440, 46452 - 46476, 46532 - 46565, 46571 - 46594, 46596 - 46621 , 46641 - 46668, 46672 - 46686, 46694 - 46708, 46710 - 46742, 46758 - 46785, 46787 - 46836, 46843 - 46887, 46893 - 46910, 46912 - 46933, 46935 - 46951 , 46953 - 46967, 46977 - 47011 , 47013 - 47029, 47035 - 47075, 47077 - 47092, 47094 - 47118, 47120 - 47139, 47141 - 47161 , 47163 - 47190, 47207 - 47222, 47224 - 47251 , 47253 - 47268, 47270 - 47427, 47436 - 47451 , 47477 - 47518, 47527 - 47541 , 47543 - 47599, 47608 - 47626, 47648 - 47694, 47707 - 47723, 47731 - 47769, 47796 - 47819, 47849 - 47864, 47866 - 47880, 47931 - 47953, 47967 - 47988, 48017 - 48035, 48066 - 48081 , 48092 - 48107, 48113 - 48136, 48126 - 48162, 48164 - 48187, 48189 - 48217, 48214 - 48248, 48250 - 48269, 48267 - 48281 , 48271 - 48289, 48278 - 48315, 48305 - 48321 , 48314 - 48337, 48339 - 48361 , 48353 - 48369, 48372 - 48392, 48381 - 48397, 48393 - 48408, 48401 - 48425, 48465 - 48490, 48481 - 48497, 48512 - 48527, 48527 - 48542, 48546 - 48560, 48550 - 48607, 48597 - 48617, 48615 - 48639, 48641 - 48668, 48673 - 48707, 48716 - 48734, 48758 - 48795, 48828 - 48883, 48905 - 48930, 48936 - 48957, 48983 - 48997, 48995 - 49030, 49042 - 49067, 49083 - 49098, 49100 - 49150, 49166 - 49192, 49222 - 49320, 49322 - 49355, 49357 - 49376, 49395 - 49420, 49443 - 49461 , 49472 - 49536, 49544 - 49597, 49599 - 49631 , 49633 - 49652, 49654 - 49671 , 49673 - 49699, 49701 - 49723, 49725 - 49785, 49787 - 49809, 49826 - 49843, 49853 - 49878, 49892 - 49907, 49929 - 49962, 49981 - 50001 , 50003 - 50036, 50038 - 50069, 50071 - 50126, 50135 - 50153, 50155 - 50173, 50182 - 50240, 50242 - 50270, 50327 - 50351 , 50353 - 50386, 50394 - 50413, 50430 - 50445, 50435 - 50449, 50447 - 50484, 50498 - 50515, 50529 - 50552, 50554 - 50626, 50628 - 50642, 50651 - 50665, 50670 - 50700, 50706 - 50745, 50747 - 50786, 50788 - 50816, 50818 - 50833, 50854 - 50883, 50898 - 50917, 50932 - 50946, 50948 - 50962, 50970 - 50984, 50978 - 51004, 51016 - 51054, 51053 - 51088, 51091 - 51 123, 51 128 - 51 173, 51238 - 51254, 51256 - 51282, 51284 - 51309, 51311 - 51331 , 51346 - 51384, 51387 - 51408, 51414 - 51434, 51474 - 51490, 51493 - 51507, 51546 - 51568, 51579 - 51594, 51596 - 51616, 51618 - 51644, 51662 - 51706, 51715 - 51731 , 51739 - 51762, 51773 - 51812, 51814 - 51856, 51858 - 51873, 51898 - 51931 , 51933 - 51948, 51950 - 51976, 51988 - 52014, 52016 - 52048, 52050 - 52091 , 52093 - 52115, 52136 - 52158, 52164 - 52187, 52191 - 52242, 52276 - 52304, 52314 - 52328, 52339 - 52358, 52368 - 52384, 52396 - 52420, 52429 - 52484, 52487 - 52503, 52508 - 52567, 52574 - 52594, 52616 - 52637, 52639 - 52666, 52700 - 52716, 52718 - 52737, 52741 - 52797, 52799 - 52817, 52823 - 52837, 52834 - 52851 , 52856 - 52881 , 52883 - 52904, 52918 - 52934, 52936 - 52957, 52956 - 52972, 52974 - 52996, 52999 - 53013, 53032 - 53050, 53066 - 53094, 53099 - 53133, 53136 - 53182, 53191 - 53206, 53233 - 53247, 53256 - 53273, 53285 - 53337, 53339 - 53390, 53402 - 53419, 53421 - 53435, 53476 - 53508, 53510 - 53529, 53531 - 53569, 53588 - 53609, 53606 - 53638, 53640 - 53659, 53662 - 53676, 53678 - 53696, 53727 - 53761 , 53763 - 53781 , 53775 - 53789, 53791 - 53819, 53833 - 53847, 53849 - 53876, 53895 - 53924, 53926 - 53942, 53944 - 53969, 53971 - 54024, 54034 - 54055, 54049 - 54086, 54090 - 54112, 54114 - 54134, 54136 - 54161 , 54175 - 54210, 54226 - 54270, 54275 - 54292, 54321 - 54343, 54383 - 54410, 54424 - 54441 , 54458 - 54505, 54495 - 54515, 54526 - 54545, 54547 - 54584, 54589 - 54609, 54616 - 54643, 54642 - 54667, 54662 - 54707, 54697 - 5471 1 , 54705 - 54743, 54745 - 54759, 54761 - 54800, 54826 - 54861 , 54886 - 54983, 54985 - 55012, 55014 - 55040, 55047 - 55102, 55104 - 55122, 55128 - 55180, 55189 - 55203, 55205 - 55236, 55289 - 55305, 55307 - 55336, 55338 - 55379, 55383 - 55424, 55426 - 55456, 55459 - 55484, 55486 - 55520, 55569 - 55586, 55588 - 55617, 55623 - 55643, 55649 - 55663, 55665 - 55706, 55708 - 55725, 55748 - 55775, 55777 - 55797, 55807 - 55830, 55851 - 55874, 55884 - 55909, 55924 - 55971 , 55986 - 5601 1 , 56013 - 56066, 56081 - 56273, 56275 - 56323, 56323 - 56348, 56353 - 56392, 56394 - 56413, 56416 - 56439, 56441 - 56470, 56485 - 56504, 56511 - 56546, 56548 - 56577, 56593 - 56632, 56634 - 56698, 56706 - 56723, 56725 - 56754, 56791 - 56834, 56836 - 56865, 56880 - 56930, 56943 - 56979, 56981 - 57000, 57005 - 57021 , 57036 - 57053, 57063 - 57122, 57137 - 57201 , 57296 - 57321 , 57367 - 57392, 57401 - 57426, 57428 - 57457, 57459 - 57480, 57498 - 57568, 57570 - 57593, 57595 - 57616, 57632 - 57654, 57661 - 57686, 57724 - 57738, 57743 - 57813, 57817 - 57834, 57836 - 57884, 57979 - 58014, 58016 - 58035, 58055 - 58070, 58077 - 58110, 58112 - 58142, 58174 - 58220, 58222 - 58236, 58238 - 58294, 58296 - 58318, 58320 - 58335, 58347 - 58374, 58385 - 58409, 58406 - 58466, 58479 - 58557, 58564 - 58582, 58583 - 58619, 58623 - 58683, 58701 - 58744, 58762 - 58794, 58796 - 58822, 58824 - 58855, 58889 - 58907, 58918 - 58990, 5901 1 - 59061 , 59120 - 59167, 59203 - 59223, 59225 - 59242, 59263 - 59283, 59297 - 59327, 59332 - 59361 , 59364 - 59421 , 59423 - 59441 , 59443 - 59458, 59460 - 59486, 59500 - 59535, 59549 - 59580, 59586 - 59612, 59618 - 59649, 59651 - 59672, 59708 - 59723, 59725 - 59749, 59751 - 59770, 59780 - 59805, 59807 - 59829, 59831 - 59846, 59848 - 59998, 60002 - 60028, 60032 - 60050, 60067 - 60082, 60084 - 60103, 60105 - 60122, 60135 - 60156, 60172 - 60208, 60218 - 60293, 60292 - 60309, 60309 - 60330, 60374 - 60388, 60385 - 60433, 60423 - 60441 , 60436 - 60455, 60498 - 60518, 60518 - 60541 , 60560 - 60589, 60609 - 60674, 60664 - 60680, 60682 - 60698, 60717 - 60742, 60757 - 60796, 60798 - 60817, 60825 - 60856, 60914 - 60957, 60972 - 61012, 61014 - 61031 , 61050 - 61077, 61071 - 61087, 61089 - 61103, 61101 - 61 119, 61 120 - 61 154, 61 149 - 61 171 , 61 180 - 61 199, 61 190 - 61212, 61213 - 61238, 61235 - 61257, 61247 - 61275, 61285 - 61312, 61315 - 61332, 61322 - 61337, 61332 - 61354, 61350 - 61378, 61381 - 61400, 61415 - 61445, 61453 - 61482, 61484 - 61540, 61551 - 61575, 61579 - 61642, 61644 - 61658, 61660 - 61684, 61686 - 61704, 61720 - 61743, 61745 - 61806, 61839 - 61900, 61902 - 61928, 61930 - 61963, 61965 - 61981 , 61983 - 6201 1 , 62025 - 62045, 62047 - 62073, 62093 - 62117, 62132 - 62148, 62176 - 62199, 62228 - 62251 , 62247 - 62277, 62307 - 62324, 62319 - 62336, 62338 - 62361 , 62363 - 62409, 62411 - 62437, 62439 - 62458, 62474 - 62492, 62500 - 62562, 62565 - 62604, 62613 - 62656, 62658 - 62687, 62683 - 62697, 62702 - 62751 , 62740 - 62793, 62796 - 62816, 62806 - 62823, 62826 - 62851 , 62846 - 62863, 62861 - 62893, 62900 - 62927, 62930 - 62955, 63008 - 63042, 63044 - 63078, 63080 - 63098, 63100 - 63119, 63121 - 63138, 63139 - 63153, 63165 - 63179, 63181 - 63195, 63197 - 63228, 63230 - 63244, 63246 - 63309, 63315 - 63359, 63361 - 63399, 63426 - 63457, 63474 - 63501 , 63503 - 63526, 63528 - 63576, 63577 - 63596, 63598 - 63638, 63653 - 63669, 63689 - 63707, 63709 - 63738, 63754 - 63793, 63807 - 63835, 63837 - 63854, 63856 - 63873, 63876 - 63979, 63997 - 64020, 64016 - 64033, 64035 - 64058, 64060 - 64075, 64090 - 64145, 64186 - 64207, 64223 - 64240, 64242 - 64260, 64280 - 64299, 64325 - 64342, 64359 - 64396, 64407 - 64422, 64412 - 64426, 64424 - 64444, 64467 - 64484, 64477 - 64511 , 64517 - 64532, 64525 - 64590, 64600 - 64623, 64625 - 64646, 64662 - 64709, 64720 - 64782, 64796 - 64821 , 64823 - 64844, 64846 - 64860, 64862 - 64890, 64917 - 64947, 64949 - 64963, 64979 - 65001 , 65029 - 65053, 65059 - 65083, 65086 - 65125, 65156 - 65171 , 65173 - 65200, 65202 - 65216, 65223 - 65258, 65279 - 65294, 65302 - 65370, 65372 - 65441 , 65464 - 65486, 65493 - 65516, 65527 - 65580, 65582 - 65596, 65599 - 65620, 65688 - 65718, 65720 - 65747, 65765 - 65787, 65815 - 65839, 65841 - 65865, 65889 - 65910, 65912 - 65954, 65968 - 66002, 66012 - 66060, 66062 - 66084, 66086 - 66142, 66144 - 66158, 66207 - 66226, 66228 - 66243, 66259 - 66310, 66312 - 66329, 66332 - 66355, 66372 - 66396, 66417 - 66437, 66476 - 6651 1 , 66549 - 66588, 66591 - 66629, 66633 - 66653, 66655 - 66669, 66671 - 66690, 66725 - 66758, 66760 - 66779, 66781 - 66797, 66830 - 66890, 66893 - 66907, 66925 - 66944, 66955 - 66971 , 66973 - 66998, 67027 - 67042, 67044 - 67067, 67096 - 67148, 67149 - 67180, 67182 - 67201 , 67203 - 67220, 67245 - 67274, 67282 - 67297, 67310 - 67337, 67339 - 67368, 67381 - 67399, 67401 - 67425, 67427 - 67463, 67465 - 67486, 67498 - 67573, 67590 - 67613, 67616 - 67638, 67627 - 67642, 67641 - 67663, 67672 - 67785, 67792 - 67840, 67853 - 67909, 67919 - 67936, 67977 - 68005, 68007 - 68021 , 68023 - 68038, 68059 - 68077, 68101 - 68224, 68238 - 68259, 68275 - 68320, 68350 - 68365, 68379 - 68410, 68414 - 68439, 68441 - 68461 , 68473 - 68488, 68490 - 68529, 68531 - 68573, 68590 - 68614, 68616 - 68644, 68673 - 68703, 68704 - 68738, 68775 - 68791 , 68793 - 68820, 68845 - 68890, 68912 - 68975, 68983 - 69024, 69036 - 69058, 69057 - 69072, 69074 - 69089, 69091 - 69129, 69131 - 69155, 69174 - 69189, 6921 1 - 69227, 69229 - 69288, 69315 - 69360, 69362 - 69438, 69464 - 69485, 69494 - 69529, 69541 - 69566, 69568 - 69616, 69618 - 69655, 69688 - 69706, 69714 - 69735, 69738 - 69778, 69780 - 69808, 69832 - 69862, 69861 - 69877, 69876 - 69890, 69992 - 70029, 70021 - 70038, 70080 - 70099, 70106 - 70128, 70130 - 70156, 70176 - 70190, 70196 - 70223, 70225 - 70260, 70260 - 70319, 70321 - 70407, 70433 - 70454, 70493 - 70519, 70522 - 70546, 70557 - 70614, 70616 - 70650, 70652 - 70667, 70669 - 70685, 70681 - 70734, 70750 - 70765, 70767 - 70796, 70802 - 70816, 70830 - 70862, 70867 - 70900, 70909 - 70940, 70942 - 70975, 70975 - 71043, 71058 - 71088, 71094 - 71 115, 71 145 - 71 163, 71 180 - 71200, 71221 - 71237, 71244 - 71264, 71266 - 71287, 71289 - 71325, 71357 - 71391 , 71443 - 71461 , 71482 - 71501 , 71532 - 71559, 71572 - 71624, 71626 - 71667, 71680 - 71694, 71726 - 71766, 71774 - 71808, 71858 - 71872, 71867 - 71881 , 71888 - 71920, 71943 - 71961 , 71965 - 71982, 71984 - 72003, 72008 - 72024, 72048 - 72066, 721 16 - 72136, 72138 - 72182, 72190 - 72230, 72287 - 72312, 72325 - 72340, 72385 - 72433, 72435 - 72450, 72456 - 72489, 72499 - 72525, 72541 - 72577, 72609 - 72652, 72652 - 72667, 72683 - 72698, 72708 - 72739, 72768 - 72785, 72788 - 72820, 72822 - 72838, 72840 - 72872, 72895 - 72909, 72921 - 72936, 72940 - 72985, 73067 - 73081 , 73100 - 73120, 73132 - 73164, 73169 - 73201 , 73199 - 73213, 73203 - 73238 73267 - 73283, 73298 - 73328, 73317 - 73354, 73356 - 73377, 73400 - 73428 73430 - 73445, 73447 - 73462, 73471 - 73485, 73504 - 73605, 73633 - 73680 73680 - 73706, 73755 - 73785, 73813 - 73830, 73832 - 73857, 73891 - 73917 73919 - 73940, 73948 - 73976, 73984 - 74008, 74010 - 74036, 74038 - 74060 74070 - 74098, 74100 - 74116, 74118 - 74158, 74209 - 74239, 74260 - 74290 74292 - 74343, 74355 - 74372, 74453 - 74502, 74504 - 74532, 74545 - 74577 74589 - 74606, 74603 - 74631 , 74624 - 74645, 74634 - 74648, 74650 - 74670 74672 - 74701 , 74718 - 74756, 74759 - 74774, 74764 - 74810, 74813 - 74843 74833 - 74850, 74865 - 74888, 74914 - 74963, 74974 - 74992, 74994 - 75015 7501 1 - 75030, 75028 - 75072, 75079 - 75100, 75108 - 75122, 7511 1 - 75137 75128 - 75142, 75143 - 75157, 75148 - 75162, 75152 - 75169, 75171 - 75217 75206 - 75240, 75242 - 75282, 75296 - 75317, 75332 - 75347, 75337 - 75357 75364 - 75389, 75403 - 75439, 75470 - 75497, 75499 - 75525, 75565 - 75624 75627 - 75651 , 75653 - 75672, 75704 - 75729, 75731 - 75759, 75760 - 75775 75777 - 75793, 75795 - 75827, 75829 - 75844, 75848 - 75874, 75898 - 75916 75915 - 75937, 75940 - 75968, 76002 - 76016, 76018 - 76062, 76073 - 76097 76117 - 76148, 76150 - 76167, 76169 - 76191 , 76193 - 76207, 76242 - 76256 76253 - 76273, 76275 - 76347, 76349 - 76367, 76390 - 76404, 76393 - 76417 76419 - 76433, 76435 - 76470, 76489 - 76505, 76515 - 76530, 76532 - 76597 76599 - 76646, 76663 - 76681 , 76701 - 76716, 76749 - 76786, 76797 - 7681 1 76864 - 76885, 76897 - 76916, 76918 - 76965, 76994 - 77019, 77021 - 77070 77072 - 77147, 77161 - 77208, 77212 - 77227, 77250 - 77274, 77293 - 77331 77333 - 77359, 77361 - 77377, 77384 - 77459, 77465 - 77520, 77522 - 77537 77553 - 77578, 77580 - 77609, 77672 - 77690, 77692 - 77727, 77759 - 77804 77812 - 77843, 77861 - 77877, 77913 - 77936, 77951 - 77980, 77994 - 78025 78027 - 78073, 78083 - 78115, 78132 - 78148, 78172 - 78196, 78198 - 78221 78228 - 78245, 78247 - 78263, 78265 - 78285, 78287 - 78331 , 78367 - 78391 78399 - 78428, 78430 - 78459, 78478 - 78500, 78510 - 78534, 78550 - 78570 78572 - 78588, 78598 - 78630, 78658 - 78694, 78696 - 78723, 78734 - 78768 78770 - 78791 , 78799 - 78813, 78815 - 78856, 78858 - 78883, 78897 - 78941 78943 - 78980, 78982 - 79002, 78998 - 79012, 79020 - 79046, 79086 - 79112 79117 - 79147, 79149 - 79169, 79171 - 79191 , 79193 - 79231 , 79259 - 79274 79347 - 79361 , 79391 - 7941 1 , 79427 - 79462, 79464 - 79492, 79494 - 79520 79540 - 79566, 79586 - 79609, 79615 - 79637, 79665 - 79682, 79684 - 79700 79701 - 79717, 79719 - 79786, 79792 - 79825, 79827 - 79848, 79852 - 79880, 79882 - 79944, 79958 - 79975, 80000 - 80051 , 80053 - 80067, 80074 - 80091 , 80093 - 80124, 80137 - 80162, 80187 - 80208, 80218 - 80246, 80276 - 80301 , 80322 - 80340, 80352 - 80366, 80419 - 80433, 80439 - 80453, 80458 - 80476, 80499 - 80518, 80520 - 80548, 80550 - 80578, 80616 - 80635, 80637 - 80674, 80687 - 80714, 80716 - 80736, 80742 - 80763, 80765 - 80817, 80828 - 80879, 80958 - 80979, 81029 - 81054, 81 144 - 81173, 81175 - 81203, 81207 - 81224, 81245 - 81264, 81270 - 81296, 81298 - 81322, 81324 - 81359, 81374 - 81389, 81391 - 81440, 81442 - 81461 , 81481 - 81498, 81500 - 81523, 81548 - 81567, 81590 - 81639, 81633 - 81668, 81676 - 81736, 81735 - 81793, 81828 - 81862, 81864 - 81886, 81888 - 81905, 81907 - 81949, 81984 - 82044, 82058 - 82072, 82074 - 82101 , 82119 - 82175, 82177 - 82192, 82204 - 82222, 82235 - 82252, 82302 - 82323, 82327 - 82358, 82377 - 82400, 82432 - 82446, 82460 - 82476, 82477 - 82492, 82493 - 82570, 82572 - 82640, 82650 - 82664, 82676 - 82691 , 82685 - 82736, 82727 - 82741 , 82735 - 82749, 82747 - 82789, 82783 - 82858, 82861 - 82898, 82900 - 82921 , 82938 - 82955, 82965 - 82988, 83004 - 83047, 83070 - 83097, 83092 - 83171 , 83173 - 83275, 83291 - 83314, 83340 - 83366, 83368 - 83383, 83427 - 83483, 83501 - 83545, 83571 - 83609, 8361 1 - 83626, 83634 - 83684, 83687 - 83707, 83709 - 83733, 83735 - 83759, 83851 - 83903, 83917 - 83933, 83967 - 83983, 83986 - 84025, 84047 - 84079, 84090 - 84229, 85639 - 85654, 85643 - 85674, 85809 - 85824, 85873 - 85892, 85882 - 85899, 86600 - 86622, 86643 - 86665, 86671 - 86686, 86710 - 86735, 86758 - 86775, 86802 - 86819, 87013 - 87052, 87060 - 87094, 87107 - 87121 , 87123 - 87150, 88108 - 88253, 88273 - 88291 , 88284 - 88316, 88310 - 88324, 88326 - 88358, 88358 - 88394, 88396 - 88415, 88424 - 88438, 89475 - 89493, 89484 - 8951 1 , 89502 - 89519, 89518 - 89548, 89548 - 89572, 89567 - 89587, 89589 - 89605, 89616 - 89633, 89693 - 89714, 89704 - 89719, 90241 - 90261 , 90261 - 90275, 90291 - 9031 1 , 90313 - 90330, 90343 - 90364, 90401 - 90415, 90419 - 90436, 90425 - 90446, 90457 - 90471 , 90461 - 90475, 90480 - 90500, 90504 - 90541 , 90544 - 90558, 90552 - 90573, 90563 - 90593, 90602 - 90624, 90634 - 90649, 90651 - 90668, 90712 - 90732, 90721 - 90741 , 90730 - 90749, 90760 - 90783, 90785 - 90806, 90816 - 90835, 90824 - 90849, 93450 - 93475, 93502 - 93516, 93580 - 93599, 93603 - 93621 , 93630 - 93655, 93645 - 93661 , 93666 - 93686, 93691 - 93705, 93714 - 93740, 93960 - 93974, 94410 - 94428, 94480 - 94495, 94555 - 94569, 94561 - 94581 , 94586 - 94601 , 94620 - 94640, 96242 - 96256, 96246 - 96262, 96270 - 96286, 96292 - 96338, 96355 - 96376, 96379 - 96396, 96387 - 96408, 96398 - 96435, 96440 - 96455, 96448 - 96469, 96736 - 96750, 96766 - 96796, 96797 - 96829, 96831 - 96850, 96839 - 96882, 96892 - 96912, 96927 - 96941, 96933 - 96953, 96943 - 96961 , 96964 - 96980, 96983 - 97006, 97147 - 97190, 97185 - 97199, 97204 - 97231 , 97242 - 97257, 97265 - 97302, 97304 - 97326, 97332 - 97351 , 97352 - 97388, 97390 - 97417, 97407 - 97438, 97440 - 97461, 98217 - 98258, 98259 - 98277, 98279 - 98295, 98310 - 98348, 98341 - 98356, 98345 - 98366, 98360 - 98377, 98383 - 98401, 98403 - 98418, 98425 - 98442, 98445 - 98464, 98470 - 98494, 98483 - 98503, 99116 - 99186, 99188 - 99213, 99208 - 99223, 99215 - 99268, 99259 - 99276, 99283 - 99312, 99302 - 99345, 99347 - 99373, 99367 - 99422, 100868 - 100882, 102411 - 102425, 102783 - 102797, 103436 - 103450, 104811 - 104825, 104853 - 104872, 104874 - 104891, 104919 - 104961, 104972 - 104986, 104975- 104992, 104988 - 105007, 105001 - 105041, 105051 - 105065, 105068 - 105104, 105106 - 105124, 105126 - 105156, 105158 - 105188, 105192 - 105221, 105219 - 105261, 105276- 105292, 105312- 105330, 105362- 105390, 105388 - 105404, 105649 - 105688, 105686 - 105700, 105690 - 105707, 105702 - 105723, 105737 - 105762, 105793 - 105812, 105817 - 105836, 105843 - 105877, 105909 - 105928, 106311 - 106331, 106324 - 106340, 106329 - 106363, 106385- 106404, 106399 - 106437, 106464 - 106497, 106486 - 106506, 106509 - 106531, 106525 - 106540, 106533 - 106549, 106550 - 106569, 106580 - 106620, 107575 - 107606, 107609 - 107664, 107666 - 107683, 107690 - 107705, 107694 - 107708, 107699 - 107716, 107709 - 107733, 107769 - 107791, 107795 - 107823, 109491 - 109523, 109532 - 109557, 109576 - 109613, 109615 - 109637, 109639 - 109654, 109657 - 109671, 109667 - 109732, 109736 - 109763, 109764 - 109779, 109845 - 109859, 109853 - 109875, 109887- 109901, 109901 - 109921, 109924- 109940, 109929 - 109962, 109953 - 109972, 110010 - 110072, 110065- 110103, 110105- 110120, 110208 - 110222, 110237- 110254, 110297- 110313, 110358- 110374, 110379 - 110398, 110418 - 110437, 110466 - 110490, 110518 - 110535, 110526 - 110545, 110548 - 110565, 110567- 110581, 110583- 110601, 110616- 110630, 110639 - 110661, 110730 - 110745, 110780 - 110809, 110811 - 110840, 112148 - 112163, 112158- 112172, 112178- 112199, 112201 - 112249, 112240- 112258,
112267 - 112294, 112299 - 112369, 112383 - 112443, 112632 - 112649, 112689 - 112721, 112726- 112741, 112762- 112789, 112790- 112813, 112822- 112855, 112846 - 112867, 112873 - 112889, 112916 - 112930, 112919 - 112950, 112969 - 112986, 113000 - 113027, 113027- 113046, 113036- 113050, 113062- 113078, 1 13069 - 1 13089, 113091 - 1 13114, 1131 12 - 1 13126, 1131 16 - 1 13130, 113123 - 1 13139, 113134 - 113195, 1 13190 - 113215, 1 1321 1 - 113228, 1 13217 - 1 13247, and 113708 - 113725.
6. The antisense oligonucleotide according to any one of claims 1 - 5, 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.
7. The antisense oligonucleotide according to claim 6, 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.
8. The antisense oligonucleotide according to claim 6 or 7, wherein region G comprises 5 - 16 contiguous DNA nucleosides.
9. The antisense oligonucleotide according to any one of claims 1 - 8, wherein the antisense oligonucleotide is a LNA gapmer oligonucleotide.
10. The antisense oligonucleotide according to any one of claims 6 - 9, wherein the LNA nucleosides are beta-D-oxy LNA nucleosides.
1 1. The antisense oligonucleotide according to any one of claims 1 - 10, wherein the internucleoside linkages between the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.
12. The antisense oligonucleotide according to any one of claims 1 - 11 , wherein the oligonucleotide comprises a contiguous nucleotide sequence selected from the group consisting of: SEQ ID NO 3, SEQ ID NO 8, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO 18, and SEQ ID NO 20, or selected from the group consisting of SEQ ID NO 1 , SEQ ID NO: 2, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 10, and SEQ ID NO 11 , SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16 and SEQ ID NO 19.
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:
TTtaagtgttcGACC (SEQ ID NO 1 ), TTGtgataaagcCGT (SEQ ID NO 2),
CGCatatatcaaGTA (SEQ ID NO 3), CGtatgagaggCAAG (SEQ ID NO 4),
GGAT atacagtcgCG (SEQ ID NO 5), TAatgtcactaCGGG (SEQ ID NO 6), CCGAttactgattTA (SEQ ID NO 7), TCgataatgtaAGGC (SEQ ID NO 8),
TTAgtagtcgcTCG (SEQ ID NO 9), GGctattgacgATTC (SEQ ID NO 10),
ATGAggcgaagttTA (SEQ ID NO 11 ), ATCaacgcataCGC (SEQ ID NO 12),
CCAtgttaaacgCAT (SEQ ID NO 13), ATCAgaaggtaggCG (SEQ ID NO 14),
CGAtaatgtaagGCA (SEQ ID NO 15), GACaaattaccattTCA (SEQ ID NO 16), ACatttaataagcAGGA (SEQ ID NO 17), TAataagcaggaaCACC (SEQ ID NO 18), GATgttgtagttGAG (SEQ ID NO 19) and CATttaataagcaGGA (SEQ ID NO 20);
such as selected from the group consisting of
CGCatatatcaaGTA (SEQ ID NO 3), TCgataatgtaAGGC (SEQ ID NO 8),
CCAtgttaaacgCAT (SEQ ID NO 13), CGAtaatgtaagGCA (SEQ ID NO 15),
ACatttaataagcAGGA (SEQ ID NO 17), TAataagcaggaaCACC (SEQ ID NO 18) and CATttaataagcaGGA (SEQ ID NO 20),
wherein a capital letter represents a LNA nucleoside, a lower case letter represents a DNA nucleoside.
14. The antisense oligonucleotide according to any one of claims 1 - 13, wherein the oligonucleotide comprises or consists of a contiguous nucleotide sequence, selected from the group consisting of: TTtaagtgttmcGACC (SEQ ID NO 1 ), TTGtgataaagcCGT (SEQ ID NO 2), CGCatatatcaaGTA (SEQ ID NO 3), CGtatgagaggCAAG (SEQ ID NO 4), GGATatacagtmcgCG (SEQ ID NO 5), TAatgtcactaCGGG (SEQ ID NO 6), CCGAttactgattTA (SEQ ID NO 7), TCgataatgtaAGGC (SEQ ID NO 8),
TTAgtagrcgcTCG (SEQ ID NO 9), GGctattgamcgATTC (SEQ ID NO 10),
ATGAggmcgaagttTA (SEQ ID NO 1 1 ), ATCaamcgcataCGC (SEQ ID NO 12), CCAtgttaaamcgCAT (SEQ ID NO 13), ATCAgaaggtaggCG (SEQ ID NO 14), CGAtaatgtaagGCA (SEQ ID NO 15), GACaaattaccattTCA (SEQ ID NO 16), ACatttaataagcAGGA (SEQ ID NO 17), TAataagcaggaaCACC (SEQ ID NO 18), GATgttgtagttGAG (SEQ ID NO 19) and CATttaataagcaGGA (SEQ ID NO 20);
such as selected from the group consisting of
CGCatatatcaaGTA (SEQ ID NO 3), TCgataatgtaAGGC (SEQ ID NO 8),
CCAtgttaaamcgCAT (SEQ ID NO 13), CGAtaatgtaagGCA (SEQ ID NO 15),
ACatttaataagcAGGA (SEQ ID NO 17), TAataagcaggaaCACC (SEQ ID NO 18), and CATttaataagcaGGA (SEQ ID NO 20),
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 mc is 5-methyl cytosine DNA, and wherein the internucleoside linkages between the nucleosides are phosphorothioate internucleoside linkages.
15. A conjugate comprising the oligonucleotide according to any one of claims 1 - 14, and at least one conjugate moiety covalently attached to said oligonucleotide.
16. A pharmaceutical composition comprising the oligonucleotide of claim 1-14 or the conjugate of claim 14 and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.
17. An in vivo or in vitro method for modulating SPTLC1 expression in a target cell which is expressing SPTLC1, said method comprising administering an oligonucleotide of any one of claims 1-14, the conjugate according to claim 15, or the pharmaceutical composition of claim 16 in an effective amount to said cell.
18. 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 - 14 or the conjugate according to claim 15 or the pharmaceutical composition of claim 16 to a subject suffering from or susceptible to the disease.
19. The method of claim 18, wherein the disease is selected from the group consisting of hypertension such as cancer, hepatitis C, diabetes (such as type 2 diabetes), opiate/opioid-antinociceptive tolerance, Niemann-Pick type C disease, HIV infection and amphetamine/methamphetamine-induced toxicity.
20. The oligonucleotide of any one of claims 1 - 14 or the conjugate according to claim 15 or the pharmaceutical composition of claim 16 for use in medicine.
21. The oligonucleotide of any one of claims 1 - 14 or the conjugate according to claim 15 or the pharmaceutical composition of claim 16 for use in the treatment or prevention of a disease selected from the group consisting of hypertension such as cancer, hepatitis C, diabetes (such as type 2 diabetes), opiate/opioid-antinociceptive tolerance, Niemann-Pick type C disease, HIV infection and
amphetamine/methamphetamine-induced toxicity.
22. Use of the oligonucleotide of claim 1 - 14 or the conjugate according to claim 15 or the pharmaceutical composition of claim 16, for the preparation of a medicament for treatment or prevention of a disease selected from the group consisting of hypertension such as cancer, hepatitis C, diabetes (such as type 2 diabetes), opiate/opioid-antinociceptive tolerance, Niemann-Pick type C disease, HIV infection and amphetamine/methamphetamine-induced toxicity.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024187097A3 (en) * 2023-03-09 2024-11-07 Leal Therapeutics, Inc. Compositions and methods for modulating sptlc1
US12391947B2 (en) * 2018-11-29 2025-08-19 University Of Massachusetts Modulation of SPTLC1 via recombinant adeno-associated vectors

Citations (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998039352A1 (en) 1997-03-07 1998-09-11 Takeshi Imanishi Novel bicyclonucleoside and oligonucleotide analogues
WO1999014226A2 (en) 1997-09-12 1999-03-25 Exiqon A/S Bi- and tri-cyclic nucleoside, nucleotide and oligonucleotide analogues
WO2000047599A1 (en) 1999-02-12 2000-08-17 Sankyo Company, Limited Novel nucleosides and oligonucleotide analogues
WO2000066604A2 (en) 1999-05-04 2000-11-09 Exiqon A/S L-ribo-lna analogues
WO2001023613A1 (en) 1999-09-30 2001-04-05 Isis Pharmaceuticals, Inc. Human rnase h and oligonucleotide compositions thereof
WO2002048325A2 (en) 2000-12-15 2002-06-20 Bayer Aktiengesellschaft Regulation of human serine palmitoyltransferase
WO2004046160A2 (en) 2002-11-18 2004-06-03 Santaris Pharma A/S Amino-lna, thio-lna and alpha-l-oxy-ln
WO2007031091A2 (en) 2005-09-15 2007-03-22 Santaris Pharma A/S Rna antagonist compounds for the modulation of p21 ras expression
WO2007090071A2 (en) 2006-01-27 2007-08-09 Isis Pharmaceuticals, Inc. 6-modified bicyclic nucleic acid analogs
WO2007134181A2 (en) 2006-05-11 2007-11-22 Isis Pharmaceuticals, Inc. 5'-modified bicyclic nucleic acid analogs
WO2008049085A1 (en) 2006-10-18 2008-04-24 Isis Pharmaceuticals, Inc. Antisense compounds
WO2008122037A2 (en) 2007-04-02 2008-10-09 Saint Louis University Inhibitors of the ceramide metabolic pathway as adjuncts to opiates
WO2008150729A2 (en) 2007-05-30 2008-12-11 Isis Pharmaceuticals, Inc. N-substituted-aminomethylene bridged bicyclic nucleic acid analogs
WO2008154401A2 (en) 2007-06-08 2008-12-18 Isis Pharmaceuticals, Inc. Carbocyclic bicyclic nucleic acid analogs
WO2009001097A2 (en) 2007-06-27 2008-12-31 Isis Innovation Limited Substrate reduction therapy
WO2009006478A2 (en) 2007-07-05 2009-01-08 Isis Pharmaceuticals, Inc. 6-disubstituted bicyclic nucleic acid analogs
WO2009067647A1 (en) 2007-11-21 2009-05-28 Isis Pharmaceuticals, Inc. Carbocyclic alpha-l-bicyclic nucleic acid analogs
WO2010036698A1 (en) 2008-09-24 2010-04-01 Isis Pharmaceuticals, Inc. Substituted alpha-l-bicyclic nucleosides
WO2010077578A1 (en) 2008-12-09 2010-07-08 Isis Pharmaceuticals, Inc. Bis-modified bicyclic nucleic acid analogs
WO2011017521A2 (en) 2009-08-06 2011-02-10 Isis Pharmaceuticals, Inc. Bicyclic cyclohexose nucleic acid analogs
WO2011156202A1 (en) 2010-06-08 2011-12-15 Isis Pharmaceuticals, Inc. Substituted 2 '-amino and 2 '-thio-bicyclic nucleosides and oligomeric compounds prepared therefrom
WO2012109395A1 (en) 2011-02-08 2012-08-16 Isis Pharmaceuticals, Inc. Oligomeric compounds comprising bicyclic nucleotides and uses thereof
WO2013022984A1 (en) 2011-08-11 2013-02-14 Isis Pharmaceuticals, Inc. Selective antisense compounds and uses thereof
WO2013149250A1 (en) 2012-03-30 2013-10-03 The Regents Of The University Of California Methods of treatment, diagnosis and monitoring for methamphetamine toxicity which target ceramide metabolic pathways and cellular senescence
WO2013154798A1 (en) 2012-04-09 2013-10-17 Isis Pharmaceuticals, Inc. Tricyclic nucleic acid analogs
WO2014076195A1 (en) 2012-11-15 2014-05-22 Santaris Pharma A/S Oligonucleotide conjugates
WO2015113922A1 (en) 2014-01-30 2015-08-06 Roche Innovation Center Copenhagen A/S Poly oligomer compound with biocleavable conjugates
WO2017153753A1 (en) * 2016-03-11 2017-09-14 Ucl Business Plc Allele-specific gene suppression

Patent Citations (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998039352A1 (en) 1997-03-07 1998-09-11 Takeshi Imanishi Novel bicyclonucleoside and oligonucleotide analogues
WO1999014226A2 (en) 1997-09-12 1999-03-25 Exiqon A/S Bi- and tri-cyclic nucleoside, nucleotide and oligonucleotide analogues
WO2000047599A1 (en) 1999-02-12 2000-08-17 Sankyo Company, Limited Novel nucleosides and oligonucleotide analogues
WO2000066604A2 (en) 1999-05-04 2000-11-09 Exiqon A/S L-ribo-lna analogues
WO2001023613A1 (en) 1999-09-30 2001-04-05 Isis Pharmaceuticals, Inc. Human rnase h and oligonucleotide compositions thereof
WO2002048325A2 (en) 2000-12-15 2002-06-20 Bayer Aktiengesellschaft Regulation of human serine palmitoyltransferase
WO2004046160A2 (en) 2002-11-18 2004-06-03 Santaris Pharma A/S Amino-lna, thio-lna and alpha-l-oxy-ln
WO2007031091A2 (en) 2005-09-15 2007-03-22 Santaris Pharma A/S Rna antagonist compounds for the modulation of p21 ras expression
WO2007090071A2 (en) 2006-01-27 2007-08-09 Isis Pharmaceuticals, Inc. 6-modified bicyclic nucleic acid analogs
WO2007134181A2 (en) 2006-05-11 2007-11-22 Isis Pharmaceuticals, Inc. 5'-modified bicyclic nucleic acid analogs
WO2008049085A1 (en) 2006-10-18 2008-04-24 Isis Pharmaceuticals, Inc. Antisense compounds
WO2008122037A2 (en) 2007-04-02 2008-10-09 Saint Louis University Inhibitors of the ceramide metabolic pathway as adjuncts to opiates
WO2008150729A2 (en) 2007-05-30 2008-12-11 Isis Pharmaceuticals, Inc. N-substituted-aminomethylene bridged bicyclic nucleic acid analogs
WO2008154401A2 (en) 2007-06-08 2008-12-18 Isis Pharmaceuticals, Inc. Carbocyclic bicyclic nucleic acid analogs
WO2009001097A2 (en) 2007-06-27 2008-12-31 Isis Innovation Limited Substrate reduction therapy
WO2009006478A2 (en) 2007-07-05 2009-01-08 Isis Pharmaceuticals, Inc. 6-disubstituted bicyclic nucleic acid analogs
WO2009067647A1 (en) 2007-11-21 2009-05-28 Isis Pharmaceuticals, Inc. Carbocyclic alpha-l-bicyclic nucleic acid analogs
WO2010036698A1 (en) 2008-09-24 2010-04-01 Isis Pharmaceuticals, Inc. Substituted alpha-l-bicyclic nucleosides
WO2010077578A1 (en) 2008-12-09 2010-07-08 Isis Pharmaceuticals, Inc. Bis-modified bicyclic nucleic acid analogs
WO2011017521A2 (en) 2009-08-06 2011-02-10 Isis Pharmaceuticals, Inc. Bicyclic cyclohexose nucleic acid analogs
WO2011156202A1 (en) 2010-06-08 2011-12-15 Isis Pharmaceuticals, Inc. Substituted 2 '-amino and 2 '-thio-bicyclic nucleosides and oligomeric compounds prepared therefrom
WO2012109395A1 (en) 2011-02-08 2012-08-16 Isis Pharmaceuticals, Inc. Oligomeric compounds comprising bicyclic nucleotides and uses thereof
WO2013022984A1 (en) 2011-08-11 2013-02-14 Isis Pharmaceuticals, Inc. Selective antisense compounds and uses thereof
EP2742135A1 (en) 2011-08-11 2014-06-18 Isis Pharmaceuticals, Inc. Linkage modified gapped oligomeric compounds and uses thereof
WO2013149250A1 (en) 2012-03-30 2013-10-03 The Regents Of The University Of California Methods of treatment, diagnosis and monitoring for methamphetamine toxicity which target ceramide metabolic pathways and cellular senescence
WO2013154798A1 (en) 2012-04-09 2013-10-17 Isis Pharmaceuticals, Inc. Tricyclic nucleic acid analogs
WO2014076195A1 (en) 2012-11-15 2014-05-22 Santaris Pharma A/S Oligonucleotide conjugates
WO2015113922A1 (en) 2014-01-30 2015-08-06 Roche Innovation Center Copenhagen A/S Poly oligomer compound with biocleavable conjugates
WO2017153753A1 (en) * 2016-03-11 2017-09-14 Ucl Business Plc Allele-specific gene suppression

Non-Patent Citations (29)

* Cited by examiner, † Cited by third party
Title
"Remington's Pharmaceutical Sciences", 1985, MACK PUBLISHING COMPANY
ANSEL: "Pharmaceutical Dosage Forms and Drug Delivery Systems", 1995, pages: 196,1456 - 1457
AUER-GRUMBACH ET AL., EUR J MED GENET, vol. 56, no. 5, May 2013 (2013-05-01), pages 266 - 269
BASTIN, ORGANIC PROCESS RESEARCH & DEVELOPMENT, vol. 4, 2000, pages 427 - 435
BERGSTROM, CURRENT PROTOCOLS IN NUCLEIC ACID CHEMISTRY, 2009
CARUTHERS ET AL., METHODS IN ENZYMOLOGY, vol. 154, 1987, pages 287 - 313
DELEAVEYDAMHA, CHEMISTRY AND BIOLOGY, vol. 19, 2012, pages 937
FLUITER ET AL., MOL. BIOSYST., vol. 10, 2009, pages 1039
FREIERALTMANN, NUCL. ACID RES., vol. 25, 1997, pages 4429 - 4443
GASTROENTEROLOGY, vol. 145, no. 4, 2013, pages 865 - 73
HANSEN ET AL., CHEM. COMM., 1965, pages 36 - 38
HIRAO ET AL., ACCOUNTS OF CHEMICAL RESEARCH, vol. 45, 2012, pages 2055
HOLDGATE ET AL., DRUG DISCOV TODAY, 2005
LANGER, SCIENCE, vol. 249, 1990, pages 1527 - 1533
LEE ET AL., CANCER BIOLOGY & THERAPY, vol. 13, no. 2, 2002, pages 92 - 100
MANGOS ET AL., J. AM. CHEM. SOC., vol. 125, 2003, pages 654 - 661
MCTIGUE ET AL., BIOCHEMISTRY, vol. 43, 2004, pages 5388 - 5405
MERGNYLACROIX, OLIGONUCLEOTIDES, vol. 13, 2003, pages 515 - 537
MITSUOKA ET AL., NUCLEIC ACIDS RESEARCH, vol. 37, no. 4, 2009, pages 1225 - 1238
MORITA ET AL., BIOORGANIC & MED.CHEM. LETT., vol. 12, pages 73 - 76
PERRY ET AL., J. BIOL. CHEM., vol. 275, 2002, pages 9078 - 9084
RUKOV ET AL., NUCL. ACIDS RES., vol. 43, 2015, pages 8476 - 8487
SANTALUCIA, PROC NATL ACAD SCI USA., vol. 95, 1998, pages 1460 - 1465
SETH ET AL., J. ORG. CHEM., vol. 75, no. 5, 2010, pages 1569 - 81
SUGIMOTO ET AL., BIOCHEMISTRY, vol. 34, 1995, pages 11211 - 11216
UHLMANN, CURR. OPINION IN DRUG DEVELOPMENT, vol. 3, no. 2, 2000, pages 293 - 213
VESTER ET AL., BIOORG. MED. CHEM. LETT., vol. 18, 2008, pages 2296 - 2300
WANSETH, J. MEDICAL CHEMISTRY, vol. 59, 2016, pages 9645 - 9667
YAGUSHI, BIOCHEM BIOPHYS RES COMMUN., vol. 484, no. 3, 2017, pages 493 - 500

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12391947B2 (en) * 2018-11-29 2025-08-19 University Of Massachusetts Modulation of SPTLC1 via recombinant adeno-associated vectors
WO2024187097A3 (en) * 2023-03-09 2024-11-07 Leal Therapeutics, Inc. Compositions and methods for modulating sptlc1

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