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Definitions

• compositions, and methods for reducing expression of human plasma prekallikrein (PKK) mRNA and protein in an animal are provided.
• PPK human plasma prekallikrein
• Plasma prekallikrein is the precursor of plasma kallikrein (PK), which is encoded by the KLKBl gene.
• PKK is a glycoprotein that participates in the surface- dependent activation of blood coagulation, fibrinolysis, kinin generation, and inflammation.
• PKK is converted to PK by Factor Xlla by the cleavage of an internal Arg-Ile peptide bond.
• PK liberates kinins from kininogens and also generates plasmin from plasminogen.
• PK is a member of the kinin-kallikrein pathway, which consists of several proteins that play a role in inflammation, blood pressure control, coagulation, and pain.
• compounds useful for modulating expression of PKK mRNA and protein are antisense compounds.
• the antisense compounds are antisense oligonucleotides.
• modulation can occur in a cell or tissue.
• the cell or tissue is in an animal.
• the animal is a human.
• PKK mRNA levels are reduced.
• PKK protein levels are reduced. Such reduction can occur in a time- dependent manner or in a dose- dependent manner.
• PKK associated diseases, disorders, and conditions are inflammatory diseases.
• the inflammatory disease may be an acute or chronic inflammatory disease.
• such inflammatory diseases may include hereditary angioedema (HAE), edema, angioedema, swelling, angioedema of the lids, ocular edema, macular edema, and cerebral edema.
• HAE hereditary angioedema
• thromboembolic diseases may include thrombosis, embolism, thromboembolism, deep vein thrombosis, pulmonary embolism, myocardial infarction, stroke, and infarct.
• Such diseases, disorders, and conditions can have one or more risk factors, causes, or outcomes in common.
• Certain risk factors and causes for development of an inflammatory disease include genetic predisposition to an inflammatory disease and environmental factors.
• the subject has a mutated complement 1 esterase inhibitor (Cl-INH) gene or mutated Factor 12 gene.
• Cl-INH mutated complement 1 esterase inhibitor
• the subject has taken or is on angiotensin-converting enzyme inhibitors (ACE inhibitors) or angiotensin II receptor blockers (ARBs).
• ACE inhibitors angiotensin-converting enzyme inhibitors
• ARBs angiotensin II receptor blockers
• the subject has had an allergic reaction leading to angioedema.
• the subject has type I HAE.
• the subject has type II HAE.
• the subject has type III HAE.
• Certain outcomes associated with development of an inflammatory disease include edema/s welling in various body parts including the extremities (i.e., hands, feet, arms, legs), the intestines (abdomen), the face, the genitals, the larynx (i.e., voice box); vascular permeability; vascular leakage; generalized inflammation; abdominal pain; bloating; vomiting; diarrhea; itchy skin; respiratory (asthmatic) reactions; rhinitis; anaphylaxis; bronchoconstriction; hypotension; coma; and death.
• Certain risk factors and causes for development of a thromboembolic disease include genetic predisposition to a thromboembolic disease, immobility, surgery (particularly orthopedic surgery), malignancy, pregnancy, older age, use of oral contraceptives, atrial fibrillation, previous thromboembolic condition, chronic inflammatory disease, and inherited or acquired prothrombotic clotting disorders.
• Certain outcomes associated with development of a thromboembolic condition include decreased blood flow through an affected vessel, death of tissue, and death.
• methods of treatment include administering a PKK antisense compound to an individual in need thereof. In certain embodiments, methods of treatment include administering a PKK antisense oligonucleotide to an individual in need thereof.
• Figure 1 is a Western blot quantification of HMWK from blood samples as described in Example 11.
• Figure 2 is a Western blot quantification of HMWK from blood samples as described in Example 14.
• 2'-0-methoxyethyl refers to an O-methoxyethyl modification of the 2' position of a furanose ring.
• a 2 '-O-methoxyethyl modified sugar is a modified sugar.
• 2 '-O-methoxyethyl modified 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 furanose ring other than H or OH.
• 2' substituted nucleosides include nucleosides with bicyclic sugar modifications.
• 2'-deoxynucleoside means a nucleoside comprising a hydrogen at the 2' position of the sugar portion of the nucleoside.
• 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.
• administering refers to the co-administration of two pharmaceutical agents in any manner in which the pharmacological effects of both are manifest in the patient at the same time.
• Concomitant administration does not require that both pharmaceutical agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration.
• the effects of both pharmaceutical agents need not manifest themselves at the same time. The effects need only be overlapping for a period of time and need not be coextensive.
• administering means providing a pharmaceutical agent to an animal, and includes, but is not limited to administering by a medical professional and self-administering.
• “Amelioration” refers to a lessening, slowing, stopping, or reversing of at least one indicator of the severity 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 compound means an oligomeric compound that 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 or 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 mechanisms are all those mechanisms involving hybridization of a compound with a 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 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.
• 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 means a furanose ring modified by the bridging of two atoms.
• a bicyclic sugar is a modified sugar.
• Bicyclic nucleoside (also bicyclic nucleic acid or BNA) means a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system.
• the bridge connects the 4'-carbon and the 2'-carbon of the sugar ring.
• 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 nucleoside having a 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'.
• cEt modified nucleoside (also “constrained ethyl nucleoside”) means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH 3 )-0-2' bridge.
• “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'- O-methoxyethyl nucleosides is chemically distinct from a region having nucleosides without 2'-0- methoxyethyl modifications.
• Chimeric antisense compound means an antisense compound that has at least two chemically distinct regions, each position having a plurality of subunits.
• Co-administration means administration of two or more pharmaceutical agents to an individual. The two or more pharmaceutical agents may be in a single pharmaceutical composition, or may be in separate pharmaceutical compositions. Each of the two or more pharmaceutical agents may be administered through the same or different routes of administration. Co-administration encompasses parallel or sequential administration.
• “Complementarity” means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.
• Contiguous nucleobases means nucleobases immediately adjacent to each other.
• Designing or “Designed to” refer to the process of creating an oligomeric compound that specifically hybridizes with a selected nucleic acid molecule.
• “Diluent” means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable.
• the diluent may be a liquid, e.g. saline solution.
• Dose means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period.
• a dose may be administered in one, two, or more boluses, tablets, or injections.
• the desired dose requires a volume not easily accommodated by a single injection, therefore, two or more injections may be used to achieve the desired dose.
• the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses may be stated as the amount of pharmaceutical agent per hour, day, week, or month.
• Downstream refers to the relative direction toward the 3 ' end or C-terminal end of a nucleic acid.
• Effective amount in the context of modulating an activity or of treating or preventing a condition means the administration of that amount of pharmaceutical agent to a subject in need of such modulation, treatment, or prophylaxis, either in a single dose or as part of a series, that is effective for modulation of that effect, or for treatment or prophylaxis or improvement of that condition.
• 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 a "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 target nucleic acid.
• complementary nucleic acid molecules include, but are not limited to, an antisense oligonucleotide and a nucleic acid target.
• Identifying an animal having an inflammatory disease means identifying an animal having been diagnosed with an inflammatory disease or predisposed to develop an inflammatory disease. Individuals predisposed to develop an inflammatory disease include those having one or more risk factors for developing an inflammatory disease including environmental factors, having a personal or family history, or genetic predisposition to one or more inflammatory disease. Such identification may be accomplished by any method including evaluating an individual's medical history and standard clinical tests or assessments, such as genetic testing.
• Identifying an animal having a PKK associated disease means identifying an animal having been diagnosed with a PKK associated disease or predisposed to develop a PKK associated disease. Individuals predisposed to develop a PKK associated disease include those having one or more risk factors for developing a PKK associated disease including having a personal or family history, or genetic predisposition of one or more PKK associated diseases. Such identification may be accomplished by any method including evaluating an individual's medical history and standard clinical tests or assessments, such as genetic testing.
• Identifying an animal having a thromboembolic disease means identifying an animal having been diagnosed with a thromboembolic disease or predisposed to develop a thromboembolic disease.
• Individuals predisposed to develop a thromboembolic disease include those having one or more risk factors for developing a thromboembolic disease including having a personal or family history, or genetic predisposition of one or more thromboembolic diseases, immobility, surgery (particularly orthopedic surgery), malignancy, pregnancy, older age, use of oral contraceptives, atrial fibrillation, previous thromboembolic condition, chronic inflammatory disease, and inherited or acquired prothrombotic clotting disorders.
• identification may be accomplished by any method including evaluating an individual's medical history and standard clinical tests or assessments, such as genetic testing.
• 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.
• “Individual” means a human or non-human animal selected for treatment or therapy.
• “Inhibiting PKK” means reducing the level or expression of a PKK mRNA and/or protein.
• PKK mRNA and/or protein levels are inhibited in the presence of an antisense compound targeting PKK, including an antisense oligonucleotide targeting PKK, as compared to expression of PKK mRNA and/or protein levels in the absence of a PKK antisense compound, such as an antisense
• “Inhibiting the expression or activity” refers to a reduction or 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.
• Linked nucleosides means adjacent nucleosides linked together by an internucleoside linkage.
• LNA Locked nucleic acid
• LNA Low-power nucleic acid
• LNA nucleosides means nucleic acid monomers having a bridge connecting two carbon atoms between the 4' and 2'position of the nucleoside sugar unit, thereby forming a bicyclic sugar.
• bicyclic sugar include, but are not limited to A) a-L- Methyleneoxy (4'-CH 2 -0-2') LNA , (B) ⁇ -D-Methyleneoxy (4'-CH 2 -0-2') LNA , (C) Ethyleneoxy (4'-
• Examples of 4'- 2' bridging groups encompassed within the definition of LNA include, but are not limited to one of formulae: -[C(Ri)(R 2 )] n -, -[C(Ri)(R 2 )] n -0-, -C(RiR 2 )-N(R -O- or -C(RiR 2 )-O-N(R -.
• bridging groups encompassed with the definition of LNA are 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(Ri)-2' and 4'-CH 2 -N(R 1 )-0-2'- bridges, wherein each Ri and R 2 is, independently, H, a protecting group or Ci-Ci 2 alkyl.
• LNAs in which the 2'- hydroxyl group of the ribosyl sugar ring is connected to the 4' carbon atom of the sugar ring, thereby forming a methyleneoxy (4'-CH 2 -0-2') bridge to form the bicyclic sugar moiety.
• the bridge can also be a methylene (-CH 2 -) group connecting the 2' oxygen atom and the 4' carbon atom, for which the term methyleneoxy (4'- CH 2 -0-2') LNA is used.
• ethyleneoxy (4'-CH 2 CH 2 -0-2') LNA is used, a -L- methyleneoxy (4'-CH 2 - 0-2'), an isomer of methyleneoxy (4'-CH 2 -0-2') LNA is also encompassed within the definition of LNA, as used herein.
• 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 (also known as 5-methyluracil), 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, and/or modified nucleobase.
• Modified oligonucleotide means an oligonucleotide comprising at least one modified
• Modified sugar means substitution and/or any change from a natural sugar moiety.
• “Monomer” means a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occurring 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, double-stranded nucleic acids, small interfering ribonucleic acids (siRNA), and microRNAs (miRNA).
• RNA ribonucleic acids
• DNA deoxyribonucleic acids
• siRNA small interfering ribonucleic acids
• miRNA microRNAs
• Nucleobase means a heterocyclic moiety capable of pairing with a base of another nucleic acid.
• nucleobase 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 (U).
• 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.
• Off-target effect refers to an unwanted or deleterious biological effect associated with modulation of RNA or protein expression of a gene other than the intended target nucleic acid.
• Oligomer means a polymer of linked monomeric subunits which is capable of hybridizing to at least a region of a nucleic acid molecule.
• 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 (e.g., bolus injection) or infusion.
• Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g., intrathecal or intracerebroventricular administration.
• peptide means a molecule formed by linking at least two amino acids by amide bonds.
• peptide refers to polypeptides and proteins.
• “Pharmaceutical agent” means a substance that provides a therapeutic benefit when administered to an individual.
• an antisense oligonucleotide targeted to PKK is a pharmaceutical agent.
• “Pharmaceutical composition” means a mixture of substances suitable for administering to a subject.
• a pharmaceutical composition may comprise an antisense oligonucleotide and a sterile aqueous solution.
• “Pharmaceutically acceptable derivative” encompasses pharmaceutically acceptable salts, conjugates, prodrugs or isomers of the compounds described herein.
• “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.
• PKA plasma prekallikrein
• Plasma prekallikrein is the precursor of plasma kallikrein (PK), which is encoded by the KLKBl gene.
• PKK associated disease means any disease associated with any PKK nucleic acid or expression product thereof. Such diseases may include an inflammatory disease or a thromboembolic disease. Such diseases may include hereditary angioedema (HAE).
• HAE hereditary angioedema
• PKK mPvNA means any messenger RNA expression product of a DNA sequence encoding PKK.
• PKK nucleic acid means any nucleic acid encoding PKK.
• a DNA sequence encoding PKK for example, in certain embodiments, a DNA sequence encoding PKK.
• PKK nucleic acid includes a DNA sequence encoding PKK, an RNA sequence transcribed from DNA encoding PKK (including genomic DNA comprising introns and exons), and an mRNA sequence encoding PKK.
• PKK mRNA means an mRNA encoding a PKK protein.
• PKK protein means the polypeptide expression product of a PKK nucleic acid.
• 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 or “preventing” refers to delaying or forestalling the onset or development of a disease, disorder, or condition for a period of time from minutes to days, weeks to months, or indefinitely.
• Prodrug means a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
• “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.
• Salts mean a 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. “Segments” are defined as smaller or sub-portions of regions within a target nucleic acid.
• Side effects means physiological responses attributable to a treatment other than desired effects.
• side effects include, without limitation, injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system
• Single-stranded oligonucleotide means an oligonucleotide which is not hybridized to a complementary strand.
• Sites are defined as unique nucleobase positions within a target nucleic acid.
• Specifically hybridizable or “specifically hybridizes” 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 or “targeted” means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.
• Target nucleic acid 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” or “treating” or “treatment” refers to administering a composition to effect an improvement of the disease or condition.
• "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).
• Upstream refers to the relative direction toward the 5 ' end or N-terminal end of a nucleic acid.
• Wild segment means a plurality of nucleosides modified to impart to an oligonucleotide properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.
• Certain embodiments provide compounds, compositions, and methods for inhibiting plasma prekallikrein (PKK) mRNA and protein expression. Certain embodiments provide compounds,
• compositions, and methods for decreasing PKK mRNA and protein levels are provided.
• Certain embodiments provide antisense compounds targeted to a plasma prekallikrein (PKK) nucleic acid.
• PKK nucleic acid is the sequence set forth in GENBANK Accession No. NM 000892.3 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No. DC412984.1
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 30-2226.
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of the nucleobase sequence of SEQ ID NO: 570.
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of the nucleobase sequence of SEQ ID NO: 705.
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 consecutive nucleobases of the nucleobase sequence of SEQ ID NO: 1666.
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 20 linked nucleosides and having the nucleobase sequence of SEQ ID NO: 570.
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 20 linked nucleosides and having the nucleobase sequence of SEQ ID NO: 705.
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 16 linked nucleosides and having the nucleobase sequence of SEQ ID NO: 1666.
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 62, 72, 103, 213, 312, 334-339, 344, 345, 346, 348, 349, 351, 369, 373, 381, 382, 383, 385, 387-391, 399, 411, 412, 414, 416, 444, 446-449, 452, 453, 454, 459, 460, 462-472, 473, 476, 477, 479, 480, 481, 484, 489-495, 497, 500, 504, 506, 522, 526, 535, 558, 559, 560, 564, 566, 568- 571, 573, 576,
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 62, 72, 103, 213, 334-339, 344, 346, 348, 349, 351, 381, 382, 383, 385, 389, 390, 391, 446, 448, 452, 453, 454, 466-473, 476, 481, 484, 491, 492, 494, 495, 497, 504, 526, 558, 559, 566, 568-571, 576, 578, 587, 595, 597, 598, 600-604, 607, 610, 613, 618, 619, 624, 635, 638, 639, 645, 652, 656, 657, 658
• the modified oligonucleotide achieves at least 85%> mRNA inhibition of PKK.
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 334, 346, 351, 382, 390, 391, 446, 448, 452, 453, 468, 469, 470, 471, 472, 476, 481, 491, 495, 504, 558, 566, 568, 570, 571, 578, 587, 597, 598, 600, 604, 613, 635, 638, 645, 656, 658, 660, 674, 675, 684, 704, 705, 880, 901-905, 909, 922, 931, 951, 954, 956, 990, 1005, 1020, 1032, 1037, 1040, 1041, 1045, 10
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 334, 391, 448, 468, 469, 568, 570, 598, 635, 658, 674, 684, 705, 901, 903, 904, 922, 990, 1267, 1291, 1420, 1430, 1431, 1434, 1435, 1436, 1537, 1538, and 1540.
• the modified oligonucleotide achieves at least 95%> mRNA inhibition of PKK.
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 334, 338, 346, 349, 382, 383, 390, 448, 452, 453, 454, 495, 526, 559, 570, 587, 598, 635, 660, 705, 901, 903, 904, 908, 923, 931, 955, 974, 988, 990, 1020, 1039, 1040, 1111, 1117, 1267, 1291, 1349, 1352, 1367, 1389, 1393, 1399, 1401, 1408, 1411, 1426, 1499, 1516, 1535, 1544, 1548, 1563, 1564, 1568, 1569, 1598, 1616
• the modified oligonucleotide achieves an IC 5 o ( ⁇ ) of 0.4 or less.
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 334, 346, 349, 382, 453, 454, 495, 526, 570, 587, 598, 635, 660, 901, 903, 904, 931, 955, 990, 1020, 1111, 1267, 1349, 1352, 1367, 1389, 1399, 1408, 1411, 1426, 1516, 1535, 1544, 1548, 1563, 1564, 1568, 1569, 1598, 1616, 1617, 1623, 1643, 1661, 1665, 1666, 1673, 1695, 1804, 1876, 1881, 2019, 2044, 2100, 2101, 2115, and 2116
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 334, 346, 382, 453, 495, 526, 570, 587, 598, 635, 901, 904, 931, 955, 1020, 1111, 1349, 1352, 1389, 1426, 1516, 1535, 1544, 1548, 1564, 1569, 1598, 1616, 1617, 1665, 1666, 1804, 1876, 1881, 2019, 2044, 2101, and 2116.
• the modified oligonucleotide achieves an IC 5 o ( ⁇ ) of 0.2 or less.
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 334, 495, 587, 598, 635, 1349, 1352, 1389, 1516, 1544, 1548, 1569, 1598, 1617, 1665, 1666, 1804, 1881, and 2019.
• the modified oligonucleotide achieves an IC 5 o ( ⁇ ) of less than 0.2.
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases complementary to an equal length portion of nucleobases 27427-27466 of SEQ ID NO: 10.
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases complementary to an equal length portion of nucleobases 33183-33242 of SEQ ID NO: 10.
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases complementary to an equal length portion of nucleobases 30570-30610 of SEQ ID NO: 10.
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases complementary to an equal length portion of nucleobases 27427-27520 of SEQ ID NO: 10.
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases complementary to an equal length portion of nucleobases 33085-33247 of SEQ ID NO: 10.
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases complementary to an equal length portion of nucleobases 30475-30639 of SEQ ID NO: 10.
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases complementary to an equal length portion of nucleobases 27362-27524 of SEQ ID NO: 10.
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases complementary to an equal length portion of nucleobases 33101-33240 of SEQ ID NO: 10.
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases complementary to an equal length portion of nucleobases 30463-30638 of SEQ ID NO: 10.
• Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases complementary to an equal length portion of ex on 9, exon 12, or exon 14 of a PKK nucleic acid.
• the nucleobase sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to SEQ ID NO: 10.
• the compound consists of a single-stranded modified oligonucleotide.
• At least one internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage.
• At least one modified internucleoside linkage of the modified oligonucleotide is a phosphorothioate internucleoside linkage.
• each internucleoside linkage of the modified oligonucleotide is a phosphorothioate linkage.
• At least one nucleoside of the modified oligonucleotide comprises a modified nucleobase.
• the modified nucleobase is a 5-methylcytosine.
• the modified oligonucleotide comprises at least one modified sugar.
• the modified sugar is a 2' modified sugar, a BNA, or a THP.
• the modified sugar is any of a 2'-0-methoxyethyl, 2'-0-methyl, a constrained ethyl, a LNA, or a 3'-fluoro-HNA.
• the compound comprises at least one 2'-0-methoxyethyl nucleoside, 2'-0- methyl nucleoside, constrained ethyl nucleoside, LNA nucleoside, or 3'-fluoro-HNA nucleoside.
• the modified oligonucleotide comprises:
• a 3' wing segment consisting of 5 linked nucleosides
• each nucleoside of each wing segment comprises a modified sugar
• the modified oligonucleotide consists of 20 linked nucleosides.
• the modified oligonucleotide consists of 19 linked nucleosides.
• the modified oligonucleotide consists of 18 linked nucleosides.
• Certain embodiments provide compounds consisting of a modified oligonucleotide according to the following formula: Tes Ges mCes Aes Aes Gds Tds mCds Tds mCds Tds Tds Gds Gds mCds Aes Aes mCes Ae; wherein,
• A an adenine
• mC a 5'-methylcytosine
• G a guanine
• e a 2'-0-methoxyethyl modified nucleoside
• s a phosphorothioate internucleoside linkage.
• Certain embodiments provide compounds consisting of a modified oligonucleotide according to the following formula: mCes mCes mCes mCes mCes Tds Tds mCds Tds Tds Tds Ads Tds Ads Gds mCes mCes Aes Ges mCe; wherein,
• A an adenine
• mC a 5'-methylcytosine
• G a guanine
• T a thymine
• e a 2'-0-methoxyethyl modified nucleoside
• s a phosphorothioate intemucleoside linkage.
• Certain embodiments provide compounds consisting of a modified oligonucleotide according to the following formula: mCes Ges Aks Tds Ads Tds mCds Ads Tds Gds Ads Tds Tds mCks mCks mCe; wherein,
• A an adenine
• mC a 5'-methylcytosine
• G a guanine
• e a 2'-0-methoxyethyl modified nucleoside
• k a cEt modified nucleoside
• s a phosphorothioate intemucleoside linkage.
• compositions comprising the compound of any preceding claim or salt thereof and at least one of a pharmaceutically acceptable carrier or diluent.
• Certain embodiments provide methods comprising administering to an animal the compound or composition of any preceding claim.
• the animal is a human.
• administering the compound prevents, treats, or ameliorates a PKK associated disease, disorder or condition.
• the PKK associated disease, disorder or condition is a hereditary angioedema (HAE), edema, angioedema, swelling, angioedema of the lids, ocular edema, macular edema, cerebral edema, thrombosis, embolism, thromboembolism, deep vein thrombosis, pulmonary embolism, myocardial infarction, stroke, or infarct.
• HAE hereditary angioedema
• edema angioedema
• swelling angioedema of the lids
• ocular edema macular edema
• cerebral edema cerebral edema
• thrombosis embolism
• thromboembolism thromboembolism
• deep vein thrombosis deep vein thrombosis
• pulmonary embolism myocardial infarction, stroke, or infarct.
• Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs.
• An oligomeric compound may be "antisense" to a target nucleic acid, meaning that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
• an antisense compound has a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.
• an antisense oligonucleotide has a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.
• an antisense compound targeted to a PKK nucleic acid is 12 to 30 subunits in length. In certain embodiments, an antisense compound targeted to PKK nucleic acid is 12 to 25 subunits in length. In certain embodiments, an antisense compound targeted to PKK nucleic acid is 12 to 22 subunits in length. In certain embodiments, an antisense compound targeted to PKK nucleic acid is 14 to 20 subunits in length. In certain embodiments, an antisense compound targeted to PKK nucleic acid is 15 to 25 subunits in length. In certain embodiments, an antisense compound targeted to PKK nucleic acid is 18 to 22 subunits in length.
• an antisense compound targeted to PKK nucleic acid is 19 to 21 subunits in length.
• 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 30, 18 to 50, 19 to 30, 19 to 50, or 20 to 30 linked subunits in length.
• an antisense compound targeted to a PKK nucleic acid is 12 subunits in length. In certain embodiments, an antisense compound targeted to a PKK nucleic acid is 13 subunits in length. In certain embodiments, an antisense compound targeted to a PKK nucleic acid is 14 subunits in length. In certain embodiments, an antisense compound targeted to a PKK nucleic acid is 15 subunits in length. In certain embodiments, an antisense compound targeted to a PKK nucleic acid is 16 subunits in length. In certain embodiments, an antisense compound targeted to a PKK nucleic acid is 17 subunits in length.
• an antisense compound targeted to a PKK nucleic acid is 18 subunits in length. In certain embodiments, an antisense compound targeted to a PKK nucleic acid is 19 subunits in length. In certain embodiments, an antisense compound targeted to a PKK nucleic acid is 20 subunits in length. In certain embodiments, an antisense compound targeted to a PKK nucleic acid is 21 subunits in length. In certain embodiments, an antisense compound targeted to a PKK nucleic acid is 22 subunits in length. In certain embodiments, an antisense compound targeted to a PKK nucleic acid is 23 subunits in length.
• an antisense compound targeted to a PKK nucleic acid is 24 subunits in length. In certain embodiments, an antisense compound targeted to a PKK nucleic acid is 25 subunits in length. In certain embodiments, an antisense compound targeted to a PKK nucleic acid is 26 subunits in length. In certain embodiments, an antisense compound targeted to a PKK nucleic acid is 27 subunits in length. In certain embodiments, an antisense compound targeted to a PKK nucleic acid is 28 subunits in length. In certain embodiments, an antisense compound targeted to a PKK nucleic acid is 29 subunits in length.
• an antisense compound targeted to a PKK nucleic acid is 30 subunits in length.
• the antisense compound targeted to a PKK nucleic acid is 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 nucleosides.
• antisense oligonucleotides targeted to a PKK nucleic acid may be shortened or truncated.
• 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 a PKK 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 J. Natl. Cancer Inst. 93:463-471, March 2001
• oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA 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 targeted to a PKK nucleic acid 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 optionally serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.
• Antisense compounds having a gapmer motif are considered chimeric antisense compounds.
• a gapmer an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region.
• the gap segment In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides.
• the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region.
• wings may include several modified sugar moieties, including, for example 2'-MOE.
• wings may include several modified and unmodified sugar moieties.
• wings may include various combinations of 2'-MOE 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, "Y” 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. In certain embodiments, "X" and “Z” are the same; in other embodiments they are different.
• gapmers provided herein include, for example 20-mers having a motif of 5-
• Nucleotide sequences that encode human plasma prekallikrein include, without limitation, the following: GENBANK Accession No. NM 000892.3 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No. DC412984.1 (incorporated herein as SEQ ID NO: 2), GENBANK Accession No.
• CN265612.1 (incorporated herein as SEQ ID NO: 3), GENBANK Accession No. AK297672.1 (incorporated herein as SEQ ID NO: 4), GENBANK Accession No. DC413312.1 (incorporated herein as SEQ ID NO: 5), GENBANK Accession No. AV688858.2 (incorporated herein as SEQ ID NO: 6), GENBANK Accession No. CD652077.1 (incorporated herein as SEQ ID NO: 7), GENBANK Accession No. BC143911.1 (incorporated herein as SEQ ID NO: 8), GENBANK Accession No. CB162532.1 (incorporated herein as SEQ ID NO: 9), GENBANK Accession No. NT 016354.19 truncated from nucleobases 111693001 to 111730000
• SEQ ID NO: 15 GENBANK Accession No. XM 002804276.1 (incorporated herein as SEQ ID NO: 17), and GENBANK Accession No. NW 001118167.1 truncated from nucleobases 2358000 to 2391000 (incorporated herein as SEQ ID NO: 18).
• 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 No) indicate a combination of nucleobase sequence and motif.
• a target region is a structurally defined region of the target nucleic acid.
• a target region may encompass a 3' UTR, a 5' UTR, an ex on, an intron, an ex on/intron junction, a coding region, a translation initiation region, translation termination region, or other defined nucleic acid region.
• the structurally defined regions for PKK can be obtained by accession number from sequence databases such as NCBI and such information is incorporated herein by reference.
• a target region may encompass the sequence from a 5' target site of one target segment within the target region to a 3' target site of another target segment within the same target region.
• Targeting includes determination of at least one target segment to which an antisense compound hybridizes, such that a desired effect occurs.
• the desired effect is a reduction in mRNA target nucleic acid levels.
• the desired effect is reduction of levels of protein encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid.
• a target region may contain one or more target segments. Multiple target segments within a target region may be overlapping. Alternatively, they may be non- overlapping. In certain embodiments, target segments within a target region are separated by no more than about 300 nucleotides. In certain emodiments, target segments within a target region are separated by a number of nucleotides that is, is about, is no more than, is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on the target nucleic acid, or is a range defined by any two of the preceeding values.
• target segments within a target region are separated by no more than, or no more than about, 5 nucleotides on the target nucleic acid. In certain embodiments, target segments are contiguous. Contemplated are target regions defined by a range having a starting nucleic acid that is any of the 5' target sites or 3' target sites listed herein.
• Suitable target segments may be found within a 5' UTR, a coding region, a 3' UTR, an intron, an ex on, or an exon/intr on junction.
• Target segments containing a start codon or a stop codon are also suitable target segments.
• a suitable target segment may specifcally exclude a certain structurally defined region such as the start codon or stop codon.
• the determination of suitable target segments may include a comparison of the sequence of a target nucleic acid to other sequences throughout the genome.
• the BLAST algorithm may be used to identify regions of similarity amongst different nucleic acids. This comparison can prevent the selection of antisense compound sequences that may hybridize in a non-specific manner to sequences other than a selected target nucleic acid (i.e., non-target or off-target sequences).
• reductions in PKK mRNA levels are indicative of inhibition of PKK expression.
• Reductions in levels of a PKK protein are also indicative of inhibition of target mRNA expression.
• phenotypic changes are indicative of inhibition of PKK expression.
• reduced or prevented inflammation can be indicative of inhibition of PKK expression.
• reduced or prevented edema/swelling can be indicative of inhibition of PKK expression.
• reduced or prevented vascular permeability can be indicative of inhibition of PKK expression.
• reduced or prevented vascular leakage can be indicative of inhibition of PKK expression.
• vascular permeability is measured by quanification of a dye, such as Evans Blue.
• hybridization occurs between an antisense compound disclosed herein and a target 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 target 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 PKK nucleic acid).
• Non-complementary nucleobases between an antisense compound and a PKK 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 PKK 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%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to an PKK 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.
• 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 ah, 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 plasma prekallikrein 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
• a 20 nucleobase portion of a 30 nucleobase antisense compound can be "fully complementary" to a target sequence that is 400 nucleobases long.
• the 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the antisense compound.
• the entire 30 nucleobase antisense compound may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.
• non-complementary nucleobase may be at the 5' end or 3' end of the antisense compound.
• the non-complementary nucleobase or nucleobases may be at an internal position of the antisense compound.
• two or more non-complementary nucleobases may be contiguous (i.e. linked) or non-contiguous.
• a non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.
• antisense compounds that are, or are up to 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 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 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. 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
• 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.
• 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.
• 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 portion of the antisense oligonucleotide is compared to an equal length portion of the target nucleic acid.
• 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,
• antisense compounds targeted to a plasma prekallikrein nucleic acid comprise one or more modified internucleoside linkages.
• the modified internucleoside linkages In certain embodiments, 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, C1-C12 alkyl or a protecting group) and combinations thereof.
• substitutent groups including 5' and 2' substituent groups
• BNA bicyclic nucleic acids
• R, Ri and R 2 are each independently H, C1-C12 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 LNA is substituted with for example a 5'-methyl or a 5'-vinyl group).
• nucleosides having modified sugar moieties include without limitation nucleosides comprising 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH 3 , 2'-OCH 2 CH 3 , 2'-OCH 2 CH 2 F and - 0(CH 2 ) 2 0CH 3 substituent groups.
• bicyclic 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.
• 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 formulas: 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;
• the bridge of a bicyclic sugar moiety is -[C(R a )(R b )] n -, -[C(R a )(R b )] n -0- , -C(R a R b )-N(R)-0- or -C(R a R b )-0-N(R)-.
• 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'-(CH2)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)
• Bx is the base moiety and R is independently H, a protecting group, Ci-Ci 2 alkyl or Ci-Ci 2 alkoxy.
• bicyclic nucleosides are provided having Formula I:
• Bx is a heterocyclic base moiety
• R c is Ci-Ci 2 alkyl or an amino protecting group
• 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.
• bicyclic nucleosides are provided having Formula II:
• 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;
• 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 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;
• bicyclic nucleosides are provided having Formula IV:
• 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, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
• each q a , q b , q c 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 Cp 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 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;
• q g and q h are each, inde endently, H, halogen, C1-C12 alkyl or substituted C1-C12 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 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;
• q g and q h are each, independently, H, halogen, C1-C12 alkyl or substituted C1-C12 alkyl.
• 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' modifications are selected from substituents including, but not limited to: 0[(CH 2 ) n O] m CH 3 , 0(CH 2 ) n NH 2 , 0(CH 2 ) n CH 3 , 0(CH 2 ) n F, 0(CH 2 ) n ONH 2 ,
• OCH 2 C( 0)N(H)CH 3, and 0(CH 2 ) n ON[(CH 2 ) n CH 3 ] 2 , where n and m are from 1 to about 10.
• Other - 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 , S0 2 CH 3 , ON0 2 , N0 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, 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 anti
• 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 -1954) or fluoro HNA (F-HNA) having a tetrahydropyran
• sugar surrogates are selected having Formula VII:
• Bx is a heterocyclic base moiety
• T a and T b 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 T b is H, a hydroxyl protecting group, a linked conjugate group or a 5' or 3'-terminal group;
• qi, q 2 , q 3 , q 4 , qs, q 6 and q 7 are each independently, H, Ci-Ce alkyl, substituted Ci-Ce alkyl, C 2 -C6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C6 alkynyl or substituted C 2 -C6 alkynyl; and each of Ri and R 2 is selected from hydrogen, hydroxyl, halogen, subsitituted or unsubstituted alkoxy, NJiJ 2 , SJi, N 3 , and CN, wherein X is O, S or NJi and each J J 2 and J 3 is, independently, H or Ci-Cg alkyl.
• the modified THP nucleosides of Formula VII are provided wherein qi, q 2 , q 3 , q 4 , q 5 , q 6 and q 7 are each H. In certain embodiments, at least one of qi, q 2 , q 3 , q 4 , qs, q 6 and q 7 is other than H. In certain embodiments, at least one of qi, q 2 , q 3 , q 4 , qs, q 6 and q 7 is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of Ri and R 2 IS fluoro.
• 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).
• morpholino means a
• 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 al, 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. Soc, 2008, 130(6), 1979-1984; Horvath et al,
• modified cyclohexenyl nucleosides have Formula X.
• Bx is a heterocyclic base moiety
• T 3 and T are each, independently, an internucleoside linking group linking the cyclohexenyl nucleoside analog to an antisense compound or one of T 3 and T 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, q2, q 3 , q 4 , qs, q6, q7, qs and q9 are each, independently, H, Ci-Ce alkyl, substituted Ci-Ce alkyl, C2- e alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-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 '-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, -OCF 3 , 0-(CH 2 ) 2 -0-CH 3 , 2'-0(CH 2 ) 2 SCH 3 , 0-(CH 2 ) 2 -0- N(R m )(R n ), or where each R m and R n is, independently, H or substituted or unsubstituted C
• 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.
• MOE or "2'-MOE” or “2'-OCH 2 CH 2 OCH 3 " or “2'-0-methoxyethyl” each refers to a nucleoside comprising a sugar comprising a -OCH 2 CH 2 OCH 3 group at the 2' position of the sugar ring.
• oligonucleotide refers to a compound comprising a plurality of linked nucleosides.
• an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).
• 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.
• 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.
• 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.
• the effects of antisense compounds on the level, activity, or expression of PKK nucleic acids can be tested in vitro in a variety of cell types.
• Cell types used for such analyses are available from commercial vendors (e.g., American Type Culture Collection, Manassus, VA; Zen-Bio, Inc., Research Triangle Park, NC; Clonetics Corporation, Walkersville, MD) and are cultured according to the vendor's instructions using commercially available reagents (e.g., Life Technologies, Carlsbad, CA).
• Illustrative cell types include, but are not limited to, HepaRGTMT cells and mouse primary hepatocytes.
• 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 (Life Technologies, Carlsbad, CA). Antisense
• oligonucleotides may be mixed with LIPOFECTIN in OPTI-MEM 1 (Life Technologies, 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 tiM antisense oligonucleotide.
• Another reagent used to introduce antisense oligonucleotides into cultured cells includes
• LIPOFECT AMINE in OPTI-MEM 1 reduced serum medium (Life Technologies, Carlsbad, CA) to achieve the desired concentration of antisense oligonucleotide and a LIPOFECT AMINE concentration that may range from 2 to 12 ug/mL per 100 tiM 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 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 (Life Technologies, Carlsbad, CA) according to the manufacturer's recommended protocols.
• Target nucleic acid levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitaive real-time PCR.
• RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Quantitative real-time PCR can be conveniently accomplished using the commercially available ABI PRISM 7600, 7700, or 7900 Sequence Detection System, available from PE- Applied Biosystems, Foster City, CA and used according to manufacturer's instructions. Quantitative Real-Time PCR Analysis of Target RNA Levels
• Quantitation of target RNA levels may be accomplished by quantitative real-time PCR using the ABI PRISM 7600, 7700, or 7900 Sequence Detection System (PE- Applied Biosystems, Foster City, CA) according to manufacturer's instructions. Methods of quantitative real-time PCR are well known in the art.
• RNA Prior to real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction, which produces complementary DNA (cDNA) that is then used as the substrate for the real-time PCR amplification.
• RT and real-time PCR reactions are performed sequentially in the same sample well.
• RT and real-time PCR reagents may be obtained from Life Technologies (Carlsbad, CA). RT real-time-PCR reactions are carried out by methods well known to those skilled in the art.
• Gene (or RNA) target quantities obtained by real time PCR are normalized using either the expression level of a gene whose expression is constant, such as cyclophilin A, or by quantifying total RNA using RIBOGREEN (Life Technologies, Inc. Carlsbad, CA). Cyclophilin A expression is quantified by real time PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RIBOGREEN RNA quantification reagent (Invetrogen, Inc. Eugene, OR). Methods of RNA quantification by RIBOGREEN are taught in Jones, L.J., et al, (Analytical Biochemistry, 1998, 265, 368- 374). A CYTOFLUOR 4000 instrument (PE Applied Biosystems) is used to measure RIBOGREEN fluorescence.
• Probes and primers are designed to hybridize to a PKK nucleic acid.
• Methods for designing realtime PCR probes and primers are well known in the art, and may include the use of software such as PRIMER EXPRESS Software (Applied Biosystems, Foster City, CA).
• Antisense inhibition of PKK nucleic acids can be assessed by measuring PKK protein levels. Protein levels of PKK can be evaluated or quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (for example, caspase activity assays), immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS).
• Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, MI), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.
• Antisense compounds for example, antisense oligonucleotides, are tested in animals to assess their ability to inhibit expression of PKK and produce phenotypic changes.
• such phenotypic changes include those associated with an inflammatory disease, such as, reduced inflammation, edema/swelling, vascular permeability, and vascular leakage.
• inflammation is measured by measuring the increase or decrease of edema, temperature, pain, color of tissue, and abdominal function in the animal.
• such phenotypic changes include those associated with a thromboembolic disease, such as, prolonged aPTT, prolonged aPTT time in conjunction with a normal PT, decreased quantity of Platelet Factor 4 (PF-4), and reduced formation of thrombus or increased time for thrombus formation.
• PF-4 Platelet Factor 4
• Testing may be performed in normal animals, or in experimental disease models.
• antisense oligonucleotides are formulated in a pharmaceutically acceptable diluent, such as phosphate-buffered saline.
• Administration includes parenteral routes of administration, such as intraperitoneal, intravenous, and subcutaneous. Calculation of antisense oligonucleotide dosage and dosing frequency is within the abilities of those skilled in the art, and depends upon factors such as route of administration and animal body weight.
• RNA is isolated from liver tissue and changes in PKK nucleic acid expression are measured.
• the invention provides methods of treating an individual comprising administering one or more pharmaceutical compositions as described herein.
• the individual has an inflammatory disease.
• the individual is at risk for developing an inflammatory condition, including, but not limited to hereditary angioedema (HAE), edema, angioedema, swelling, angioedema of the lids, ocular edema, macular edema, and cerebral edema.
• HAE hereditary angioedema
• the individual has been identified as in need of anti-inflammation therapy.
• an abnormal code can lead to a deficiency in Cl-INH (i.e., type I HAE), an inability of existing Cl-INH to function properly (type II HAE), or hyperfunctional Factor 12 (i.e., type III HAE).
• the individual has a thromboembolic disease. In certain embodiments, the individual has a thromboembolic disease.
• the individual is at risk for a blood clotting disorder, including, but not limited to, infarct, thrombosis, embolism, thromboembolism such as deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.
• a blood clotting disorder including, but not limited to, infarct, thrombosis, embolism, thromboembolism such as deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.
• the individual has been identified as in need of anticoagulation therapy. Examples of such individuals include, but are not limited to, those undergoing major orthopedic surgery (e.g
• the invention provides methods for prophylactically reducing PKK expression in an individual. Certain embodiments include treating an individual in need thereof by administering to an individual a therapeutically effective amount of an antisense compound targeted to a PKK nucleic acid.
• administration of a therapeutically effective amount of an antisense compound targeted to a PKK nucleic acid is accompanied by monitoring of PKK levels in the serum of an individual, to determine an individual's response to administration of the antisense compound.
• An individual's response to administration of the antisense compound is used by a physician to determine the amount and duration of therapeutic intervention.
• administering results in reduction of PKK expression by at least 15, 20, 25, 30, 35, 40, 45, 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, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%, or a range defined by any two of these values.
• pharmaceutical compositions comprising an antisense compound targeted to PKK are used for the preparation of a medicament for treating a patient suffering or susceptible to an inflammatory disease or thromboembolic disease.
• ISIS 546254 is characterized as a 5-10-5 MOE gapmer, having a sequence of (from 5' to 3') TGCAAGTCTCTTGGCAAACA (incorporated herein as SEQ ID NO: 570), wherein each internucleoside linkage is a phosphorothioate linkage, each cytosine is a 5'-methylcytosine, each of nucleosides 1-5 and 16-20 are 2'-0-methoxyethyl modified nucleosides, and each of nucleosides 6-15 are 2'- deoxynucleosides.
• mC a 5'-methylcytosine
• G a guanine
• e a 2'-0-methoxyethyl modified nucleoside
• s a phosphorothioate internucleoside linkage.
• ISIS 546254 is described by the following chemical structure:
• ISIS 546254 achieved 95% inhibition of human PKK mRNA in cultured HepaRGTM cells (density of 20,000 cells per well) when transfected using electr op oration with 5,000 nM antisense oligonucleotide after a treatment period of 24 hours and measured by quantitative real-time PCR using human primer probe set RTS3454 and adjusted according to total RNA content, as measured by RIBOGREEN® .
• ISIS 546254 achieved an IC 50 of 0.2 ⁇ and 0.3 ⁇ in a 4 point dose response curve (0.19 ⁇ , 0.56 ⁇ , 1.67 ⁇ , and 5.0 ⁇ ) in cultured HepaRGTM cells (density of 20,000 cells per well) when transfected using electr op oration after a treatment period of 16 and measured by quantitative real-time PCR using human primer probe set RTS3454 and adjusted according to total RNA content, as measured by RIBOGREEN ® .
• ISIS 546254 achieved 31%, 55%, 84%, and 83% human PKK mRNA inhibition and 0%, 36%, 51%, and 76% human PKK protein inhibition in transgenic mice harboring the human PKK gene sequence when injected subcutaneously twice a week for 3 weeks with 2.5 mg/kg/week, 5.0 mg/kg/week, 10 mg/kg/week or 20 mg/kg/week with ISIS 546254.
• ISISI 546254 is effective for inhibiting PKK mRNA and protein expression and is tolerable in primates.
• ISIS 546343 is characterized as a 5-10-5 MOE gapmer, having a sequence of (from 5' to 3') CCCCCTTCTTTATAGCCAGC (incorporated herein as SEQ ID NO: 705), wherein each internucleoside linkage is a phosphorothioate linkage, each cytosine is a 5'-methylcytosine, each of nucleosides 1-5 and 16-20 are 2'-0-methoxyethyl modified nucleosides, and each of nucleosides 6-15 are 2' deoxynucleosides.
• ISIS 546343 is described by the following chemical notation: mCes mCes mCes mCes mCes Tds Tds mCds Tds Tds Ads Tds Ads Gds mCes mCes Aes Ges mCe; wherein,
• A an adenine
• mC a 5'-methylcytosine
• G a guanine
• e a 2'-0-methoxyethyl modified nucleoside
• s a phosphorothioate internucleoside linkage.
• ISIS 546343 is described by the following chemical structure:
• ISIS 546343 achieved 97% and 91 % human PKK mRNA inhibition in cultured HepaRGTM cells (density of 20,000 cells per well) when transfected using electr op oration with 5,000 tiM antisense oligonucleotide after a treatment period of 24 hours and measured by quantitative real-time PCR using human primer probe set RTS3454 and adjusted according to total RNA content, as measured by RIBOGREEN®.
• ISIS 546343 achieved an IC 50 of 0.4 ⁇ in a 4 point dose response curve (0.19 ⁇ , 0.56 ⁇ , 1.67 ⁇ , and 5.0 ⁇ ) in cultured HepaRGTM cells (density of 20,000 cells per well) when transfected using electr op oration after a treatment period of 16 and measured by quantitative real-time PCR using human primer probe set RTS3454 and adjusted according to total RNA content, as measured by RIBOGREEN ® .
• ISIS 546343 achieved 46%>, 66%>, and 86%> human PKK mRNA inhibition and 0%>, 38%>, and 79%> human PKK protein inhibition in transgenic mice harboring the human PKK gene sequence when injected subcutaneously twice a week for 3 weeks with 2.5 mg/kg/week, 5.0 mg/kg/week, 10 mg/kg/week or 20 mg/kg/week with ISIS 546343.
• ISISI 546343 is effective for inhibiting PKK mRNA and protein expression and is tolerable in primates.
• ISIS 548048 is characterized as a modified antisense oligonucleotide having the nucleobase sequence (from 5' to 3') CGATATCATGATTCCC (incorporated herein as SEQ ID NO: 1666), consisting of a combination of sixteen 2'-deoxynucleosides, 2'-0-methoxyethyl modified nucleosides, and cEt modified nucleosides, wherein each of nucleosides 1, 2, and 16 are 2'-0-methoxyethyl modified nucleosides, wherein each of nucleosides 3, 14, and 15 are cEt modified nucleosides, wherein each of nucleosides 4-13 are 2'-deoxynucleosides, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage, and wherein each cytosine is a 5'-methylcytosine.
• ISIS 548048 is described by the following chemical notation: mCes Ges Aks Tds Ads Tds mCds Ads Tds Gds Ads Tds Tds mCks mCks mCe; wherein,
• A an adenine
• mC a 5'-methylcytosine
• G a guanine
• e a 2'-0-methoxyethyl modified nucleoside
• k a cEt modified nucleoside
• s a phosphorothioate internucleoside linkage.
• ISIS 548048 is described by the following chemical structure:
• ISIS 548048 achieved 84% mRNA inhibition in cultured HepaRGTM cells (density of 20,000 cells per well) when transfected using
• ISIS 548048 achieved an IC 50 of ⁇ . ⁇ ⁇ in a 4 point dose response curve (0.11 ⁇ , 0.33 ⁇ , 1.00 ⁇ , and 3.00 ⁇ ) in cultured HepaRGTM cells (density of 20,000 cells per well) when transfected using electroporation after a treatment period of 16 and measured by quantitative real-time PCR using human primer probe set RTS3454 and adjusted according to total RNA content, as measured by RIBOGREEN ® .
• ISIS 548048 achieved 7%, 77%, 72% and 80% human PKK mRNA inhibition and 23%, 70%, 89%, and 98% human PKK protein inhibition in transgenic mice harboring the human PKK gene sequence when injected subcutaneously twice a week for 3 weeks with 2.5 mg/kg/week, 5.0 mg/kg/week, 10 mg/kg/week or 20 mg/kg/week with ISIS 548048.
• ISISI 548048 is effective for inhibiting PKK mRNA and protein expression and is tolerable in primates.
• antisense oligonucletoides are designed to target nucleobases 27427-27466 of SEQ ID NO: 10 (GENBANK Accession No. NT 016354.19 truncated from nucleobases 111693001 to 111730000).
• nucleobases 27427-27466 of SEQ ID NO: 10 are a hotspot region.
• nucleobases 27427-27466 of SEQ ID NO: 10 are targeted by antisense
• the antisense oligonucleotides are 15, 16, 17, 18, 19, or 20 nucleobases in length.
• the antisense oligonucleotides are gapmers.
• the gapmers are 5-10-5 MOE gapmers, 4-9-4 MOE gapmers, 4-10-4 MOE gapmers, 4-10-3 MOE gapmers, 3-10-4 MOE gapmers, or 3-10-3 MOE gapmers.
• the gapmers are 5- 10-5 MOE and cEt gapmers, 4-9-4 MOE and cEt gapmers, 4-10-4 MOE and cEt gapmers, 4-10-3 MOE and cEt gapmers, 3-10-4 MOE and cEt gapmers, or 3-10-3 MOE and cEt gapmers.
• the nucleosides of the antisense olignonucleotides are linked by phosphorothioate internucleoside linkages.
• nucleobases 27427-27466 of SEQ ID NO: 10 are targeted by the following ISIS numbers: 530993, 530994, 530995, 546251, 546252, 546253, 546254, 546255, 546256, 547410, 547411, 547978, 547979, 547980, and 547981.
• nucleobases nucleobases 27427-27466 of SEQ ID NO: 10 are targeted by the following SEQ ID NOs: 94, 95, 96, 566, 567, 568, 569, 570, 571, 572, 573, 1597, 1598, 1599, and 1600.
• antisense oligonucleotides targeting nucleobases 27427-27466 of SEQ ID NO: 10 achieve at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%
• antisense oligonucletoides are designed to target nucleobases 33183-33242 of SEQ ID NO: 10 (GENBANK Accession No. NT 016354.19 truncated from nucleobases 111693001 to 111730000).
• nucleobases 33183-33242 of SEQ ID NO: 10 are a hotspot region.
• nucleobases 33183-33242 of SEQ ID NO: 10 are targeted by antisense
• the antisense oligonucleotides are 15, 16, 17, 18, 19, or 20 nucleobases in length.
• the antisense oligonucleotides are gapmers.
• the gapmers are 5-10-5 MOE gapmers, 4-9-4 MOE gapmers, 4-10-4 MOE gapmers, 4-10-3 MOE gapmers, 3-10-4 MOE gapmers, or 3-10-3 MOE gapmers.
• the gapmers are 5- 10-5 MOE and cEt gapmers, 4-9-4 MOE and cEt gapmers, 4-10-4 MOE and cEt gapmers, 4-10-3 MOE and cEt gapmers, 3-10-4 MOE and cEt gapmers, or 3-10-3 MOE and cEt gapmers.
• the nucleosides of the antisense olignonucleotides are linked by phosphorothioate internucleoside linkages.
• nucleobases 33183-33242 of SEQ ID NO: 10 are targeted by the following ISIS numbers: 531052, 531053, 531054, 531055, 531056, 531057, 531158, 546343, 546345, 547480, 547481, 547482, and 547483.
• nucleobases nucleobases 33183-33242 of SEQ ID NO: 10 are targeted by the following SEQ ID NOs: 155, 156, 157, 158, 159, 160, 261, 702, 703, 704, 705, 706, and 707.
• antisense oligonucleotides targeting nucleobases 33183-33242 of SEQ ID NO: 10 achieve at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%,
• nucleobases 30570-30610 of SEP ID NO: 10 are designed to target nucleobases 30570-30610 of SEQ ID NO: 10 (GENBANK Accession No. NT 016354.19 truncated from nucleobases 111693001 to 111730000).
• nucleobases 30570-30610 of SEQ ID NO: 10 are a hotspot region.
• nucleobases 30570-30610 of SEQ ID NO: 10 are targeted by antisense
• the antisense oligonucleotides are 15, 16, 17, 18, 19, or 20 nucleobases in length.
• the antisense oligonucleotides are gapmers.
• the gapmers are 5-10-5 MOE gapmers, 4-9-4 MOE gapmers, 4-10-4 MOE gapmers, 4-10-3 MOE gapmers, 3-10-4 MOE gapmers, or 3-10-3 MOE gapmers.
• the gapmers are 5- 10-5 MOE and cEt gapmers, 4-9-4 MOE and cEt gapmers, 4-10-4 MOE and cEt gapmers, 4-10-3 MOE and cEt gapmers, 3-10-4 MOE and cEt gapmers, or 3-10-3 MOE and cEt gapmers.
• the nucleosides of the antisense olignonucleotides are linked by phosphorothioate internucleoside linkages.
• nucleobases 30570-30610 of SEQ ID NO: 10 are targeted by the following ISIS numbers: 531026, 546309, 546310, 546311, 546313, 547453, 547454, 547455, 547456, 547457, 547458, 548046, 548047, 548048, 548049, and 548050.
• nucleobases nucleobases 30570-30610 of SEQ ID NO: 10 are targeted by the following SEQ ID NOs: 129, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 1664, 1665, 1666, 1667, and 1668.
• antisense oligonucleotides targeting nucleobases 30570-30610 of SEQ ID NO: 10 achieve at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least
• antisense oligonucletoides are designed to target nucleobases 27427-27520 of SEQ ID NO: 10 (GENBANK Accession No. NT 016354.19 truncated from nucleobases 111693001 to 111730000).
• nucleobases 27427-27520 of SEQ ID NO: 10 are a hotspot region.
• nucleobases 27427-27520 of SEQ ID NO: 10 are targeted by antisense oligonucleotides.
• the antisense oligonucleotides are 15, 16, 17, 18, 19, or 20 nucleobases in length.
• the antisense oligonucleotides are gapmers.
• the gapmers are 5-10-5 MOE gapmers, 4-9-4 MOE gapmers, 4-10-4 MOE gapmers, 4-10-3 MOE gapmers, 3-10-4 MOE gapmers, or 3-10-3 MOE gapmers.
• the gapmers are 5- 10-5 MOE and cEt gapmers, 4-9-4 MOE and cEt gapmers, 4-10-4 MOE and cEt gapmers, 4-10-3 MOE and cEt gapmers, 3-10-4 MOE and cEt gapmers, or 3-10-3 MOE and cEt gapmers.
• the nucleosides of the antisense olignonucleotides are linked by phosphorothioate internucleoside linkages.
• nucleobases 27427-27520 of SEQ ID NO: 10 are targeted by the following ISIS numbers: 530993-530999, 546251-546256, 546258-546260, 546263, 546265-546268, 547410-547417, and 547978-547992.
• nucleobases nucleobases 27427-27520 of SEQ ID NO: 10 are targeted by the following SEQ ID NOs: 94-100, 566-587, and 1597-1611.
• antisense oligonucleotides targeting nucleobases 27427-27520 of SEQ ID NO: 10 achieve at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%
• antisense oligonucletoides are designed to target nucleobases 33085-33247 of SEQ ID NO: 10 (GENBANK Accession No. NT 016354.19 truncated from nucleobases 111693001 to 111730000).
• nucleobases 33085-33247 of SEQ ID NO: 10 are a hotspot region.
• nucleobases 33085-33247 of SEQ ID NO: 10 are targeted by antisense
• the antisense oligonucleotides are 15, 16, 17, 18, 19, or 20 nucleobases in length.
• the antisense oligonucleotides are gapmers.
• the gapmers are 5-10-5 MOE gapmers, 4-9-4 MOE gapmers, 4-10-4 MOE gapmers, 4-10-3 MOE gapmers, 3-10-4 MOE gapmers, or 3-10-3 MOE gapmers.
• the gapmers are 5- 10-5 MOE and cEt gapmers, 4-9-4 MOE and cEt gapmers, 4-10-4 MOE and cEt gapmers, 4-10-3 MOE and cEt gapmers, 3-10-4 MOE and cEt gapmers, or 3-10-3 MOE and cEt gapmers.
• the nucleosides of the antisense olignonucleotides are linked by phosphorothioate internucleoside linkages.
• nucleobases 33085-33247 of SEQ ID NO: 10 are targeted by the following ISIS numbers: 531041-531158, 546336, 546339, 546340, 546343, 546345, 547474-547483, 547778, 548077- 548082, and 548677-548678.
• nucleobases nucleobases 33085-33247 of SEQ ID NO: 10 are targeted by the following SEQ ID NOs: 144-160, 261, 693-707, 1256, 1320-1325, 2214, and 2215.
• antisense oligonucleotides targeting nucleobases 33085-33247 of SEQ ID NO: 10 achieve at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%
• antisense oligonucletoides are designed to target nucleobases 30475-30639 of SEQ ID NO: 10 (GENBANK Accession No. NT 016354.19 truncated from nucleobases 111693001 to 111730000).
• nucleobases 30475-30639 of SEQ ID NO: 10 are a hotspot region.
• nucleobases 30475-30639 of SEQ ID NO: 10 are targeted by antisense
• the antisense oligonucleotides are 15, 16, 17, 18, 19, or 20 nucleobases in length.
• the antisense oligonucleotides are gapmers.
• the gapmers are 5-10-5 MOE gapmers, 4-9-4 MOE gapmers, 4-10-4 MOE gapmers, 4-10-3 MOE gapmers, 3-10-4 MOE gapmers, or 3-10-3 MOE gapmers.
• the gapmers are 5- 10-5 MOE and cEt gapmers, 4-9-4 MOE and cEt gapmers, 4-10-4 MOE and cEt gapmers, 4-10-3 MOE and cEt gapmers, 3-10-4 MOE and cEt gapmers, or 3-10-3 MOE and cEt gapmers.
• the nucleosides of the antisense olignonucleotides are linked by phosphorothioate internucleoside linkages.
• nucleobases 30475-30639 of SEQ ID NO: 10 are targeted by the following ISIS numbers: 531021-531029, 531146, 546297, 546299-546304, 546306-546311, 546313, 546316-546319, 547444-547462, 548031, 548032, and 548034-548056.
• nucleobases nucleobases 30475-30639 of SEQ ID NO: 10 are targeted by the following SEQ ID NOs: 124-132, 249, 633-669, and 1650-1674.
• antisense oligonucleotides targeting nucleobases 30475-30639 of SEQ ID NO: 10 achieve at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%
• antisense oligonucletoides are designed to target nucleobases 27362-27524 of SEQ ID NO: 10 (GENBANK Accession No. NT 016354.19 truncated from nucleobases 111693001 to 111730000).
• nucleobases 27362-27524 correspond to exon 9 of PKK (GENBANK Accession No. NT 016354.19 truncated from nucleobases 111693001 to 111730000).
• nucleobases 27362-27524 of SEQ ID NO: 10 are a hotspot region. In certain embodiments, nucleobases 27362-27524 of SEQ ID NO: 10 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 15, 16, 17, 18, 19, or 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers, 4-9-4 MOE gapmers, 4-10-4 MOE gapmers, 4-10-3 MOE gapmers, 3-10-4 MOE gapmers, or 3-10-3 MOE gapmers.
• the gapmers are 5-10-5 MOE and cEt gapmers, 4-9-4 MOE and cEt gapmers, 4-10-4 MOE and cEt gapmers, 4-10-3 MOE and cEt gapmers, 3-10-4 MOE and cEt gapmers, or 3-10-3 MOE and cEt gapmers.
• the nucleosides of the antisense olignonucleotides are linked by phosphorothioate internucleoside linkages.
• nucleobases 27361-27524 of SEQ ID NO: 10 are targeted by the following ISIS numbers: 530985-530999, 546244, 546247-546256, 546258-546260, 546263, 546265-546268, 547403- 547417, 547723, 547968-547970, and 547972-547992.
• nucleobases nucleobases 27361-27524 of SEQ ID NO: 10 are targeted by the following SEQ ID NOs: 86-100, 554-587, 1217, and 1588-1611.
• antisense oligonucleotides targeting nucleobases 27362-27524 of SEQ ID NO: 10 achieve at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%,
• antisense oligonucletoides are designed to target nucleobases 33101-33240 of SEQ ID NO: 10 (GENBANK Accession No. NT 016354.19 truncated from nucleobases 111693001 to 111730000).
• nucleobases 33101-33240 correspond to exon 14 of PKK (GENBANK Accession No. NT 016354.19 truncated from nucleobases 111693001 to 111730000).
• nucleobases 33101-33240 of SEQ ID NO: 10 are a hotspot region. In certain embodiments, nucleobases 33101-33240 of SEQ ID NO: 10 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 15, 16, 17, 18, 19, or 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers, 4-9-4 MOE gapmers, 4-10-4 MOE gapmers, 4-10-3 MOE gapmers, 3-10-4 MOE gapmers, or 3-10-3 MOE gapmers.
• the gapmers are 5-10-5 MOE and cEt gapmers, 4-9-4 MOE and cEt gapmers, 4-10-4 MOE and cEt gapmers, 4-10-3 MOE and cEt gapmers, 3-10-4 MOE and cEt gapmers, or 3-10-3 MOE and cEt gapmers.
• the nucleosides of the antisense olignonucleotides are linked by phosphorothioate internucleoside linkages.
• nucleobases 33101-33240 of SEQ ID NO: 10 are targeted by the following:
• nucleobases nucleobases 33101-33240 of SEQ ID NO: 10 are targeted by the following SEQ ID NOs: 144-160, 261, 693-707, 1320-1325, and 2215.
• antisense oligonucleotides targeting nucleobases 33101-33240 of SEQ ID NO: 1 In certain embodiments, antisense oligonucleotides targeting nucleobases 33101-33240 of SEQ ID NO: 1
• NO: 10 achieve at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%,
• antisense oligonucletoides are designed to target nucleobases 30463-30638 of SEQ ID NO: 10 (GENBANK Accession No. NT 016354.19 truncated from nucleobases 111693001 to
• nucleobases 30463-30638 correspond to exon 12 of PKK (GENBANK Accession No. NT 016354.19 truncated from nucleobases 111693001 to 111730000). In certain embodiments,
• nucleobases 30463-30638 of SEQ ID NO: 10 are a hotspot region. In certain embodiments, nucleobases 30463-30638 of SEQ ID NO: 10 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 15, 16, 17, 18, 19, or 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers, 4-9-4 MOE gapmers, 4-10-4 MOE gapmers, 4-10-3 MOE gapmers, 3-10-4 MOE gapmers, or 3-10-3 MOE gapmers.
• the gapmers are 5-10-5 MOE and cEt gapmers, 4-9-4 MOE and cEt gapmers, 4-10-4 MOE and cEt gapmers, 4-10-3 MOE and cEt gapmers, 3-10-4 MOE and cEt gapmers, or 3-10-3 MOE and cEt gapmers.
• the nucleosides of the antisense olignonucleotides are linked by phosphorothioate internucleoside linkages.
• nucleobases 30463-30638 of SEQ ID NO: 10 are targeted by the following ISIS numbers: 531021-531029, 531146, 546297, 546299-546304, 546306-546311, 546313, 546316-546319, 547444-547462, 548031, 548032, and 548034-548056.
• nucleobases nucleobases 30463-30638 of SEQ ID NO: 10 are targeted by the following SEQ ID NOs: 124-132, 249, 633-669, and 1650-1674.
• antisense oligonucleotides targeting nucleobases 30463-30638 of SEQ ID NO: 10 achieve at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%
• Example 1 Antisense inhibition of human PKK in HepaRGTM T cells by antisense oligonucleotides with 2'-MOE sugar modifications
• HepaRGTM cells which are terminally differentiated hepatic cells derived from a human hepatic progenitor cell line and retain many characteristics of primary human hepatocytes (Lubberstedt M. et al., J. Pharmacol. Toxicol. Methods 2011 63: 59-68), were used in the screen.
• the chimeric antisense oligonucleotides in the tables below were designed as 5-10-5 MOE gapmers.
• the 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'-0-methoxyethyl modification.
• the internucleoside linkages throughout each gapmer are phosphorothioate 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 human gene sequence.
• “Stop site” indicates the 3'- most nucleoside to which the gapmer is targeted in the human gene sequence.
• Each gapmer listed in the tables below is targeted to either the human PKK mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM 000892.3) or the human PKK genomic sequence, designated herein as SEQ ID NO: 10 (GENBANK Accession No. NT O 16354.19 truncated from nucleotides 111693001 to 111730000).
• 'n/a' indicates that the antisense oligonucleotide does not target that particular gene sequence.
• Human primer probe set RTS3454 forward sequence CCAAAAAAGGTGCACCAGTAACA, designated herein as SEQ ID NO: 20; reverse sequence CCTCCGGGACTGTACTTTAATAGG, designated herein as SEQ ID NO: 21 ; probe sequence CACGCAAACATTTCACAAGGCAGAGTACC, designated herein as SEQ ID NO: 22
• RTS3454 forward sequence CCAAAAAAGGTGCACCAGTAACA, designated herein as SEQ ID NO: 20
• reverse sequence CCTCCGGGACTGTACTTTAATAGG designated herein as SEQ ID NO: 21
• probe sequence CACGCAAACATTTCACAAGGCAGAGTACC designated herein as SEQ ID NO: 22
• PKK mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®.
• the antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Results are presented as percent inhibition of PKK, relative to untreated control cells.

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