WO2015054579A1 - Treatment of acute kidney injury with phd1 modulators - Google Patents

Treatment of acute kidney injury with phd1 modulators Download PDF

Info

Publication number
WO2015054579A1
WO2015054579A1 PCT/US2014/060044 US2014060044W WO2015054579A1 WO 2015054579 A1 WO2015054579 A1 WO 2015054579A1 US 2014060044 W US2014060044 W US 2014060044W WO 2015054579 A1 WO2015054579 A1 WO 2015054579A1
Authority
WO
WIPO (PCT)
Prior art keywords
modified
certain embodiments
specific inhibitor
phdl
subject
Prior art date
Application number
PCT/US2014/060044
Other languages
French (fr)
Inventor
Gene Hung
Original Assignee
Isis Pharmaceuticals, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Isis Pharmaceuticals, Inc. filed Critical Isis Pharmaceuticals, Inc.
Publication of WO2015054579A1 publication Critical patent/WO2015054579A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/11—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)
    • C12Y114/11029—Hypoxia-inducible factor-proline dioxygenase (1.14.11.29)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C12N2310/3341—5-Methylcytosine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications

Definitions

  • the present embodiments provide methods, compounds, and compositions for treating, preventing, or ameliorating acute kidney injury (AKI) by administering a PHD1 specific inhibitor to a subject.
  • AKI acute kidney injury
  • AKI Acute kidney injury
  • AKI is characterized by rapid reduction of kidney function and can be caused by decreased blood flow to the kidney resulting from low blood volume, low blood pressure, heart failure, or local changes to the blood vessels supplying the kidney.
  • Renal ischemia refers to blood deficiency in the kidney and can lead to reduction of glomerular filtration rate (GFR), an indication of kidney function and health. Renal ischemia can be caused by hypoperfusion stemming from reduced cardiac output. For instance, cardiac surgery mediated hypoperfusion is associated with increased morbidity and mortality from AKI.
  • PHDs Prolyl hydroxylases
  • HIFs hypoxia-inducible factors
  • Certain embodiments provided herein relate to methods, compounds, and compositions for treating, preventing, or ameliorating acute kidney injury (AKI) in a subject comprising administering to the subject a specific inhibitor of prolyl hydroxylase 1 (PHD1), thereby treating, preventing, or ameliorating the acute kidney injury.
  • the acute kidney injury can be caused by hypoxia and/or renal ischemia.
  • the renal ischemia is caused by cardiac-mediated hypoperfusion, which in certain embodiments can be caused by cardiac surgery.
  • Certain embodiments relate to methods, compounds, and compositions for improving renal function and/or pathology in a subject having or at risk of having AKI by administering a specific inhibitor of PHD 1.
  • the specific inhibitor of PHD1 is an antisense compound, such as a single- stranded modified oligonucleotide targeted to PHD1.
  • antisense compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase.
  • Antisense compounds described by ISIS number indicate a combination of nucleobase sequence, chemical modification, and motif. Unless otherwise indicated, the following terms have the following meanings:
  • 2'-0-methoxyethyl refers to an O-methoxy-ethyl modification at the 2' position of a sugar ring, e.g. a furanose ring.
  • a 2'-0-methoxyethyl modified sugar is a modified sugar.
  • 2'-MOE nucleoside (also 2'-0-methoxyethyl nucleoside) means a nucleoside comprising a 2'-
  • 2 '-substituted nucleoside means a nucleoside comprising a substituent at the 2 '-position of the furanosyl ring other than H or OH.
  • 2' substituted nucleosides include nucleosides with bicyclic sugar modifications.
  • 3' target site refers to the nucleotide of a target nucleic acid which is complementary to the 3'- most nucleotide of a particular antisense compound.
  • 5' target site refers to the nucleotide of a target nucleic acid which is complementary to the 5'- most nucleotide of a particular antisense compound.
  • 5-methylcytosine means a cytosine modified with a methyl group attached to the 5 position.
  • a 5-methylcytosine is a modified nucleobase.
  • “About” means within ⁇ 10% of a value. For example, if it is stated, “the compounds affected at least about 70% inhibition of PHD1", it is implied that PHD1 levels are inhibited within a range of 60%> and 80%.
  • administering refers to routes of introducing an antisense compound provided herein to a subject to perform its intended function.
  • routes of administration includes, but is not limited to parenteral administration, such as subcutaneous, intravenous, or intramuscular injection or infusion.
  • “Amelioration” refers to a lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition.
  • amelioration includes a delay or slowing in the progression of one or more indicators of a condition or disease.
  • the severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art.
  • Animal refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.
  • Antisense activity means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.
  • Antisense compound means an oligomeric compound that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
  • antisense compounds include single- stranded and double- stranded compounds, such as, antisense oligonucleotides, siRNAs, shRNAs, ssRNAs, and occupancy-based compounds.
  • Antisense inhibition means reduction of target nucleic acid levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels in the absence of the antisense compound.
  • Antisense mechanisms are all those mechanisms involving hybridization of a compound with target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing.
  • Antisense oligonucleotide means a single- stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.
  • Base complementarity refers to the capacity for the precise base pairing of nucleobases of an antisense oligonucleotide with corresponding nucleobases in a target nucleic acid (i.e., hybridization), and is mediated by Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen binding between corresponding nucleobases.
  • Bocyclic sugar moiety means a modified sugar moiety comprising a 4 to 7 membered ring
  • the 4 to 7 membered ring is a sugar ring.
  • the 4 to 7 membered ring is a furanosyl.
  • the bridge connects the 2'-carbon and the 4'-carbon of the furanosyl.
  • Cap structure or "terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an antisense compound.
  • cEt or "constrained ethyl” means a bicyclic sugar moiety comprising a bridge connecting the 4'-carbon and the 2'-carbon, wherein the bridge has the formula: 4'-CH(CH 3 )-0-2'.
  • Consstrained ethyl nucleoside (also cEt nucleoside) means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH 3 )-0-2' bridge.
  • PHD1 means any nucleic acid or protein of PHD1.
  • PHD1 nucleic acid means any nucleic acid encoding PHD1.
  • a PHD1 nucleic acid includes a DNA sequence encoding PHD1, an RNA sequence transcribed from DNA encoding PHD1 (including genomic DNA comprising introns and exons), including a non-protein encoding (i.e. non-coding) RNA sequence, and an mRNA sequence encoding PHD1.
  • PHD1 mRNA means an mRNA encoding a PHD1 protein.
  • PHD1 specific inhibitor refers to any agent capable of specifically inhibiting PHD1 RNA and/or PHD1 protein expression or activity at the molecular level.
  • PHD1 specific inhibitors include nucleic acids (including antisense compounds), peptides, antibodies, small molecules, and other agents capable of inhibiting the expression of PHD1 RNA and/or PHD1 protein.
  • “Chemically distinct region” refers to a region of an antisense compound that is in some way chemically different than another region of the same antisense compound. For example, a region having 2'-0-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2'-0- methoxyethyl modifications.
  • Chimeric antisense compounds means antisense compounds that have at least 2 chemically distinct regions, each position having a plurality of subunits.
  • “Complementarity” means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.
  • Contiguous nucleobases means nucleobases immediately adjacent to each other.
  • Deoxyribonucleotide means a nucleotide having a hydrogen at the 2' position of the sugar portion of the nucleotide. Deoxyribonucleotides may be modified with any of a variety of substituents.
  • Designing or “Designed to” refer to the process of designing an oligomeric compound that specifically hybridizes with a selected nucleic acid molecule.
  • Effective amount means 1he amount of active pharmaceutical agent sufficient to effectuate a desired physiological outcome in an individual in need of the agent.
  • the effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.
  • “Efficacy” means the ability to produce a desired effect. “Expression” includes all the functions by which a gene's coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to the products of transcription and translation.
  • “Fully complementary” or “100% complementary” means each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid.
  • a first nucleic acid is an antisense compound and a target nucleic acid is a second nucleic acid.
  • Gapmer means a chimeric antisense compound in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions.
  • the internal region may be referred to as the "gap” and the external regions may be referred to as the "wings.”
  • Hybridization means the annealing of complementary nucleic acid molecules.
  • complementary nucleic acid molecules include, but are not limited to, an antisense compound and a nucleic acid target.
  • complementary nucleic acid molecules include, but are not limited to, an antisense oligonucleotide and a nucleic acid target.
  • Identifying an animal having, or at risk for having, a disease, disorder and/or condition means identifying an animal having been diagnosed with the disease, disorder and/or condition or identifying an animal predisposed to develop the disease, disorder and/or condition. Such identification may be accomplished by any method including evaluating an individual's medical history and standard clinical tests or assessments.
  • “Individual” means a human or non-human animal selected for treatment or therapy.
  • “Inhibiting the expression or activity” refers to a reduction, blockade of the expression or activity and does not necessarily indicate a total elimination of expression or activity.
  • Internucleoside linkage refers to the chemical bond between nucleosides.
  • Lengthened antisense oligonucleotides are those that have one or more additional nucleosides relative to an antisense oligonucleotide disclosed herein.
  • Linked deoxynucleoside means a nucleic acid base (A, G, C, T, U) substituted by deoxyribose linked by a phosphate ester to form a nucleotide.
  • Linked nucleosides means adjacent nucleosides linked together by an internucleoside linkage.
  • mis or non-complementary nucleobase refers to the case when a nucleobase of a first nucleic acid is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid.
  • Modified internucleoside linkage refers to a substitution or any change from a naturally occurring internucleoside bond (i.e. a phosphodiester internucleoside bond).
  • Modified nucleobase means any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil.
  • An "unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleoside means a nucleoside having, independently, a modified sugar moiety and/or modified nucleobase.
  • Modified nucleotide means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, or modified nucleobase.
  • Modified oligonucleotide means an oligonucleotide comprising at least one modified internucleoside linkage, a modified sugar, and/or a modified nucleobase.
  • Modified sugar means substitution and/or any change from a natural sugar moiety.
  • Modulating refers to changing or adjusting a feature in a cell, tissue, organ or organism.
  • modulating PHD1 mRNA can mean to increase or decrease the level of PHD 1 mRNA and/or PHD1 protein in a cell, tissue, organ or organism.
  • a “modulator” effects the change in the cell, tissue, organ or organism.
  • a PHD1 antisense compound can be a modulator that decreases the amount of PHD 1 mRNA and/or PHD1 protein in a cell, tissue, organ or organism.
  • “Monomer” refers to a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occuring or modified.
  • Microtif means the pattern of unmodified and modified nucleosides in an antisense compound.
  • Natural sugar moiety means a sugar moiety found in DNA (2'-H) or RNA (2'-OH).
  • Naturally occurring internucleoside linkage means a 3' to 5' phosphodiester linkage.
  • Non-complementary nucleobase refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.
  • Nucleic acid refers to molecules composed of monomeric nucleotides.
  • a nucleic acid includes, but is not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single- stranded nucleic acids, and double- stranded nucleic acids.
  • RNA ribonucleic acids
  • DNA deoxyribonucleic acids
  • Single- stranded nucleic acids single- stranded nucleic acids
  • double- stranded nucleic acids double- stranded nucleic acids.
  • Nucleobase means a heterocyclic moiety capable of pairing with a base of another nucleic acid.
  • Nucleobase complementarity refers to a nucleobase that is capable of base pairing with another nucleobase.
  • adenine (A) is complementary to thymine (T).
  • adenine (A) is complementary to uracil (IS).
  • complementary nucleobase refers to a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid.
  • nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid
  • the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair.
  • Nucleobase sequence means the order of contiguous nucleobases independent of any sugar, linkage, and/or nucleobase modification.
  • Nucleoside means a nucleobase linked to a sugar.
  • Nucleoside mimetic includes those structures used to replace the sugar or the sugar and the base and not necessarily the linkage at one or more positions of an oligomeric compound such as for example nucleoside mimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics, e.g., non furanose sugar units.
  • Sugar surrogate overlaps with the slightly broader term nucleoside mimetic but is intended to indicate replacement of the sugar unit (furanose ring) only.
  • the tetrahydropyranyl rings provided herein are illustrative of an example of a sugar surrogate wherein the furanose sugar group has been replaced with a tetrahydropyranyl ring system.
  • Mimetic refers to groups that are substituted for a sugar, a nucleobase, and/ or internucleoside linkage. Generally, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target.
  • Nucleotide means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.
  • Olemeric compound means a polymer of linked monomeric subunits which is capable of hybridizing to at least a region of a nucleic acid molecule.
  • Oligonucleoside means an oligonucleotide in which the internucleoside linkages do not contain a phosphorus atom.
  • Oligonucleotide means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another.
  • Parenteral administration means administration through injection or infusion.
  • Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g. intrathecal or intracerebroventricular administration.
  • “Pharmaceutical composition” means a mixture of substances suitable for administering to an individual.
  • a pharmaceutical composition may comprise one or more active pharmaceutical agents and a sterile aqueous solution.
  • “Pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.
  • Phosphorothioate linkage means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom.
  • a phosphorothioate linkage is a modified internucleoside linkage.
  • Portion means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound
  • Prevent refers to delaying or forestalling the onset, development or progression of a disease, disorder, or condition for a period of time from minutes to indefinitely. Prevent also means reducing the risk of developing a disease, disorder, or condition.
  • “Prophylactically effective amount” refers to an amount of a pharmaceutical agent that provides a prophylactic or preventative benefit to an animal.
  • Regular is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.
  • “Ribonucleotide” means a nucleotide having a hydroxy at the 2' position of the sugar portion of the nucleotide. Ribonucleotides may be modified with any of a variety of substituents.
  • “Segments” are defined as smaller or sub-portions of regions within a target nucleic acid.
  • Side effects means physiological disease and/or conditions attributable to a treatment other than the desired effects.
  • side effects include injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, myopathies, and malaise.
  • increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality.
  • increased bilirubin may indicate liver toxicity or liver function abnormality.
  • Sites are defined as unique nucleobase positions within a target nucleic acid. “Slows progression” means decrease in the development of the said disease.
  • Specifically hybridizable refers to an antisense compound having a sufficient degree of complementarity between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays and therapeutic treatments.
  • Stringent hybridization conditions or “stringent conditions” refer to conditions under which an oligomeric compound will hybridize to its target sequence, but to a minimal number of other sequences.
  • Subject means a human or non-human animal selected for treatment or therapy.
  • Target refers to a protein, the modulation of which is desired.
  • Target gene refers to a gene encoding a target.
  • 'Targeting means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.
  • 'Target nucleic acid means a nucleic acid capable of being targeted by antisense compounds.
  • 'Target region means a portion of a target nucleic acid to which one or more antisense compounds is targeted.
  • 'Target segment means the sequence of nucleotides of a target nucleic acid to which an antisense compound is targeted.
  • 5' target site refers to the 5'-most nucleotide of a target segment.
  • 3' target site refers to the 3 '-most nucleotide of a target segment.
  • 'Therapeutically effective amount means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual.
  • 'Treat refers to administering a pharmaceutical composition to an animal in order to effect an alteration or improvement of a disease, disorder, or condition in the animal.
  • one or more pharmaceutical compositions can be administered to the animal.
  • "Unmodified" nucleobases mean the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Unmodified nucleotide means a nucleotide composed of naturally occuring nucleobases, sugar moieties, and internucleoside linkages.
  • an unmodified nucleotide is an RNA nucleotide (i.e. ⁇ -D-ribonucleosides) or a DNA nucleotide (i.e. ⁇ -D-deoxyribonucleoside).
  • a method of treating, preventing, or ameliorating acute kidney injury (AKI) in a subject comprisies administering to the subject a specific inhibitor of prolyl hydroxylase 1 (PHD1), thereby treating, preventing, or ameliorating the acute kidney injury.
  • the subject's kidney is hypoxic.
  • the acute kidney injury is caused by renal ischemia.
  • the renal ischemia is caused by cardiac-mediated hypoperfusion.
  • the cardiac-mediated hypoperfusion is caused by cardiac surgery, such as bypass surgery, coronary artery bypass grafting, valvular surgery, or combined coronary artery bypass grafting and valvular surgery.
  • administering the specific inhibitor of PHD 1 improves renal function. In certain embodiments, administering the specific inhibitor of PHD 1 improves tubular epithelial cell injury in the subject. In certain embodiments, administering the specific inhibitor of PHD 1 improves glomerular filtration rate (GFR). In certain embodiments, administering the specific inhibitor of PHD 1 decreases levels of blood urea nitrogen (BUN) and/or creatinine, which are markers of renal function that increase with impaired renal function. In certain embodiments, administering the specific inhibitor of PHD1 decreases levels of neutrophil gelatinase-associated lipocalin (NGAL) and/or kidney injury molecule 1 (KIM-1), which are biomarkers whose levels positively correlate with AKI.
  • BUN blood urea nitrogen
  • KIM-1 kidney injury molecule 1
  • a method of inhibiting expression of PHD 1 in a subject having, or at risk of having, acute kidney injury comprises administering a specific inhibitor of PHD 1 to the subject, thereby inhibiting expression of PHD1 in the subject.
  • administering the inhibitor reduces expression of PHD1 in the kidney.
  • the subject's kidney is hypoxic.
  • the acute kidney injury is caused by renal ischemia.
  • the renal ischemia is caused by cardiac-mediated hypoperfusion.
  • the cardiac- mediated hypoperfusion is caused by cardiac surgery, such as bypass surgery, coronary artery bypass grafting, valvular surgery, or combined coronary artery bypass grafting and valvular surgery.
  • the renal ischemia is caused by gastric bypass surgery, orthotropic liver transplant, abdominal aortic aneurysm repair, contrast agent induced hypoxia, or drug induced hypoxia.
  • reducing expression of PHDl in the kidney by administering the specific inhibitor of PHDl improves renal function.
  • reducing expression of PHDl in the kidney by administering the specific inhibitor of PHDl improves tubular epithelial cell injury in the subject.
  • reducing expression of PHDl in the kidney by administering the specific inhibitor of PHDl improves glomerular filtration rate (GFR).
  • GFR glomerular filtration rate
  • reducing expression of PHDl in the kidney by administering the specific inhibitor of PHDl decreases levels of blood urea nitrogen (BUN) and/or creatinine. In certain embodiments, reducing expression of PHDl in the kidney by administering the specific inhibitor of PHDl decreases levels of neutrophil gelatinase-associated lipocalin (NGAL) and/or kidney injury molecule 1 (KIM-1).
  • BUN blood urea nitrogen
  • KIM-1 kidney injury molecule 1
  • a method of reducing or inhibiting tubular epithelial cell injury in the kidney of a subject having, or at risk of having, acute kidney injury comprises administering a specific inhibitor of PHDl to the subject, thereby reducing or inhibiting tubular epithelial cell injury in the kidney of the subject.
  • the subject's kidney is hypoxic.
  • the acute kidney injury is caused by renal ischemia.
  • the renal ischemia is caused by cardiac-mediated hypoperfusion.
  • the cardiac-mediated hypoperfusion is caused by cardiac surgery, such as bypass surgery, coronary artery bypass grafting, valvular surgery, or combined coronary artery bypass grafting and valvular surgery.
  • the renal ischemia is caused by gastric bypass surgery, orthotropic liver transplant, abdominal aortic aneurysm repair, contrast agent induced hypoxia, or drug induced hypoxia.
  • a method of preventing or protecting a subject from acute kidney injury comprises administering a specific inhibitor of PHDl to the subject prior to the subject undergoing cardiac surgery. In certain embodiments, a method of preventing or protecting a subject from acute kidney injury comprises administering a specific inhibitor of PHDl to the subject prior to performing cardiac surgery on the patient. In certain embodiments, a method of preventing or protecting a subject from acute kidney injury comprises identifying a subject in need of cardiac surgery and administering a specific inhibitor of PHDl to the subject prior to the subject undergoing cardiac surgery. In certain embodiments, the cardiac surgery is bypass surgery, coronary artery bypass grafting, valvular surgery, or combined coronary artery bypass grafting and valvular surgery.
  • administering the specific inhibitor of PHDl to the subject prior to the subject undergoing cardiac surgery is sufficient to prevent acute kidney injury, protect against acute kidney injury, minimize acute kidney injury, or reduce acute kidney injury in the subject following cardiac surgery.
  • the specific inhibitor of PHD l is administered multiple times prior to the subject undergoing cardiac surgery.
  • a method of preventing or protecting a subject from acute kidney injury comprises administering a specific inhibitor of PHDl to the subject prior to the subject undergoing a surgery or operation that causes renal ischemia. In certain embodiments, a method of preventing or protecting a subject from acute kidney injury comprises administering a specific inhibitor of PHD l to the subject prior to performing surgery or operation that causes renal ischemia on the patient. In certain embodiments, a method of preventing or protecting a subject from acute kidney injury comprises identifying a subject in need of surgery or operation and administering a specific inhibitor of PHDl to the subject prior to the subject undergoing surgery or operation. In certain embodiments, the surgery or operation is gastric bypass surgery, orthotopic liver transplant, or abdominal aortic aneurysm repair.
  • administering the specific inhibitor of PHD l to the subject prior to the subject undergoing surgery or operation is sufficient to prevent acute kidney injury, protect against acute kidney injury, minimize acute kidney injury, or reduce acute kidney injury in the subject following surgery or operation.
  • the specific inhibitor of PHD l is administered multiple times prior to the subject undergoing surgery or operation.
  • a method of preventing or protecting a subject from acute kidney injury comprises administering to the subject a specific inhibitor of PHD l prior to administering a drug that induces ischemia.
  • a method of preventing or protecting a subject from acute kidney injury comprises identifying a subject in need of a drug known to induce ischemia and administering a specific inhibitor of PHD l to the subject prior to administering the drug known to induce ischemia.
  • administering the specific inhibitor of PHDl to the subject prior to administering a drug known to induce ischemia is sufficient to prevent acute kidney injury, protect against acute kidney injury, minimize acute kidney injury, or reduce acute kidney injury in the subject following administration of the drug known to induce ischemia.
  • the specific inhibitor of PHDl is administered multiple times prior to administering the drug known to induce ischemia.
  • drugs that induce ischemia include cyclosporine A and its structural analogues; angiotensin converting enzyme (ACE) inhibitors, such as, but not limited to, Benazepril, Captopril, Enalapril, Fosinopril, Lisinopril, Moexipril, Perindopril, Quinapril, Ramipril, and Trandolapril; and non-steroidal anti-inflammatory drugs (NSAIDs), such as, but not limited to, Aspirin (acetylsalicylic acid), Diflunisal, Salsalate, Ibuprofen, Dexibuprofen, Naproxen, Fenoprofen, Ketoprofen, Dexketoprofen, Flurbiprofen, Oxaprozin, Loxoprofen, Indome
  • ACE angiotensin converting enzyme
  • the acute kidney injury is caused by hypoxia.
  • the acute kidney injury is caused by renal ischemia.
  • the renal ischemia is caused by cardiac-mediated hypoperfusion.
  • the cardiac-mediated hypoperfusion is caused by cardiac surgery, such as bypass surgery, coronary artery bypass grafting, valvular surgery, or combined coronary artery bypass grafting and valvular surgery.
  • Certain embodiments provide a use of a PHD1 specific inhibitor for the preparation of a medicament for treating acute kidney injury.
  • the acute kidney injury is caused by hypoxia.
  • the acute kidney injury is caused by renal ischemia.
  • the renal ischemia is caused by cardiac-mediated hypoperfusion.
  • the cardiac-mediated hypoperfusion is caused by cardiac surgery, such as bypass surgery, coronary artery bypass grafting, valvular surgery, or combined coronary artery bypass grafting and valvular surgery.
  • the inhibitor is an antisense compound targeted to PHD1.
  • the antisense compound comprises at least one modified sugar, such as a 2'-0-methoxyethyl group or a bicyclic sugar, such as 4'-CH(CH 3 )-0-2', 4'-CH 2 -0-2' or 4'-(CH 2 ) 2 - 0-2'.
  • the antisense compound comprises at least one modified internucleoside linkage, such as a phosphorothioate internucleoside linkage.
  • the antisense compound comprises at least one modified nucleobase, such as a 5-methylcytosine.
  • the antisense compound is a single- stranded modified oligonucleotide.
  • the modified oligonucleotide comprises a gap segment consisting of linked deoxynucleosides; a 5' wing segment consisting of linked nucleosides; and a 3' wing segment consisting of linked nucleosides; wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
  • the PHD1 specific inhibitor is an antisense compound comprising a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 7-59.
  • the antisense compound can be a single- stranded oligonucleotide.
  • Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleo sides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs.
  • An oligomeric compound may be "antisense" to a target nucleic acid, meaning that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
  • an antisense compound has a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.
  • an antisense oligonucleotide has a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.
  • an antisense compound is 10 to 30 subunits in length. In certain embodiments, an antisense compound is 12 to 30 subunits in length. In certain embodiments, an antisense compound is 12 to 22 subunits in length. In certain embodiments, an antisense compound is 14 to 30 subunits in length. In certain embodiments, an antisense compound is 14 to 20 subunits in length. In certain embodiments, an antisense compoun is 15 to 30 subunits in length. In certain embodiments, an antisense compound is 15 to 20 subunits in length. In certain embodiments, an antisense compound is 16 to 30 subunits in length. In certain embodiments, an antisense compound is 16 to 20 subunits in length.
  • an antisense compound is 17 to 30 subunits in length. In certain embodiments, an antisense compound is 17 to 20 subunits in length. In certain embodiments, an antisense compound is 18 to 30 subunits in length. In certain embodiments, an antisense compound is 18 to 21 subunits in length. In certain embodiments, an antisense compound is 18 to 20 subunits in length. In certain embodiments, an antisense compound is 20 to 30 subunits in length.
  • antisense compounds are from 12 to 30 linked subunits, 14 to 30 linked subunits, 14 to 20 subunits, 15 to 30 subunits, 15 to 20 subunits, 16 to 30 subunits, 16 to 20 subunits, 17 to 30 subunits, 17 to 20 subunits, 18 to 30 subunits, 18 to 20 subunits, 18 to 21 subunits, 20 to 30 subunits, or 12 to 22 linked subunits, respectively.
  • an antisense compound is 14 subunits in length.
  • an antisense compound is 16 subunits in length.
  • an antisense compound is 17 subunits in length.
  • an antisense compound is 18 subunits in length.
  • an antisense compound is 19 subunits in length. In certain embodiments, an antisense compound is 20 subunits in length. In other embodiments, the antisense compound is 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to 30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linked subunits.
  • the antisense compounds are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a range defined by any two of the above values.
  • the antisense compound is an antisense oligonucleotide, and the linked subunits are nucleotides.
  • antisense oligonucleotides may be shortened or truncated. For example, a single subunit may be deleted from the 5' end (5' truncation), or alternatively from the 3' end (3' truncation).
  • a shortened or truncated antisense compound targeted to an PHD1 nucleic acid may have two subunits deleted from the 5' end, or alternatively may have two subunits deleted from the 3' end, of the antisense compound.
  • the deleted nucleosides may be dispersed throughout the antisense compound, for example, in an antisense compound having one nucleoside deleted from the 5' end and one nucleoside deleted from the 3' end.
  • the additional subunit may be located at the 5' or 3' end of the antisense compound.
  • the added subunits may be adjacent to each other, for example, in an antisense compound having two subunits added to the 5' end (5' addition), or alternatively to the 3' end (3' addition), of the antisense compound.
  • the added subunits may be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit added to the 5' end and one subunit added to the 3' end.
  • an antisense compound such as an antisense oligonucleotide
  • an antisense oligonucleotide it is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity.
  • an antisense compound such as an antisense oligonucleotide
  • a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model.
  • Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.
  • Gautschi et al. demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl- xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti- tumor activity in vivo.
  • antisense compounds have chemically modified subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.
  • Chimeric antisense compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased inhibitory activity.
  • a second region of a chimeric antisense compound may confer another desired property e.g., serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.
  • Antisense activity may result from any mechanism involving the hybridization of the antisense compound (e.g., oligonucleotide) with a target nucleic acid, wherein the hybridization ultimately results in a biological effect.
  • the amount and/or activity of the target nucleic acid is modulated.
  • the amount and/or activity of the target nucleic acid is reduced.
  • hybridization of the antisense compound to the target nucleic acid ultimately results in target nucleic acid degradation.
  • hybridization of the antisense compound to the target nucleic acid does not result in target nucleic acid degradation.
  • the presence of the antisense compound hybridized with the target nucleic acid results in a modulation of antisense activity.
  • antisense compounds having a particular chemical motif or pattern of chemical modifications are particularly suited to exploit one or more mechanisms.
  • antisense compounds function through more than one mechanism and/or through mechanisms that have not been elucidated. Accordingly, the antisense compounds described herein are not limited by particular mechanism.
  • Antisense mechanisms include, without limitation, RNase H mediated antisense; RNAi mechanisms, which utilize the RISC pathway and include, without limitation, siRNA, ssRNA and microRNA mechanisms; and occupancy based mechanisms. Certain antisense compounds may act through more than one such mechanism and/or through additional mechanisms.
  • antisense activity results at least in part from degradation of target RNA by RNase H.
  • RNase H is a cellular endonuclease that cleaves the RNA strand of an RNADNA duplex. It is known in the art that single- stranded antisense compounds which are "DNA-like" elicit RNase H activity in mammalian cells. Accordingly, antisense compounds comprising at least a portion of DNA or DNA-like nucleosides may activate RNase H, resulting in cleavage of the target nucleic acid.
  • antisense compounds that utilize RNase H comprise one or more modified nucleosides. In certain embodiments, such antisense compounds comprise at least one block of 1-8 modified nucleosides.
  • the modified nucleosides do not support RNase H activity.
  • such antisense compounds are gapmers, as described herein.
  • the gap of the gapmer comprises DNA nucleosides.
  • the gap of the gapmer comprises DNA-like nucleosides.
  • the gap of the gapmer comprises DNA nucleosides and DNA-like nucleosides.
  • Certain antisense compounds having a gapmer motif are considered chimeric antisense compounds.
  • a gapmer an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region.
  • the gap segment In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides.
  • the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region.
  • sugar moieties that are used to differentiate the regions of a gapmer may in some embodiments include ⁇ -D- ribonucleosides, ⁇ -D-deoxyribonucleosides, 2'-modified nucleosides (such 2'-modified nucleosides may include 2'-MOE and 2'-0-CH 3 , among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a constrained ethyl).
  • nucleosides in the wings may include several modified sugar moieties, including, for example 2'-MOE and bicyclic sugar moieties such as constrained ethyl or LNA.
  • wings may include several modified and unmodified sugar moieties.
  • wings may include various combinations of 2'-MOE nucleosides, bicyclic sugar moieties such as constrained ethyl nucleosides or LNA nucleosides, and 2'-deoxynucleosides.
  • Each distinct region may comprise uniform sugar moieties, variant, or alternating sugar moieties.
  • the wing-gap-wing motif is frequently described as "X-Y-Z", where "X” represents the length of the 5'-wing, ' ⁇ " represents the length of the gap, and “Z” represents the length of the 3'-wing.
  • "X” and “Z” may comprise uniform, variant, or alternating sugar moieties.
  • "X" and "Y” may include one or more 2'-deoxynucleosides.”Y” may comprise 2'-deoxynucleosides.
  • a gapmer described as "X-Y-Z” has a configuration such that the gap is positioned immediately adjacent to each of the 5'-wing and the 3 ' wing. Thus, no intervening nucleotides exist between the 5'- wing and gap, or the gap and the 3 '-wing.
  • Any of the antisense compounds described herein can have a gapmer motif.
  • "X” and “Z” are the same; in other embodiments they are different.
  • ' ⁇ " is between 8 and 15 nucleosides.
  • X, Y, or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleosides.
  • the antisense compound targeted to a PHD1 nucleic acid has a gapmer motif in which the gap consists of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 linked nucleosides.
  • the antisense oligonucleotide has a sugar motif described by Formula A as follows: (J) m -(B) n -(J) p -(B) r -(A) t -(D) g -(A) v -(B) w -(J) x -(B) y -(J) z
  • each A is independently a 2'-substituted nucleoside
  • each B is independently a bicyclic nucleoside
  • each J is independently either a 2'-substituted nucleoside or a 2'-deoxynucleoside;
  • each D is a 2'-deoxynucleoside
  • At least one of m, n, and r is other than 0;
  • At least one of w and y is other than 0;
  • antisense compounds are interfering RNA compounds (RNAi), which include double- stranded RNA compounds (also referred to as short-interfering RNA or siRNA) and single- stranded RNAi compounds (or ssRNA). Such compounds work at least in part through the RISC pathway to degrade and/or sequester a target nucleic acid (thus, include microRNA/microRNA-mimic compounds). In certain embodiments, antisense compounds comprise modifications that make them particularly suited for such mechanisms.
  • RNAi interfering RNA compounds
  • siRNA double- stranded RNA compounds
  • ssRNAi compounds single- stranded RNAi compounds
  • antisense compounds including those particularly suited for use as single- stranded RNAi compounds (ssRNA) comprise a modified 5'-terminal end.
  • the 5'-terminal end comprises a modified phosphate moiety.
  • such modified phosphate is stabilized (e.g., resistant to degradation/cleavage compared to unmodified 5'- phosphate).
  • such 5'-terminal nucleosides stabilize the 5 '-phosphorous moiety.
  • Certain modified 5'-terminal nucleosides may be found in the art, for example in WO/201 1/139702.
  • the 5'-nucleoside of an ssRNA compound has Formula lie:
  • Ti is an optionally protected phosphorus moiety
  • T 2 is an internucleoside linking group linking the compound of Formula lie to the oligomeric compound
  • A has one of the formula
  • Qi and Q 2 are each, independently, H, halogen, Ci-Ce alkyl, substituted Ci-Ce alkyl, d-Ce alkoxy, substituted Ci-Ce alkoxy, C 2 -C6 alkenyl, substituted C 2 -C6 alkenyl, C 2 -C6 alkynyl, substituted C 2 -C6 alkynyl or N(R 3 )(R4);
  • Q 3 is O, S, N(R 5 ) or C(R6)(R 7 );
  • each R 3 , R4 R 5 , R 6 and R 7 is, independently, H, C -C 6 alkyl, substituted C -C 6 alkyl or C -C 6 alkoxy;
  • Ri 4 is H, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci-Ce alkoxy, substituted Ci-Ce alkoxy, C 2 -C6 alkenyl, substituted C 2 -C6 alkenyl, C 2 -C6 alkynyl or substituted C 2 -C6 alkynyl;
  • Ri 5 , Ri 6 , Ri 7 and Ris are each, independently, H, halogen, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci- Ce alkoxy, substituted Ci-Ce alkoxy, C 2 -C6 alkenyl, substituted C 2 -C6 alkenyl, C 2 -C6 alkynyl or substituted C 2 -C 6 alkynyl;
  • Bx ! is a heterocyclic base moiety
  • Bx 2 is a heterocyclic base moiety and Bx ! is H, halogen, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci-Ce alkoxy, substituted Ci-Ce alkoxy, C 2 -C6 alkenyl, substituted C 2 -C6 alkenyl, C 2 -C6 alkynyl or substituted C 2 -C6 alkynyl;
  • each R 19 , R 20 and R 2 i is, independently, H, C 1 -C6 alkyl, substituted C 1 -C6 alkyl, C 1 -C6 alkoxy, substituted C 1 -C6 alkoxy, C 2 -C6 alkenyl, substituted C 2 -C6 alkenyl, C 2 -C6 alkynyl or substituted C 2 -C6 alkynyl;
  • G is H, OH, halogen or
  • each R 8 and R 9 is, independently, H, halogen, C 1 -C6 alkyl or substituted C 1 -C6 alkyl;
  • Xi is O, S or N(Ei);
  • Z is H, halogen, C 1 -C6 alkyl, substituted C 1 -C6 alkyl, C 2 -C6 alkenyl, substituted C 2 -C6 alkenyl, C 2 - Ce alkynyl, substituted C 2 -C6 alkynyl or N(E 2 )(E 3 ) ;
  • Ei, E 2 and E 3 are each, independently, H, C 1 -C6 alkyl or substituted C 1 -C6 alkyl;
  • n is from 1 to about 6;
  • n 0 or 1 ;
  • j is 0 or 1 ;
  • each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJi, N(Ji)(J 2 ), and
  • X 2 is O, S or NJ 3 ;
  • each Ji, J 2 and J 3 is, independently, H or C 1 -C6 alkyl
  • said oligomeric compound comprises from 8 to 40 monomeric subunits and is hybridizable to at least a portion of a target nucleic acid.
  • J 4 , J 5 , Je and J 7 are each H. In certain embodiments, J 4 forms a bridge with one of J 5 or J 7 .
  • Qi and (3 ⁇ 4 are each, independently, H, halogen, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci-Ce alkoxy or substituted Ci-Ce alkoxy.
  • Qi and Q 2 are each H.
  • Qi and Q 2 are each, independently, H or halogen.
  • Qi and Q 2 is H and the other of Qj and Q 2 is F, CH 3 or OCH 3 .
  • Ti has the formula:
  • R a and R c are each, independently, protected hydroxyl, protected thiol, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci-Ce alkoxy, substituted Ci-Ce alkoxy, protected amino or substituted amino; and
  • R b is O or S. In certain embodiments, R b is O and R a and R c are each, independently, OCH 3 ,
  • G is F, OCH 3 or 0(CH 2 ) 2 -OCH 3 .
  • G is 0(CH 2 ) 2 -OCH 3 .
  • antisense compounds including those particularly suitable for ssRNA comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar modification motif.
  • Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications.
  • the oligonucleotides comprise or consist of a region having uniform sugar modifications.
  • each nucleoside of the region comprises the same RNA-like sugar modification.
  • each nucleoside of the region is a 2'-F nucleoside.
  • each nucleoside of the region is a 2'-OMe nucleoside. In certain embodiments, each nucleoside of the region is a 2'-MOE nucleoside. In certain embodiments, each nucleoside of the region is a cEt nucleoside. In certain embodiments, each nucleoside of the region is an LNA nucleoside. In certain embodiments, the uniform region constitutes all or essentially all of the oligonucleotide. In certain embodiments, the region constitutes the entire oligonucleotide except for 1-4 terminal nucleosides.
  • oligonucleotides comprise one or more regions of alternating sugar modifications, wherein the nucleosides alternate between nucleotides having a sugar modification of a first type and nucleotides having a sugar modification of a second type.
  • nucleosides of both types are RNA-like nucleosides.
  • the alternating nucleosides are selected from: 2'-OMe, 2'-F, 2'-MOE, LNA, and cEt.
  • the alternating modificatios are 2'-F and 2'-OMe. Such regions may be contiguous or may be interupted by differently modified nucleosides or conjugated nucleosides.
  • the alternating region of alternating modifications each consist of a single nucleoside (i.e., the patern is (AB) x A y wheren A is a nucleoside having a sugar modification of a first type and B is a nucleoside having a sugar modification of a second type; x is 1-20 and y is 0 or 1).
  • one or more alternating regions in an alternating motif includes more than a single nucleoside of a type.
  • oligonucleotides may include one or more regions of any of the following nucleoside motifs:
  • A is a nucleoside of a first type and B is a nucleoside of a second type.
  • a and B are each selected from 2'-F, 2'-OMe, BNA, and MOE.
  • oligonucleotides having such an alternating motif also comprise a modified 5' terminal nucleoside, such as those of formula lie or He.
  • oligonucleotides comprise a region having a 2-2-3 motif. Such regions comprises the following motif:
  • A is a first type of modifed nucleosde
  • B and C are nucleosides that are differently modified than A, however, B and C may have the same or different modifications as one another;
  • x and y are from 1 to 15.
  • A is a 2'-OMe modified nucleoside.
  • B and C are both 2'-F modified nucleosides.
  • A is a 2'-OMe modified nucleoside and B and C are both 2'-F modified nucleosides.
  • oligonucleo sides have the following sugar motif:
  • Q is a nucleoside comprising a stabilized phosphate moiety.
  • Q is a nucleoside having Formula lie or He;
  • A is a first type of modifed nucleoside
  • B is a second type of modified nucleoside
  • D is a modified nucleoside comprising a modification different from the nucleoside adjacent to it. Thus, if y is 0, then D must be differently modified than B and if y is 1, then D must be differently modified than A. In certain embodiments, D differs from both A and B.
  • X is 5-15;
  • Y is O or 1;
  • Z is 0-4.
  • oligonucleo sides have the following sugar motif:
  • Q is a nucleoside comprising a stabilized phosphate moiety.
  • Q is a nucleoside having Formula lie or He;
  • A is a first type of modifed nucleoside
  • D is a modified nucleoside comprising a modification different from A.
  • X is 11-30;
  • Z is 0-4.
  • A, B, C, and D in the above motifs are selected from: 2'-OMe, 2'-F, 2'- MOE, LNA, and cEt.
  • D represents terminal nucleosides. In certain embodiments, such terminal nucleosides are not designed to hybridize to the target nucleic acid (though one or more might hybridize by chance).
  • the nucleobase of each D nucleoside is adenine, regardless of the identity of the nucleobase at the corresponding position of the target nucleic acid. In certain embodiments the nucleobase of each D nucleoside is thymine.
  • antisense compounds comprising those particularly suited for use as ssRNA comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif.
  • oligonucleotides comprise a region having an alternating internucleoside linkage motif.
  • oligonucleotides comprise a region of uniformly modified internucleoside linkages.
  • the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages.
  • the oligonucleotide is uniformly linked by phosphorothioate internucleoside linkages.
  • each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate.
  • each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.
  • the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages.
  • the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least one 12 consecutive phosphoro- thioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3' end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3' end of the oligonucleotide.
  • Oligonucleotides having any of the various sugar motifs described herein may have any linkage motif.
  • the oligonucleotides including but not limited to those described above, may have a linkage motif selected from non- limiting the table below:
  • antisense compounds are double- stranded RNAi compounds (siRNA).
  • siRNA double- stranded RNAi compounds
  • one or both strands may comprise any modification motif described above for ssRNA.
  • ssRNA compounds may be unmodified RNA.
  • siRNA compounds may comprise unmodified RNA nucleosides, but modified internucleoside linkages.
  • compositions comprising a first and a second oligomeric compound that are fully or at least partially hybridized to form a duplex region and further comprising a region that is complementary to and hybridizes to a nucleic acid target. It is suitable that such a composition comprise a first oligomeric compound that is an antisense strand having full or partial complementarity to a nucleic acid target and a second oligomeric compound that is a sense strand having one or more regions of complementarity to and forming at least one duplex region with the first oligomeric compound.
  • compositions of several embodiments modulate gene expression by hybridizing to a nucleic acid target resulting in loss of its normal function.
  • the target nucleic acid is PHD1.
  • the degradation of the targeted PHD1 is facilitated by an activated RISC complex that is formed with compositions of the invention.
  • compositions of the present invention are directed to double- stranded compositions wherein one of the strands is useful in, for example, influencing the preferential loading of the opposite strand into the RISC (or cleavage) complex.
  • the compositions are useful for targeting selected nucleic acid molecules and modulating the expression of one or more genes.
  • the compositions of the present invention hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA.
  • compositions of the present invention can be modified to fulfill a particular role in for example the siRNA pathway.
  • Using a different motif in each strand or the same motif with different chemical modifications in each strand permits targeting the antisense strand for the RISC complex while inhibiting the incorporation of the sense strand.
  • each strand can be independently modified such that it is enhanced for its particular role.
  • the antisense strand can be modified at the 5'-end to enhance its role in one region of the RISC while the 3 '-end can be modified differentially to enhance its role in a different region of the RISC.
  • the double- stranded oligonucleotide molecules can be a double- stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • the double- stranded oligonucleotide molecules can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e.
  • each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double- stranded structure, for example wherein the double- stranded region is about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 15 to about 25 or more nucleotides of the double- stranded oligonucleotide molecule are complementary to the target nucleic acid or a portion thereof).
  • the double- stranded oligonucleotide is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siRNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s).
  • the double- stranded oligonucleotide can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • the double- stranded oligonucleotide can be a circular single- stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion Ihereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi.
  • the double- stranded oligonucleotide comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions.
  • the double- stranded oligonucleotide comprises nucleotide sequence that is complementary to nucleotide sequence of a target gene.
  • the double- stranded oligonucleotide interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene.
  • double- stranded oligonucleotides need not be limited to those molecules containing only RNA, but further encompasses chemically modified nucleotides and non-nucleotides.
  • the short interfering nucleic acid molecules lack 2'-hydroxy (2'-OH) containing nucleotides.
  • short interfering nucleic acids optionally do not include any ribonucleotides (e.g., nucleotides having a 2'-OH group).
  • double- stranded oligonucleotides that do not require the presence of ribonucleotides within the molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2'-OH groups.
  • double- stranded oligonucleotides can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions.
  • siRNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double- stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically modified siRNA, post- transcriptional gene silencing RNA (ptgsRNA), and others.
  • siRNA short interfering RNA
  • dsRNA double- stranded RNA
  • miRNA micro-RNA
  • shRNA short hairpin RNA
  • ptgsRNA post- transcriptional gene silencing RNA
  • RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics.
  • sequence specific RNA interference such as post transcriptional gene silencing, translational inhibition, or epigenetics.
  • double- stranded oligonucleotides can be used to epigenetically silence genes at both the post- transcriptional level and the pre-transcriptional level.
  • epigenetic regulation of gene expression by siRNA molecules of the invention can result from siRNA mediated modification of chromatin structure or methylation pattern to alter gene expression (see, for example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237).
  • compositions of several embodiments provided herein can target PHD1 by a dsRNA-mediated gene silencing or RNAi mechanism, including, e.g., "hairpin” or stem-loop double- stranded RNA effector molecules in which a single RNA strand with self- complementary sequences is capable of assuming a double- stranded conformation, or duplex dsRNA effector molecules comprising two separate strands of RNA.
  • a dsRNA-mediated gene silencing or RNAi mechanism including, e.g., "hairpin" or stem-loop double- stranded RNA effector molecules in which a single RNA strand with self- complementary sequences is capable of assuming a double- stranded conformation, or duplex dsRNA effector molecules comprising two separate strands of RNA.
  • the dsRNA consists entirely of ribonucleotides or consists of a mixture of ribonucleotides and deoxynucleotides, such as the RNA/DNA hybrids disclosed, for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.
  • the dsRNA or dsRNA effector molecule may be a single molecule with a region of self-complementarity such that nucleotides in one segment of the molecule base pair with nucleotides in another segment of the molecule.
  • a dsRNA that consists of a single molecule consists entirely of ribonucleotides or includes a region of ribonucleotides that is complementary to a region of deoxyribonucleotides.
  • the dsRNA may include two different strands that have a region of com lementarity to each other.
  • both strands consist entirely of ribonucleotides, one strand consists entirely of ribonucleotides and one strand consists entirely of deoxyribonucleotides, or one or both strands contain a mixture of ribonucleotides and deoxyribonucleotides.
  • the regions of complementarity are at least 70, 80, 90, 95, 98, or 100% complementary to each other and to a target nucleic acid sequence.
  • the region of the dsRNA that is present in a double- stranded conformation includes at least 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 200, 500, 1000, 2000 or 5000 nucleotides or includes all of the nucleotides in a cDNA or other target nucleic acid sequence being represented in the dsRNA.
  • the dsRNA does not contain any single stranded regions, such as single stranded ends, or the dsRNA is a hairpin.
  • the dsRNA has one or more single stranded regions or overhangs.
  • RNA/DNA hybrids include a DNA strand or region that is an antisense strand or region (e.g, has at least 70, 80, 90, 95, 98, or 100% complementarity to a target nucleic acid) and an RNA strand or region that is a sense strand or region (e.g, has at least 70, 80, 90, 95, 98, or 100% identity to a target nucleic acid), and vice versa.
  • an antisense strand or region e.g, has at least 70, 80, 90, 95, 98, or 100% complementarity to a target nucleic acid
  • RNA strand or region that is a sense strand or region e.g, has at least 70, 80, 90, 95, 98, or 100% identity to a target nucleic acid
  • the RNA/DNA hybrid is made in vitro using enzymatic or chemical synthetic methods such as those described herein or those described in WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.
  • a DNA strand synthesized in vitro is complexed with an RNA strand made in vivo or in vitro before, after, or concurrent with the transformation of the DNA strand into the cell.
  • the dsRNA is a single circular nucleic acid containing a sense and an antisense region, or the dsRNA includes a circular nucleic acid and either a second circular nucleic acid or a linear nucleic acid (see, for example, WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.)
  • Exemplary circular nucleic acids include lariat structures in which the free 5' phosphoryl group of a nucleotide becomes linked to the 2' hydroxyl group of another nucleotide in a loop back fashion.
  • the dsRNA includes one or more modified nucleotides in which the 2' position in the sugar contains a halogen (such as fluorine group) or contains an alkoxy group (such as a methoxy group) which increases the half-life of the dsRNA in vitro or in vivo compared to the corresponding dsRNA in which the corresponding 2' position contains a hydrogen or an hydroxyl group.
  • the dsRNA includes one or more linkages between adjacent nucleotides other than a naturally-occurring phosphodiester linkage. Examples of such linkages include phosphoramide, phosphorothioate, and phosphorodithioate linkages.
  • the dsRNAs may also be chemically modified nucleic acid molecules as taught in U.S. Pat. No. 6,673,661.
  • the dsRNA contains one or two capped strands, as disclosed, for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.
  • the dsRNA can be any of the at least partially dsRNA molecules disclosed in WO 00/63364, as well as any of the dsRNA molecules described in U.S. Provisional Application 60/399,998; and U.S. Provisional Application 60/419,532, and PCT/US2003/033466, the teaching of which is hereby incorporated by reference. Any of the dsRNAs may be expressed in vitro or in vivo using the methods described herein or standard methods, such as those described in WO 00/63364.
  • antisense compounds are not expected to result in cleavage or the target nucleic acid via RNase H or to result in cleavage or sequestration through the RISC pathway.
  • antisense activity may result from occupancy, wherein the presence of the hybridized antisense compound disrupts the activity of the target nucleic acid.
  • the antisense compound may be uniformly modified or may comprise a mix of modifications and/or modified and unmodified nucleosides.
  • Nucleotide sequences that encode PHD1 include, without limitation, the following: GENBANK Accession No. NM 053208.4 (incorporated herein as SEQ ID NO: 1); the complement of GENBANK Accession No. NT 039413.7 truncated from nucleotides 24942670 to 24952750 (incorporated herein SEQ ID NO: 2); GENBANK Accession No. NM 080732.3 (incorporated herein SEQ ID NO: 3); GENBANK Accession No. NM 053046.3 (incorporated herein SEQ ID NO: 4); GENBANK Accession No. AL133009.1 (incorporated herein SEQ ID NO: 5); and GENBANK Accession No. NT 011 109.16 truncated from nucleotides 13572265 to 13583555 (incorporated herein SEQ ID NO: 6).
  • hybridization occurs between an antisense compound disclosed herein and a PHD1 nucleic acid.
  • the most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.
  • Hybridization can occur under varying conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.
  • the antisense compounds provided herein are specifically hybridizable with a PHD1 nucleic acid.
  • An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., antisense inhibition of a target nucleic acid, such as a PHD1 nucleic acid).
  • Non-complementary nucleobases between an antisense compound and a PHD1 nucleic acid may be tolerated provided that the antisense compound remains able to specifically hybridize to a target nucleic acid.
  • an antisense compound may hybridize over one or more segments of a PHD1 nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).
  • the antisense compounds provided herein, or a specified portion thereof are, or are at least, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%o, 97%), 98%), 99%), or 100% complementary to a PHD1 nucleic acid, a target region, target segment, or specified portion thereof. Percent complementarity of an antisense compound with a target nucleic acid can be determined using routine methods.
  • an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize would represent 90 percent complementarity.
  • the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases.
  • an antisense compound which is 18 nucleobases in length having four noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention.
  • Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).
  • the antisense compounds provided herein, or specified portions thereof are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof.
  • an antisense compound may be fully complementary to a PHD1 nucleic acid, or a target region, or a target segment or target sequence thereof.
  • "fully complementary" means each nucleobase of an antisense compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid.
  • a 20 nucleobase antisense compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the antisense compound.
  • Fully complementary can also be used in reference to a specified portion of the first and /or the second nucleic acid.
  • a 20 nucleobase portion of a 30 nucleobase antisense compound can be "fully complementary" to a target sequence that is 400 nucleobases long.
  • the 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the antisense compound.
  • the entire 30 nucleobase antisense compound may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.
  • non-complementary nucleobase may be at the 5' end or 3' end of 1he antisense compound.
  • the non-complementary nucleobase or nucleobases may be at an internal position of the antisense compound.
  • two or more non-complementary nucleobases may be contiguous (i.e. linked) or non-contiguous.
  • a non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.
  • antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a PHD1 nucleic acid, or specified portion thereof.
  • antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a PHD1 nucleic acid, or specified portion thereof.
  • the antisense compounds provided also include those which are complementary to a portion of a target nucleic acid.
  • portion refers to a defined number of contiguous (i.e. linked) nucleobases within a region or segment of a target nucleic acid.
  • a “portion” can also refer to a defined number of contiguous nucleobases of an antisense compound.
  • the antisense compounds are complementary to at least an 8 nucleobase portion of a target segment.
  • the antisense compounds are complementary to at least a 9 nucleobase portion of a target segment.
  • the antisense compounds are complementary to at least a 10 nucleobase portion of a target segment.
  • the antisense compounds are complementary to at least an 11 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 12 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 13 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 14 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 15 nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values. Identity
  • the antisense compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific Isis number, or portion thereof.
  • an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability.
  • a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine.
  • Shortened and lengthened versions of the antisense compounds described herein as well as compounds having non-identical bases relative to the antisense compounds provided herein also are contemplated.
  • the non-identical bases may be adjacent to each other or dispersed throughout the antisense compound. Percent identity of an antisense compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.
  • the antisense compounds, or portions thereof are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of the antisense compounds or SEQ ID NOs, or a portion thereof, disclosed herein.
  • a portion of the antisense compound is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid. In certain embodiments, a portion of the antisense oligonucleotide is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.
  • a nucleoside is a base-sugar combination.
  • the nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety.
  • Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3' or 5' hydroxyl moiety of the sugar.
  • Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.
  • Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.
  • Chemically modified nucleosides may also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides.
  • RNA and DNA The naturally occuring internucleoside linkage of RNA and DNA is a 3' to 5' phosphodiester linkage.
  • Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.
  • Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom.
  • Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.
  • antisense compounds targeted to a PHD1 nucleic acid comprise one or more modified internucleoside linkages.
  • the modified internucleoside linkages are phosphorothioate linkages.
  • each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.
  • Antisense compounds can optionally contain one or more nucleosides wherein the sugar group has been modified.
  • Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the antisense compounds.
  • nucleosides comprise chemically modified ribofuranose ring moieties.
  • Examples of chemically modified ribofuranose rings include without limitation, addition of substitutent groups (including 5' and 2' substituent groups, bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA), replacement of the ribosyl ring oxygen atom with S, N(R), or C(Ri)(R 2 ) (R, Ri and R 2 are each independently H, C 1 -C 12 alkyl or a protecting group) and combinations thereof.
  • substitutent groups including 5' and 2' substituent groups, bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA)
  • BNA bicyclic nucleic acids
  • R, Ri and R 2 are each independently H, C 1 -C 12 alkyl or a protecting group
  • Examples of chemically modified sugars include 2'-F-5'-methyl substituted nucleoside (see PCT International Application WO 2008/101157 Published on 8/21/08 for other disclosed 5',2'-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2'-position (see published U.S. Patent Application US2005-0130923, published on June 16, 2005) or alternatively 5'-substitution of a BNA (see PCT International Application WO 2007/134181 Published on 11/22/07 wherein 4'-(CH 2 )-0-2' (LNA) is substituted with for example a 5'-methyl or a 5'-vinyl group).
  • 2'-F-5'-methyl substituted nucleoside see PCT International Application WO 2008/101157 Published on 8/21/08 for other disclosed 5',2'-bis substituted nucleosides
  • nucleosides having modified sugar moieties include without limitation nucleosides comprising 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH 3 , 2'-OCH 2 CH 3 , 2'-OCH 2 CH 2 F and 2'- 0(CH 2 ) 2 OCH 3 substituent groups.
  • '3 ⁇ 4icyclic nucleosides refer to modified nucleosides comprising a bicyclic sugar moiety.
  • bicyclic nucleosides include without limitation nucleosides comprising a bridge between the 4' and the 2' ribosyl ring atoms.
  • antisense compounds provided herein include one or more bicyclic nucleosides comprising a 4' to 2' bridge.
  • 4' to 2' bridged bicyclic nucleosides include but are not limited to one of the formulae: 4'-(CH 2 )-0-2' (LNA) ; 4'- (CH 2 )-S-2'; 4'-(CH 2 ) 2 -0-2' (ENA) ; 4'-CH(CH 3 )-0-2' (also referred to as constrained ethyl or cEt) and 4'- CH(CH 2 OCH 3 )-0-2' (and analogs thereof see U.S.
  • Patent 7,427,672 issued on September 23, 2008
  • 4'-CH 2 -C- (H)(CH 3 )-2' see Chattopadhyaya et al, J. Org. Chem., 2009, 74, 118-134
  • Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example a-L-ribofuranose and ⁇ -D-ribofuranose (see PCT international application PCT/DK98/00393, published on March 25, 1999 as WO 99/14226).
  • x 0, 1, or 2;
  • n 1, 2, 3, or 4;
  • each R a and R b is, independently, H, a protecting group, hydroxyl, C 1 -C 12 alkyl, substituted d-
  • the bridge of a bicyclic sugar moiety is -[C(R a )(Rb)] n -, -[C(R a )(Rb)] n -0-
  • the bridge is 4'-CH 2 -2', 4'-(CH 2 ) 2 -2', 4'-(CH 2 ) 3 -2', 4'-CH 2 -0-2', 4'-(CH 2 ) 2 -0-2', 4'-CH 2 -0-N(R)-2' and 4'-CH 2 -N(R)-0-2'- wherein each R is, independently, H, a protecting group or C1-C12 alkyl.
  • bicyclic nucleosides are further defined by isomeric configuration.
  • a nucleoside comprising a 4'-2' methylene-oxy bridge may be in the a-L configuration or in the ⁇ -D configuration.
  • a-L-methyleneoxy (4'-CH 2 -0-2') BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et ah, Nucleic Acids Research, 2003, 21, 6365-6372).
  • bicyclic nucleosides include, but are not limited to, (A) a-L- methyleneoxy (4'-CH 2 -0-2') BNA , (B) ⁇ -D-methyleneoxy (4'-CH 2 -0-2') BNA , (C) ethyleneoxy (4'- (CH 2 ) 2 -0-2') BNA , (D) aminooxy (4'-CH 2 -0-N(R)-2') BNA, (E) oxyamino (4'-CH 2 -N(R)-0-2') BNA, and (F) methyl(methyleneoxy) (4'-CH(CH 3 )-0-2') BNA, (G) methylene-thio (4'-CH 2 -S-2') BNA, (H) methylene-amino (4'-CH 2 -N(R)-2') BNA, (I) methyl carbocyclic (4'-CH2-CH(CH 3 )-2') BNA, (J
  • Bx is the base moiety and R is independently H, a protecting group, C 1 -C 12 alkyl or Q- C 12 alkoxy.
  • bicyclic nucleosides having Formula I:
  • Bx is a heterocyclic base moiety
  • -Qa-Qb-Qc- is -CH 2 -N(R C )-CH 2 -, -CH 2 -0-N(Rc)-, - ⁇ 2 - ⁇ (3 ⁇ 4)-0- or - ⁇ (3 ⁇ 4)-
  • R c is C 1 -C 12 alkyl or an amino protecting group
  • T a and T are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium.
  • bicyclic nucleosides are provided having Formula II:
  • Bx is a heterocyclic base moiety
  • T a and T are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
  • Z a is Ci-Ce alkyl, C 2 -C6 alkenyl, C 2 -C6 alkynyl, substituted Ci-Ce alkyl, substituted C 2 -C6 alkenyl, substituted C 2 -C6 alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thio.
  • bicyclic nucleosides are provided having Formula III:
  • Bx is a heterocyclic base moiety
  • T a and T are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
  • Bx is a heterocyclic base moiety
  • T a and T b are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
  • R d is Ci-Ce alkyl, substituted Ci-Ce alkyl, C 2 -C6 alkenyl, substituted C 2 -C6 alkenyl, C 2 -C6 alkynyl or substituted C 2 -C6 alkynyl;
  • each q a , q b , q c and q d is, independently, H, halogen, Ci-Ce alkyl, substituted Ci-Ce alkyl, C 2 -C6 alkenyl, substituted C 2 -C6 alkenyl, C 2 -C6 alkynyl or substituted C 2 -C6 alkynyl, Ci-Ce alkoxyl, substituted Ci-Ce alkoxyl, acyl, substituted acyl, Ci-Ce aminoalkyl or substituted Ci-Ce aminoalkyl;
  • bicyclic nucleosides are provided having Formula V:
  • Bx is a heterocyclic base moiety
  • T a and T are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
  • q g and q are each, independently, H, halogen, C 1 -C 12 alkyl or substituted C 1 -C 12 alkyl.
  • BNA methyleneoxy (4'-CH 2 -0-2') BNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.
  • bicyclic nucleosides are provided having Formula VI:
  • Bx is a heterocyclic base moiety
  • T a and ⁇ are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
  • 4 '-2' bicyclic nucleoside or “4' to 2' bicyclic nucleoside” refers to a bicyclic nucleoside comprising a furanose ring comprising a bridge connecting two carbon atoms of the furanose ring connects the 2' carbon atom and the 4' carbon atom of the sugar ring.
  • nucleosides refer to nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties.
  • sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position.
  • 2'-modified sugar means a furanosyl sugar modified at the 2' position.
  • modifications include substituents selected from: a halide, including, but not limited to substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl.
  • 2'- substituent groups can also be selected from: C1-C12 alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, CI, Br, CN, F, CF 3 , OCF 3 , SOCH 3 , SO2CH3, ON0 2 , N0 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, amino alkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving pharmacokinetic properties, or a group for improving the pharmacodynamic properties of an antisense compound, and other substituents having similar properties.
  • modifed nucleosides comprise a 2'-MOE side chain (Baker et al, J. Biol. Chem., 1997, 272, 11944-12000).
  • 2'-MOE substitution have been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2'- O-methyl, O-propyl, and O-aminopropyl.
  • Oligonucleotides having the 2'-MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, Helv. Chim.
  • a "modified tetrahydropyran nucleoside” or “modified THP nucleoside” means a nucleoside having a six-membered tetrahydropyran "sugar” substituted in for the pentofuranosyl residue in normal nucleosides (a sugar surrogate).
  • Modified THP nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, Bioorg. Med. Chem., 2002, 10, 841-854) or fluoro HNA (F-HNA) having a tetr
  • Bx is a heterocyclic base moiety
  • T a and T are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T a and T b is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T a and ⁇ , is H, a hydroxyl protecting group, a linked conjugate group or a 5' or 3'-terminal group;
  • the modified THP nucleosides of Formula VII are provided wherein q 1; q 2 , q 3 , q4, qs, qe and q7 are each H. In certain embodiments, at least one of qi, q 2 , q 3 , q4, qs, qe and q7 is other than H. In certain embodiments, at least one of qi, q 2 , q 3 , q4, qs, qe and q7 is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of Ri and R 2 is fluoro. In certain embodiments, Ri is fluoro and R 2 is H; Ri is methoxy and R 2 is H, and Ri is methoxyethoxy and R 2 is H.
  • sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom.
  • nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds has been reported (see for example: Braasch et al., Biochemistry, 2002, 41, 4503- 4510; and U.S. Patents 5,698,685; 5, 166,315; 5, 185,444; and 5,034,506).
  • morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure.
  • sugar surrogates are referred to herein as "modifed morpholinos.”
  • Patent Application US2005-0130923, published on June 16, 2005) or alternatively 5'-substitution of a bicyclic nucleic acid see PCT International Application WO 2007/134181, published on 11/22/07 wherein a 4'-CH 2 -0-2' bicyclic nucleoside is further substituted at the 5' position with a 5'-methyl or a 5'-vinyl group.
  • PCT International Application WO 2007/134181 published on 11/22/07 wherein a 4'-CH 2 -0-2' bicyclic nucleoside is further substituted at the 5' position with a 5'-methyl or a 5'-vinyl group.
  • carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described ⁇ see, e.g., Srivastava et ah, J. Am. Chem. Soc. 2007, 129(26), 8362-8379).
  • antisense compounds comprise one or more modified cyclohexenyl nucleosides, which is a nucleoside having a six-membered cyclohexenyl in place of the pentofuranosyl residue in naturally occurring nucleosides.
  • Modified cyclohexenyl nucleosides include, but are not limited to those described in the art (see for example commonly owned, published PCT Application WO 2010/036696, published on April 10, 2010, Robeyns et al, J. Am. Chem.
  • Bx is a heterocyclic base moiety
  • T 3 and T 4 are each, independently, an internucleoside linking group linking the cyclohexenyl nucleoside analog to an antisense compound or one of T 3 and T 4 is an internucleoside linking group linking the tetrahydropyran nucleoside analog to an antisense compound and the other of T 3 and T is H, a hydroxyl protecting group, a linked conjugate group, or a 5'-or 3'-terminal group; and
  • qi, q 2 , q 3 , q 4 , qs, q6, q7, qs and qg are each, independently, H, Ci-Ce alkyl, substituted Ci-Ce alkyl, C 2 -C6 alkenyl, substituted C 2 -C6 alkenyl, C 2 -C6 alkynyl, substituted C 2 -C6 alkynyl or other sugar substituent group.
  • 2'-modified or “2 '-substituted” refers to a nucleoside comprising a sugar comprising a substituent at the 2' position other than H or OH.
  • 2'-F refers to a nucleoside comprising a sugar comprising a fluoro group at the 2' position of the sugar ring.
  • 2'-OMe or “2'-OCH 3 " or “2'-0-methyl” each refers to a nucleoside comprising a sugar comprising an -OCH 3 group at the 2' position of the sugar ring.
  • oligonucleotide refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleo sides (RNA) and/or deoxyribonucleosides (DNA).
  • RNA ribonucleo sides
  • DNA deoxyribonucleosides
  • bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see for example review article: Leumann, Bioorg. Med. Chem., 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to enhance activity.
  • nucleobase moieties In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.
  • antisense compounds comprise one or more nucleosides having modified sugar moieties.
  • the modified sugar moiety is 2'-MOE.
  • the 2'-MOE modified nucleosides are arranged in a gapmer motif.
  • the modified sugar moiety is a bicyclic nucleoside having a (4'-CH(CH 3 )-0-2') bridging group.
  • the (4'-CH(CH 3 )-0-2') modified nucleosides are arranged throughout the wings of a gapmer motif.
  • Nucleobase (or base) modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications can impart nuclease stability, binding affinity or some other beneficial biological property to antisense compounds. Modified nucleobases include synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of an antisense compound for a target nucleic acid.
  • 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).
  • Additional modified nucleobases include 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- amino adenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C ⁇ C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5- trifluoromethyl and other 5-substit
  • Heterocyclic base moieties can also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7- deaza- adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
  • Nucleobases that are particularly useful for increasing the binding affinity of antisense compounds include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
  • antisense compounds targeted to a PHDl nucleic acid comprise one or more modified nucleobases.
  • shortened or gap-widened antisense oligonucleotides targeted to a PHDl nucleic acid comprise one or more modified nucleobases.
  • the modified nucleobase is 5-methylcytosine.
  • each cytosine is a 5-methylcytosine.
  • Antisense compounds may be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides.
  • Typical conjugate groups include cholesterol moieties and lipid moieties.
  • Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • Antisense compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of antisense compounds to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the antisense compound having terminal nucleic acid from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5'-terminus (5'- cap), or at the 3'-terminus (3'-cap), or can be present on both termini. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3' and 5'-stabilizing groups that can be used to cap one or both ends of an antisense compound to impart nuclease stability include those disclosed in WO 03/004602 published on January 16, 2003.
  • antisense compounds are modified by attachment of one or more conjugate groups.
  • conjugate groups modify one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge and clearance.
  • Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional conjugate linking moiety or conjugate linking group to a parent compound such as an oligonucleotide.
  • Conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes.
  • Certain conjugate groups have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci.
  • Acids Res., 1990, 18, 3777-3783 a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino- carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).
  • Described herein are methods for treatment of cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.
  • Cells may be treated with antisense oligonucleotides when the cells reach approximately 60- 80% confluency in culture.
  • One reagent commonly used to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN (Invitrogen, Carlsbad, CA).
  • Antisense oligonucleotides may be mixed with LIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, CA) to achieve the desired final concentration of antisense oligonucleotide and a LIPOFECTIN concentration that may range from 2 to 12 ug/mL per 100 nM antisense oligonucleotide.
  • Another reagent used to introduce antisense oligonucleotides into cultured cells includes LIPOFECTAMINE (Invitrogen, Carlsbad, CA).
  • Antisense oligonucleotide is mixed with LIPOFECTAMINE in OPTI-MEM 1 reduced serum medium (Invitrogen, Carlsbad, CA) to achieve the desired concentration of antisense oligonucleotide and a LIPOFECTAMINE concentration that may range from 2 to 12 ug/mL per 100 nM antisense oligonucleotide.
  • Another technique used to introduce antisense oligonucleotides into cultured cells includes electroporation.
  • Yet another technique used to introduce antisense oligonucleotides into cultured cells includes free uptake of the oligonucleotides by the cells.
  • Cells are treated with antisense oligonucleotides by routine methods. Cells may be harvested 16-
  • RNA or protein levels of target nucleic acids are measured by methods known in the art and described herein.
  • the data are presented as the average of the replicate treatments.
  • the concentration of antisense oligonucleotide used varies from cell line to cell line. Methods to determine the optimal antisense oligonucleotide concentration for a particular cell line are well known in the art. Antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with LIPOFECTAMINE. Antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation. RNA Isolation
  • RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using the TRIZOL Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's recommended protocols.
  • Antisense compounds may be admixed 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.
  • An antisense compound targeted to PHD1 nucleic acid can be utilized in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier.
  • a pharmaceutically acceptable diluent is water, such as sterile water suitable for injection.
  • employed in the methods described herein is a pharmaceutical composition comprising an antisense compound targeted to PHD1 nucleic acid and a pharmaceutically acceptable diluent.
  • the pharmaceutically acceptable diluent is water.
  • the antisense compound is an antisense oligonucleotide provided herein.
  • compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
  • a prodrug can include the incorporation of additional nucleosides at one or both ends of an antisense compound which are cleaved by endogenous nucleases within the body, to form the active antisense compound.
  • the compounds or compositions further comprise a pharmaceutically acceptable carrier or diluent.
  • Example 1 Antisense inhibition of mouse PHDl in mouse primary hepatocytes by MOE gapmers
  • Antisense oligonucleotides were designed targeting mouse PHDl nucleic acid and were tested for their effects on PHDl mRNA in vitro.
  • the antisense oligonucleotides were tested at a 5,000 nM concentration in mouse primary hepatocytes cultured at a density of 10,000 cells per well and the antisense oligonucleotides were introduced to the cells by free uptake. After a treatment period of approximately 24 hours, RNA was isolated from the cells and PHDl mRNA levels were measured by quantitative real-time PCR.
  • Primer probe set RTS3551 (forward sequence TGAATCAGAACTGGGATGTTAAGG, designated herein as SEQ ID NO: 60; reverse sequence GGCTCGATGTTGGCTACCA, designated herein as SEQ ID NO: 61 ; probe sequence ATCTTCCCCGAGGGTCGGCCA, designated herein as SEQ ID NO: 62) was used to measure mRNA levels. PHDl mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of PHDl mRNA, relative to untreated control cells.
  • the newly designed chimeric antisense oligonucleotides in the Table below were designed as 5- 10-5 MOE gapmers.
  • the 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2'-deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising five nucleosides each.
  • Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment has a 2'-MOE modification.
  • “Start site” indicates the 5 '-most nucleoside to which the gapmer is targeted in the mouse gene sequence.
  • “Stop site” indicates the 3 '-most nucleoside to which the gapmer is targeted in the mouse gene sequence.
  • Each gapmer listed in the Table below is targeted to the mouse PHDl mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM 053208.4) Table 1
  • Antisense oligonucleotides were designed targeting mouse PHDl nucleic acid and were tested for their effects on PHDl mRNA in vitro.
  • the antisense oligonucleotides were tested at a 3,000 nM concentration in mouse b.END cells cultured at a density of 20,000 cells per well and the antisense oligonucleotides were introduced to the cells by electroporation. After a treatment period of approximately 24 hours, RNA was isolated from the cells and PHDl mRNA levels were measured by quantitative real-time PCR.
  • Primer probe set RTS3551 forward sequence
  • TGAATCAGAACTGGGATGTTAAGG designated herein as SEQ ID NO: 60
  • reverse sequence GGCTCGATGTTGGCTACCA designated herein as SEQ ID NO: 61
  • probe sequence ATCTTCCCCGAGGGTCGGCCA designated herein as SEQ ID NO: 62
  • the 3-10-3 cEt gapmers are 16 nucleosides in length, wherein the central gap segment comprises of ten 2'-deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising three nucleosides each.
  • Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment has a cEt modification.
  • Stop site indicates the 3 '-most nucleoside to which the gapmer is targeted in the mouse gene sequence.
  • Each gapmer listed in the Table below is targeted to SEQ ID NO: 2, which is the complement of the mouse PHDl genomic sequence (GENBANK Accession No. NT l 87034.1 truncated from nucleotides 24158658 to 24166802).
  • Example 3 Effect of antisense inhibition of PHD1 in a mouse kidney ischemia model
  • ISIS 576146 described in the study above, was tested in two different studies in a mouse model for kidney ischemia.
  • the basic protocol for both studies is as follows: C57BL/6 mice were treated with ISIS 576146 administered subcutaneously at 100 mg/kg/week for 2.5 weeks. Another group of mice was administered PBS for 2.5 weeks. This PBS set served as a control group.
  • Kidney ischemia was induced approximately 24 hours after the last dose of ISIS oligonucleotide by a hanging weight system as described in Grenz et al. (J Vis Exp. 2011 ; (53): 2549, which is incorporated by reference herein in its entirety) for all groups of mice.
  • Mice were anesthetized with pentobarbital at a dose of 70 mg/kg and maintained with approximately 10 mg/kg/h sodium pentobarbital. Once pain reflexes were absent (tested by squeezing the toes with tweezers) mice were placed on a temperature-controlled heating table (RT, Effenberg, Munich, Germany to maintain body temperature at 37.0°C) in a left lateral decubitus position.
  • RT temperature-controlled heating table
  • mice The lower and upper extremities of the mice were attached to the table with tape.
  • a right flank incision was performed with a scissor and a coagulation electrode (Erbe, ICC50, Germany) to prevent bleeding of muscle and skin vessels.
  • the renal pedicle including renal artery and renal vein were ligated with suture and the right kidney was removed.
  • the surgical wound was closed using continuous suture of the muscle wall and skin with a suture.
  • the mice were placed in a right lateral decubitus position and a left flank incision was performed.
  • the left kidney was removed from the connective tissues, avoiding the adrenal gland and vessels.
  • the kidney was turned with its ventral side down and collected in a sterile cup.
  • the kidney holder was restrained ventrally to have tension on the renal vessels.
  • the renal artery was dissected from adjacent tissues with forceps. A suture was placed under the renal artery over a small pole, and a weight of approximately 1 gram was attached to each end. While the weights were unsupported, the renal artery was immediately occluded. In contrast, when the weights were supported, blood flow was immediately restored to the organ. Successful occlusion was confirmed by a change of color from red to white.
  • Ischemia time was 30 minutes to allow recoverable kidney injury. After a 24-hour reperfusion, the kidney was replaced out of the sterile cup. The kidney holder was unfixed and the tension on the kidney was released. The cranial pole was gently removed out of the cup with forceps and the caudal pole followed immediately.
  • GFR glomerular filtration rate
  • mice treated with ISIS oligonucleotide or PBS and that underwent ischemia- reperfusion were administered a dose of 8 ⁇ /g FITC-inulin via tail vein injection.
  • the mice were then bled retro-orbitally 5, 15, 45, and 90 min after the injection.
  • the collected blood plasma was diluted 1 : 40 with 0.5 M HEPES and fluorescence was measured on a plate reader at 485/538 nm.
  • mice treated with an antisense oligonucleotide targeting PHDl show better clearance of FITC-inulin in the plasma compared to the PBS control group, and the clearance was similar to mice without kidney ischemia.
  • Plasma levels of transaminases, bilirubin, creatinine and BUN were measured using an automated clinical chemistry analyzer. The results are presented in the Table below and indicate that mice treated with an antisense oligonucleotide targeting PHDl do not show the increases in any markers as seen in the PBS control group.
  • results indicate that treatment with an antisense oligonucleotide did not cause adverse changes in liver function (as evidenced by ALT and AST levels).
  • results also indicate that the adverse increase in the kidney marker levels of BUN and creatinine due to kidney injury and ischemia in the PBS control were reduced due to antisense inhibition of PHDl.
  • GFR is measured as described in Lorenz JN et al., Am J Physiol. 1999;276(1 Pt 2):F172-F177 and Conger JD et al., Kidney Int. 1989;35(5): 1126—1132, which are incorporated by reference herein in their entireties. The results are presented in the Table below and indicate that GFR is increased in mice after treatment with an antisense oligonucleotide targeting PHDl.
  • Plasma and urine levels of transaminases, bilirubin, creatinine and BUN were measured using an automated clinical chemistry analyzer. The results are presented in the Table below and indicate that there were no changes in levels as a result of the treatment.
  • Urine electrolytes were measured by using crown ether electrodes for sodium and potassium and a molecular oriented PVC membrane for chloride. The results are presented in the Table below and indicate that there was no change in electrolyte concentrations as a result of the treatment.
  • Urine electrolytes (mEq/L) in a mouse kidney ischemia model

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present embodiments provide methods, compounds, and compositions for treating, preventing, or ameliorating acute kidney injury (AKI) by administering a PHD1 specific inhibitor to a subject.

Description

TREATMENT OF ACUTE KIDNEY INJURY WITH PHD1 MODULATORS
Sequence Listing
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0223WOSEQ_ST25.txt created October 10, 2014, which is 68 kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
Field
The present embodiments provide methods, compounds, and compositions for treating, preventing, or ameliorating acute kidney injury (AKI) by administering a PHD1 specific inhibitor to a subject.
Background Acute kidney injury (AKI) is one of the most common complications with no treatment. AKI is characterized by rapid reduction of kidney function and can be caused by decreased blood flow to the kidney resulting from low blood volume, low blood pressure, heart failure, or local changes to the blood vessels supplying the kidney. Renal ischemia refers to blood deficiency in the kidney and can lead to reduction of glomerular filtration rate (GFR), an indication of kidney function and health. Renal ischemia can be caused by hypoperfusion stemming from reduced cardiac output. For instance, cardiac surgery mediated hypoperfusion is associated with increased morbidity and mortality from AKI.
During renal ischemia, inadequate blood supply to the kidney can result in low oxygen levels, a condition known as hypoxia. Prolyl hydroxylases (PHDs) are enzymes that sense oxygen levels and mediate the transcriptional response to hypoxia by regulating the stability of hypoxia-inducible factors (HIFs). Under normoxic conditions, prolyl hydroxylases (PHD1-3) promote the hydryxolation of proline residues on HIF proteins, resulting in the ubiquitin-mediated destruction of HIF by Von Hippel Lindau (VHL) ubiquitin ligase. Under hypoxia, PHDs are inhibited, which leads to the stabilization of HIFs. Summary
Certain embodiments provided herein relate to methods, compounds, and compositions for treating, preventing, or ameliorating acute kidney injury (AKI) in a subject comprising administering to the subject a specific inhibitor of prolyl hydroxylase 1 (PHD1), thereby treating, preventing, or ameliorating the acute kidney injury. In certain embodiments, the acute kidney injury can be caused by hypoxia and/or renal ischemia. In certain embodiments, the renal ischemia is caused by cardiac-mediated hypoperfusion, which in certain embodiments can be caused by cardiac surgery.
Certain embodiments relate to methods, compounds, and compositions for improving renal function and/or pathology in a subject having or at risk of having AKI by administering a specific inhibitor of PHD 1.
In certain embodiments, the specific inhibitor of PHD1 is an antisense compound, such as a single- stranded modified oligonucleotide targeted to PHD1.
Detailed Description
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference for the portions of the document discussed herein, as well as in their entirety.
It is understood that the sequence set forth in each SEQ ID NO in the examples contained herein is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, antisense compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Antisense compounds described by ISIS number (ISIS #) indicate a combination of nucleobase sequence, chemical modification, and motif. Unless otherwise indicated, the following terms have the following meanings:
"2'-0-methoxyethyl" (also 2'-MOE and 2'-0(CH2)2-OCH3) refers to an O-methoxy-ethyl modification at the 2' position of a sugar ring, e.g. a furanose ring. A 2'-0-methoxyethyl modified sugar is a modified sugar.
"2'-MOE nucleoside" (also 2'-0-methoxyethyl nucleoside) means a nucleoside comprising a 2'-
MOE modified sugar moiety.
"2 '-substituted nucleoside" means a nucleoside comprising a substituent at the 2 '-position of the furanosyl ring other than H or OH. In certain embodiments, 2' substituted nucleosides include nucleosides with bicyclic sugar modifications.
"3' target site" refers to the nucleotide of a target nucleic acid which is complementary to the 3'- most nucleotide of a particular antisense compound.
"5' target site" refers to the nucleotide of a target nucleic acid which is complementary to the 5'- most nucleotide of a particular antisense compound.
"5-methylcytosine" means a cytosine modified with a methyl group attached to the 5 position. A 5-methylcytosine is a modified nucleobase.
"About" means within ±10% of a value. For example, if it is stated, "the compounds affected at least about 70% inhibition of PHD1", it is implied that PHD1 levels are inhibited within a range of 60%> and 80%.
"Administration" or "administering" refers to routes of introducing an antisense compound provided herein to a subject to perform its intended function. An example of a route of administration that can be used includes, but is not limited to parenteral administration, such as subcutaneous, intravenous, or intramuscular injection or infusion.
"Amelioration" refers to a lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition. In certain embodiments, amelioration includes a delay or slowing in the progression of one or more indicators of a condition or disease. The severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art.
"Animal" refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.
"Antisense activity" means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.
"Antisense compound" means an oligomeric compound that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. Examples of antisense compounds include single- stranded and double- stranded compounds, such as, antisense oligonucleotides, siRNAs, shRNAs, ssRNAs, and occupancy-based compounds.
"Antisense inhibition" means reduction of target nucleic acid levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels in the absence of the antisense compound.
"Antisense mechanisms" are all those mechanisms involving hybridization of a compound with target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing.
"Antisense oligonucleotide" means a single- stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.
"Base complementarity" refers to the capacity for the precise base pairing of nucleobases of an antisense oligonucleotide with corresponding nucleobases in a target nucleic acid (i.e., hybridization), and is mediated by Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen binding between corresponding nucleobases.
"Bicyclic sugar moiety" means a modified sugar moiety comprising a 4 to 7 membered ring
(including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4 to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2'-carbon and the 4'-carbon of the furanosyl.
"Cap structure" or "terminal cap moiety" means chemical modifications, which have been incorporated at either terminus of an antisense compound.
"cEt" or "constrained ethyl" means a bicyclic sugar moiety comprising a bridge connecting the 4'-carbon and the 2'-carbon, wherein the bridge has the formula: 4'-CH(CH3)-0-2'.
"Constrained ethyl nucleoside" (also cEt nucleoside) means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH3)-0-2' bridge. "PHD1" means any nucleic acid or protein of PHD1. "PHD1 nucleic acid" means any nucleic acid encoding PHD1. For example, in certain embodiments, a PHD1 nucleic acid includes a DNA sequence encoding PHD1, an RNA sequence transcribed from DNA encoding PHD1 (including genomic DNA comprising introns and exons), including a non-protein encoding (i.e. non-coding) RNA sequence, and an mRNA sequence encoding PHD1. "PHD1 mRNA" means an mRNA encoding a PHD1 protein.
"PHD1 specific inhibitor" refers to any agent capable of specifically inhibiting PHD1 RNA and/or PHD1 protein expression or activity at the molecular level. For example, PHD1 specific inhibitors include nucleic acids (including antisense compounds), peptides, antibodies, small molecules, and other agents capable of inhibiting the expression of PHD1 RNA and/or PHD1 protein.
"Chemically distinct region" refers to a region of an antisense compound that is in some way chemically different than another region of the same antisense compound. For example, a region having 2'-0-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2'-0- methoxyethyl modifications.
"Chimeric antisense compounds" means antisense compounds that have at least 2 chemically distinct regions, each position having a plurality of subunits.
"Complementarity" means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.
"Comprise," "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.
"Contiguous nucleobases" means nucleobases immediately adjacent to each other.
"Deoxyribonucleotide" means a nucleotide having a hydrogen at the 2' position of the sugar portion of the nucleotide. Deoxyribonucleotides may be modified with any of a variety of substituents.
"Designing" or "Designed to" refer to the process of designing an oligomeric compound that specifically hybridizes with a selected nucleic acid molecule.
"Effective amount" means 1he amount of active pharmaceutical agent sufficient to effectuate a desired physiological outcome in an individual in need of the agent. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.
"Efficacy" means the ability to produce a desired effect. "Expression" includes all the functions by which a gene's coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to the products of transcription and translation.
"Fully complementary" or "100% complementary" means each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid. In certain embodiments, a first nucleic acid is an antisense compound and a target nucleic acid is a second nucleic acid.
"Gapmer" means a chimeric antisense compound in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as the "gap" and the external regions may be referred to as the "wings."
"Hybridization" means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense compound and a nucleic acid target. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense oligonucleotide and a nucleic acid target.
"Identifying an animal having, or at risk for having, a disease, disorder and/or condition" means identifying an animal having been diagnosed with the disease, disorder and/or condition or identifying an animal predisposed to develop the disease, disorder and/or condition. Such identification may be accomplished by any method including evaluating an individual's medical history and standard clinical tests or assessments.
"Immediately adjacent" means there are no intervening elements between the immediately adjacent elements.
"Individual" means a human or non-human animal selected for treatment or therapy.
"Inhibiting the expression or activity" refers to a reduction, blockade of the expression or activity and does not necessarily indicate a total elimination of expression or activity.
"Internucleoside linkage" refers to the chemical bond between nucleosides.
"Lengthened" antisense oligonucleotides are those that have one or more additional nucleosides relative to an antisense oligonucleotide disclosed herein.
"Linked deoxynucleoside" means a nucleic acid base (A, G, C, T, U) substituted by deoxyribose linked by a phosphate ester to form a nucleotide.
"Linked nucleosides" means adjacent nucleosides linked together by an internucleoside linkage. "Mismatch" or "non-complementary nucleobase" refers to the case when a nucleobase of a first nucleic acid is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid.
"Modified internucleoside linkage" refers to a substitution or any change from a naturally occurring internucleoside bond (i.e. a phosphodiester internucleoside bond).
"Modified nucleobase" means any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. An "unmodified nucleobase" means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
"Modified nucleoside" means a nucleoside having, independently, a modified sugar moiety and/or modified nucleobase.
"Modified nucleotide" means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, or modified nucleobase.
"Modified oligonucleotide" means an oligonucleotide comprising at least one modified internucleoside linkage, a modified sugar, and/or a modified nucleobase.
"Modified sugar" means substitution and/or any change from a natural sugar moiety.
"Modulating" refers to changing or adjusting a feature in a cell, tissue, organ or organism. For example, modulating PHD1 mRNA can mean to increase or decrease the level of PHD 1 mRNA and/or PHD1 protein in a cell, tissue, organ or organism. A "modulator" effects the change in the cell, tissue, organ or organism. For example, a PHD1 antisense compound can be a modulator that decreases the amount of PHD 1 mRNA and/or PHD1 protein in a cell, tissue, organ or organism.
"Monomer" refers to a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occuring or modified.
"Motif means the pattern of unmodified and modified nucleosides in an antisense compound.
"Natural sugar moiety" means a sugar moiety found in DNA (2'-H) or RNA (2'-OH).
"Naturally occurring internucleoside linkage" means a 3' to 5' phosphodiester linkage.
"Non-complementary nucleobase" refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.
"Nucleic acid" refers to molecules composed of monomeric nucleotides. A nucleic acid includes, but is not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single- stranded nucleic acids, and double- stranded nucleic acids. "Nucleobase" means a heterocyclic moiety capable of pairing with a base of another nucleic acid.
"Nucleobase complementarity" refers to a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (IS). In certain embodiments, complementary nucleobase refers to a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair.
"Nucleobase sequence" means the order of contiguous nucleobases independent of any sugar, linkage, and/or nucleobase modification.
"Nucleoside" means a nucleobase linked to a sugar.
"Nucleoside mimetic" includes those structures used to replace the sugar or the sugar and the base and not necessarily the linkage at one or more positions of an oligomeric compound such as for example nucleoside mimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics, e.g., non furanose sugar units. Nucleotide mimetic includes those structures used to replace the nucleoside and the linkage at one or more positions of an oligomeric compound such as for example peptide nucleic acids or morpholinos (morpholinos linked by -N(H)-C(=0)-0- or other non- phosphodiester linkage). Sugar surrogate overlaps with the slightly broader term nucleoside mimetic but is intended to indicate replacement of the sugar unit (furanose ring) only. The tetrahydropyranyl rings provided herein are illustrative of an example of a sugar surrogate wherein the furanose sugar group has been replaced with a tetrahydropyranyl ring system. "Mimetic" refers to groups that are substituted for a sugar, a nucleobase, and/ or internucleoside linkage. Generally, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target.
"Nucleotide" means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.
"Oligomeric compound" means a polymer of linked monomeric subunits which is capable of hybridizing to at least a region of a nucleic acid molecule.
"Oligonucleoside" means an oligonucleotide in which the internucleoside linkages do not contain a phosphorus atom. "Oligonucleotide" means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another.
"Parenteral administration" means administration through injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g. intrathecal or intracerebroventricular administration.
"Pharmaceutical composition" means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition may comprise one or more active pharmaceutical agents and a sterile aqueous solution.
"Pharmaceutically acceptable salts" means physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.
"Phosphorothioate linkage" means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.
"Portion" means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound
"Prevent" refers to delaying or forestalling the onset, development or progression of a disease, disorder, or condition for a period of time from minutes to indefinitely. Prevent also means reducing the risk of developing a disease, disorder, or condition.
"Prophylactically effective amount" refers to an amount of a pharmaceutical agent that provides a prophylactic or preventative benefit to an animal.
"Region" is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.
"Ribonucleotide" means a nucleotide having a hydroxy at the 2' position of the sugar portion of the nucleotide. Ribonucleotides may be modified with any of a variety of substituents.
"Segments" are defined as smaller or sub-portions of regions within a target nucleic acid.
"Side effects" means physiological disease and/or conditions attributable to a treatment other than the desired effects. In certain embodiments, side effects include injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, myopathies, and malaise. For example, increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality. For example, increased bilirubin may indicate liver toxicity or liver function abnormality.
"Sites," as used herein, are defined as unique nucleobase positions within a target nucleic acid. "Slows progression" means decrease in the development of the said disease.
"Specifically hybridizable" refers to an antisense compound having a sufficient degree of complementarity between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays and therapeutic treatments. "Stringent hybridization conditions" or "stringent conditions" refer to conditions under which an oligomeric compound will hybridize to its target sequence, but to a minimal number of other sequences.
"Subject" means a human or non-human animal selected for treatment or therapy.
"Target" refers to a protein, the modulation of which is desired.
"Target gene" refers to a gene encoding a target.
'Targeting" means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.
'Target nucleic acid," "target RNA," "target RNA transcript" and "nucleic acid target" all mean a nucleic acid capable of being targeted by antisense compounds.
'Target region" means a portion of a target nucleic acid to which one or more antisense compounds is targeted.
'Target segment" means the sequence of nucleotides of a target nucleic acid to which an antisense compound is targeted. "5' target site" refers to the 5'-most nucleotide of a target segment. "3' target site" refers to the 3 '-most nucleotide of a target segment. 'Therapeutically effective amount" means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual.
'Treat" refers to administering a pharmaceutical composition to an animal in order to effect an alteration or improvement of a disease, disorder, or condition in the animal. In certain embodiments, one or more pharmaceutical compositions can be administered to the animal. "Unmodified" nucleobases mean the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
"Unmodified nucleotide" means a nucleotide composed of naturally occuring nucleobases, sugar moieties, and internucleoside linkages. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e. β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).
Certain Embodiments
In certain embodiments, a method of treating, preventing, or ameliorating acute kidney injury (AKI) in a subject comprisies administering to the subject a specific inhibitor of prolyl hydroxylase 1 (PHD1), thereby treating, preventing, or ameliorating the acute kidney injury. In certain embodiments, the subject's kidney is hypoxic. In certain embodiments, the acute kidney injury is caused by renal ischemia. In certain embodimens, the renal ischemia is caused by cardiac-mediated hypoperfusion. In certain embodiments, the cardiac-mediated hypoperfusion is caused by cardiac surgery, such as bypass surgery, coronary artery bypass grafting, valvular surgery, or combined coronary artery bypass grafting and valvular surgery. It has been estimated that acute renal failure occurs in up to 30% of patients who undergo cardiac surgery and is associated with morbidity and mortality. Furthermore, dialysis is required in approximately 1% of all cardiac surgery patients, most of whom remain dependent on dialysis long- term. Rosner et al., Clin J Am Soc Nephrol. 2006 Jan;l(l):19-32.
In certain embodiments, administering the specific inhibitor of PHD 1 improves renal function. In certain embodiments, administering the specific inhibitor of PHD 1 improves tubular epithelial cell injury in the subject. In certain embodiments, administering the specific inhibitor of PHD 1 improves glomerular filtration rate (GFR). In certain embodiments, administering the specific inhibitor of PHD 1 decreases levels of blood urea nitrogen (BUN) and/or creatinine, which are markers of renal function that increase with impaired renal function. In certain embodiments, administering the specific inhibitor of PHD1 decreases levels of neutrophil gelatinase-associated lipocalin (NGAL) and/or kidney injury molecule 1 (KIM-1), which are biomarkers whose levels positively correlate with AKI.
In certain embodiments, a method of inhibiting expression of PHD 1 in a subject having, or at risk of having, acute kidney injury (AKI) comprises administering a specific inhibitor of PHD 1 to the subject, thereby inhibiting expression of PHD1 in the subject. In certain embodiments, administering the inhibitor reduces expression of PHD1 in the kidney. In certain embodiments, the subject's kidney is hypoxic. In certain embodiments, the acute kidney injury is caused by renal ischemia. In certain embodiments, the renal ischemia is caused by cardiac-mediated hypoperfusion. In certain embodiments, the cardiac- mediated hypoperfusion is caused by cardiac surgery, such as bypass surgery, coronary artery bypass grafting, valvular surgery, or combined coronary artery bypass grafting and valvular surgery. In certain embodiments, the renal ischemia is caused by gastric bypass surgery, orthotropic liver transplant, abdominal aortic aneurysm repair, contrast agent induced hypoxia, or drug induced hypoxia. In certain embodiments, reducing expression of PHDl in the kidney by administering the specific inhibitor of PHDl improves renal function. In certain embodiments, reducing expression of PHDl in the kidney by administering the specific inhibitor of PHDl improves tubular epithelial cell injury in the subject. In certain embodiments, reducing expression of PHDl in the kidney by administering the specific inhibitor of PHDl improves glomerular filtration rate (GFR). In certain embodiments, reducing expression of PHDl in the kidney by administering the specific inhibitor of PHDl decreases levels of blood urea nitrogen (BUN) and/or creatinine. In certain embodiments, reducing expression of PHDl in the kidney by administering the specific inhibitor of PHDl decreases levels of neutrophil gelatinase-associated lipocalin (NGAL) and/or kidney injury molecule 1 (KIM-1).
In certain embodiments, a method of reducing or inhibiting tubular epithelial cell injury in the kidney of a subject having, or at risk of having, acute kidney injury comprises administering a specific inhibitor of PHDl to the subject, thereby reducing or inhibiting tubular epithelial cell injury in the kidney of the subject. In certain embodiments, the subject's kidney is hypoxic. In certain embodiments, the acute kidney injury is caused by renal ischemia. In certain embodiments, the renal ischemia is caused by cardiac-mediated hypoperfusion. In certain embodiments, the cardiac-mediated hypoperfusion is caused by cardiac surgery, such as bypass surgery, coronary artery bypass grafting, valvular surgery, or combined coronary artery bypass grafting and valvular surgery. In certain embodiments, the renal ischemia is caused by gastric bypass surgery, orthotropic liver transplant, abdominal aortic aneurysm repair, contrast agent induced hypoxia, or drug induced hypoxia.
In certain embodiments, a method of preventing or protecting a subject from acute kidney injury comprises administering a specific inhibitor of PHDl to the subject prior to the subject undergoing cardiac surgery. In certain embodiments, a method of preventing or protecting a subject from acute kidney injury comprises administering a specific inhibitor of PHDl to the subject prior to performing cardiac surgery on the patient. In certain embodiments, a method of preventing or protecting a subject from acute kidney injury comprises identifying a subject in need of cardiac surgery and administering a specific inhibitor of PHDl to the subject prior to the subject undergoing cardiac surgery. In certain embodiments, the cardiac surgery is bypass surgery, coronary artery bypass grafting, valvular surgery, or combined coronary artery bypass grafting and valvular surgery. In certain embodiments, administering the specific inhibitor of PHDl to the subject prior to the subject undergoing cardiac surgery is sufficient to prevent acute kidney injury, protect against acute kidney injury, minimize acute kidney injury, or reduce acute kidney injury in the subject following cardiac surgery. In certain embodiments, the specific inhibitor of PHD l is administered multiple times prior to the subject undergoing cardiac surgery.
In certain embodiments, a method of preventing or protecting a subject from acute kidney injury comprises administering a specific inhibitor of PHDl to the subject prior to the subject undergoing a surgery or operation that causes renal ischemia. In certain embodiments, a method of preventing or protecting a subject from acute kidney injury comprises administering a specific inhibitor of PHD l to the subject prior to performing surgery or operation that causes renal ischemia on the patient. In certain embodiments, a method of preventing or protecting a subject from acute kidney injury comprises identifying a subject in need of surgery or operation and administering a specific inhibitor of PHDl to the subject prior to the subject undergoing surgery or operation. In certain embodiments, the surgery or operation is gastric bypass surgery, orthotopic liver transplant, or abdominal aortic aneurysm repair. In certain embodiments, administering the specific inhibitor of PHD l to the subject prior to the subject undergoing surgery or operation is sufficient to prevent acute kidney injury, protect against acute kidney injury, minimize acute kidney injury, or reduce acute kidney injury in the subject following surgery or operation. In certain embodiments, the specific inhibitor of PHD l is administered multiple times prior to the subject undergoing surgery or operation.
In certain embodiments, a method of preventing or protecting a subject from acute kidney injury comprises administering to the subject a specific inhibitor of PHD l prior to administering a drug that induces ischemia. In certain embodiments, a method of preventing or protecting a subject from acute kidney injury comprises identifying a subject in need of a drug known to induce ischemia and administering a specific inhibitor of PHD l to the subject prior to administering the drug known to induce ischemia. In certain embodiments, administering the specific inhibitor of PHDl to the subject prior to administering a drug known to induce ischemia is sufficient to prevent acute kidney injury, protect against acute kidney injury, minimize acute kidney injury, or reduce acute kidney injury in the subject following administration of the drug known to induce ischemia. In certain embodiments, the specific inhibitor of PHDl is administered multiple times prior to administering the drug known to induce ischemia. Several types of drugs that induce ischemia are known in the art. Non-limiting examples include cyclosporine A and its structural analogues; angiotensin converting enzyme (ACE) inhibitors, such as, but not limited to, Benazepril, Captopril, Enalapril, Fosinopril, Lisinopril, Moexipril, Perindopril, Quinapril, Ramipril, and Trandolapril; and non-steroidal anti-inflammatory drugs (NSAIDs), such as, but not limited to, Aspirin (acetylsalicylic acid), Diflunisal, Salsalate, Ibuprofen, Dexibuprofen, Naproxen, Fenoprofen, Ketoprofen, Dexketoprofen, Flurbiprofen, Oxaprozin, Loxoprofen, Indomethacin, Tolmetin, Sulindac, Etodolac, Ketorolac, Diclofenac, Nabumetone, Piroxicam, Meloxicam, Tenoxicam, Droxicam, Lornoxicam, Isoxicam, Mefenamic acid, Meclofenamic acid, Flufenamic acid, Tolfenamic acid, and Celecoxib. Certain embodiments provide a PHD1 specific inhibitor for treating, preventing, or ameliorating acute kidney injury. In certain embodiments, the acute kidney injury is caused by hypoxia. In certain embodiments, the acute kidney injury is caused by renal ischemia. In certain embodiments, the renal ischemia is caused by cardiac-mediated hypoperfusion. In certain embodiments, the cardiac-mediated hypoperfusion is caused by cardiac surgery, such as bypass surgery, coronary artery bypass grafting, valvular surgery, or combined coronary artery bypass grafting and valvular surgery.
Certain embodiments provide a use of a PHD1 specific inhibitor for the preparation of a medicament for treating acute kidney injury. In certain embodiments, the acute kidney injury is caused by hypoxia. In certain embodiments, the acute kidney injury is caused by renal ischemia. In certain embodiments, the renal ischemia is caused by cardiac-mediated hypoperfusion. In certain embodiments, the cardiac-mediated hypoperfusion is caused by cardiac surgery, such as bypass surgery, coronary artery bypass grafting, valvular surgery, or combined coronary artery bypass grafting and valvular surgery.
In certain embodiments of any of the foregoing methods or uses of a PHD1 specific inhibitor for treating, preventing, or ameliorating acute kidney injury, the inhibitor is an antisense compound targeted to PHD1. In certain embodiments, the antisense compound comprises at least one modified sugar, such as a 2'-0-methoxyethyl group or a bicyclic sugar, such as 4'-CH(CH3)-0-2', 4'-CH2-0-2' or 4'-(CH2)2- 0-2'. In certain embodiments, the antisense compound comprises at least one modified internucleoside linkage, such as a phosphorothioate internucleoside linkage. In certain embodiments, the antisense compound comprises at least one modified nucleobase, such as a 5-methylcytosine. In certain embodiments, the antisense compound is a single- stranded modified oligonucleotide. In certain embodiments, the modified oligonucleotide comprises a gap segment consisting of linked deoxynucleosides; a 5' wing segment consisting of linked nucleosides; and a 3' wing segment consisting of linked nucleosides; wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
In certain embodiments, the PHD1 specific inhibitor is an antisense compound comprising a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 7-59. In any of the foregoing embodiments, the antisense compound can be a single- stranded oligonucleotide. Antisense compounds
Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleo sides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs. An oligomeric compound may be "antisense" to a target nucleic acid, meaning that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted. In certain such embodiments, an antisense oligonucleotide has a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.
In certain embodiments, an antisense compound is 10 to 30 subunits in length. In certain embodiments, an antisense compound is 12 to 30 subunits in length. In certain embodiments, an antisense compound is 12 to 22 subunits in length. In certain embodiments, an antisense compound is 14 to 30 subunits in length. In certain embodiments, an antisense compound is 14 to 20 subunits in length. In certain embodiments, an antisense compoun is 15 to 30 subunits in length. In certain embodiments, an antisense compound is 15 to 20 subunits in length. In certain embodiments, an antisense compound is 16 to 30 subunits in length. In certain embodiments, an antisense compound is 16 to 20 subunits in length. In certain embodiments, an antisense compound is 17 to 30 subunits in length. In certain embodiments, an antisense compound is 17 to 20 subunits in length. In certain embodiments, an antisense compound is 18 to 30 subunits in length. In certain embodiments, an antisense compound is 18 to 21 subunits in length. In certain embodiments, an antisense compound is 18 to 20 subunits in length. In certain embodiments, an antisense compound is 20 to 30 subunits in length. In other words, such antisense compounds are from 12 to 30 linked subunits, 14 to 30 linked subunits, 14 to 20 subunits, 15 to 30 subunits, 15 to 20 subunits, 16 to 30 subunits, 16 to 20 subunits, 17 to 30 subunits, 17 to 20 subunits, 18 to 30 subunits, 18 to 20 subunits, 18 to 21 subunits, 20 to 30 subunits, or 12 to 22 linked subunits, respectively. In certain embodiments, an antisense compound is 14 subunits in length. In certain embodiments, an antisense compound is 16 subunits in length. In certain embodiments, an antisense compound is 17 subunits in length. In certain embodiments, an antisense compound is 18 subunits in length. In certain embodiments, an antisense compound is 19 subunits in length. In certain embodiments, an antisense compound is 20 subunits in length. In other embodiments, the antisense compound is 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to 30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linked subunits. In certain such embodiments, the antisense compounds are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a range defined by any two of the above values. In some embodiments the antisense compound is an antisense oligonucleotide, and the linked subunits are nucleotides. In certain embodiments antisense oligonucleotides may be shortened or truncated. For example, a single subunit may be deleted from the 5' end (5' truncation), or alternatively from the 3' end (3' truncation). A shortened or truncated antisense compound targeted to an PHD1 nucleic acid may have two subunits deleted from the 5' end, or alternatively may have two subunits deleted from the 3' end, of the antisense compound. Alternatively, the deleted nucleosides may be dispersed throughout the antisense compound, for example, in an antisense compound having one nucleoside deleted from the 5' end and one nucleoside deleted from the 3' end.
When a single additional subunit is present in a lengthened antisense compound, the additional subunit may be located at the 5' or 3' end of the antisense compound. When two or more additional subunits are present, the added subunits may be adjacent to each other, for example, in an antisense compound having two subunits added to the 5' end (5' addition), or alternatively to the 3' end (3' addition), of the antisense compound. Alternatively, the added subunits may be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit added to the 5' end and one subunit added to the 3' end.
It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.
Gautschi et al. (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl- xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti- tumor activity in vivo.
Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358,1988) tested a series of tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42 nucleobase antisense oligonucleotides comprised of the sequence of two or three of the tandem antisense oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase antisense oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase antisense oligonucleotides. Certain Antisense Compound Motifs and Mechanisms
In certain embodiments, antisense compounds have chemically modified subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.
Chimeric antisense compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased inhibitory activity. A second region of a chimeric antisense compound may confer another desired property e.g., serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.
Antisense activity may result from any mechanism involving the hybridization of the antisense compound (e.g., oligonucleotide) with a target nucleic acid, wherein the hybridization ultimately results in a biological effect. In certain embodiments, the amount and/or activity of the target nucleic acid is modulated. In certain embodiments, the amount and/or activity of the target nucleic acid is reduced. In certain embodiments, hybridization of the antisense compound to the target nucleic acid ultimately results in target nucleic acid degradation. In certain embodiments, hybridization of the antisense compound to the target nucleic acid does not result in target nucleic acid degradation. In certain such embodiments, the presence of the antisense compound hybridized with the target nucleic acid (occupancy) results in a modulation of antisense activity. In certain embodiments, antisense compounds having a particular chemical motif or pattern of chemical modifications are particularly suited to exploit one or more mechanisms. In certain embodiments, antisense compounds function through more than one mechanism and/or through mechanisms that have not been elucidated. Accordingly, the antisense compounds described herein are not limited by particular mechanism.
Antisense mechanisms include, without limitation, RNase H mediated antisense; RNAi mechanisms, which utilize the RISC pathway and include, without limitation, siRNA, ssRNA and microRNA mechanisms; and occupancy based mechanisms. Certain antisense compounds may act through more than one such mechanism and/or through additional mechanisms.
RNase H-Mediated Antisense
In certain embodiments, antisense activity results at least in part from degradation of target RNA by RNase H. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNADNA duplex. It is known in the art that single- stranded antisense compounds which are "DNA-like" elicit RNase H activity in mammalian cells. Accordingly, antisense compounds comprising at least a portion of DNA or DNA-like nucleosides may activate RNase H, resulting in cleavage of the target nucleic acid. In certain embodiments, antisense compounds that utilize RNase H comprise one or more modified nucleosides. In certain embodiments, such antisense compounds comprise at least one block of 1-8 modified nucleosides. In certain such embodiments, the modified nucleosides do not support RNase H activity. In certain embodiments, such antisense compounds are gapmers, as described herein. In certain such embodiments, the gap of the gapmer comprises DNA nucleosides. In certain such embodiments, the gap of the gapmer comprises DNA-like nucleosides. In certain such embodiments, the gap of the gapmer comprises DNA nucleosides and DNA-like nucleosides.
Certain antisense compounds having a gapmer motif are considered chimeric antisense compounds. In a gapmer an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region. In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides. In certain embodiments, the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region. The types of sugar moieties that are used to differentiate the regions of a gapmer may in some embodiments include β-D- ribonucleosides, β-D-deoxyribonucleosides, 2'-modified nucleosides (such 2'-modified nucleosides may include 2'-MOE and 2'-0-CH3, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a constrained ethyl). In certain embodiments, nucleosides in the wings may include several modified sugar moieties, including, for example 2'-MOE and bicyclic sugar moieties such as constrained ethyl or LNA. In certain embodiments, wings may include several modified and unmodified sugar moieties. In certain embodiments, wings may include various combinations of 2'-MOE nucleosides, bicyclic sugar moieties such as constrained ethyl nucleosides or LNA nucleosides, and 2'-deoxynucleosides.
Each distinct region may comprise uniform sugar moieties, variant, or alternating sugar moieties. The wing-gap-wing motif is frequently described as "X-Y-Z", where "X" represents the length of the 5'-wing, 'Ύ" represents the length of the gap, and "Z" represents the length of the 3'-wing. "X" and "Z" may comprise uniform, variant, or alternating sugar moieties. In certain embodiments, "X" and "Y" may include one or more 2'-deoxynucleosides."Y" may comprise 2'-deoxynucleosides. As used herein, a gapmer described as "X-Y-Z" has a configuration such that the gap is positioned immediately adjacent to each of the 5'-wing and the 3 ' wing. Thus, no intervening nucleotides exist between the 5'- wing and gap, or the gap and the 3 '-wing. Any of the antisense compounds described herein can have a gapmer motif. In certain embodiments, "X" and "Z" are the same; in other embodiments they are different. In certain embodiments, 'Ύ" is between 8 and 15 nucleosides. X, Y, or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleosides. In certain embodiments, the antisense compound targeted to a PHD1 nucleic acid has a gapmer motif in which the gap consists of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 linked nucleosides.
In certain embodiments, the antisense oligonucleotide has a sugar motif described by Formula A as follows: (J)m-(B)n-(J)p-(B)r-(A)t-(D)g-(A)v-(B)w-(J)x-(B)y-(J)z
wherein:
each A is independently a 2'-substituted nucleoside;
each B is independently a bicyclic nucleoside;
each J is independently either a 2'-substituted nucleoside or a 2'-deoxynucleoside;
each D is a 2'-deoxynucleoside;
m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; g is 6-14; provided that:
at least one of m, n, and r is other than 0;
at least one of w and y is other than 0;
the sum of m, n, p, r, and t is from 2 to 5; and
the sum of v, w, x, y, and z is from 2 to 5.
RNAi Compounds
In certain embodiments, antisense compounds are interfering RNA compounds (RNAi), which include double- stranded RNA compounds (also referred to as short-interfering RNA or siRNA) and single- stranded RNAi compounds (or ssRNA). Such compounds work at least in part through the RISC pathway to degrade and/or sequester a target nucleic acid (thus, include microRNA/microRNA-mimic compounds). In certain embodiments, antisense compounds comprise modifications that make them particularly suited for such mechanisms.
i. ssRNA compounds
In certain embodiments, antisense compounds including those particularly suited for use as single- stranded RNAi compounds (ssRNA) comprise a modified 5'-terminal end. In certain such embodiments, the 5'-terminal end comprises a modified phosphate moiety. In certain embodiments, such modified phosphate is stabilized (e.g., resistant to degradation/cleavage compared to unmodified 5'- phosphate). In certain embodiments, such 5'-terminal nucleosides stabilize the 5 '-phosphorous moiety. Certain modified 5'-terminal nucleosides may be found in the art, for example in WO/201 1/139702.
In certain embodiments, the 5'-nucleoside of an ssRNA compound has Formula lie:
Figure imgf000021_0001
lie
wherein:
Ti is an optionally protected phosphorus moiety;
T2 is an internucleoside linking group linking the compound of Formula lie to the oligomeric compound;
A has one of the formula
Figure imgf000021_0002
Qi and Q2 are each, independently, H, halogen, Ci-Ce alkyl, substituted Ci-Ce alkyl, d-Ce alkoxy, substituted Ci-Ce alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or N(R3)(R4);
Q3 is O, S, N(R5) or C(R6)(R7);
each R3, R4 R5, R6 and R7 is, independently, H, C -C6 alkyl, substituted C -C6 alkyl or C -C6 alkoxy;
M3 is O, S, NR14, C(R15)(R16), C(R15)(R16)C(R17)(Ri8), C(R15)=C(R17), OC(R15)(R16) or OC(R15)(Bx2) ;
Ri4 is H, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci-Ce alkoxy, substituted Ci-Ce alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
Ri5, Ri6, Ri7 and Ris are each, independently, H, halogen, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci- Ce alkoxy, substituted Ci-Ce alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
Bx! is a heterocyclic base moiety;
or if Bx2 is present then Bx2 is a heterocyclic base moiety and Bx! is H, halogen, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci-Ce alkoxy, substituted Ci-Ce alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
J4, J5, Je and J7 are each, independently, H, halogen, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci-Ce alkoxy, substituted Ci-Ce alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; or J4 forms a bridge with one of J5 or J7 wherein said bridge comprises from 1 to 3 linked biradical groups selected from O, S, NR19, C(R20)(R2i), C(R2o)=C(R2i),
Figure imgf000022_0001
and C(=0) and the other two of J5, Je and J7 are each, ind endently, H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, Ci- Ce alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
each R19, R20 and R2i is, independently, H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
G is H, OH, halogen or
Figure imgf000022_0002
each R8 and R9 is, independently, H, halogen, C1-C6 alkyl or substituted C1-C6 alkyl;
Xi is O, S or N(Ei);
Z is H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2- Ce alkynyl, substituted C2-C6 alkynyl or N(E2)(E3) ;
Ei, E2 and E3 are each, independently, H, C1-C6 alkyl or substituted C1-C6 alkyl;
n is from 1 to about 6;
m is 0 or 1 ;
j is 0 or 1 ;
each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJi, N(Ji)(J2),
Figure imgf000022_0003
and
Figure imgf000022_0004
X2 is O, S or NJ3;
each Ji, J2 and J3 is, independently, H or C1-C6 alkyl;
when j is 1 then Z is other than halogen or N(E2)(E3) ; and
wherein said oligomeric compound comprises from 8 to 40 monomeric subunits and is hybridizable to at least a portion of a target nucleic acid.
In certain embodiments, M3 is O, CH=CH, OCH2 or OC(H)(Bx2). In certain embodiments, M3 is
O.
In certain embodiments, J4, J5, Je and J7 are each H. In certain embodiments, J4 forms a bridge with one of J5 or J7.
In certain embodiments A has one of the formulas:
Figure imgf000022_0005
wherein: Qi and (¾ are each, independently, H, halogen, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci-Ce alkoxy or substituted Ci-Ce alkoxy. In certain embodiments, Qi and Q2 are each H. In certain embodiments, Qi and Q2 are each, independently, H or halogen. In certain embodiments, Qi and Q2 is H and the other of Qj and Q2 is F, CH3 or OCH3.
In certain embodiments, Ti has the formula:
Ra
Rb= -|
Rc
wherein:
Ra and Rc are each, independently, protected hydroxyl, protected thiol, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci-Ce alkoxy, substituted Ci-Ce alkoxy, protected amino or substituted amino; and
Rb is O or S. In certain embodiments, Rb is O and Ra and Rc are each, independently, OCH3,
Figure imgf000023_0001
In certain embodiments, G is halogen, OCH3, OCH2F, OCHF2, OCF3, OCH2CH3, 0(CH2)2F, OCH2CHF2, OCH2CF3, OCH2-CH=CH2, 0(CH2)2-OCH3, 0(CH2)2-SCH3, 0(CH2)2-OCF3, 0(CH2)3- N(R10)(R„), O(CH2)2-ON(R10)(Rn), O(CH2)2-O(CH2)2-N(R10)(R„), OCH2C(=O)-N(R10)(R„), OCH2C(=0)-N(R12)-(CH2)2-N(R1o)(R„) or 0(CH2)2-N(R12)-C(=NR13)[N(R1o)(R„)] wherein R10, R„, R12 and Ri3 are each, independently, H or Ci-Ce alkyl. In certain embodiments, G is halogen, OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2-CH=CH2, 0(CH2)2-OCH3, 0(CH2)2-0(CH2)2-N(CH3)2, OCH2C(=0)- N(H)CH3, OCH2C(=0)-N(H)-(CH2)2-N(CH3)2 or OCH2-N(H)-C(=NH)NH2. In certain embodiments, G is F, OCH3 or 0(CH2)2-OCH3. In certain embodiments, G is 0(CH2)2-OCH3.
In c al nucleoside has Formula He:
Figure imgf000023_0002
He
In certain embodiments, antisense compounds, including those particularly suitable for ssRNA comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar modification motif. Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications.
In certain embodiments, the oligonucleotides comprise or consist of a region having uniform sugar modifications. In certain such embodiments, each nucleoside of the region comprises the same RNA-like sugar modification. In certain embodiments, each nucleoside of the region is a 2'-F nucleoside.
In certain embodiments, each nucleoside of the region is a 2'-OMe nucleoside. In certain embodiments, each nucleoside of the region is a 2'-MOE nucleoside. In certain embodiments, each nucleoside of the region is a cEt nucleoside. In certain embodiments, each nucleoside of the region is an LNA nucleoside. In certain embodiments, the uniform region constitutes all or essentially all of the oligonucleotide. In certain embodiments, the region constitutes the entire oligonucleotide except for 1-4 terminal nucleosides.
In certain embodiments, oligonucleotides comprise one or more regions of alternating sugar modifications, wherein the nucleosides alternate between nucleotides having a sugar modification of a first type and nucleotides having a sugar modification of a second type. In certain embodiments, nucleosides of both types are RNA-like nucleosides. In certain embodiments the alternating nucleosides are selected from: 2'-OMe, 2'-F, 2'-MOE, LNA, and cEt. In certain embodiments, the alternating modificatios are 2'-F and 2'-OMe. Such regions may be contiguous or may be interupted by differently modified nucleosides or conjugated nucleosides.
In certain embodiments, the alternating region of alternating modifications each consist of a single nucleoside (i.e., the patern is (AB)xAy wheren A is a nucleoside having a sugar modification of a first type and B is a nucleoside having a sugar modification of a second type; x is 1-20 and y is 0 or 1). In certan embodiments, one or more alternating regions in an alternating motif includes more than a single nucleoside of a type. For example, oligonucleotides may include one or more regions of any of the following nucleoside motifs:
AABBAA;
ABBABB;
AABAAB;
ABBABAABB;
ABABAA;
AABABAB;
ABABAA;
ABBAABBABABAA;
BABBAABBABABAA; or
ABABBAABBABABAA;
wherein A is a nucleoside of a first type and B is a nucleoside of a second type. In certain embodiments, A and B are each selected from 2'-F, 2'-OMe, BNA, and MOE.
In certain embodiments, oligonucleotides having such an alternating motif also comprise a modified 5' terminal nucleoside, such as those of formula lie or He. In certain embodiments, oligonucleotides comprise a region having a 2-2-3 motif. Such regions comprises the following motif:
-(A)2-(B)x-(A)2-(C)y-(A)3- wherein: A is a first type of modifed nucleosde;
B and C, are nucleosides that are differently modified than A, however, B and C may have the same or different modifications as one another;
x and y are from 1 to 15.
In certain embodiments, A is a 2'-OMe modified nucleoside. In certain embodiments, B and C are both 2'-F modified nucleosides. In certain embodiments, A is a 2'-OMe modified nucleoside and B and C are both 2'-F modified nucleosides.
In certain embodiments, oligonucleo sides have the following sugar motif:
5'- (Q)- (AB)xAy-(D)z
wherein:
Q is a nucleoside comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having Formula lie or He;
A is a first type of modifed nucleoside;
B is a second type of modified nucleoside;
D is a modified nucleoside comprising a modification different from the nucleoside adjacent to it. Thus, if y is 0, then D must be differently modified than B and if y is 1, then D must be differently modified than A. In certain embodiments, D differs from both A and B.
X is 5-15;
Y is O or 1;
Z is 0-4.
In certain embodiments, oligonucleo sides have the following sugar motif:
5'- (Q)- (A)X-(D)Z
wherein:
Q is a nucleoside comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having Formula lie or He;
A is a first type of modifed nucleoside;
D is a modified nucleoside comprising a modification different from A.
X is 11-30;
Z is 0-4.
In certain embodiments A, B, C, and D in the above motifs are selected from: 2'-OMe, 2'-F, 2'- MOE, LNA, and cEt. In certain embodiments, D represents terminal nucleosides. In certain embodiments, such terminal nucleosides are not designed to hybridize to the target nucleic acid (though one or more might hybridize by chance). In certiain embodiments, the nucleobase of each D nucleoside is adenine, regardless of the identity of the nucleobase at the corresponding position of the target nucleic acid. In certain embodiments the nucleobase of each D nucleoside is thymine.
In certain embodiments, antisense compounds, including those particularly suited for use as ssRNA comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.
In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least one 12 consecutive phosphoro- thioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3' end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3' end of the oligonucleotide.
Oligonucleotides having any of the various sugar motifs described herein, may have any linkage motif. For example, the oligonucleotides, including but not limited to those described above, may have a linkage motif selected from non- limiting the table below:
Figure imgf000026_0001
ii. siRNA compounds
In certain embodiments, antisense compounds are double- stranded RNAi compounds (siRNA). In such embodiments, one or both strands may comprise any modification motif described above for ssRNA. In certain embodiments, ssRNA compounds may be unmodified RNA. In certain embodiments, siRNA compounds may comprise unmodified RNA nucleosides, but modified internucleoside linkages.
Several embodiments relate to double- stranded compositions wherein each strand comprises a motif defined by the location of one or more modified or unmodified nucleosides. In certain embodiments, compositions are provided comprising a first and a second oligomeric compound that are fully or at least partially hybridized to form a duplex region and further comprising a region that is complementary to and hybridizes to a nucleic acid target. It is suitable that such a composition comprise a first oligomeric compound that is an antisense strand having full or partial complementarity to a nucleic acid target and a second oligomeric compound that is a sense strand having one or more regions of complementarity to and forming at least one duplex region with the first oligomeric compound.
The compositions of several embodiments modulate gene expression by hybridizing to a nucleic acid target resulting in loss of its normal function. In some embodiments, the target nucleic acid is PHD1. In certain embodiment, the degradation of the targeted PHD1 is facilitated by an activated RISC complex that is formed with compositions of the invention.
Several embodiments are directed to double- stranded compositions wherein one of the strands is useful in, for example, influencing the preferential loading of the opposite strand into the RISC (or cleavage) complex. The compositions are useful for targeting selected nucleic acid molecules and modulating the expression of one or more genes. In some embodiments, the compositions of the present invention hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA.
Certain embodiments are drawn to double- stranded compositions wherein both the strands comprises a hemimer motif, a fully modified motif, a positionally modified motif or an alternating motif. Each strand of the compositions of the present invention can be modified to fulfill a particular role in for example the siRNA pathway. Using a different motif in each strand or the same motif with different chemical modifications in each strand permits targeting the antisense strand for the RISC complex while inhibiting the incorporation of the sense strand. Within this model, each strand can be independently modified such that it is enhanced for its particular role. The antisense strand can be modified at the 5'-end to enhance its role in one region of the RISC while the 3 '-end can be modified differentially to enhance its role in a different region of the RISC.
The double- stranded oligonucleotide molecules can be a double- stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The double- stranded oligonucleotide molecules can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e. each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double- stranded structure, for example wherein the double- stranded region is about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 15 to about 25 or more nucleotides of the double- stranded oligonucleotide molecule are complementary to the target nucleic acid or a portion thereof). Alternatively, the double- stranded oligonucleotide is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siRNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s).
The double- stranded oligonucleotide can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The double- stranded oligonucleotide can be a circular single- stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion Ihereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi.
In certain embodiments, the double- stranded oligonucleotide comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions. In certain embodiments, the double- stranded oligonucleotide comprises nucleotide sequence that is complementary to nucleotide sequence of a target gene. In another embodiment, the double- stranded oligonucleotide interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene.
As used herein, double- stranded oligonucleotides need not be limited to those molecules containing only RNA, but further encompasses chemically modified nucleotides and non-nucleotides. In certain embodiments, the short interfering nucleic acid molecules lack 2'-hydroxy (2'-OH) containing nucleotides. In certain embodiments short interfering nucleic acids optionally do not include any ribonucleotides (e.g., nucleotides having a 2'-OH group). Such double- stranded oligonucleotides that do not require the presence of ribonucleotides within the molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2'-OH groups. Optionally, double- stranded oligonucleotides can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. As used herein, the term siRNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double- stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically modified siRNA, post- transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics. For example, double- stranded oligonucleotides can be used to epigenetically silence genes at both the post- transcriptional level and the pre-transcriptional level. In a non-limiting example, epigenetic regulation of gene expression by siRNA molecules of the invention can result from siRNA mediated modification of chromatin structure or methylation pattern to alter gene expression (see, for example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237).
It is contemplated that compounds and compositions of several embodiments provided herein can target PHD1 by a dsRNA-mediated gene silencing or RNAi mechanism, including, e.g., "hairpin" or stem-loop double- stranded RNA effector molecules in which a single RNA strand with self- complementary sequences is capable of assuming a double- stranded conformation, or duplex dsRNA effector molecules comprising two separate strands of RNA. In various embodiments, the dsRNA consists entirely of ribonucleotides or consists of a mixture of ribonucleotides and deoxynucleotides, such as the RNA/DNA hybrids disclosed, for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999. The dsRNA or dsRNA effector molecule may be a single molecule with a region of self-complementarity such that nucleotides in one segment of the molecule base pair with nucleotides in another segment of the molecule. In various embodiments, a dsRNA that consists of a single molecule consists entirely of ribonucleotides or includes a region of ribonucleotides that is complementary to a region of deoxyribonucleotides. Alternatively, the dsRNA may include two different strands that have a region of com lementarity to each other.
In various embodiments, both strands consist entirely of ribonucleotides, one strand consists entirely of ribonucleotides and one strand consists entirely of deoxyribonucleotides, or one or both strands contain a mixture of ribonucleotides and deoxyribonucleotides. In certain embodiments, the regions of complementarity are at least 70, 80, 90, 95, 98, or 100% complementary to each other and to a target nucleic acid sequence. In certain embodiments, the region of the dsRNA that is present in a double- stranded conformation includes at least 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 200, 500, 1000, 2000 or 5000 nucleotides or includes all of the nucleotides in a cDNA or other target nucleic acid sequence being represented in the dsRNA. In some embodiments, the dsRNA does not contain any single stranded regions, such as single stranded ends, or the dsRNA is a hairpin. In other embodiments, the dsRNA has one or more single stranded regions or overhangs. In certain embodiments, RNA/DNA hybrids include a DNA strand or region that is an antisense strand or region (e.g, has at least 70, 80, 90, 95, 98, or 100% complementarity to a target nucleic acid) and an RNA strand or region that is a sense strand or region (e.g, has at least 70, 80, 90, 95, 98, or 100% identity to a target nucleic acid), and vice versa.
In various embodiments, the RNA/DNA hybrid is made in vitro using enzymatic or chemical synthetic methods such as those described herein or those described in WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999. In other embodiments, a DNA strand synthesized in vitro is complexed with an RNA strand made in vivo or in vitro before, after, or concurrent with the transformation of the DNA strand into the cell. In yet other embodiments, the dsRNA is a single circular nucleic acid containing a sense and an antisense region, or the dsRNA includes a circular nucleic acid and either a second circular nucleic acid or a linear nucleic acid (see, for example, WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.) Exemplary circular nucleic acids include lariat structures in which the free 5' phosphoryl group of a nucleotide becomes linked to the 2' hydroxyl group of another nucleotide in a loop back fashion.
In other embodiments, the dsRNA includes one or more modified nucleotides in which the 2' position in the sugar contains a halogen (such as fluorine group) or contains an alkoxy group (such as a methoxy group) which increases the half-life of the dsRNA in vitro or in vivo compared to the corresponding dsRNA in which the corresponding 2' position contains a hydrogen or an hydroxyl group. In yet other embodiments, the dsRNA includes one or more linkages between adjacent nucleotides other than a naturally-occurring phosphodiester linkage. Examples of such linkages include phosphoramide, phosphorothioate, and phosphorodithioate linkages. The dsRNAs may also be chemically modified nucleic acid molecules as taught in U.S. Pat. No. 6,673,661. In other embodiments, the dsRNA contains one or two capped strands, as disclosed, for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.
In other embodiments, the dsRNA can be any of the at least partially dsRNA molecules disclosed in WO 00/63364, as well as any of the dsRNA molecules described in U.S. Provisional Application 60/399,998; and U.S. Provisional Application 60/419,532, and PCT/US2003/033466, the teaching of which is hereby incorporated by reference. Any of the dsRNAs may be expressed in vitro or in vivo using the methods described herein or standard methods, such as those described in WO 00/63364.
Occupancy
In certain embodiments, antisense compounds are not expected to result in cleavage or the target nucleic acid via RNase H or to result in cleavage or sequestration through the RISC pathway. In certain such embodiments, antisense activity may result from occupancy, wherein the presence of the hybridized antisense compound disrupts the activity of the target nucleic acid. In certain such embodiments, the antisense compound may be uniformly modified or may comprise a mix of modifications and/or modified and unmodified nucleosides.
Target Nucleic Acids, Target Regions and Nucleotide Sequences
Nucleotide sequences that encode PHD1 include, without limitation, the following: GENBANK Accession No. NM 053208.4 (incorporated herein as SEQ ID NO: 1); the complement of GENBANK Accession No. NT 039413.7 truncated from nucleotides 24942670 to 24952750 (incorporated herein SEQ ID NO: 2); GENBANK Accession No. NM 080732.3 (incorporated herein SEQ ID NO: 3); GENBANK Accession No. NM 053046.3 (incorporated herein SEQ ID NO: 4); GENBANK Accession No. AL133009.1 (incorporated herein SEQ ID NO: 5); and GENBANK Accession No. NT 011 109.16 truncated from nucleotides 13572265 to 13583555 (incorporated herein SEQ ID NO: 6).
Hybridization
In some embodiments, hybridization occurs between an antisense compound disclosed herein and a PHD1 nucleic acid. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules. Hybridization can occur under varying conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.
Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In certain embodiments, the antisense compounds provided herein are specifically hybridizable with a PHD1 nucleic acid.
Complementarity
An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., antisense inhibition of a target nucleic acid, such as a PHD1 nucleic acid).
Non-complementary nucleobases between an antisense compound and a PHD1 nucleic acid may be tolerated provided that the antisense compound remains able to specifically hybridize to a target nucleic acid. Moreover, an antisense compound may hybridize over one or more segments of a PHD1 nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).
In certain embodiments, the antisense compounds provided herein, or a specified portion thereof, are, or are at least, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%o, 97%), 98%), 99%), or 100% complementary to a PHD1 nucleic acid, a target region, target segment, or specified portion thereof. Percent complementarity of an antisense compound with a target nucleic acid can be determined using routine methods.
For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having four noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).
In certain embodiments, the antisense compounds provided herein, or specified portions thereof, are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof. For example, an antisense compound may be fully complementary to a PHD1 nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, "fully complementary" means each nucleobase of an antisense compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 20 nucleobase antisense compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the antisense compound. Fully complementary can also be used in reference to a specified portion of the first and /or the second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase antisense compound can be "fully complementary" to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the antisense compound. At the same time, the entire 30 nucleobase antisense compound may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.
The location of a non-complementary nucleobase may be at the 5' end or 3' end of 1he antisense compound. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e. linked) or non-contiguous. In one embodiment, a non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.
In certain embodiments, antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a PHD1 nucleic acid, or specified portion thereof.
In certain embodiments, antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a PHD1 nucleic acid, or specified portion thereof.
The antisense compounds provided also include those which are complementary to a portion of a target nucleic acid. As used herein, "portion" refers to a defined number of contiguous (i.e. linked) nucleobases within a region or segment of a target nucleic acid. A "portion" can also refer to a defined number of contiguous nucleobases of an antisense compound. In certain embodiments, the antisense compounds, are complementary to at least an 8 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 9 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 10 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least an 11 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 12 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 13 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 14 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 15 nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values. Identity
The antisense compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific Isis number, or portion thereof. As used herein, an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the antisense compounds described herein as well as compounds having non-identical bases relative to the antisense compounds provided herein also are contemplated. The non-identical bases may be adjacent to each other or dispersed throughout the antisense compound. Percent identity of an antisense compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.
In certain embodiments, the antisense compounds, or portions thereof, are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of the antisense compounds or SEQ ID NOs, or a portion thereof, disclosed herein.
In certain embodiments, a portion of the antisense compound is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid. In certain embodiments, a portion of the antisense oligonucleotide is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.
Modifications
A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3' or 5' hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.
Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.
Chemically modified nucleosides may also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides.
Modified Internucleoside Linkages
The naturally occuring internucleoside linkage of RNA and DNA is a 3' to 5' phosphodiester linkage. Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.
Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known. In certain embodiments, antisense compounds targeted to a PHD1 nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.
Modified Sugar Moieties
Antisense compounds can optionally contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the antisense compounds. In certain embodiments, nucleosides comprise chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include without limitation, addition of substitutent groups (including 5' and 2' substituent groups, bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA), replacement of the ribosyl ring oxygen atom with S, N(R), or C(Ri)(R2) (R, Ri and R2 are each independently H, C1-C12 alkyl or a protecting group) and combinations thereof. Examples of chemically modified sugars include 2'-F-5'-methyl substituted nucleoside (see PCT International Application WO 2008/101157 Published on 8/21/08 for other disclosed 5',2'-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2'-position (see published U.S. Patent Application US2005-0130923, published on June 16, 2005) or alternatively 5'-substitution of a BNA (see PCT International Application WO 2007/134181 Published on 11/22/07 wherein 4'-(CH2)-0-2' (LNA) is substituted with for example a 5'-methyl or a 5'-vinyl group).
Examples of nucleosides having modified sugar moieties include without limitation nucleosides comprising 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3, 2'-OCH2CH3, 2'-OCH2CH2F and 2'- 0(CH2)2OCH3 substituent groups. The substituent at the 2' position can also be selected from allyl, amino, azido, thio, O-allyl, O-CrC10 alkyl, OCF3, OCH2F, 0(CH2)2SCH3, 0(CH2)2-0-N(Rm)(Rn), 0-CH2- C(=0)-N(Rm)(Rn), and 0-CH2-C(=0)-N(R1)-(CH2)2-N(Rm)(Rn), where each ¾ Rm and Rn is, independently, H or substituted or unsubstituted
Figure imgf000036_0001
alkyl.
As used herein, '¾icyclic nucleosides" refer to modified nucleosides comprising a bicyclic sugar moiety. Examples of bicyclic nucleosides include without limitation nucleosides comprising a bridge between the 4' and the 2' ribosyl ring atoms. In certain embodiments, antisense compounds provided herein include one or more bicyclic nucleosides comprising a 4' to 2' bridge. Examples of such 4' to 2' bridged bicyclic nucleosides, include but are not limited to one of the formulae: 4'-(CH2)-0-2' (LNA) ; 4'- (CH2)-S-2'; 4'-(CH2)2-0-2' (ENA) ; 4'-CH(CH3)-0-2' (also referred to as constrained ethyl or cEt) and 4'- CH(CH2OCH3)-0-2' (and analogs thereof see U.S. Patent 7,399,845, issued on July 15, 2008); 4'- C(CH3)(CH3)-0-2' (and analogs thereof see published International Application WO/2009/006478, published January 8, 2009); 4'-CH2-N(OCH3)-2' (and analogs thereof see published International Application WO/2008/150729, published December 11, 2008); 4'-CH2-0-N(CH3)-2' (see published U.S. Patent Application US 2004-0171570, published September 2, 2004 ); 4'-CH2-N(R)-0-2', wherein R is H, C1-C12 alkyl, or a protecting group (see U.S. Patent 7,427,672, issued on September 23, 2008); 4'-CH2-C- (H)(CH3)-2' (see Chattopadhyaya et al, J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and analogs thereof see published International Application WO 2008/154401, published on December 8, 2008).
Further reports related to bicyclic nucleosides can also be found in published literature (see for example: Singh et al, Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607- 3630; Wahlestedt et al, Proc. Natl. Acad. Sci. U. S. A , 2000, 97, 5633-5638; Kumar et al, Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379; Elayadi et al, Curr. Opinion Invest. Drugs, 2001, 2, 558-561 ; Braasch et al, Chem. Biol, 2001, 8, 1-7; and Orum et al, Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Patent Nos. 6,268,490; 6,525,191 ; 6,670,461 ; 6,770,748; 6,794,499; 7,034,133; 7,053,207; 7,399,845; 7,547,684; and 7,696,345; U.S. Patent Publication No. US2008-0039618; US 2009-0012281 ; U.S. Patent Serial Nos. 60/989,574; 61/026,995; 61/026,998; 61/056,564; 61/086,231 ; 61/097,787; and 61/099,844; Published PCT International applications WO 1994/014226; WO 2004/106356; WO 2005/021570; WO 2007/134181 ; WO 2008/150729; WO 2008/154401 ; and WO 2009/006478. Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example a-L-ribofuranose and β-D-ribofuranose (see PCT international application PCT/DK98/00393, published on March 25, 1999 as WO 99/14226).
In certain embodiments, bicyclic sugar moieties of BNA nucleosides include, but are not limited to, compounds having at least one bridge between the 4' and the 2' position of the pentofuranosyl sugar moiety wherein such bridges independently comprises 1 or from 2 to 4 linked groups independently selected from -[C(Ra)(Rb)]n-, -C(Ra)=C(Rb)-, -C(Ra)=N-, -C(=0)-, -C(=NRa)-, -C(=S)-, -0-, -Si(Ra)2-, - S(=0)x-, and -N(Ra)-;
wherein:
x is 0, 1, or 2;
n is 1, 2, 3, or 4;
each Ra and Rb is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted d-
C12 alkyl, C2-Ci2 alkenyl, substituted C2-Ci2 alkenyl, C2-Ci2 alkynyl, substituted C2-Ci2 alkynyl, Cs-C2o aryl, substituted Cs-C2o aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJi, NJi J2, S Ji, N3, COOJi, acyl (C(=0)-H), substituted acyl, CN, sulfonyl (S(=O)2-J , or sulfoxyl (S(=O)-J ; and each J! and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-Q2 alkenyl, substituted C2-C12 alkenyl, C2-Q2 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=0)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl or a protecting group.
In certain embodiments, the bridge of a bicyclic sugar moiety is -[C(Ra)(Rb)]n-, -[C(Ra)(Rb)]n-0-
, -C(RaRb)-N(R)-0- or -C(RaRb)-0-N(R)-. In certain embodiments, the bridge is 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-0-2', 4'-(CH2)2-0-2', 4'-CH2-0-N(R)-2' and 4'-CH2-N(R)-0-2'- wherein each R is, independently, H, a protecting group or C1-C12 alkyl.
In certain embodiments, bicyclic nucleosides are further defined by isomeric configuration. For example, a nucleoside comprising a 4'-2' methylene-oxy bridge, may be in the a-L configuration or in the β-D configuration. Previously, a-L-methyleneoxy (4'-CH2-0-2') BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et ah, Nucleic Acids Research, 2003, 21, 6365-6372).
In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) a-L- methyleneoxy (4'-CH2-0-2') BNA , (B) β-D-methyleneoxy (4'-CH2-0-2') BNA , (C) ethyleneoxy (4'- (CH2)2-0-2') BNA , (D) aminooxy (4'-CH2-0-N(R)-2') BNA, (E) oxyamino (4'-CH2-N(R)-0-2') BNA, and (F) methyl(methyleneoxy) (4'-CH(CH3)-0-2') BNA, (G) methylene-thio (4'-CH2-S-2') BNA, (H) methylene-amino (4'-CH2-N(R)-2') BNA, (I) methyl carbocyclic (4'-CH2-CH(CH3)-2') BNA, (J) propylene carbocyclic (4'-(CH2)3-2') BNA and (K) vinyl BNA as depicted below:
Figure imgf000038_0001
wherein Bx is the base moiety and R is independently H, a protecting group, C1-C12 alkyl or Q- C12 alkoxy.
odiments, bicyclic nucleosides are provided having Formula I:
Figure imgf000039_0001
wherein:
Bx is a heterocyclic base moiety;
-Qa-Qb-Qc- is -CH2-N(RC)-CH2-,
Figure imgf000039_0002
-CH2-0-N(Rc)-, -ΟΗ2-Ν(¾)-0- or -Ν(¾)-
0-CH2;
Rc is C1-C12 alkyl or an amino protecting group; and
Ta and T are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium.
In certain embodiments, bicyclic nucleosides are provided having Formula II:
Figure imgf000039_0003
wherein:
Bx is a heterocyclic base moiety;
Ta and T are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
Za is Ci-Ce alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-Ce alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thio.
In one embodiment, each of the substituted groups is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJc, NJcJd, SJC, N3, OC(=X)Jc, and NJeC(=X)NJcJd, wherein each Jc, Jd and Je is, independently, H, C -C6 alkyl, or substituted Ci-Ce alkyl and X is O or NJC.
In certain embodiments, bicyclic nucleosides are provided having Formula III:
Figure imgf000040_0001
wherein:
Bx is a heterocyclic base moiety;
Ta and T are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
Zb is Ci-Ce alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-Ce alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl or substituted acyl (C(=0)-).
In c leosides are provided having Formula IV:
Figure imgf000040_0002
wherein:
Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
Rd is Ci-Ce alkyl, substituted Ci-Ce alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
each qa, qb, qc and qd is, independently, H, halogen, Ci-Ce alkyl, substituted Ci-Ce alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl, Ci-Ce alkoxyl, substituted Ci-Ce alkoxyl, acyl, substituted acyl, Ci-Ce aminoalkyl or substituted Ci-Ce aminoalkyl;
In certain embodiments, bicyclic nucleosides are provided having Formula V:
Figure imgf000041_0001
wherein:
Bx is a heterocyclic base moiety;
Ta and T are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
qa, qb, qe and qf are each, independently, hydrogen, halogen, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C1-C12 alkoxy, substituted CrC12 alkoxy, OJj, SJj, SOJj, S02Jj, NJjJk, N3, CN, C(=0)OJj, C(=0)NJjJk, C(=0)Jj, 0-C(=0)- NJjJk, N(H)C(=NH)NJjJk, N(H)C(=0)NJjJk or N(H)C(=S)NJjJk;
or qe and qf together are =C(qg)(q );
qg and q are each, independently, H, halogen, C1-C12 alkyl or substituted C1-C12 alkyl.
The synthesis and preparation of the methyleneoxy (4'-CH2-0-2') BNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.
Analogs of methyleneoxy (4'-CH2-0-2') BNA and 2'-thio-BNAs, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs comprising oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226 ). Furthermore, synthesis of 2'-amino-BNA, a novel comformationally restricted high-affinity oligonucleotide analog has been described in the art (Singh et al, J. Org. Chem., 1998, 63, 10035-10039). In addition, 2'-amino- and 2'-methylamino-BNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.
In certain embodiments, bicyclic nucleosides are provided having Formula VI:
Figure imgf000042_0001
wherein:
Bx is a heterocyclic base moiety;
Ta and Ί , are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
each qi, ¾, qk and qi is, independently, H, halogen, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C1-C12 alkoxyl, substituted CrC12 alkoxyl, OJj, SJj, SOJj, S02Jj, NJjJk, N3, CN, C(=0)OJj, C(=0)NJjJk, C(=0)Jj, 0-C(=0)NJjJk, N(H)C(=NH)NJjJk, N(H)C(=0)NJjJk or N(H)C(=S)NJjJk; and
qi and ¾ or qi and qk together are =C(qg)(q ), wherein qg and q are each, ind endently, H, halogen, C1-C12 alkyl or substituted C1-C12 alkyl.
One carbocyclic bicyclic nucleoside having a 4'-(CH2)3-2' bridge and the alkenyl analog bridge 4'- CH=CH-CH2-2' have been described (Freier et al, Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al, J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (Srivastava et al, J. Am. Chem. Soc, 2007, 129(26), 8362-8379).
As used herein, "4 '-2' bicyclic nucleoside" or "4' to 2' bicyclic nucleoside" refers to a bicyclic nucleoside comprising a furanose ring comprising a bridge connecting two carbon atoms of the furanose ring connects the 2' carbon atom and the 4' carbon atom of the sugar ring.
As used herein, "monocylic nucleosides" refer to nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties. In certain embodiments, the sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position.
As used herein, "2'-modified sugar" means a furanosyl sugar modified at the 2' position. In certain embodiments, such modifications include substituents selected from: a halide, including, but not limited to substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In certain embodiments, 2' modifications are selected from substituents including, but not limited to: 0[(CH2)nO]mCH3, 0(CH2)nNH2, 0(CH2)nCH3, 0(CH2)nF, 0(CH2)nONH2, OCH2C(=0)N(H)CH3, and 0(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10. Other 2'- substituent groups can also be selected from: C1-C12 alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, CI, Br, CN, F, CF3, OCF3, SOCH3, SO2CH3, ON02, N02, N3, NH2, heterocycloalkyl, heterocycloalkaryl, amino alkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving pharmacokinetic properties, or a group for improving the pharmacodynamic properties of an antisense compound, and other substituents having similar properties. In certain embodiments, modifed nucleosides comprise a 2'-MOE side chain (Baker et al, J. Biol. Chem., 1997, 272, 11944-12000). Such 2'-MOE substitution have been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2'- O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the 2'-MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).
As used herein, a "modified tetrahydropyran nucleoside" or "modified THP nucleoside" means a nucleoside having a six-membered tetrahydropyran "sugar" substituted in for the pentofuranosyl residue in normal nucleosides (a sugar surrogate). Modified THP nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, Bioorg. Med. Chem., 2002, 10, 841-854) or fluoro HNA (F-HNA) having a tetr
Figure imgf000043_0001
In certain embo s are selected having Formula VII:
Figure imgf000043_0002
VII
wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula VII:
Bx is a heterocyclic base moiety;
Ta and T are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of Ta and Tb is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of Ta and Ί , is H, a hydroxyl protecting group, a linked conjugate group or a 5' or 3'-terminal group;
qi, q2, q3, q4, qs, q6 and q7 are each independently, H, Ci-Ce alkyl, substituted Ci-Ce alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; and each of Ri and R2 is selected from hydrogen, hydroxyl, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJi, N3, OC{=X)ih OC(=X)NJ!J2, NJ3C(=X)NJ!J2 and CN, wherein X is O, S or NJj and each Jj, J2 and J3 is, independently, H or Ci-Ce alkyl.
In certain embodiments, the modified THP nucleosides of Formula VII are provided wherein q1; q2, q3, q4, qs, qe and q7 are each H. In certain embodiments, at least one of qi, q2, q3, q4, qs, qe and q7 is other than H. In certain embodiments, at least one of qi, q2, q3, q4, qs, qe and q7 is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of Ri and R2 is fluoro. In certain embodiments, Ri is fluoro and R2 is H; Ri is methoxy and R2 is H, and Ri is methoxyethoxy and R2 is H.
In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds has been reported (see for example: Braasch et al., Biochemistry, 2002, 41, 4503- 4510; and U.S. Patents 5,698,685; 5, 166,315; 5, 185,444; and 5,034,506). As used here, the term "morp surrogate having the following formula:
Figure imgf000044_0001
In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as "modifed morpholinos."
Combinations of modifications are also provided without limitation, such as 2'-F-5'-methyl substituted nucleosides (see PCT International Application WO 2008/101157 published on 8/21/08 for other disclosed 5', 2'-bis substituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2'-position (see published U.S. Patent Application US2005-0130923, published on June 16, 2005) or alternatively 5'-substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/134181, published on 11/22/07 wherein a 4'-CH2-0-2' bicyclic nucleoside is further substituted at the 5' position with a 5'-methyl or a 5'-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described {see, e.g., Srivastava et ah, J. Am. Chem. Soc. 2007, 129(26), 8362-8379). In certain embodiments, antisense compounds comprise one or more modified cyclohexenyl nucleosides, which is a nucleoside having a six-membered cyclohexenyl in place of the pentofuranosyl residue in naturally occurring nucleosides. Modified cyclohexenyl nucleosides include, but are not limited to those described in the art (see for example commonly owned, published PCT Application WO 2010/036696, published on April 10, 2010, Robeyns et al, J. Am. Chem. Soc., 2008, 130(6), 1979- 1984; Horvath et al, Tetrahedron Letters, 2007, 48, 3621-3623; Nauwelaerts et ah, J. Am. Chem. Soc., 2007, 129(30), 9340-9348; Gu et al.,, Nucleosides, Nucleotides & Nucleic Acids, 2005, 24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005, 33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F: Structural Biology and Crystallization Communications, 2005, F61(6), 585-586; Gu et al, Tetrahedron, 2004, 60(9), 21 11-2123; Gu et al, Oligonucleotides, 2003, 13(6), 479- 489; Wang et al, J. Org. Chem., 2003, 68, 4499-4505; Verbeure et al, Nucleic Acids Research, 2001, 29(24), 4941-4947; Wang et al, J. Org. Chem., 2001, 66, 8478-82; Wang et al, Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7), 785-788; Wang et al, J. Am. Chem., 2000, 122, 8595-8602; Published PCT application, WO 06/047842; and Published PCT Application WO 01/049687; the text of each is incorporated by reference herein, in their entirety). Certain modified cyclohexenyl nucleosides have Formula X.
Figure imgf000045_0001
X
wherein independently for each of said at least one cyclohexenyl nucleoside analog of Formula
X:
Bx is a heterocyclic base moiety;
T3 and T4 are each, independently, an internucleoside linking group linking the cyclohexenyl nucleoside analog to an antisense compound or one of T3 and T4 is an internucleoside linking group linking the tetrahydropyran nucleoside analog to an antisense compound and the other of T3 and T is H, a hydroxyl protecting group, a linked conjugate group, or a 5'-or 3'-terminal group; and
qi, q2, q3, q4, qs, q6, q7, qs and qg are each, independently, H, Ci-Ce alkyl, substituted Ci-Ce alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or other sugar substituent group.
As used herein, "2'-modified" or "2 '-substituted" refers to a nucleoside comprising a sugar comprising a substituent at the 2' position other than H or OH. 2'-modified nucleosides, include, but are not limited to, bicyclic nucleosides wherein the bridge connecting two carbon atoms of the sugar ring connects the 2' carbon and another carbon of the sugar ring; and nucleosides with non-bridging 2'substituents, such as allyl, amino, azido, thio, O-allyl, O-Ci-Cio alkyl, -OCF3, 0-(CH2)2-0-CH3, 2'- 0(CH2)2SCH3, 0-(CH2)2-0-N(Rm)(Rn), or 0-CH2-C(=0)-N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted Ci-Cio alkyl. 2'-modifed nucleosides may further comprise other modifications, for example at other positions of the sugar and/or at the nucleobase.
As used herein, "2'-F" refers to a nucleoside comprising a sugar comprising a fluoro group at the 2' position of the sugar ring.
As used herein, "2'-OMe" or "2'-OCH3" or "2'-0-methyl" each refers to a nucleoside comprising a sugar comprising an -OCH3 group at the 2' position of the sugar ring.
As used herein, "oligonucleotide" refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleo sides (RNA) and/or deoxyribonucleosides (DNA).
Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see for example review article: Leumann, Bioorg. Med. Chem., 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to enhance activity.
Methods for the preparations of modified sugars are well known to those skilled in the art. Some representative U.S. patents that teach the preparation of such modified sugars include without limitation, U.S.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811 ; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,670,633; 5,700,920; 5,792,847 and 6,600,032 and International Application PCT/US2005/019219, filed June 2, 2005 and published as WO 2005/121371 on December 22, 2005, and each of which is herein incorporated by reference in its entirety.
In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.
In certain embodiments, antisense compounds comprise one or more nucleosides having modified sugar moieties. In certain embodiments, the modified sugar moiety is 2'-MOE. In certain embodiments, the 2'-MOE modified nucleosides are arranged in a gapmer motif. In certain embodiments, the modified sugar moiety is a bicyclic nucleoside having a (4'-CH(CH3)-0-2') bridging group. In certain embodiments, the (4'-CH(CH3)-0-2') modified nucleosides are arranged throughout the wings of a gapmer motif. Modified Nucleobases
Nucleobase (or base) modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications can impart nuclease stability, binding affinity or some other beneficial biological property to antisense compounds. Modified nucleobases include synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of an antisense compound for a target nucleic acid. For example, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).
Additional modified nucleobases include 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- amino adenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5- trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F- adenine, 2- amino- adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3- deazaguanine and 3-deazaadenine.
Heterocyclic base moieties can also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7- deaza- adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Nucleobases that are particularly useful for increasing the binding affinity of antisense compounds include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
In certain embodiments, antisense compounds targeted to a PHDl nucleic acid comprise one or more modified nucleobases. In certain embodiments, shortened or gap-widened antisense oligonucleotides targeted to a PHDl nucleic acid comprise one or more modified nucleobases. In certain embodiments, the modified nucleobase is 5-methylcytosine. In certain embodiments, each cytosine is a 5-methylcytosine.
Conjugated Antisense compounds
Antisense compounds may be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides. Typical conjugate groups include cholesterol moieties and lipid moieties. Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
Antisense compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of antisense compounds to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the antisense compound having terminal nucleic acid from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5'-terminus (5'- cap), or at the 3'-terminus (3'-cap), or can be present on both termini. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3' and 5'-stabilizing groups that can be used to cap one or both ends of an antisense compound to impart nuclease stability include those disclosed in WO 03/004602 published on January 16, 2003.
In certain embodiments, antisense compounds, including, but not limited to those particularly suited for use as ssRNA, are modified by attachment of one or more conjugate groups. In general, conjugate groups modify one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional conjugate linking moiety or conjugate linking group to a parent compound such as an oligonucleotide. Conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes. Certain conjugate groups have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let, 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let, 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison- Behmoaras et al., EMBO J., 1991, 10, 1 111- 1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl- ammonium l,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino- carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).
For additional conjugates including those useful for ssRNA and their placement within antisense compounds, see e.g., US Application No.; 61/583,963.
In vitro testing of antisense oligonucleotides
Described herein are methods for treatment of cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.
Cells may be treated with antisense oligonucleotides when the cells reach approximately 60- 80% confluency in culture.
One reagent commonly used to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN (Invitrogen, Carlsbad, CA). Antisense oligonucleotides may be mixed with LIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, CA) to achieve the desired final concentration of antisense oligonucleotide and a LIPOFECTIN concentration that may range from 2 to 12 ug/mL per 100 nM antisense oligonucleotide.
Another reagent used to introduce antisense oligonucleotides into cultured cells includes LIPOFECTAMINE (Invitrogen, Carlsbad, CA). Antisense oligonucleotide is mixed with LIPOFECTAMINE in OPTI-MEM 1 reduced serum medium (Invitrogen, Carlsbad, CA) to achieve the desired concentration of antisense oligonucleotide and a LIPOFECTAMINE concentration that may range from 2 to 12 ug/mL per 100 nM antisense oligonucleotide.
Another technique used to introduce antisense oligonucleotides into cultured cells includes electroporation.
Yet another technique used to introduce antisense oligonucleotides into cultured cells includes free uptake of the oligonucleotides by the cells.
Cells are treated with antisense oligonucleotides by routine methods. Cells may be harvested 16-
24 hours after antisense oligonucleotide treatment, at which time RNA or protein levels of target nucleic acids are measured by methods known in the art and described herein. In general, when treatments are performed in multiple replicates, the data are presented as the average of the replicate treatments.
The concentration of antisense oligonucleotide used varies from cell line to cell line. Methods to determine the optimal antisense oligonucleotide concentration for a particular cell line are well known in the art. Antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with LIPOFECTAMINE. Antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation. RNA Isolation
RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using the TRIZOL Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's recommended protocols.
Compositions and Methods for Formulating Pharmaceutical Compositions
Antisense compounds may be admixed 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.
An antisense compound targeted to PHD1 nucleic acid can be utilized in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutically acceptable diluent is water, such as sterile water suitable for injection. Accordingly, in one embodiment, employed in the methods described herein is a pharmaceutical composition comprising an antisense compound targeted to PHD1 nucleic acid and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is water. In certain embodiments, the antisense compound is an antisense oligonucleotide provided herein.
Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
A prodrug can include the incorporation of additional nucleosides at one or both ends of an antisense compound which are cleaved by endogenous nucleases within the body, to form the active antisense compound.
In certain embodiments, the compounds or compositions further comprise a pharmaceutically acceptable carrier or diluent. EXAMPLES
Non-limiting disclosure and incorporation by reference
While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references recited in the present application is incorporated herein by reference in its entirety.
Example 1 : Antisense inhibition of mouse PHDl in mouse primary hepatocytes by MOE gapmers
Antisense oligonucleotides were designed targeting mouse PHDl nucleic acid and were tested for their effects on PHDl mRNA in vitro. The antisense oligonucleotides were tested at a 5,000 nM concentration in mouse primary hepatocytes cultured at a density of 10,000 cells per well and the antisense oligonucleotides were introduced to the cells by free uptake. After a treatment period of approximately 24 hours, RNA was isolated from the cells and PHDl mRNA levels were measured by quantitative real-time PCR. Primer probe set RTS3551 (forward sequence TGAATCAGAACTGGGATGTTAAGG, designated herein as SEQ ID NO: 60; reverse sequence GGCTCGATGTTGGCTACCA, designated herein as SEQ ID NO: 61 ; probe sequence ATCTTCCCCGAGGGTCGGCCA, designated herein as SEQ ID NO: 62) was used to measure mRNA levels. PHDl mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of PHDl mRNA, relative to untreated control cells.
The newly designed chimeric antisense oligonucleotides in the Table below were designed as 5- 10-5 MOE gapmers. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2'-deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising five nucleosides each. Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment has a 2'-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P=S) linkages. All cytosine residues throughout each gapmer are 5- methylcyto sines. "Start site" indicates the 5 '-most nucleoside to which the gapmer is targeted in the mouse gene sequence. "Stop site" indicates the 3 '-most nucleoside to which the gapmer is targeted in the mouse gene sequence. Each gapmer listed in the Table below is targeted to the mouse PHDl mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM 053208.4) Table 1
Inhibition of PHDl mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1
Figure imgf000052_0001
Example 2: Antisense inhibition of mouse PHDl in by b.END cells by cEt gapmers
Antisense oligonucleotides were designed targeting mouse PHDl nucleic acid and were tested for their effects on PHDl mRNA in vitro. The antisense oligonucleotides were tested at a 3,000 nM concentration in mouse b.END cells cultured at a density of 20,000 cells per well and the antisense oligonucleotides were introduced to the cells by electroporation. After a treatment period of approximately 24 hours, RNA was isolated from the cells and PHDl mRNA levels were measured by quantitative real-time PCR. Primer probe set RTS3551 (forward sequence
TGAATCAGAACTGGGATGTTAAGG, designated herein as SEQ ID NO: 60; reverse sequence GGCTCGATGTTGGCTACCA, designated herein as SEQ ID NO: 61 ; probe sequence ATCTTCCCCGAGGGTCGGCCA, designated herein as SEQ ID NO: 62) was used to measure mRNA levels. PHDl mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of PHDl mRNA, relative to untreated control cells.
The newly designed chimeric antisense oligonucleotides in the Table below were designed as 3-
10-3 cEt gapmers. The 3-10-3 cEt gapmers are 16 nucleosides in length, wherein the central gap segment comprises of ten 2'-deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising three nucleosides each. Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment has a cEt modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P=S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. "Start site" indicates the 5 '-most nucleoside to which the gapmer is targeted in the mouse gene sequence. "Stop site" indicates the 3 '-most nucleoside to which the gapmer is targeted in the mouse gene sequence. Each gapmer listed in the Table below is targeted to SEQ ID NO: 2, which is the complement of the mouse PHDl genomic sequence (GENBANK Accession No. NT l 87034.1 truncated from nucleotides 24158658 to 24166802).
Table 2
Inhibition of PHDl mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 2
Figure imgf000053_0001
576112 8683 GAT ACTT AGT GCC CTC 51 46
576114 8741 AAAACCC AGC AC CGCT 22 47
576116 8775 ATGACCGCCAGCCACA 37 48
576118 8807 TACACTC AAC GGAC CA 46 49
576122 8868 ACACCTTTC CAGC AAG 25 50
576124 8898 ACGAGGGTGAGATGAC 0 51
576127 8967 CCAGTCCATACACTGC 16 52
576129 9001 TCATGGCACCCTACTC 16 53
576146 7128 AAATGATGGTTTGTGC 48 54
576153 8615 AGC GGGC ACT GAGGCA 42 55
576155 8688 TCAGT GAT ACTT AGT G 47 56
576157 8785 GCCACCAGTCATGACC 41 57
576160 8892 GTGAGATGAC ACC CAC 13 58
576175 8802 TCAACGGACCAGACAC 52 59
Example 3: Effect of antisense inhibition of PHD1 in a mouse kidney ischemia model
ISIS 576146, described in the study above, was tested in two different studies in a mouse model for kidney ischemia. The basic protocol for both studies is as follows: C57BL/6 mice were treated with ISIS 576146 administered subcutaneously at 100 mg/kg/week for 2.5 weeks. Another group of mice was administered PBS for 2.5 weeks. This PBS set served as a control group.
Kidney ischemia was induced approximately 24 hours after the last dose of ISIS oligonucleotide by a hanging weight system as described in Grenz et al. (J Vis Exp. 2011 ; (53): 2549, which is incorporated by reference herein in its entirety) for all groups of mice. Mice were anesthetized with pentobarbital at a dose of 70 mg/kg and maintained with approximately 10 mg/kg/h sodium pentobarbital. Once pain reflexes were absent (tested by squeezing the toes with tweezers) mice were placed on a temperature-controlled heating table (RT, Effenberg, Munich, Germany to maintain body temperature at 37.0°C) in a left lateral decubitus position. The lower and upper extremities of the mice were attached to the table with tape. A right flank incision was performed with a scissor and a coagulation electrode (Erbe, ICC50, Germany) to prevent bleeding of muscle and skin vessels. The renal pedicle including renal artery and renal vein were ligated with suture and the right kidney was removed. The surgical wound was closed using continuous suture of the muscle wall and skin with a suture. The mice were placed in a right lateral decubitus position and a left flank incision was performed. The left kidney was removed from the connective tissues, avoiding the adrenal gland and vessels. The kidney was turned with its ventral side down and collected in a sterile cup. The kidney holder was restrained ventrally to have tension on the renal vessels. The renal artery was dissected from adjacent tissues with forceps. A suture was placed under the renal artery over a small pole, and a weight of approximately 1 gram was attached to each end. While the weights were unsupported, the renal artery was immediately occluded. In contrast, when the weights were supported, blood flow was immediately restored to the organ. Successful occlusion was confirmed by a change of color from red to white.
Ischemia time was 30 minutes to allow recoverable kidney injury. After a 24-hour reperfusion, the kidney was replaced out of the sterile cup. The kidney holder was unfixed and the tension on the kidney was released. The cranial pole was gently removed out of the cup with forceps and the caudal pole followed immediately.
Study 1
FITC-inuUn clearance through IV injection
A reduction in glomerular filtration rate (GFR) is a primary characteristic of ischemic acute renal failure (Kim, S.J. et al., Ren. Fail. 2000. 22: 129-141). Measurement of FITC-inulin clearance is a well- known method of estimating glomerular filtration (Sohtell, M. et al., Acta Physiol. Scand. 1983. 119: 313-316) and this method was employed to study the effect of antisense inhibition of PHDl on AKI in this model.
Groups of mice treated with ISIS oligonucleotide or PBS and that underwent ischemia- reperfusion, and a group of mice which did not undergo the surgery (the sham group), were administered a dose of 8 μΕ/g FITC-inulin via tail vein injection. The mice were then bled retro-orbitally 5, 15, 45, and 90 min after the injection. The collected blood plasma was diluted 1 : 40 with 0.5 M HEPES and fluorescence was measured on a plate reader at 485/538 nm. The results are presented in the Table below and indicate that mice treated with an antisense oligonucleotide targeting PHDl show better clearance of FITC-inulin in the plasma compared to the PBS control group, and the clearance was similar to mice without kidney ischemia.
Therefore, antisense inhibition of PHDl improved GFR, indicating that administration of PHDl ASO is beneficial in treating, preventing or ameliorating AKI.. Table 3
FITC-inulin clearance in a mouse kidney ischemia model
Figure imgf000056_0001
Plasma chemistries
Plasma levels of transaminases, bilirubin, creatinine and BUN were measured using an automated clinical chemistry analyzer. The results are presented in the Table below and indicate that mice treated with an antisense oligonucleotide targeting PHDl do not show the increases in any markers as seen in the PBS control group.
The results indicate that treatment with an antisense oligonucleotide did not cause adverse changes in liver function (as evidenced by ALT and AST levels). The results also indicate that the adverse increase in the kidney marker levels of BUN and creatinine due to kidney injury and ischemia in the PBS control were reduced due to antisense inhibition of PHDl.
Table 4
Plasma chemistry in a mouse kidney ischemia model
Figure imgf000056_0002
RNA levels
Levels of PHDl, NGAL, EPO, and KIM1 mRNA in ischemic and non-ischemic kidney were measured. The results presented in the Table below are normalized to RIBOGREEN©. Treatment of the mice with an antisense oligonucleotide results in inhibition of PHDl expression in both ischemic and non-ischemic kidneys. The effect of antisense inhibition of PHDl in ischemic kidney results in increased expression of EPO, and decreased expression of NGAL and KIM1, compared to the control and to nonischemic kidney expressions. EPO is effective in attenuating renal ischemia/reperfusion injury (Ates, E. et al., ANZ J. Surg. 2005. 75: 1 100-1105). Hence, increase in levels of EPO as a result of antisense inhibition of PHDl is beneficial outcome in treating, preventing or ameliorating AKI. Similarly, NGAL and KIMl levels are widely used as a quantitative estimate of renal injury (Abassi, Z. et al., Invest. Urol. 2013. 189: 1559- 1566). Hence, decrease in levels of these two markers is an indication that antisense inhibition of PHDl is beneficial in treating, preventing or ameliorating AKI.
Table 5
Non-ischemic kidney mRNA levels in a mouse kidney ischemia model
Figure imgf000057_0001
Table 6
Ischemic kidney mRNA levels in a mouse kidney ischemia model
Figure imgf000057_0002
Study 2
Measurement of GFR
GFR is measured as described in Lorenz JN et al., Am J Physiol. 1999;276(1 Pt 2):F172-F177 and Conger JD et al., Kidney Int. 1989;35(5): 1126—1132, which are incorporated by reference herein in their entireties. The results are presented in the Table below and indicate that GFR is increased in mice after treatment with an antisense oligonucleotide targeting PHDl.
Therefore, antisense inhibition of PHDl improved GFR, indicating that administration of PHDl ASO treated and ameliorated AKI.
Table 7
GFR analysis in a mouse kidney ischemia model
Figure imgf000057_0003
Plasma and urine chemistries
Plasma and urine levels of transaminases, bilirubin, creatinine and BUN were measured using an automated clinical chemistry analyzer. The results are presented in the Table below and indicate that there were no changes in levels as a result of the treatment.
Urine electrolytes were measured by using crown ether electrodes for sodium and potassium and a molecular oriented PVC membrane for chloride. The results are presented in the Table below and indicate that there was no change in electrolyte concentrations as a result of the treatment.
Hence, treatment with an antisense oligonucleotide targeting PHDl did not cause any adverse effects on liver and kidney function in this model.
Table 8
Plasma chemistry in a mouse kidney ischemia model
Figure imgf000058_0001
Table 9
Urine electrolytes (mEq/L) in a mouse kidney ischemia model
Figure imgf000058_0002
RNA levels
Levels of PHDl mRNA, NGAL, EPO, and KIM1 in ischemic and non-ischemic kidney were measured. The results are presented in the Table below are normalized to RIBOGREEN©. Treatment of the mice with an antisense oligonucleotide results in inhibition of PHDl expression in both ischemic and non-ischemic kidneys. The effect of antisense inhibition of PHDl in ischemic kidney results in increased expression of EPO, and decreased expression of NGAL and KIMl, compared to the control and to nonischemic kidney expressions.
As mentioned in the earlier study described above, increase in levels of EPO as a result of antisense inhibition of PHDl is a beneficial outcome in treating, preventing or ameliorating AKI. Similarly, decrease in NGAL and KIMl levels is an indication that antisense inhibition of PHDl is beneficial in treating, preventing or ameliorating AKI.
Table 10
Non-ischemic kidney mRNA levels in a mouse kidney ischemia model
Figure imgf000059_0001
Table 11
Ischemic kidney mRNA levels in a mouse kidney ischemia
Figure imgf000059_0002

Claims

WHAT IS CLAIMED:
1. A method of treating, preventing, or ameliorating acute kidney injury (AKI) in a subject comprising administering to the subject a specific inhibitor of prolyl hydroxylase 1 (PHD1), thereby treating, preventing, or ameliorating the acute kidney injury.
2. The method of claim 1, wherein the acute kidney injury is caused by renal ischemia.
3. The method of claim 2, wherein the renal ischemia is caused by cardiac-mediated hypoperfusion.
4. The method of claim 3, wherein the cardiac-mediated hypoperfusion is caused by cardiac surgery.
5. The method of claim 2, wherein the renal ischemia is caused by gastric bypass surgery, orthotropic liver transplant, abdominal aortic aneurysm repair, or drug-induced ischemia.
6. The method of any one of claims 1-5, wherein administering the specific inhibitor of PHD1 improves renal function.
7. The method of claim 6, wherein administering the specific inhibitor of PHD 1 improves tubular epithelial cell injury in the subject.
8. The method of claim 7, wherein administering the specific inhibitor of PHD1 improves renal function, reduces BUN levels, reduces creatinine levels, improves glomerular filtration rate (GFR), reduces KIM1 levels, or reduces NGAL levels, or any combination thereof.
9. A method of inhibiting expression of PHD1 in a subject having, or at risk of having, acute kidney injury (AKI) comprising administering a specific inhibitor of PHD 1 to the subject, thereby inhibiting expression of PHD 1 in the subject.
10. The method of claim 9, wherein administering the inhibitor inhibits expression of PHD1 in the kidney.
11. The method of claim 10, wherein the acute kidney injury is caused by renal ischemia.
12. The method of claim 11, wherein the renal ischemia is caused by cardiac-mediated hypoperfusion.
13. The method of claim 12, wherein the cardiac-mediated hypoperfusion is caused by cardiac surgery.
14. The method of claim 11, wherein the renal ischemia is caused by gastric bypass surgery, orthotropic liver transplant, abdominal aortic aneurysm repair, or drug-induced ischemia.
15. A method of reducing or inhibiting tubular epithelial cell injury in the kidney of a subject having, or at risk of having, acute kidney injury comprising administering a specific inhibitor of PHDl to the subject, thereby reducing or inhibiting tubular epithelial cell injury in the kidney of the subject.
16. The method of claim 15, wherein the acute kidney injury is caused by renal ischemia.
17. The method of claim 16, wherein the renal ischemia is caused by cardiac- mediated hypoperfusion.
18. The method of claim 17, wherein the cardiac-mediated hypoperfusion is caused by cardiac surgery.
19. The method of claim 16, wherein the renal ischemia is caused by gastric bypass surgery, orthotropic liver transplant, abdominal aortic aneurysm repair, or drug-induced ischemia.
20. The method of any one of claims 15-19, wherein administering the specific inhibitor of PHDl improves renal function, reduces BUN levels, reduces creatinine levels, improves glomerular filtration rate (GFR), reduces KIM1 levels, or reduces NGAL levels, or any combination thereof.
21. A method of preventing or protecting a subject from acute kidney injury comprising administering a specific inhibitor of PHDl to the subject prior to the subject undergoing surgery or operation.
22. A method of preventing or protecting a subject from acute kidney injury comprising identifying a subject in need of surgery or operation and administering a specific inhibitor of PHDl to the subject prior to the subject undergoing surgery or operation.
23. The method of claim 21 or 22, wherein the surgery is cardiac surgery.
24. The method of claim 23, wherein the cardiac surgery is bypass surgery, coronary artery bypass grafting, valvular surgery, or combined coronary artery bypass grafting and valvular surgery.
25. The method of claim 21 or 22, wherein the surgery or operation is gastric bypass surgery, orthotropic liver transplant, or abdominal aortic aneurysm repair.
26. The method of any one of claims 21-25, wherein administering the specific inhibitor of PHDl to the subject prior to the subject undergoing surgery or operation is sufficient to prevent acute kidney injury, protect against acute kidney injury, minimize acute kidney injury, or reduce acute kidney injury in the subject following surgery or operation.
27. A method of preventing or protecting a subject from acute kidney injury comprising administering a specific inhibitor of PHD1 to the subject prior administering a drug known to induce ischemia.
28. A method of preventing or protecting a subject from acute kidney injury comprising identifying a subject in need of a drug known to induce ischemia and administering a specific inhibitor of PHD1 to the subject prior administering the drug..
29. The method of claim 27 or 28, wherein administering the specific inhibitor of PHD 1 to the subject prior to administering the drug is sufficient to prevent acute kidney injury, protect against acute kidney injury, minimize acute kidney injury, or reduce acute kidney injury in the subject following administration of the drug.
30. The method of any one of claims 21-29, wherein the specific inhibitor of PHD1 is administered multiple times prior to the subject undergoing surgery or operation, or prior to administration of the drug known to induce ischemia.
31. The method of any one of claims 1-30, wherein the inhibitor is an antisense compound targeted to PHD1.
32. The method of claim 31, wherein the antisense compound comprises at least one modified sugar.
33. The method of claim 32, wherein the modified sugar comprises a 2'-0-methoxyethyl group.
34. The method of claim 32, wherein the modified sugar comprises a bicyclic sugar.
35. The method of claim 34, wherein the bicyclic sugar comprises a group selected from: 4'- CH(CH3)-0-2', 4'-CH2-0-2' and 4'-(CH2)2-0-2'.
36. The method of any one of claims 31-35, wherein the antisense compound comprises at least one modified internucleoside linkage.
37. The method of claim 36, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.
38. The method of any one of claims 31-37, wherein the antisense compound comprises at least one modified nucleobase.
39. The method of claim 38, wherein the modified nucleobase is a 5-methylcytosine.
40. The method of any one of claims 31-39, wherein the antisense compound is a single- stranded modified oligonucleotide.
41. The method of claim 40, wherein the modified oligonucleotide comprises:
a gap segment consisting of linked deoxynucleosides;
a 5' wing segment consisting of linked nucleosides; and
a 3' wing segment consisting of linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
42. A PHDl specific inhibitor for treating, preventing, or ameliorating acute kidney injury.
43. The PHDl specific inhibitor of claim 42, wherein the acute kidney injury is caused by renal ischemia.
44. The PHDl specific inhibitor of claim 43, wherein the renal ischemia is caused by cardiac-mediated hypoperfusion.
45. The PHDl specific inhibitor of claim 44, wherein the cardiac-mediated hypoperfusion is caused by cardiac surgery.
46. The PHDl specific inhibitor of any one of claims 42-45, wherein the PHDl specific inhibitor is an antisense compound targeted to PHD 1.
47. The PHDl specific inhibitor of claim 46, wherein the antisense compound comprises at least one modified sugar.
48. The PHDl specific inhibitor of claim 47, wherein the modified sugar comprises a 2'-0- methoxyethyl group.
49. The PHDl specific inhibitor of claim 47, wherein the modified sugar comprises a bicyclic sugar.
50. The PHDl specific inhibitor of claim 49, wherein the bicyclic sugar comprises a group selected from: 4'-CH(CH3)-0-2', 4'-CH2-0-2' and 4'-(CH2)2-0-2'.
51. The PHDl specific inhibitor of any one of claims 46-50, wherein the antisense compound comprises at least one modified internucleoside linkage.
52. The PHDl specific inhibitor of claim 51, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.
53. The PHDl specific inhibitor of any one of claims 46-52, wherein the antisense compound comprises at least one modified nucleobase.
54. The PHDl specific inhibitor of claim 53, wherein the modified nucleobase is a 5- methylcytosine.
55. The PHDl specific inhibitor of any one of claims 46-54, wherein the antisense compound is a single- stranded modified oligonucleotide.
56. The PHDl specific inhibitor of claim 55, wherein the modified oligonucleotide comprises:
a gap segment consisting of linked deoxynucleosides;
a 5' wing segment consisting of linked nucleosides; and
a 3' wing segment consisting of linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
57. Use of a PHDl specific inhibitor for the preparation of a medicament for treating acute kidney injury.
58. The use of claim 57, wherein the acute kidney injury is caused by renal ischemia.
59. The use of claim 58, wherein the renal ischemia is caused by cardiac-mediated hypoperfusion.
60. The use of claim 59, wherein the cardiac-mediated hypoperfusion is caused by cardiac surgery.
61. The use of any one of claims 57-60, wherein the PHDl specific inhibitor is an antisense compound targeted to PHD 1.
62. The use of claim 61, wherein the antisense compound comprises at least one modified sugar.
63. The use of claim 62, wherein the modified sugar comprises a 2'-0-methoxyethyl group.
64. The use of claim 62, wherein the modified sugar comprises a bicyclic sugar.
65. The use of claim 64, wherein the bicyclic sugar comprises a group selected from: 4'- CH(CH3)-0-2', 4'-CH2-0-2' and 4'-(CH2)2-0-2'.
66. The use of any one of claims 57-65, wherein the antisense compound comprises at least one modified internucleoside linkage.
67. The use of claim 66, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.
68. The use of any one of claims 57-67, wherein the antisense compound comprises at least one modified nucleobase.
69. The use of claim 68, wherein the modified nucleobase is a 5-methylcytosine.
70. The use of any one of claims 57-69, wherein the antisense compound is a single- stranded modified oligonucleotide.
71. The use of claim 70, wherein the modified oligonucleotide comprises:
a gap segment consisting of linked deoxynucleosides;
a 5' wing segment consisting of linked nucleosides; and
a 3' wing segment consisting of linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
PCT/US2014/060044 2013-10-11 2014-10-10 Treatment of acute kidney injury with phd1 modulators WO2015054579A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201361890062P 2013-10-11 2013-10-11
US61/890,062 2013-10-11

Publications (1)

Publication Number Publication Date
WO2015054579A1 true WO2015054579A1 (en) 2015-04-16

Family

ID=52813664

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2014/060044 WO2015054579A1 (en) 2013-10-11 2014-10-10 Treatment of acute kidney injury with phd1 modulators

Country Status (1)

Country Link
WO (1) WO2015054579A1 (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120149712A1 (en) * 2001-12-06 2012-06-14 Fibrogen, Inc. Methods for improving kidney function
WO2012078967A2 (en) * 2010-12-10 2012-06-14 Alnylam Pharmaceuticals, Inc. Compositions and methods for increasing erythropoietin (epo) production
US20120289580A1 (en) * 2011-04-13 2012-11-15 Sanjay Bhanot Antisense modulation of ptp1b expression
WO2013134660A1 (en) * 2012-03-09 2013-09-12 Fibrogen, Inc. 4 -hydroxy- isoquinoline compounds as hif hydroxylase inhibitors

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120149712A1 (en) * 2001-12-06 2012-06-14 Fibrogen, Inc. Methods for improving kidney function
WO2012078967A2 (en) * 2010-12-10 2012-06-14 Alnylam Pharmaceuticals, Inc. Compositions and methods for increasing erythropoietin (epo) production
US20120289580A1 (en) * 2011-04-13 2012-11-15 Sanjay Bhanot Antisense modulation of ptp1b expression
WO2013134660A1 (en) * 2012-03-09 2013-09-12 Fibrogen, Inc. 4 -hydroxy- isoquinoline compounds as hif hydroxylase inhibitors

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BOVE ET AL.: "The incidence and risk of acute renal failure after cardiac surgery.", JOURNAL OF CARDIOTHORACIC AND VASCULAR ANESTHESIA, vol. 18, no. 4, August 2004 (2004-08-01), pages 442 - 445, XP004563484, DOI: doi:10.1053/j.jvca.2004.05.021 *
WANG ET AL.: "The protective effect of prolyl-hydroxylase inhibition against renal ischaemia requires application prior to ischaemia but is superior to EPO treatment.", NEPHROL. DIAL. TRANSPLANT., vol. 27, no. 3, 3 March 2012 (2012-03-03), pages 929 - 936 *

Similar Documents

Publication Publication Date Title
AU2016334114B2 (en) Compounds and methods for modulating angiotensinogen expression
AU2013280474B2 (en) Modulation of UBE3A-ATS expression
CA2859729C (en) Methods for modulating metastasis-associated-in-lung-adenocarcinoma-transcript-1(malat-1) expression
AU2014284398B2 (en) Modulators of growth hormone receptor
EP3043827A2 (en) Modulators of complement factor b
WO2013120003A1 (en) Modulation of rna by repeat targeting
WO2016138353A1 (en) Allele specific modulators of p23h rhodopsin
EP3038627A2 (en) Modulation of prekallikrein (pkk) expression
WO2016086104A1 (en) Modulation of ube3a-ats expression
US9523094B2 (en) Methods of treating kennedy's disease
WO2013130868A1 (en) Methods for modulating fibrinogen expression
WO2015054579A1 (en) Treatment of acute kidney injury with phd1 modulators

Legal Events

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

Ref document number: 14852677

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14852677

Country of ref document: EP

Kind code of ref document: A1