# Patent

## Title
Modulation of factor 7 expression

## Abstract

Disclosed herein are antisense compounds and methods for decreasing Factor 7 and treating or preventing thromboembolic complications, hyperproliferative disorders, or inflammatory conditions in an individual in need thereof. Examples of disease conditions that can be ameliorated with the administration of antisense compounds targeted to Factor 7 include thrombosis, embolism, and thromboembolism, such as, deep vein thrombosis, pulmonary embolism, myocardial infarction, stroke, cancer, rheumatoid arthritis, and fibrosis.

## Background
RELATED APPLICATIONS
This application is a 35 U.S.C. §371 national phase application of international application serial no. PCT/US2008/082526, filed on Nov. 5, 2008, which is a non-provisional of and claims priority to U.S. patent application Ser. No. 60/986,928, filed on Nov. 9, 2007, the disclosure of each of which is incorporated herein by reference in its entirety.


SEQUENCE LISTING
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0099WOSEQ.txt created Nov. 5, 2008, which is 180 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
Embodiments of the present invention provide methods, compounds, and compositions for reducing expression of Factor 7 mRNA and protein in an animal. Such methods, compounds, and compositions are useful to treat, prevent, or ameliorate thromboembolic complications, hyperproliferative disorders, and inflammatory conditions.
BACKGROUND OF THE INVENTION
The circulatory system requires mechanisms that prevent blood loss, as well as those that counteract inappropriate intravascular obstructions. Generally, coagulation comprises a cascade of reactions culminating in the conversion of soluble fibrinogen to an insoluble fibrin gel. The steps of the cascade involve the conversion of an inactive zymogen to an activated enzyme. The active enzyme then catalyzes the next step in the cascade.
Coagulation Cascade
The coagulation cascade may be initiated through two branches, the tissue factor pathway (also “extrinsic pathway”), which is the primary pathway, and the contact activation pathway (also “intrinsic pathway”).
The tissue factor pathway is initiated by the cell surface receptor tissue factor (TF, also referred to as factor III), which is expressed constitutively by extravascular cells (pericytes, cardiomyocytes, smooth muscle cells, and keratinocytes) and expressed by vascular monocytes and endothelial cells upon induction by inflammatory cytokines or endotoxin. (Drake et al., Am J Pathol 1989, 134:1087-1097). TF is the high affinity cellular receptor for coagulation factor VIIa, a serine protease. In the absence of TF, VIIa has very low catalytic activity, and binding to TF is necessary to render VIIa functional through an allosteric mechanism. (Drake et al., Am J Pathol 1989, 134:1087-1097). The TF-VIIa complex activates factor X to Xa. Xa in turn associates with its co-factor factor Va into a prothrombinase complex which in turn activates prothrombin, (also known as factor II or factor 2) to thrombin (also known as factor IIa, or factor 2a). Thrombin activates platelets, converts fibrinogen to fibrin and promotes fibrin cross-linking by activating factor XIII, thus forming a stable plug at sites where TF is exposed on extravascular cells. In addition, thrombin reinforces the coagulation cascade response by activating factors V and VIII.
The contact activation pathway is triggered by activation of factor XII to XIIa. Factor XIIa converts XI to XIa, and XIa converts IX to IXa. IXa associates with its cofactor VIIIa to convert X to Xa. The two pathways converge at this point as factor Xa associates factor Va to activate prothrombin (factor II) to thrombin (factor IIa).
Inhibition of Coagulation
At least three mechanisms keep the coagulation cascade in check, namely the action of activated protein C, antithrombin, and tissue factor pathway inhibitor. Activated protein C is a serine protease that degrades cofactors Va and VIIIa. Protein C is activated by thrombin with thrombomodulin, and requires coenzyme Protein S to function. Antithrombin is a serine protease inhibitor (serpin) that inhibits serine proteases: thrombin, Xa, XIIa, XIa and IXa. Tissue factor pathway inhibitor inhibits the action of Xa and the TF-VIIa complex. (Schwartz A L et al., Trends Cardiovasc Med. 1997; 7:234-239.)
Disease
Thrombosis is the pathological development of blood clots, and an embolism occurs when a blood clot migrates to another part of the body and interferes with organ function. Thromboembolism may cause conditions such as deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke. Significantly, thromboembolism is a major cause of morbidity affecting over 2 million Americans every year. (Adcock et al. American Journal of Clinical Pathology. 1997; 108:434-49). While most cases of thrombosis are due to acquired extrinsic problems, for example, surgery, cancer, immobility, some cases are due to a genetic predisposition, for example, antiphospholipid syndrome and the autosomal dominant condition, Factor V Leiden. (Bertina R M et al. Nature 1994; 369:64-67.)
Treatment
The most commonly used anticoagulants, warfarin, heparin, and low molecular weight heparin (LMWH) all possess significant drawbacks.
Warfarin is typically used to treat patients suffering from atrial fibrillation. The drug interacts with vitamin K—dependent coagulation factors which include factors II, VII, IX and X. Anticoagulant proteins C and S are also inhibited by warfarin. Drug therapy using warfarin is further complicated by the fact that warfarin interacts with other medications, including drugs used to treat atrial fibrillation, such as amiodarone. Because therapy with warfarin is difficult to predict, patients must be carefully monitored in order to detect any signs of anomalous bleeding.
Heparin functions by activating antithrombin which inhibits both thrombin and factor X. (Bjork I, Lindahl U. Mol Cell Biochem. 1982 48: 161-182.) Treatment with heparin may cause an immunological reaction that makes platelets aggregate within blood vessels that can lead to thrombosis. This side effect is known as heparin-induced thrombocytopenia (HIT) and requires patient monitoring. Prolonged treatment with heparin may also lead to osteoporosis. LMWH can also inhibit Factor 2, but to a lesser degree than unfractioned heparin (UFH). LMWH has been implicated in the development of HIT.
Thus, current anticoagulant agents lack predictability and specificity and, therefore, require careful patient monitoring to prevent adverse side effects, such as bleeding complications. There are currently no anticoagulants which target only the intrinsic or extrinsic pathway.
SUMMARY OF THE INVENTION
Provided herein are antisense compounds, compositions, and methods for the treatment and prevention of clotting disorders.
Antisense compounds described herein may comprise an oligonucleotide consisting of 12 to 30 nucleosides targeted to a Factor 7 nucleic acid. In certain embodiments, the Factor 7 nucleic acid may be any of the sequences as set forth in nucleotides 1255000 to 1273000 of GENBANK® Accession No. NT—027140.6, GENBANK® Accession No. NM—019616.2, and GENBANK® Accession No. DB184141.1.
The antisense compound may be a single-stranded or double-stranded oligonucleotide. The antisense compound may be 100, 95, 90, 85, 80, 75, or 70% complementary to the Factor 7 nucleic acid.
The antisense oligonucleotide may be modified, wherein at least one internucleoside linkage is a modified internucleoside linkage. The internucleoside linkage may be a phosphorothioate internucleoside linkage.
The antisense oligonucleotide may be modified, wherein at least one nucleoside comprises a modified sugar. The modified sugar may be a bicyclic sugar. The modified sugar may comprise a 2′-O-methoxyethyl.
The antisense oligonucleotide may be modified, wherein at least one nucleoside comprises a modified nucleobase. The modified nucleobase may be a 5-methylcytosine.
The antisense oligonucleotide may be a 5-10-5 MOE gapmer. The antisense oligonucleotide may consist of 20 linked nucleosides.
Compositions described herein may comprise an oligonucleotide consisting of 12 to 30 linked nucleosides, targeted to a Factor 7 nucleic acid or a salt thereof and a pharmaceutically acceptable carrier or diluent.
The composition may be a single-stranded or double-stranded oligonucleotide.
Methods described herein may comprise administering to an animal a compound comprising an oligonucleotide consisting of 12 to 30 linked nucleosides targeted to a Factor 7 nucleic acid.
Administration of the compound may slow or stop coagulation. The compound may be co-administered with any of aspirin, clopidogrel, dipyridamole, heparin, lepirudin, ticlopidine, and warfarin. Administration of the compound and a second drug may be concomitant.
Administration of the compound and/or the second drug may be by parenteral administration. Parenteral administration may be any of subcutaneous or intravenous administration.
In another embodiment, the methods described herein may also comprise identifying a human with a clotting disorder and administering to a human a therapeutically effective amount of a compound comprising an oligonucleotide consisting of 12 to 30 linked nucleosides targeted to a Factor 7 nucleic acid.
Also described is a compound comprising an antisense oligonucleotide consisting of 12 to 30 linked nucleosides that will bind within the range of nucleobases 1147 to 1227, 9169 to 9278, 10982 to 11058, 11075 to 11117, 12084 to 12117, 12387 to 13796, 13847 to 13907, 14017 to 14051, 14093 to 14134, 14172 to 14287, 14331 to 14402, 14664 to 14746, 15098 to 15570, 15609 to 15819, 15899 to 15905, or 15957 to 15982 of SEQ ID NO: 1, encoding a Factor 7 nucleic acid.
The antisense oligonucleotide may be 90, 95, or 100% complementary to SEQ ID NO: 1, encoding a Factor 7 nucleic acid. The antisense oligonucleotide may be fully complementary to SEQ ID NO: 1.
The antisense oligonucleotide may hybridize exclusively within the range of nucleobases 1147 to 1227, 9169 to 9278, 10982 to 11058, 11075 to 11117, 12084 to 12117, 12387 to 13796, 13847 to 13907, 14017 to 14051, 14093 to 14134, 14172 to 14287, 14331 to 14402, 14664 to 14746, 15098 to 15570, 15609 to 15819, 15899 to 15905, or 15957 to 15982 of SEQ ID NO: 1, encoding a Factor 7 nucleic acid.
Also described is a compound comprising an antisense oligonucleotide consisting of 12 to 30 linked nucleosides that will bind within the range of nucleobases 102 to 131 or 652 to 682 of SEQ ID NO: 2, encoding a Factor 7 nucleic acid.
The antisense oligonucleotide may be 90, 95, or 100% complementary to SEQ ID NO: 2, encoding a Factor 7 nucleic acid. The antisense oligonucleotide may be fully complementary to SEQ ID NO: 2.
The compound may hybridize exclusively within the range of nucleobases 102 to 131 or 652 to 682 of SEQ ID NO: 2, encoding a Factor 7 nucleic acid.
Embodiments of the present invention provide, a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides comprising a nucleobase sequence comprising at least 12 contiguous nucleobases of a nucleobase sequence of SEQ ID NOs: 4 to 159 and 168 to 611.
In certain embodiments, the nucleobase sequence is SEQ ID NO: 53.
In certain embodiments, the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to the nucleobase sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 167.
In certain embodiments, the nucleobase sequence of the modified oligonucleotide is at least 90% complementary to the nucleobase sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 167.
In certain embodiments, the nucleobase sequence of the modified oligonucleotide is 100% complementary to the nucleobase sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 167.
In certain embodiments, the modified oligonucleotide comprises:

    (i) a gap segment consisting of linked deoxynucleosides;
    (ii) a 5′ wing segment consisting of linked nucleosides;
    (iii) a 3′ wing segment consisting of linked nucleosides; wherein the gap segment is positioned immediately adjacent to and between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.


In certain embodiments, the modified oligonucleotide comprises:

    (i) a gap segment consisting of ten linked deoxynucleosides;
    (ii) a 5′ wing segment consisting of five linked nucleosides;
    (iii) a 3′ wing segment consisting of five linked nucleosides; wherein the gap segment is positioned immediately adjacent and between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; and wherein each internucleoside linkage is a phosphorothioate linkage.


In certain embodiments, the modified oligonucleotide comprises:

    (i) a gap segment consisting of fourteen linked deoxynucleosides;
    (ii) a 5′ wing segment consisting of three linked nucleosides;
    (iii) a 3′ wing segment consisting of three linked nucleosides; wherein the gap segment is positioned immediately adjacent and between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; and wherein each internucleoside linkage is a phosphorothioate linkage.


In certain embodiments, the modified oligonucleotide comprises:

    (i) a gap segment consisting of thirteen linked deoxynucleosides;
    (ii) a 5′ wing segment consisting of two linked nucleosides;
    (iii) a 3′ wing segment consisting of five linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; and wherein each internucleoside linkage is a phosphorothioate linkage.


In certain embodiments, the modified oligonucleotide comprises:

    (i) a gap segment consisting of twelve linked deoxynucleosides;
    (ii) a 5′ wing segment consisting of two linked nucleosides;
    (iii) a 3′ wing segment consisting of two linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; and wherein each internueleoside linkage is a phosphorothioate linkage.


Embodiments of the present invention provide a composition comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides comprising a nucleobase sequence comprising at least 12 contiguous nucleobases of a nucleobase sequence of any of SEQ ID NOs: 4 to 159 and 168 to 611 or a salt thereof and a pharmaceutically acceptable carrier or diluent.
In certain embodiments, the nucleobase sequence is SEQ ID NO: 53.
Embodiments of the present invention provide a method comprising administering to an animal a modified oligonucleotide consisting of 12 to 30 linked nucleosides comprising a nucleobase sequence comprising at least 12 contiguous nucleobases of a nucleobase sequence of any of SEQ ID NOs: 4 to 159 and 168 to 611.
In certain embodiments, the nucleobase sequence is SEQ ID NO: 53.
In certain embodiments, the animal is a human.
In certain embodiments, the administering prevents deep vein thrombosis.
In certain embodiments, the administering prevents pulmonary embolism.
In certain embodiments, the administering treats a hyperproliferative disorder.
In certain embodiments, the administering treats an inflammatory condition.
Embodiments of the present invention provide a method comprising identifying an animal at risk for having thromboembolic complications and administering to the at risk animal a therapeutically effective amount of a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified oligonucleotide is complementary to a Factor 7 nucleic acid.
In certain embodiments, the thromboembolic complication is deep vein thrombosis, pulmonary embolism, or a combination thereof.
Embodiments of the present invention provide a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence, wherein the nucleobase sequence comprises an at least 12 consecutive nucleobase portion complementary to a equal number of nucleobases of nucleotides 15128 to 15223 of SEQ ID NO: 1, wherein the modified oligonucleotide is at least 80% complementary to SEQ ID NO: 1.
In certain embodiments, the modified oligonucleotide has a nucleobase sequence of SEQ ID NO: 53.


DETAILED DESCRIPTION OF THE INVENTION
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or”, unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference for the portions of the document discussed herein, as well as in their entirety.
Definitions
Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Where permitted, all patents, applications, published applications and other publications, GENBANK Accession Numbers and associated sequence information obtainable through databases such as National Center for Biotechnology Information (NCBI) and other data referred to throughout in the disclosure herein are incorporated by reference for the portions of the document discussed herein, as well as in their entirety.
Unless otherwise indicated, the following terms have the following meanings:
“2′-O-methoxyethyl” (also 2′-MOE and 2′-O(CH2)2—OCH3) refers to an O-methoxy-ethyl modification of the 2′ position of a furanosyl ring. A 2′-O-methoxyethyl modified sugar is a modified sugar.
“2′-O-methoxyethyl nucleotide” means a nucleotide comprising a 2′-O-methoxyethyl modified sugar moiety.
“5-methylcytosine” means a cytosine modified with a methyl group attached to the 5′ position. A 5-methylcytosine is a modified nucleobase.
“Active antisense compounds” means antisense compounds that reduce target nucleic acid levels or protein levels.
“Administered concomitantly” refers to the co-administration of two 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 agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of both 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 individual, and includes, but is not limited to administering by a medical professional and self-administering.
“Amelioration” refers to a lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition. 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.
“Antidote compound” refers to a compound capable decreasing the intensity or duration of any antisense activity.
“Antidote oligonucleotide” means an antidote compound comprising an oligonucleotide that is complementary to and capable of hybridizing with an antisense compound.
“Antidote protein” means an antidote compound comprising a peptide.
“Antibody” refers to a molecule characterized by reacting specifically with an antigen in some way, where the antibody and the antigen are each defined in terms of the other. Antibody may refer to a complete antibody molecule or any fragment or region thereof, such as the heavy chain, the light chain, Fab region, and Fc region.
“Antisense activity” means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.
“Antisense compound” means an oligomeric compound that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
“Antisense inhibition” means reduction of target nucleic acid levels or target protein levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound.
“Antisense oligonucleotide” means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.
“Bicyclic sugar” means a furosyl ring modified by the bridging of two non-geminal ring atoms. A bicyclic sugar is a modified sugar.
“Bicyclic nucleic acid” or “BNA” or “bicyclic nucleoside” or bicyclic nucleotide” refers to a nucleoside or nucleotide wherein the furanose portion of the nucleoside or nucleotide includes a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system. As used herein, unless otherwise indicated, the term “methyleneoxy BNA” alone refers to β-D-methyleneoxy BNA.
“Cap structure” or “terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an antisense compound.
“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 nucleotides is chemically distinct from a region having nucleotides without 2′-O-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 administration in parallel or sequentially.
“Coagulation factor” means any of factors I, II, III, IV, V, VII, VIII, IX, X, XI, XII, or XIII in the blood coagulation cascade. “Coagulation factor nucleic acid” means any nucleic acid encoding a coagulation factor. For example, in certain embodiments, a coagulation factor nucleic acid includes, without limitation, a DNA sequence encoding a coagulation factor (including genomic DNA comprising introns and exons), an RNA sequence transcribed from DNA encoding a coagulation factor, and an mRNA sequence encoding a coagulation factor. “Coagulation factor mRNA” means an mRNA encoding a coagulation factor protein.
“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.
“Diluent” means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable. For example, in drugs that are injected 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. In certain embodiments, a dose may be administered in one, two, or more boluses, tablets, or injections. For example, in certain embodiments where subcutaneous administration is desired, 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. In certain embodiments, 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.
“Efficacy” means the ability to produce a desired effect. “Effective amount” means the amount of active pharmaceutical agent sufficient to effectuate a desired physiological outcome in an individual in need of the agent. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.
“Factor 7 nucleic acid” or “Factor VII nucleic acid” means any nucleic acid encoding Factor 7. For example, in certain embodiments, a Factor 7 nucleic acid includes, without limitation, a DNA sequence encoding Factor 7, an RNA sequence transcribed from DNA encoding Factor 7 (including genomic DNA comprising introns and exons), and an mRNA sequence encoding Factor 7. “Factor 7 mRNA” means an mRNA encoding a Factor 7 protein.
“Factor 7 specific inhibitor” refers to any agent capable of specifically inhibiting the expression of Factor 7 mRNA and/or Factor 7 protein at the molecular level. For example, Factor 7 specific inhibitors include nucleic acids (including antisense compounds), peptides, antibodies, small molecules, and other agents capable of inhibiting the expression of Factor 7 mRNA and/or Factor 7 protein. In certain embodiments, by specifically modulating Factor 7 mRNA expression and/or Factor 7 protein expression, Factor 7 specific inhibitors may affect other components of the coagulation cascade including downstream components. Similarly, in certain embodiments, Factor 7 specific inhibitors may affect other molecular processes in an animal.
“Factor 7 specific inhibitor antidote” means a compound capable of decreasing the effect of a Factor 7 specific inhibitor. In certain embodiments, a Factor 7 specific inhibitor antidote is selected from a Factor 7 peptide; a Factor 7 antidote oligonucleotide; including a Factor 7 antidote compound complementary to a Factor 7 antisense compound; and any compound or protein that affects the intrinsic or extrinsic coagulation pathway.
“Fully complementary” or “100% complementary” means each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid. In certain embodiments, a first nucleic acid is an antisense compound and a target nucleic acid is a second nucleic acid. In certain such embodiments, an antisense oligonucleotide is a first nucleic acid and a target nucleic acid is a second nucleic acid.
“Gapmer” means an antisense compound in which an internal position having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having one or more nucleotides that are chemically distinct from the nucleosides of the internal region. A “gap segment” means the plurality of nucleotides that make up the internal region of a gapmer. A “wing segment” means the external region of a gapmer.
“Gap-widened” means a chimeric antisense compound having a gap segment of 12 or more contiguous 2′-deoxyribonucleosides positioned between and immediately adjacent to 5′ and 3′ wing segments having from one to six nucleosides.
“Hybridization” means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense compound and a target nucleic acid. In certain such embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense oligonucleotide and a nucleic acid target.
“Hyperproliferative disorder” refers to disorders characterized by an abnormal or pathological proliferation of cells, for example, cancer, psoriasis, hyperplasia and the like.
“Identifying an animal at risk for thromboembolic complications” means identifying an animal having been diagnosed with a thromboembolic complication, or identifying an animal predisposed to develop a thromboembolic complication. Individuals predisposed to develop a thromboembolic complication include those having one or more risk factors for thromboembolic complications including immobility, surgery (particularly orthopedic surgery), malignancy, pregnancy, older age, use of oral contraceptives, and inherited or acquired prothrombotic clotting disorders. Such identification may be accomplished by any method including evaluating an individual's medical history and standard clinical tests or assessments.
“Immediately adjacent” means there are no intervening elements between the immediately adjacent elements.
“Individual” means a human or non-human animal selected for treatment or therapy.
“Individual in need thereof” refers to a human or non-human animal selected for treatment or therapy that is in need of such treatment or therapy.
“Inflammatory condition” refers to a disease, disease state, syndrome, or other condition resulting in inflammation. For example, rheumatoid arthritis and liver fibrosis are inflammatory conditions. Other examples of inflammatory conditions include sepsis, myocardial ischemia/reperfusion injury, adult respiratory distress syndrome, nephritis, graft rejection, inflammatory bowel disease, multiple sclerosis, arteriosclerosis, and vasculitis.
“Internucleoside linkage” refers to the chemical bond between nucleosides.
“Linked nucleosides” means adjacent nucleosides which are bonded together.
“Mismatch” or “non-complementary nucleobase” means a nucleobase of a first nucleic acid that is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid.
“Modified internucleoside linkage” refers to a substitution and/or any change from a naturally occurring internucleoside bond (i.e. a phosphodiester internucleoside bond).
“Modified nucleobase” refers to any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. An “unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).
“Modified nucleotide” means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, or modified nucleobase. A “modified nucleoside” means a nucleoside having, independently, a modified sugar moiety or modified nucleobase.
“Modified oligonucleotide” means an oligonucleotide comprising a modified internucleoside linkage, a modified sugar, and/or a modified nucleobase.
“Modified sugar” refers to a substitution and/or any change from a natural sugar. “Modified sugar moiety” means a sugar moiety having any substitution and/or change from a natural sugar moiety.
“Motif” means the pattern of unmodified and modified nucleosides in an antisense compound, i.e. the pattern of chemically distinct regions in an antisense compound.
“Naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage.
“Natural sugar moiety” means a sugar found in DNA (2′-H) or RNA (2′-OH).
“Nucleic acid” refers to molecules composed of monomeric nucleotides. A nucleic acid includes ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acids (siRNA), and microRNAs (miRNA).
“Nucleobase” means a heterocyclic moiety capable of pairing with a base of another nucleic acid.
“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.
“Nucleotide” means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.
“Oligomeric compound” or “oligomer” means a polymer comprising 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 or infusion. Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g. intrathecal or intracerebroventricular administration. “Subcutaneous administration” means administration just below the skin. “Intravenous administration” means administration into the veins.
“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 provides a therapeutic benefit when administered to an individual. For example, in certain embodiments, an antisense oligonucleotide targeted to Factor 7 is a pharmaceutical agent. “Active pharmaceutical agent” means the substance or substances in a pharmaceutical composition that provides a desired effect.
“Pharmaceutical composition” means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition may comprise one or more antisense oligonucleotides and a sterile aqueous solution.
“Pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.
“Phosphorothioate linkage” means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.
“Portion” means a defined number of contiguous (i.e. linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound.
“Prevent” refers to delaying or forestalling the onset or development of a disease, disorder, or condition for a period of time from minutes to indefinitely. “Prevent” also means reducing risk of developing a disease, disorder, or condition.
“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 or conditions.
“Side effects” means physiological responses attributable to a treatment other than the desired effects. In certain embodiments, side effects include, without limitation, injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, myopathies, and malaise. For example, increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality. For example, increased bilirubin may indicate liver toxicity or liver function abnormality.
“Single-stranded oligonucleotide” means an oligonucleotide which is not hybridized to a complementary strand. “Single-stranded modified oligonucleotide” means a modified oligonucleotide which is not hybridized to a complementary strand.
“Specifically hybridizable” means an antisense compound that hybridizes to a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids. For example, specifically hybridizable refers to an antisense compound having a sufficient degree of complementarity between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e. under physiological conditions in the case of in vivo assays and therapeutic treatments.
“Stringent hybridization conditions” means conditions under which a nucleic acid molecule, such as an antisense compound, will hybridize to a target nucleic acid sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will vary in different circumstances. In the context of this invention, stringent conditions under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated.
“Targeted” or “targeted to” means having a nucleobase sequence that will allow specific hybridization of an antisense compound to a target nucleic acid to induce a desired effect. In certain embodiments, a desired effect is reduction of a target nucleic acid. In certain embodiments, a desired effect is reduction of a Factor 7 mRNA.
“Targeting” means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.
“Target nucleic acid,” “target RNA,” “target RNA transcript,” and “nucleic acid target” all mean a nucleic acid capable of being targeted by antisense compounds.
“Target 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.
“Target region” or “active target region” means a portion of a target nucleic acid to which one or more antisense compounds is targeted.
“Therapeutically effective amount” means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual.
“Thromboembolic complication” means any disease, disorder, or condition involving an embolism caused by a thrombus. Examples of such diseases, disorders, and conditions include the categories of thrombosis, embolism, and thromboembolism. In certain embodiments, such disease disorders, and conditions include deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.
“Treat” refers to administering a pharmaceutical composition to effect an alteration or improvement of a disease, disorder, or condition.
“Unmodified nucleotide” means a nucleotide composed of naturally occuring nucleobases, sugar moieties, and internucleoside linkages. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e. β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).
Certain Embodiments
Embodiments of the present invention provide methods, compounds, and compositions for modulating expression of Factor 7 mRNA and protein. In certain embodiments, expression of Factor 7 mRNA and protein is decreased. In certain embodiments, Factor 7 specific inhibitors modulate expression of Factor 7 mRNA and protein. In certain embodiments, Factor 7 specific inhibitors are nucleic acids, proteins, or small molecules.
In certain embodiments, modulation can occur in a cell or tissue. In certain embodiments, the cell or tissue is in an animal. In certain embodiments, the animal is a human. In certain embodiments, Factor 7 mRNA levels are reduced. In certain embodiments, Factor 7 protein levels are reduced. Such reduction can occur in a time-dependent manner or in a dose-dependent manner.
Embodiments of the present invention provide methods, compounds, and compositions for the treatment, prevention, or amelioration of diseases, disorders, and conditions associated with Factor 7 in an individual in need thereof. In certain embodiments, such diseases, disorders, and conditions are thromboembolic complications. Such thromboembolic complications include the categories of thrombosis, embolism, and thromboembolism. In certain embodiments such thromboembolic complications include deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.
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 a thromboembolic complication include immobility, surgery (particularly orthopedic surgery), malignancy, pregnancy, older age, use of oral contraceptives, atrial fibrillation, previous thromboembolic complication, chronic inflammatory disease, and inherited or acquired prothrombotic clotting disorders. Certain outcomes associated with development of a thromboembolic complication include decreased blood flow through an affected vessel, death of tissue, and death of the individual. Certain risk factors and causes for development of a hyperproliferative disorder include genetic factors, such as gene mutations and chromosomal aberrations, which may or may not be inherited; and environmental factors, which include, but are not limited to, exposure to known mutagens, such as high energy radiation from radioactive elements, X-rays, gamma rays, microwaves, and ultraviolet light; certain industrial chemicals; pollutants such as cigarette smoke; certain pesticides; drugs, and viruses. Certain outcomes associated with development of a hyperproliferative disorder include non-malignant tumors, pre-malignant tumors and malignant tissues in an individual. Certain risk factors and causes for development of an inflammatory condition include any noxious stimulus that causes a cellular response to an underlying pathophysiologic condition, which includes but is not limited to bacterial and viral infections, and allergens. Inflammation is mediated by cytokines, which are secreted by the host macrophages, T-lymphocytes, endothelial cells. Certain outcomes associated with development of an inflammatory condition include redness, pain, swelling at the affected area, loss of function, morbidity and mortality of the individual.
In certain embodiments, methods of treatment include administering a Factor 7 specific inhibitor to an individual in need thereof.
In certain embodiments, the present invention provides methods and compounds for the preparation of a medicament for the treatment, prevention, or amelioration of a disease, disorder, or condition associated with Factor 7. Factor 7 associated diseases, disorders, and conditions include thromboembolic complications, hyperproliferative disorders, and inflammatory conditions. Thromboembolic complications include thrombosis, embolism, thromboembolism, deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke. Hyperproliferative disorders include cancer. Inflammatory conditions include rheumatoid arthritis and fibrosis.
Embodiments of the present invention provide a Factor 7 specific inhibitor for use in treating, preventing, or ameliorating a Factor 7 associated disease. In certain embodiments, Factor 7 specific inhibitors are nucleic acids (including antisense compounds), peptides, antibodies, small molecules, and other agents capable of inhibiting the expression of Factor 7 mRNA and/or Factor 7 protein.
Embodiments of the present invention provide a Factor 7 specific inhibitor, as described herein, for use in treating, preventing, or ameliorating thromboembolic complications such as thrombosis, embolism, thromboembolism, deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.
Embodiments of the present invention provide a Factor 7 specific inhibitor, as described herein, for use in treating, preventing, or ameliorating a thromboembolic complication, as described herein, by combination therapy with an additional agent or therapy, as described herein. Agents or therapies can be co-administered or administered concomitantly.
Embodiments of the present invention provide the use of a Factor 7 specific inhibitor, as described herein, in the manufacture of a medicament for treating, preventing, or ameliorating a thromboembolic complication, as described herein, by combination therapy with an additional agent or therapy, as described herein. Agents or therapies can be co-administered or administered concomitantly.
Embodiments of the present invention provide the use of a Factor 7 specific inhibitor, as described herein, in the manufacture of a medicament for treating, preventing, or ameliorating a thromboembolic complication, as described herein, in a patient who is subsequently administered an additional agent or therapy, as described herein.
Embodiments of the present invention provide a kit for treating, preventing, or ameliorating a thromboembolic complication, as described herein, wherein the kit comprises:

    (i) a Factor 7 specific inhibitor as described herein; and alternatively
    (ii) an additional agent or therapy as described herein.


A kit of the present invention may further include instructions for using the kit to treat, prevent, or ameliorate a thromboembolic complication, as described herein, by combination therapy, as described herein.
Embodiments of the present invention provide antisense compounds targeted to a Factor 7 nucleic acid. In certain embodiments, the human Factor 7 nucleic acid is any of the sequences set forth in GENBANK Accession No. NT—027140.6, truncated at 1255000 to 1273000 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No. NM—019616.2, (incorporated herein as SEQ ID NO: 2), GENBANK Accession No. DB184141.1 (incorporated herein as SEQ ID NO: 3), and GENBANK® Accession No. NM—000131.3 (incorporated herein as SEQ ID NO: 167). In certain embodiments, the rhesus monkey Factor 7 nucleic acid is any of the sequences set forth in GENBANK Accession No NW—001104507.1, truncated at nucleotides 691000 to 706000 (incorporated herein as SEQ ID NO: 162) and GENBANK Accession No. 3360—061_B (incorporated herein as SEQ ID NO: 163). In certain embodiments, the murine Factor 7 nucleic acid is the sequence set forth in GENBANK Accession No. NT—039455.6, truncated at nucleotides 10024000 to 10037000 (incorporated herein as SEQ ID NO: 160).
Antisense Compounds
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 it is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted. In certain such embodiments, an antisense oligonucleotide has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.
In certain embodiments, an antisense compound targeted to a Factor 7 nucleic acid is 12 to 30 subunits in length. In other words, antisense compounds are from 12 to 30 linked subunits. In other embodiments, the antisense compound is 8 to 80, 12 to 50, 15 to 30, 18 to 24, 19 to 22, or 20 linked subunits. In certain such embodiments, the antisense compounds are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a range defined by any two of the above values. In some embodiments the antisense compound is an antisense oligonucleotide, and the linked subunits are nucleotides.
In certain embodiments, a shortened or truncated antisense compound targeted to a Factor 7 nucleic acid has a single subunit deleted from the 5′ end (5′ truncation), or alternatively from the 3′ end (3′ truncation). A shortened or truncated antisense compound targeted to a Factor 7 nucleic acid may have two subunits deleted from the 5′ end, or alternatively may have two subunits deleted from the 3′ end, of the antisense compound. Alternatively, the deleted nucleosides may be dispersed throughout the antisense compound; for example, in an antisense compound having one nucleoside deleted from the 5′ end and one nucleoside deleted from the 3′ end.
When a single additional subunit is present in a lengthened antisense compound, the additional subunit may be located at the 5′ or 3′ end of the antisense compound. When two or more additional subunits are present, the added subunits may be adjacent to each other; for example, in an antisense compound having two subunits added to the 5′ end (5′ addition), or alternatively to the 3′ end (3′ addition), of the antisense compound. Alternatively, the added subunits may be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit added to the 5′ end and one subunit added to the 3′ end.
It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.
Gautschi et al. (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti-tumor activity in vivo.
Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of tandem 14 nucleobase antisense oligonucleotides, and 28 and 42 nucleobase antisense oligonucleotides comprised of the sequence of two or three of the tandem antisense oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase antisense oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase antisense oligonucleotides.
Antisense Compound Motifs
In certain embodiments, antisense compounds targeted to a Factor 7 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. In a gapmer, an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region. In the case of an antisense oligonucleotide having a gapmer motif, the gap segment supports cleavage of the target nucleic acid, while the wing segments comprise modified nucleosides to enhance stability, affinity, and exonuclease resistance. In certain embodiments, the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region. The types of sugar moieties that are used to differentiate the regions of a gapmer may, in some embodiments, include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modified nucleosides (such 2′-modified nucleosides may include 2′-MOE, and 2′-O—CH3, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a 4′-(CH2)n-O-2′ bridge, where n=1 or n=2). Preferably, each distinct region comprises uniform sugar moieties. The wing-gap-wing motif is frequently described as “X-Y-Z”, where “X” represents the length of the 5′ wing region, “Y” represents the length of the gap region, and “Z” represents the length of the 3′ wing region. As used herein, a gapmer described as “X-Y-Z” has a configuration such that the gap segment is positioned immediately adjacent each of the 5′ wing segment and the 3′ wing segment. Thus, no intervening nucleotides exist between the 5′ wing segment and gap segment, or the gap segment and the 3′ wing segment. Any of the antisense compounds described herein can have a gapmer motif. In some embodiments, X and Z are the same, in other embodiments they are different. In a preferred embodiment, Y is between 8 and 15 nucleotides. X, Y or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more nucleotides. Thus, gapmers of the present invention include, but are not limited to, for example, 5-10-5, 4-8-4, 4-12-3, 4-12-4, 3-14-3, 2-13-5, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1 or 2-8-2.
In certain embodiments, the antisense compound has a “wingmer” motif, having a wing-gap or gap-wing configuration, i.e. an X-Y or Y-Z configuration, as described above, for the gapmer configuration. Thus, wingmer configurations of the present invention include, but are not limited to, for example, 5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1, 10-3, 2-10, 1-10, 8-2, 2-13, or 5-13.
In certain embodiments, antisense compounds targeted to a Factor 7 nucleic acid possess a 5-10-5 gapmer motif.
In certain embodiments, antisense compounds targeted to a Factor 7 nucleic acid possess a 3-14-3 gapmer motif.
In certain embodiments, antisense compounds targeted to a Factor 7 nucleic acid possess a 2-13-5 gapmer motif.
In certain embodiments, antisense compounds targeted to a Factor 7 nucleic acid possess a 2-12-2 gapmer motif
In certain embodiments, an antisense compound targeted to a Factor 7 nucleic acid has a gap-widened motif.
In certain embodiments, a gap-widened antisense oligonucleotide targeted to a Factor 7 nucleic acid has a gap segment of fourteen 2′-deoxyribonucleotides positioned immediately adjacent to and between wing segments of three chemically modified nucleosides. In certain embodiments, the chemical modification comprises a 2′-sugar modification. In another embodiment, the chemical modification comprises a 2′-MOE sugar modification.
In certain embodiments, a gap-widened antisense oligonucleotide targeted to a Factor 7 nucleic acid has a gap segment of thirteen 2′-deoxyribonucleotides positioned immediately adjacent to and between a 5′ wing segment of two chemically modified nucleosides and a 3′ wing segment of five chemically modified nucleosides. In certain embodiments, the chemical modification comprises a 2′-sugar modification. In another embodiment, the chemical modification comprises a 2′-MOE sugar modification.
Target Nucleic Acids, Target Regions and Nucleotide Sequences
Nucleotide sequences that encode the Factor 7 gene sequence include, without limitation, the following: GENBANK® Accession No. NT—027140.6, truncated from 1255000 to 1273000, first deposited with GENBANK® on Jul. 17, 2001, incorporated herein as SEQ ID NO: 1; GENBANK Accession No. NM—019616.2, first deposited with GENBANK® on Oct. 3, 2000, and incorporated herein as SEQ ID NO: 2; GENBANK® Accession No. DB184141.1, first deposited with GENBANK® on Dec. 11, 2005, incorporated herein as SEQ ID NO: 3; GENBANK Accession No. NM—000131.3, first deposited with GENBANK® on Mar. 24, 1999, and incorporated herein as SEQ ID NO: 167; GENBANK Accession No. NT—039455.6, truncated at nucleotides 10024000 to 10037000, first deposited with GENBANK® on Feb. 24, 2003, and incorporated herein as SEQ ID NO: 160; GENBANK Accession No. NW—00104507.1, incorporated herein as SEQ ID NO: 162; and GENBANK Accession No. 3360—061_B, incorporated herein as SEQ ID NO: 163.
It is understood that the sequence set forth in each SEQ ID NO in the Examples contained herein is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, antisense compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Antisense compounds described by Isis Number (Isis No.) indicate a combination of nucleobase sequence and motif.
In certain embodiments, a target region is a structurally defined region of the target nucleic acid. For example, a target region may encompass a 3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, a coding region, a translation initiation region, a translation termination region, or other defined nucleic acid regions. The structurally defined regions for Factor 7 gene sequences can be obtained by accession number from sequence databases, such as NCBI, and such information is incorporated herein by reference. In certain embodiments, 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 target region.
Targeting includes determination of at least one target segment to which an antisense compound hybridizes, such that a desired effect occurs. In certain embodiments, the desired effect is a reduction in mRNA target nucleic acid levels. In certain embodiments, 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 embodiments, 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 preceding values. In certain embodiments, 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 exon, or an exon/intron junction. Target segments containing a start codon or a stop codon are also suitable target segments. A suitable target segment may specifically 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. For example, 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).
There may be variation in activity (e.g., as defined by percent reduction of target nucleic acid levels) of the antisense compounds within an active target region. In certain embodiments, reductions in Factor 7 mRNA levels are indicative of inhibition of Factor 7 expression. Reductions in levels of a Factor 7 protein are also indicative of inhibition of target mRNA expression. Further, phenotypic changes are indicative of inhibition of Factor 7 expression. For example, a prolonged PT time can be indicative of inhibition of Factor 7 expression. In another example, prolonged aPTT time in conjunction with a prolonged PT time can be indicative of inhibition of Factor 7 expression. In another example, a decreased level of Platelet Factor 4 (PF-4) expression can be indicative of inhibition of Factor 7 expression. In another example, reduced formation of thrombus or increased time for thrombus formation can be indicative of inhibition of Factor 7 expression.
Hybridization
In some embodiments, hybridization occurs between an antisense compound disclosed herein and a Factor 7 nucleic acid. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.
Hybridization can occur under varying conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.
Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In certain embodiments, the antisense compounds provided herein are specifically hybridizable with a Factor 7 nucleic acid.
Complementarity
An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., antisense inhibition of a target nucleic acid, such as a Factor 7 nucleic acid).
Non-complementary nucleobases between an antisense compound and a Factor 7 nucleic acid may be tolerated provided that the antisense compound remains able to specifically hybridize to a target nucleic acid. Moreover, an antisense compound may hybridize over one or more segments of a Factor 7 nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).
In certain embodiments, the antisense compounds provided herein, or a specified portion thereof, are, or are at least, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a Factor 7 nucleic acid, a target region, target segment, or specified portion thereof. Percent complementarity of an antisense compound with a target nucleic acid can be determined using routine methods.
For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining non-complementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) non-complementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).
In certain embodiments, the antisense compounds provided herein, or specified portions thereof, are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof. For example, antisense compound may be fully complementary to a Factor 7 nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, “fully complementary” means each nucleobase of an antisense compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 20 nucleobase antisense compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the antisense compound. Fully complementary can also be used in reference to a specified portion of the first and/or the second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase antisense compound can be “fully complementary” to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to each nucleobase of the 20 nucleobase portion of the antisense compound. At the same time, the entire 30 nucleobase antisense compound may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.
The location of a non-complementary nucleobase may be at the 5′ end or 3′ end of the antisense compound. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e. linked) or non-contiguous. In one embodiment, a non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.
In certain embodiments, antisense compounds that are, or are up to 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a Factor 7 nucleic acid, or specified portion thereof.
In certain embodiments, antisense compounds that are, or are up to 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a Factor 7 nucleic acid, or specified portion thereof.
The antisense compounds provided herein also include those which are complementary to a portion of a target nucleic acid. As used herein, “portion” refers to a defined number of contiguous (i.e. linked) nucleobases within a region or segment of a target nucleic acid. A “portion” can also refer to a defined number of contiguous nucleobases of an antisense compound. In certain embodiments, the antisense compounds are complementary to at least an 8 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 12 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 15 nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values.
Identity
The antisense compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific Isis number, or portion thereof. As used herein, an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA, which contains uracil in place of thymidine in a disclosed DNA sequence, would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the antisense compounds described herein, as well as compounds having non-identical bases relative to the antisense compounds provided herein, are also contemplated. The non-identical bases may be adjacent to each other or dispersed throughout the antisense compound. Percent identity of an antisense compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.
In certain embodiments, the antisense compounds, or portions thereof, are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of the antisense compounds or SEQ ID NOs, or a portion thereof, disclosed herein.
Modifications
A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.
Modifications to antisense compounds encompass substitutions or changes to intemucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.
Chemically modified nucleosides may also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides.
Modified Internucleoside Linkages
The naturally occurring internucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside linkages because of desirable properties, such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.
Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.
In certain embodiments, antisense compounds targeted to a Factor 7 nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.
Modified Sugar Moieties
Antisense compounds of the invention can optionally contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity or some other beneficial biological property to the antisense compounds. In certain embodiments, nucleosides comprise chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include, without limitation, addition of substituent 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(R1)(R)2 (R═H, C1-C12 alkyl or a protecting group) and combinations thereof. Examples of chemically modified sugars include 2′-F-5′-methyl substituted nucleoside (see PCT International Application WO 2008/101157, published on Aug. 21, 2008, for other disclosed 5′,2′-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with ‘S’ and with further substitution at the 2′-position (see U.S. Patent. Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a BNA (see PCT International Application WO 2007/134181, published on Nov. 22, 2007, wherein LNA is substituted with, for example, a 5′-methyl or a 5′-vinyl group).
Examples of nucleosides having modified sugar moieties include, without limitation, nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S, 2′-F, 2′-OCH3 and 2′-O(CH2)2OCH3 substituent groups. The substituent at the 2′ position can also be selected from allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, OCF3, O(CH2)2SCH3, O(CH2)2—O—N(Rm)(Rn), and O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl.
Examples of bicyclic nucleic acids (BNAs) include, without limitation, nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, antisense compounds provided herein include one or more BNA nucleosides, wherein the bridge comprises one of the formulas: 4′-(CH2)—O-2′ (LNA); 4′-(CH2)—S-2′; 4′-(CH2)—O-2′ (LNA); 4′-(CH2)2—O-2′ (ENA); 4′-C(CH3)2—O-2′ (see PCT/US2008/068922); 4′-CH(CH3)—O-2′ and 4′-CH(CH2OCH3)—O-2′ (see U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-CH2—N(OCH3)-2′ (see PCT/US2008/064591); 4′-CH2—O—N(CH3)-2′ (see U.S. Patent Application US2004-0171570, published Sep. 2, 2004); 4′-CH2—N(R)—O-2′ (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH2—C(CH3)-2′ and 4′-CH2—C(═CH2)-2′ (see PCT/US2008/066154); and wherein R is, independently, H, C1-C12 alkyl, or a protecting group. Each of the foregoing BNAs include various stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see PCT international application PCT/DK98/00393, published on Mar. 25, 1999 as WO 99/14226).
In certain embodiments, nucleosides are modified by replacement of the ribosyl ring with a sugar surrogate. Such modification includes, without limitation, replacement of the ribosyl ring with a surrogate ring system (sometimes referred to as DNA analogs), such as a morpholino ring, a cyclohexenyl ring, a cyclohexyl ring or a tetrahydropyranyl ring, such as one having one of the formulas:
Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see, for example, review article: Leumann, Christian J.). Such ring systems can undergo various additional substitutions to enhance activity.
Methods for the preparations of modified sugars are well known to those skilled in the art.
In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.
In certain embodiments, antisense compounds targeted to a Factor 7 nucleic acid comprise one or more nucleotides having modified sugar moieties. In certain embodiments, the modified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOE modified nucleotides are arranged in a gapmer motif.
Modified Nucleobases
Nucleobase (or base) modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications may impart nuclease stability, binding affinity or some other beneficial biological property to antisense compounds. Modified nucleobases include synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of an antisense compound for a target nucleic acid. For example, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).
Additional modified nucleobases include 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly, 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.
Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Nucleobases that are particularly useful for increasing the binding affinity of antisense compounds include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
In certain embodiments, antisense compounds targeted to a Factor 7 nucleic acid comprise one or more modified nucleobases. In certain embodiments, gap-widened antisense oligonucleotides targeted to a Factor 7 nucleic acid comprise one or more modified nucleobases. In certain embodiments, the modified nucleobase is 5-methylcytosine. In certain embodiments, each cytosine is a 5-methylcytosine.
Compositions and Methods for Formulating Pharmaceutical Compositions
Antisense oligonucleotides may be admixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
Antisense compounds targeted to a Factor 7 nucleic acid can be utilized in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier. A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS). PBS is a diluent suitable for use in compositions to be delivered parenterally. Accordingly, in one embodiment employed in the methods described herein is a pharmaceutical composition comprising an antisense compound targeted to a Factor 7 nucleic acid and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is PBS. In certain embodiments, the antisense compound is an antisense oligonucleotide.
Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
A prodrug can include the incorporation of additional nucleosides at one or both ends of an antisense compound which are cleaved by endogenous nucleases within the body, to form the active antisense compound.
Conjugated Antisense Compounds
Antisense compounds may be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides. Typical conjugate groups include cholesterol moieties and lipid moieties. Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
Antisense compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of antisense compounds to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the antisense compound having terminal nucleic acid from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be present on both termini. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizing groups that can be used to cap one or both ends of an antisense compound to impart nuclease stability include those disclosed in WO 03/004602, published on Jan. 16, 2003.
Cell Culture and Antisense Compounds Treatment
The effects of antisense compounds on the level, activity or expression of Factor 7 nucleic acids can be tested in vitro in a variety of cell types. Cell types used for such analyses are available from commerical vendors (e.g. American Type Culture Collection, Manassus, Va.; Zen-Bio, Inc., Research Triangle Park, N.C.; Clonetics Corporation, Walkersville, Md.) and cells are cultured according to the vendor's instructions using commercially available reagents (e.g. Invitrogen Life Technologies, Carlsbad, Calif.). Illustrative cell types include HepG2 cells, HepB3 cells, and primary hepatocytes.
In Vitro Testing of Antisense Oligonucleotides
Described herein are methods for treatment of cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.
In general, cells are treated with antisense oligonucleotides when the cells reach approximately 60-80% confluency in culture.
One reagent commonly used to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN® (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotides are mixed with LIPOFECTIN® in OPTI-MEM® 1 (Invitrogen, Carlsbad, Calif.) to achieve the desired final concentration of antisense oligonucleotide and a LIPOFECTIN® concentration that typically ranges 2 to 12 ug/mL per 100 nM antisense oligonucleotide.
Another reagent used to introduce antisense oligonucleotides into cultured cells includes LIPOFECTAMINE® (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE® in OPTI-MEM® 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve the desired concentration of antisense oligonucleotide and a LIPOFECTAMINE® concentration that typically ranges 2 to 12 ug/mL per 100 nM antisense oligonucleotide.
Another technique used to introduce antisense oligonucleotides into cultured cells includes electroporation.
Cells are treated with antisense oligonucleotides by routine methods. Cells are typically 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 LIPOFECTIN®. Antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation.
RNA Isolation
RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using the TRIZOL® Reagent (Invitrogen, Carlsbad, Calif.), according to the manufacturer's recommended protocols.
Analysis of Inhibition of Target Levels or Expression
Inhibition of levels or expression of a Factor 7 nucleic acid can be assayed in a variety of ways known in the art. For example, target nucleic acid levels can be quantitated by, e.g., Northern blot analysis, competitive reverse transcription polymerase chain reaction (RT-PCR), or quantitative real-time RT-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. Real-time RT-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, Calif., and used according to manufacturer's instructions.
Quantitative Real-Time RT-PCR Analysis of Target RNA Levels
Quantitation of target RNA levels may be accomplished by quantitative real-time RT-PCR using the ABI PRISM® 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. Methods of real-time RT-PCR are well known in the art.
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. The RT and real-time PCR reactions are performed sequentially in the same sample well. RT and real-time PCR reagents are obtained from Invitrogen (Carlsbad, Calif.). RT and 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 RT-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® (Invitrogen, Inc. Carlsbad, Calif.). Cyclophilin A expression is quantified by real-time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RIBOGREEN® RNA quantification reagent (Invetrogen, Inc. Eugene, Oreg.). 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 Factor 7 nucleic acid. Methods for designing real-time RT-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, Calif.).
Analysis of Protein Levels
Antisense inhibition of Factor 7 nucleic acids can be assessed by measuring Factor 7 protein levels. Protein levels of Factor 7 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, Miss.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art. Antibodies useful for the detection of human and mouse Factor 7 are commercially available.
In Vivo Testing of Antisense Compounds
Antisense compounds, for example, antisense oligonucleotides, are tested in animals to assess their ability to inhibit expression of Factor 7 and produce phenotypic changes, such as, prolonged PT, prolonged aPTT time, decreased quantity of Platelet Factor 4 (PF-4), reduced formation of thrombus or increased time for thrombus formation, and reduction of cellular proliferation. Testing may be performed in normal animals, or in experimental disease models. For administration to animals, 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. Following a period of treatment with antisense oligonucleotides, RNA is isolated from liver tissue and changes in Factor 7 nucleic acid expression are measured. Changes in Factor 7 protein levels are also measured using a thrombin generation assay. In addition, effects on clot times, e.g. PT and aPTT, are determined using plasma from treated animals.
Certain Indications
In certain embodiments, the invention provides methods of treating an individual comprising administering one or more pharmaceutical compositions of the present invention. In certain embodiments, the individual has a thromboembolic complication. In certain embodiments, the individual is at risk for a blood clotting disorder, including, but not limited to, infarction, thrombosis, embolism, thromboembolism, such as deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke. This includes individuals with an acquired problem, disease, or disorder that leads to a risk of thrombosis, for example, surgery, cancer, immobility, sepsis, atherosclerosis, atrial fibrillation, as well as genetic predisposition, for example, antiphospholipid syndrome and the autosomal dominant condition, Factor V Leiden. In certain embodiments, the individual has been identified as in need of anti-coagulation therapy. Examples of such individuals include, but are not limited to, those undergoing major orthopedic surgery (e.g., hip/knee replacement or hip fracture surgery) and patients in need of chronic treatment, such as those suffering from atrial fibrillation to prevent stroke. In certain embodiments the invention provides methods for prophylactically reducing Factor 7 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 Factor 7 nucleic acid.
In certain embodiments, pharmaceutical compositions comprising an antisense compound targeted to Factor 7 are used for the preparation of a medicament for treating a patient suffering or susceptible to a thromboembolic complication.
In certain embodiments, the binding of Factor 7 with Tissue factor to form Tissue Factor-Factor 7a complex may lead to inflammatory conditions, such as liver fibrosis, rheumatoid arthritis, and tumor growth and metastasis.
In certain embodiments, the individual has an inflammatory condition leading to a fibrosis complication. In certain embodiments, the individual is at risk of an excessive collagen deposition and fibrosis disorder, including, but not limited to, liver fibrosis, arterial sclerosis, chronic glomerulonephritis, cutis keloid formation, progressive systemic sclerosis (PSS), liver fibrosis, pulmonary fibrosis, cystic fibrosis, chronic graft versus host disease, scleroderma (local and systemic), Peyronie's disease, penis fibrosis, urethrostenosis after the test using a cystoscope, inner accretion after surgery, myelofibrosis, idiopathic retroperitoneal fibrosis. In certain embodiments, the individual has been identified as in need of anti-fibrotic therapy. This includes individuals with a genetic or acquired problem, disease, or disorder that leads to a risk of fibrosis, for example, α1-antitrypsin deficiency, copper storage disease (Wilson's disease), fructosemia, galactosemia, glycogen storage diseases (such as, types II, IV, VI, IX, and X), iron overload syndromes (such as, hemochromatosis), lipid abnormalities (such as, Gaucher's disease), peroxisomal disorders (such as, Zellweger syndrome), Tyrsoninemia, congenital hepatic fibrosis, bacterial infection (such as, brucellosis), parasitic infection (such as, echinococcosis), viral infections (such as, chronic hepatitis B, C), disorders affecting hepatic blood flow (such as, Budd Chiari syndrome, heart failure, hepatic veno-occlusive disease, and portal vein thrombosis), alcohol, and drugs (such as amiodarone, chlorpromazine, Isoniazid, Methotrexate, Methyldopa, Oxyphenisatin, and Tolbutamide). In certain embodiments, the individual has been identified as in need of anti-fibrotic therapy. In such embodiments, the tissue factor-Factor 7a (TF/F7a) complex is identified to have the major procoagulant activity in fibrosis. In certain embodiments, the invention provides methods for prophylactically reducing Factor 7 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 Factor 7 nucleic acid.
In certain embodiments, pharmaceutical compositions comprising an antisense compound targeted to Factor 7 are used for the preparation of a medicament for treating a patient suffering or susceptible to a fibrotic complication.
In certain embodiments, the individual has an inflammatory rheumatoid arthritic complication. In certain embodiments, the individual is at risk for inflammation at the joints and rheumatoid arthritis. In such embodiments, the individual suffers from pain, swelling and tenderness at the joints, fatigue, lack of appetite, low-grade fever, muscle aches and stiffness. In certain embodiments, the individual has been identified as in need of anti-inflammatory arthritic therapy. This includes individuals suffering from rheumatoid arthritis, reactive arthritis, Reiter's syndrome, psoriatic arthritis, ankylosing spondylitis, and arthritis associated with inflammatory bowel disease. In certain embodiments, the individual has been identified as in need of anti-inflammatory therapy. In such embodiments, the tissue factor-Factor 7a (TF/F7a) complex is identified to have the major procoagulant activity in inducing arthritis. In certain embodiments the invention provides methods for prophylactically reducing Factor 7 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 Factor 7 nucleic acid.
In certain embodiments, pharmaceutical compositions comprising an antisense compound targeted to Factor 7 are used for the preparation of a medicament for treating a patient suffering or susceptible to an inflammatory arthritic complication.
In certain embodiments, the individual has a malignant complication. In certain embodiments, the individual is at risk for tumor growth, angiogenesis and metastasis. In such embodiments, the individual suffering from hemostatic abnormalities, such as disseminated intravascular coagulation and venous thromboembolism, may suffer additional complications, such as primary and metastatic tumor growths. In such embodiments, the seeding of tumor metastases is a coagulation-dependent process. In such embodiments, the tissue factor-Factor 7a (TF/F7a) complex is identified to have the major procoagulant activity in cancer. In certain embodiments, the individual has been identified as in need of anti-TF/F7a therapy. In certain embodiments the invention provides methods for prophylactically reducing Factor 7 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 Factor 7 nucleic acid.
In certain embodiments, pharmaceutical compositions comprising an antisense compound targeted to Factor 7 are used for the preparation of a medicament for treating a patient suffering or susceptible to a malignant complication.
In certain embodiments, administration of a therapeutically effective amount of an antisense compound targeted to a Factor 7 nucleic acid is accompanied by monitoring of Factor 7 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.
In certain embodiments, administration of an antisense compound targeted to a Factor 7 nucleic acid results in reduction of Factor 7 expression by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values. In certain embodiments, administration of an antisense compound targeted to a Factor 7 nucleic acid results in a change in a measure of blood clotting, as measured by a standard test, for example, but not limited to, activated partial thromboplastin time (aPTT) test, prothrombin time (PT) test, thrombin time (TCT), bleeding time, or D-dimer. In certain embodiments, administration of a Factor 7 antisense compound increases the measure by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values. In some embodiments, administration of a Factor 7 antisense compound decreases the measure by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values.
In certain embodiments, pharmaceutical compositions comprising an antisense compound targeted to Factor 7 are used for the preparation of a medicament for treating a patient suffering or susceptible to a thromboembolic complication.
Certain Combination Therapies
In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with one or more other pharmaceutical agents. In certain embodiments, such one or more other pharmaceutical agents are designed to treat the same disease, disorder, or condition as the one or more pharmaceutical compositions of the present invention. In certain embodiments, such one or more other pharmaceutical agents are designed to treat a different disease, disorder, or condition as the one or more pharmaceutical compositions of the present invention. In certain embodiments, such one or more other pharmaceutical agents are designed to treat an undesired side effect of one or more pharmaceutical compositions of the present invention. In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to treat an undesired effect of that other pharmaceutical agent. In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to produce a combinational effect. In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to produce a synergistic effect.
In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at the same time. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at different times. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared together in a single formulation. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared separately.
In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include anticoagulant or antiplatelet agents. In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include, but are not limited to aspirin, clopidogrel, dipyridamole, ticlopidine, warfarin (and related coumarins), heparin, direct thrombin inhibitors (such as lepirudin, bivalirudin), apixaban, lovenox, and small molecular compounds that interfere directly with the enzymatic action of particular coagulation factors (e.g. rivaroxaban, which interferes with Factor Xa). In certain embodiments, the anticoagulant or antiplatelet agent is administered prior to administration of a pharmaceutical composition of the present invention. In certain embodiments, the anticoagulant or antiplatelet agent is administered following administration of a pharmaceutical composition of the present invention. In certain embodiments the anticoagulant or antiplatelet agent is administered at the same time as a pharmaceutical composition of the present invention. In certain embodiments the dose of a co-administered anticoagulant or antiplatelet agent is the same as the dose that would be administered if the anticoagulant or antiplatelet agent was administered alone. In certain embodiments the dose of a co-administered anticoagulant or antiplatelet agent is lower than the dose that would be administered if the anticoagulant or antiplatelet agent was administered alone. In certain embodiments the dose of a co-administered anticoagulant or antiplatelet agent is greater than the dose that would be administered if the anticoagulant or antiplatelet agent was administered alone.
In certain embodiments, the co-administration of a second compound enhances the anticoagulant effect of a first compound, such that co-administration of the compounds results in an anticoagulant effect that is greater than the effect of administering the first compound alone. In other embodiments, the co-administration results in anticoagulant effects that are additive of the effects of the compounds when administered alone. In certain embodiments, the co-administration results in anticoagulant effects that are supra-additive of the effects of the compounds when administered alone. In certain embodiments, the first compound is an antisense compound. In certain embodiments, the second compound is an antisense compound.
In certain embodiments, an antidote is administered anytime after the administration of a Factor 7 specific inhibitor. In certain embodiments, an antidote is administered anytime after the administration of an antisense oligonucleotide targeting Factor 7. In certain embodiments, the antidote is administered minutes, hours, days, weeks, or months after the administration of an antisense compound targeting Factor 7. In certain embodiments, the antidote is a complementary (e.g. a sense strand) to the antisense compound targeting Factor 7. In certain embodiments, the antidote is a Factor 7 or Factor 7a protein. In certain embodiments, the Factor 7 or Factor 7a, protein is a human Factor 7 or human Factor 7a protein.
EXAMPLES
Non-Limiting Disclosure and Incorporation by Reference
While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references recited in the present application is incorporated herein by reference in its entirety.
Example 1
Antisense Inhibition of Human Factor 7 mRNA in HepB3 Cells
Antisense oligonucleotides targeted to a Factor 7 nucleic acid were tested for their effects on Factor 7 mRNA in vitro. Cultured HepB3 cells at a density of 4,000 cells per well were transfected using lipofectin reagent with 50 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Factor 7 mRNA levels were measured by real-time RT-PCR, as described herein. Factor 7 mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented as percent inhibition of Factor 7, relative to untreated control cells.
The chimeric antisense oligonucleotides in Table 1 were designed as 5-10-5 MOE gapmers. The gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of ten 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five nucleotides each. Each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. Each gapmer listed in Table 1 is targeted to human gene sequences, SEQ ID NO: 1 (nucleotides 1255000 to 1273000 of GENBANK Accession No. NT—027140.6), SEQ ID NO: 2 (GENBANK Accession No. NM—019616.2), or SEQ ID NO: 3 (GENBANK Accession No. DB184141.1). “Human Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the specified human gene sequence. “Human Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted in the specified human gene sequence.








TABLE 1







Inhibition of human Factor 7 mRNA levels by chimeric



antisense oligonucleotides having 5-10-5 MOE wings


and deoxy gap targeted to SEQ ID NO: 1,


SEQ ID NO: 2, and SEQ ID NO: 3















Target
Target
Target






Oligo
SEQ ID
Start
Stop

%
SEQ ID


ID
NO
Site
Site
Sequence (5′ to 3′)
Inhibition
NO

















403090
1
14017
14036
AGTCCTGGGTCATCAGCCGG
69
4



 


403093
1
14231
14250
GAGACCCTGGTGTACACCCC
75
5


 


407594
1
1208
1227
CCTGCAGCCAGGCAGCCCTG
87
6


 


407595
1
2169
2188
TCCTGAGGCCTTAGCGACCC
71
7


 


407596
1
2206
2225
GAGGCCCCGGCTTCCACGGC
61
8


 


407597
1
1114
1133
TGTTGACATTCCCCATGGGA
39
9


 


407598
1
1147
1166
CCATGATGAAATCTCTGCAG
72
10


 


407599
1
1156
1175
CCTGGGAGACCATGATGAAA
73
11


 


407600
1
1190
1209
TGAAGCCCAAGCAGAAGGCA
61
12


 


407601
1
1196
1215
CAGCCCTGAAGCCCAAGCAG
52
13


 


407602
1
1207
1226
CTGCAGCCAGGCAGCCCTGA
81
14


 


407603
1
9073
9092
TAAGAAATCCAGAACAGCTT
54
15


 


407605
1
9169
9188
TTGAGGCACACTGGTCCCCA
58
16


 


407606
1
9204
9223
CTGGTCCTTGCAGGAGCCCC
89
17


 


407607
1
9209
9228
TGGAGCTGGTCCTTGCAGGA
76
18


 


407608
1
9217
9236
TATAGGACTGGAGCTGGTCC
13
19


 


407609
1
9226
9245
AGAAGCAGATATAGGACTGG
35
20


 


407610
1
9234
9253
AGGGAGGCAGAAGCAGATAT
31
21


 


407611
1
9259
9278
TCTCACAGTTCCGGCCCTCG
82
22


 


407612
1
10982
11001
AGATCAGCTGGTCATCCTTG
82
23


 


407613
1
11010
11029
TGCTCACAGCCGCCGTTCTC
66
24


 


407614
1
11018
11037
TGCAGTACTGCTCACAGCCG
78
25


 


407615
1
11088
11107
GACACCCCGTCTGCCAGCAG
65
26


 


407616
1
11093
11112
TGCAGGACACCCCGTCTGCC
64
27


 


407617
1
12084
12103
TCCACATGGATATTCAACTG
67
28


 


407618
1
12091
12110
GTATTTTTCCACATGGATAT
42
29


 


407619
1
12098
12117
AGAATAGGTATTTTTCCACA
64
30


 


407623
1
12795
12814
TCCATTCACCAACAACAGGA
69
31


 


407624
1
12842
12861
GAGACCACCCAGATGGTGTT
73
32


 


407625
1
12863
12882
TTGTCGAAACAGTGGGCCGC
100
33


 


407626
1
12871
12890
TCTTGATTTTGTCGAAACAG
82
34


 


407628
1
13777
13796
TGATGACCTGCGCCACCCGC
38
35


 


407629
1
13856
13875
TCAGTGAGGACCACGGGCTG
34
36


 


407630
1
13863
13882
CACATGGTCAGTGAGGACCA
29
37


 


407631
1
13869
13888
GGGCACCACATGGTCAGTGA
66
38


 


407632
1
13888
13907
TCCGTTCGGGCAGGCAGAGG
46
39


 


407633
1
14023
14042
GCAGGCAGTCCTGGGTCATC
71
40


 


407634
1
14032
14051
GTGACTGCTGCAGGCAGTCC
24
41


 


407635
1
14186
14205
TGGCCCCAGCTGACGATGCC
64
42


 


407636
1
14193
14212
GCAGCCCTGGCCCCAGCTGA
75
43


 


407637
1
14238
14257
GTACTGGGAGACCCTGGTGT
71
44


 


407638
1
14248
14267
GCCACTCGATGTACTGGGAG
0
45


 


407639
1
14254
14273
TTTGCAGCCACTCGATGTAC
76
46


 


407640
1
14263
14282
GCATGAGCTTTTGCAGCCAC
79
47


 


407641
1
14707
14726
CCTGAGGCCAGCAGATCACG
94
48


 


407642
1
14713
14732
CAGCAGCCTGAGGCCAGCAG
78
49


 


407643
1
15098
15117
CACACATGGAGTCAGCATCG
84
50


 


407644
1
15106
15125
GAGGACAGCACACATGGAGT
66
51


 


407645
1
15128
15147
GAGAGCTAAACAACCGCCTT
65
52


 


407646
1
15185
15204
GGTGATGCTTCTGAATTGTC
81
53


 


407647
1
15300
15319
GCAGCCGTTTATTGTGAAGC
83
54


 


407648
1
15388
15407
GCATCTCAGAGGATGAGCAC
8
55


 


407649
1
15430
15449
GAGGGTTCATTTCAGTGATG
24
56


 


407650
1
15436
15455
CCATGTGAGGGTTCATTTCA
39
57


 


407651
1
15482
15501
AGCCTCAAACATCTATCAAA
32
58


 


407652
1
15492
15511
TGGGAGCTACAGCCTCAAAC
30
59


 


407653
1
15546
15565
AATATCATTGACAAGGGCTG
21
60


 


407654
1
15630
15649
CCACTGCAGCCAGGGCCTGG
34
61


 


407655
1
15653
15672
AGAGTGCAGCTTGCCAGGTC
28
62


 


407656
1
15658
15677
CAGCAAGAGTGCAGCTTGCC
43
63


 


407657
1
15664
15683
GGGACTCAGCAAGAGTGCAG
26
64


 


407658
1
15749
15768
GCTTGCCGGAGTCTGAGTGG
16
65


 


407659
1
15778
15797
GATGGCATCGAGTCCACTCT
55
66


 


407660
1
15905
15924
GCTCTGAAGTAGATGATGCC
16
67


 


407661
1
15963
15982
TTTACAAGAGCAGGGTGCCT
47
68


 


407663
1
9112
9131
TTGCAGGCAGGACTGGTGGT
44
69


 


407664
1
5219
5238
CAGGGCGAGGCAACCCCGTG
70
70


 


407665
1
6001
6020
GTTACGAAGACTGGGAAATG
29
71


 


407666
1
8877
8896
TCCCCAGGACATCTGGAACT
52
72


 


407667
1
10897
10916
GTGGCCATGCCCTGGGTCAG
71
73


 


407668
1
12187
12206
GAAGCCTTACCTGCCATGGA
54
74


 


407669
1
12387
12406
TAGACCCTCAGTGAGTGTCG
57
75


 


407886
1
1173
1192
GCAGAGGAGCCTGAGGGCCT
72
76


 


407887
1
1201
1220
CCAGGCAGCCCTGAAGCCCA
78
77


 


407890
1
6080
6099
CAGGGAGCCCGGCCGCAGCT
60
78


 


407891
1
9176
9195
CATGGACTTGAGGCACACTG
66
79


 


407892
1
9187
9206
CCCCATTCTGGCATGGACTT
68
80


 


407893
1
9242
9261
TCGAAGGCAGGGAGGCAGAA
33
81


 


407894
1
9252
9271
GTTCCGGCCCTCGAAGGCAG
77
82


 


407895
1
10987
11006
CACACAGATCAGCTGGTCAT
72
83


 


407896
1
11029
11048
CGTGTGGTCACTGCAGTACT
73
84


 


407897
1
11039
11058
GCTTGGTGCCCGTGTGGTCA
77
85


 


407898
1
11075
11094
CCAGCAGAGAGTACCCCTCG
49
86


 


407899
1
11098
11117
GGGTGTGCAGGACACCCCGT
42
87


 


407900
1
12141
12160
CCCCACAATTCGGCCTTGGG
91
88


 


407901
1
12829
12848
TGGTGTTGATCAGGGTCCCC
89
89


 


407902
1
12837
12856
CACCCAGATGGTGTTGATCA
86
90


 


407903
1
12847
12866
CCGCGGAGACCACCCAGATG
98
91


 


407904
1
12858
12877
GAAACAGTGGGCCGCGGAGA
83
92


 


407905
1
12876
12895
CCAGTTCTTGATTTTGTCGA
40
93


 


407906
1
13847
13866
ACCACGGGCTGGTGCAGGCG
4
94


 


407907
1
13928
13947
AATGAGAAGCGCACGAAGGC
35
95


 


407908
1
13943
13962
CCCCAGCCGCTGACCAATGA
47
96


 


407909
1
14093
14112
CCATCCGAGTAGCCGGCACA
77
97


 


407910
1
14104
14123
AGTCCTTGCTGCCATCCGAG
86
98


 


407911
1
14115
14134
CCCCTTGCAGGAGTCCTTGC
74
99


 


407912
1
14149
14168
CCCGGTAGTGGGTGGCATGT
46
100


 


407913
1
14172
14191
GATGCCCGTCAGGTACCACG
74
101


 


407914
1
14181
14200
CCAGCTGACGATGCCCGTCA
82
102


 


407915
1
14198
14217
GTTGCGCAGCCCTGGCCCCA
83
103


 


407916
1
14208
14227
GTGGCCCACGGTTGCGCAGC
53
104


 


407917
1
14218
14237
ACACCCCAAAGTGGCCCACG
70
105


 


407918
1
14226
14245
CCTGGTGTACACCCCAAAGT
72
106


 


407919
1
14243
14262
TCGATGTACTGGGAGACCCT
84
107


 


407920
1
14268
14287
TGAGCGCATGAGCTTTTGCA
65
108


 


407921
1
14331
14350
CCACAGGCCAGGGCTGCTGG
65
109


 


407922
1
14354
14373
TCGACGCAGCCTTGGCTTTC
82
110


 


407923
1
14363
14382
CAGGACAGTTCGACGCAGCC
76
111


 


407924
1
14373
14392
GATTTGGTGCCAGGACAGTT
68
112


 


407925
1
14383
14402
GAATATATGGGATTTGGTGC
64
113


 


407926
1
14633
14652
CCAGGACAACCTTGGCACTC
79
114


 


407927
1
14664
14683
AGGTAAGGAGGCTCAGCTGG
55
115


 


407928
1
14677
14696
CTTGGCTGAAGGGAGGTAAG
55
116


 


407929
1
14719
14738
GCAGAGCAGCAGCCTGAGGC
53
117


 


407930
1
14727
14746
CAATGAAGGCAGAGCAGCAG
70
118


 


407931
1
15111
15130
CTTCAGAGGACAGCACACAT
56
119


 


407932
1
15141
15160
GAACCAGAAAAGTGAGAGCT
55
120


 


407933
1
15154
15173
TGATAATGGATAAGAACCAG
58
121


 


407934
1
15166
15185
CTGAAGTGAAGATGATAATG
45
122


 


407935
1
15191
15210
ATGCATGGTGATGCTTCTGA
94
123


 


407936
1
15204
15223
GGCATTCGCCACCATGCATG
91
124


 


407937
1
15237
15256
GAAGGGAGAAATACATTTGG
59
125


 


407938
1
15245
15264
CACCCAGCGAAGGGAGAAAT
60
126


 


407939
1
15255
15274
TGCAGCCCGGCACCCAGCGA
93
127


 


407940
1
15288
15307
TGTGAAGCTGGGAAGCAGGT
62
128


 


407941
1
15305
15324
GAGACGCAGCCGTTTATTGT
65
129


 


407942
1
15320
15339
CACAGGTGTGCGGAGGAGAC
41
130


 


407943
1
15328
15347
GCAGGCACCACAGGTGTGCG
34
131


 


407944
1
15338
15357
CCAGTGGGTGGCAGGCACCA
59
132


 


407945
1
15351
15370
AATCATGGGCAACCCAGTGG
56
133


 


407946
1
15361
15380
TCCAAAAATGAATCATGGGC
5
134


 


407947
1
15393
15412
AAAGAGCATCTCAGAGGATG
26
135


 


407948
1
15403
15422
TTGTGAAAGAAAAGAGCATC
8
136


 


407949
1
15418
15437
CAGTGATGTTGAAAATTGTG
9
137


 


407950
1
15441
15460
AGCTTCCATGTGAGGGTTCA
58
138


 


407951
1
15464
15483
AACAGCTTTTGTTTTTAAAA
0
139


 


407952
1
15498
15517
GGATCCTGGGAGCTACAGCC
41
140


 


407953
1
15513
15532
ACATCCAATTCCACAGGATC
20
141


 


407954
1
15523
15542
CAGGGAGAGAACATCCAATT
39
142


 


407955
1
15551
15570
TGTGAAATATCATTGACAAG
3
143


 


407956
1
15609
15628
AACTTGCATTTAGTGATGCG
19
144


 


407957
1
15617
15636
GGCCTGGGAACTTGCATTTA
52
145


 


407958
1
15754
15773
GCCGTGCTTGCCGGAGTCTG
58
146


 


407959
1
15783
15802
GCAGGGATGGCATCGAGTCC
0
147


 


407960
1
15800
15819
GTGCCCAGGACGGCCCTGCA
53
148


 


407961
1
15899
15918
AAGTAGATGATGCCTGAGTG
34
149


 


407962
1
15944
15963
TTGGAAGCAGCCCACGGCTG
41
150


 


407963
1
15957
15976
AGAGCAGGGTGCCTTGGAAG
33
151


 


407604
2
297
316
CACACTGGTCCCCATCACTG
34
152


 


407620
2
652
671
AGGACCTGCCATGGACACTC
79
153


 


407621
2
657
676
ACAACAGGACCTGCCATGGA
62
154


 


407622
2
663
682
TCACCAACAACAGGACCTGC
55
155


 


407627
2
773
792
GCCCAGCACCGCGATCAGGT
79
156


 


407888
2
102
121
CGAAGACTGCAGCCAGGCAG
34
157


 


407889
2
112
131
TCCTGGGTTACGAAGACTGC
54
158


 


407662
3
50
69
TCCTGCAGCCAGGCAGCCCT
86
159









Certain gapmers from Table 1 are 100% homologous to the rhesus monkey genomic sequence (nucleotides 691000 to 706000 of GENBANK Accession No. NW—00104507.1; incorporated herein as SEQ ID NO: 162) or the rhesus monkey mRNA sequence (GENKBANK Accession No. 3360—061_B; incorporated herein as SEQ ID NO: 163). Shown in Table 2 are the chimeric antisense oligonucleotides from Table 1, which are homologous with rhesus monkey. Gapmers are arranged by human target start site.








TABLE 2







Human/rhesus monkey cross-reactive chimeric



antisense oligonucleotides having 5-10-5 MOE


wings and deoxy gap






















Rhesus
Rhesus





Human
Human
Human

Rhesus
monkey
monkey



Target
Target
Target

Target
Target
Target
SEQ


ISIS
SEQ
Start
Stop

SEQ
Start
Stop
ID


No.
ID NO
Site
Site
Sequence (5′ to 3′)
ID No.
Site
Site
No.



















407597
1
1114
1133
TGTTGACATTCCCCATGGGA
160
537
556
9



 


407598
1
1147
1166
CCATGATGAAATCTCTGCAG
160
570
589
10


 


407599
1
1156
1175
CCTGGGAGACCATGATGAAA
160
579
598
11


 


407886
1
1173
1192
GCAGAGGAGCCTGAGGGCCT
160
596
615
76


 


407600
1
1190
1209
TGAAGCCCAAGCAGAAGGCA
160
613
632
12


 


407601
1
1196
1215
CAGCCCTGAAGCCCAAGCAG
160
619
638
13


 


407887
1
1201
1220
CCAGGCAGCCCTGAAGCCCA
160
624
643
77


 


407602
1
1207
1226
CTGCAGCCAGGCAGCCCTGA
160
630
649
14


 


407594
1
1208
1227
CCTGCAGCCAGGCAGCCCTG
160
631
650
6


 


407595
1
2169
2188
TCCTGAGGCCTTAGCGACCC
160
1628
1647
7


 


407596
1
2206
2225
GAGGCCCCGGCTTCCACGGC
160
1666
1685
8


 


407664
1
5219
5238
CAGGGCGAGGCAACCCCGTG
160
3001
3020
70


 


407666
1
8877
8896
TCCCCAGGACATCTGGAACT
160
6444
6463
72


 


407603
1
9073
9092
TAAGAAATCCAGAACAGCTT
160
6637
6656
15


 


407663
1
9112
9131
TTGCAGGCAGGACTGGTGGT
160
6676
6695
69


 


407605
1
9169
9188
TTGAGGCACACTGGTCCCCA
160
6736
6755
16


 


407891
1
9176
9195
CATGGACTTGAGGCACACTG
160
6743
6762
79


 


407892
1
9187
9206
CCCCATTCTGGCATGGACTT
160
6754
6773
80


 


407606
1
9204
9223
CTGGTCCTTGCAGGAGCCCC
160
6771
6790
17


 


407607
1
9209
9228
TGGAGCTGGTCCTTGCAGGA
160
6776
6795
18


 


407608
1
9217
9236
TATAGGACTGGAGCTGGTCC
160
6784
6803
19


 


407609
1
9226
9245
AGAAGCAGATATAGGACTGG
160
6793
6812
20


 


407610
1
9234
9253
AGGGAGGCAGAAGCAGATAT
160
6801
6820
21


 


407893
1
9242
9261
TCGAAGGCAGGGAGGCAGAA
160
6809
6828
81


 


407894
1
9252
9271
GTTCCGGCCCTCGAAGGCAG
160
6819
6838
82


 


407611
1
9259
9278
TCTCACAGTTCCGGCCCTCG
160
6826
6845
22


 


407612
1
10982
11001
AGATCAGCTGGTCATCCTTG
160
8679
8698
23


 


407895
1
10987
11006
CACACAGATCAGCTGGTCAT
160
8684
8703
83


 


407613
1
11010
11029
TGCTCACAGCCGCCGTTCTC
160
8707
8726
24


 


407614
1
11018
11037
TGCAGTACTGCTCACAGCCG
160
8715
8734
25


 


407896
1
11029
11048
CGTGTGGTCACTGCAGTACT
160
8726
8745
84


 


407897
1
11039
11058
GCTTGGTGCCCGTGTGGTCA
160
8736
8755
85


 


407898
1
11075
11094
CCAGCAGAGAGTACCCCTCG
160
8772
8791
86


 


407615
1
11088
11107
GACACCCCGTCTGCCAGCAG
160
8785
8804
26


 


407616
1
11093
11112
TGCAGGACACCCCGTCTGCC
160
8790
8809
27


 


407899
1
11098
11117
GGGTGTGCAGGACACCCCGT
160
8795
8814
158


 


407617
1
12084
12103
TCCACATGGATATTCAACTG
160
9808
9827
28


 


407618
1
12091
12110
GTATTTTTCCACATGGATAT
160
9815
9834
29


 


407619
1
12098
12117
AGAATAGGTATTTTTCCACA
160
9822
9841
30


 


407900
1
12141
12160
CCCCACAATTCGGCCTTGGG
160
9865
9884
88


 


407668
1
12187
12206
GAAGCCTTACCTGCCATGGA
160
9911
9930
74


 


407669
1
12387
12406
TAGACCCTCAGTGAGTGTCG
160
10116
10135
75


 


407623
1
12795
12814
TCCATTCACCAACAACAGGA
160
10524
10543
31


 


407901
1
12829
12848
TGGTGTTGATCAGGGTCCCC
160
10558
10577
89


 


407902
1
12837
12856
CACCCAGATGGTGTTGATCA
160
10566
10585
90


 


407624
1
12842
12861
GAGACCACCCAGATGGTGTT
160
10571
10590
32


 


407903
1
12847
12866
CCGCGGAGACCACCCAGATG
160
10576
10595
91


 


407904
1
12858
12877
GAAACAGTGGGCCGCGGAGA
160
10587
10606
92


 


407625
1
12863
12882
TTGTCGAAACAGTGGGCCGC
160
10592
10611
33


 


407626
1
12871
12890
TCTTGATTTTGTCGAAACAG
160
10600
10619
34


 


407905
1
12876
12895
CCAGTTCTTGATTTTGTCGA
160
10605
10624
93


 


407628
1
13777
13796
TGATGACCTGCGCCACCCGC
160
11499
11518
35


 


407906
1
13847
13866
ACCACGGGCTGGTGCAGGCG
160
11569
11588
94


 


407629
1
13856
13875
TCAGTGAGGACCACGGGCTG
160
11578
11597
36


 


407630
1
13863
13882
CACATGGTCAGTGAGGACCA
160
11585
11604
37


 


407631
1
13869
13888
GGGCACCACATGGTCAGTGA
160
11591
11610
38


 


407632
1
13888
13907
TCCGTTCGGGCAGGCAGAGG
160
11610
11629
39


 


407907
1
13928
13947
AATGAGAAGCGCACGAAGGC
160
11650
11669
95


 


407908
1
13943
13962
CCCCAGCCGCTGACCAATGA
160
11665
11684
96


 


403090
1
14017
14036
AGTCCTGGGTCATCAGCCGG
160
11739
11758
4


 


407633
1
14023
14042
GCAGGCAGTCCTGGGTCATC
160
11745
11764
40


 


407634
1
14032
14051
GTGACTGCTGCAGGCAGTCC
160
11754
11773
41


 


407909
1
14093
14112
CCATCCGAGTAGCCGGCACA
160
11815
11834
97


 


407910
1
14104
14123
AGTCCTTGCTGCCATCCGAG
160
11826
11845
98


 


407911
1
14115
14134
CCCCTTGCAGGAGTCCTTGC
160
11837
11856
99


 


407912
1
14149
14168
CCCGGTAGTGGGTGGCATGT
160
11871
11890
100


 


407913
1
14172
14191
GATGCCCGTCAGGTACCACG
160
11894
11913
101


 


407914
1
14181
14200
CCAGCTGACGATGCCCGTCA
160
11903
11922
102


 


407635
1
14186
14205
TGGCCCCAGCTGACGATGCC
160
11908
11927
42


 


407636
1
14193
14212
GCAGCCCTGGCCCCAGCTGA
160
11915
11934
43


 


407915
1
14198
14217
GTTGCGCAGCCCTGGCCCCA
160
11920
11939
103


 


407916
1
14208
14227
GTGGCCCACGGTTGCGCAGC
160
11930
11949
104


 


407917
1
14218
14237
ACACCCCAAAGTGGCCCACG
160
11940
11959
105


 


407918
1
14226
14245
CCTGGTGTACACCCCAAAGT
160
11948
11967
106


 


403093
1
14231
14250
GAGACCCTGGTGTACACCCC
160
11953
11972
5


 


407637
1
14238
14257
GTACTGGGAGACCCTGGTGT
160
11960
11979
44


 


407919
1
14243
14262
TCGATGTACTGGGAGACCCT
160
11965
11984
107


 


407638
1
14248
14267
GCCACTCGATGTACTGGGAG
160
11970
11989
45


 


407639
1
14254
14273
TTTGCAGCCACTCGATGTAC
160
11976
11995
46


 


407640
1
14263
14282
GCATGAGCTTTTGCAGCCAC
160
11985
12004
47


 


407920
1
14268
14287
TGAGCGCATGAGCTTTTGCA
160
11990
12009
108


 


407922
1
14354
14373
TCGACGCAGCCTTGGCTTTC
160
12076
12095
110


 


407923
1
14363
14382
CAGGACAGTTCGACGCAGCC
160
12085
12104
111


 


407924
1
14373
14392
GATTTGGTGCCAGGACAGTT
160
12095
12114
112


 


407925
1
14383
14402
GAATATATGGGATTTGGTGC
160
12105
12124
113


 


407927
1
14664
14683
AGGTAAGGAGGCTCAGCTGG
160
12384
12403
115


 


407928
1
14677
14696
CTTGGCTGAAGGGAGGTAAG
160
12397
12416
116


 


407641
1
14707
14726
CCTGAGGCCAGCAGATCACG
160
12427
12446
48


 


407642
1
14713
14732
CAGCAGCCTGAGGCCAGCAG
160
12433
12452
49


 


407929
1
14719
14738
GCAGAGCAGCAGCCTGAGGC
160
12397
12416
117


 


407930
1
14727
14746
CAATGAAGGCAGAGCAGCAG
160
12447
12466
118


 


407643
1
15098
15117
CACACATGGAGTCAGCATCG
160
12815
12834
50


 


407644
1
15106
15125
GAGGACAGCACACATGGAGT
160
12823
12842
51


 


407931
1
15111
15130
CTTCAGAGGACAGCACACAT
160
12828
12847
119


 


407645
1
15128
15147
GAGAGCTAAACAACCGCCTT
160
12845
12864
52


 


407932
1
15141
15160
GAACCAGAAAAGTGAGAGCT
160
12858
12847
120


 


407933
1
15154
15173
TGATAATGGATAAGAACCAG
160
12871
12890
121


 


407934
1
15166
15185
CTGAAGTGAAGATGATAATG
160
12883
12902
122


 


407646
1
15185
15204
GGTGATGCTTCTGAATTGTC
160
12902
12921
53


 


407935
1
15191
15210
ATGCATGGTGATGCTTCTGA
160
12908
12927
123


 


407936
1
15204
15223
GGCATTCGCCACCATGCATG
160
12921
12940
124


 


407940
1
15288
15307
TGTGAAGCTGGGAAGCAGGT
160
12985
13004
128


 


407647
1
15300
15319
GCAGCCGTTTATTGTGAAGC
160
12997
13016
54


 


407941
1
15305
15324
GAGACGCAGCCGTTTATTGT
160
13002
13021
129


 


407942
1
15320
15339
CACAGGTGTGCGGAGGAGAC
160
13017
13036
130


 


407943
1
15328
15347
GCAGGCACCACAGGTGTGCG
160
13025
13044
131


 


407944
1
15338
15357
CCAGTGGGTGGCAGGCACCA
160
13035
13054
132


 


407648
1
15388
15407
GCATCTCAGAGGATGAGCAC
160
13085
13104
136


 


407947
1
15393
15412
AAAGAGCATCTCAGAGGATG
160
13090
13109
54


 


407948
1
15403
15422
TTGTGAAAGAAAAGAGCATC
160
13100
13119
55


 


407949
1
15418
15437
CAGTGATGTTGAAAATTGTG
160
13115
13134
57


 


407649
1
15430
15449
GAGGGTTCATTTCAGTGATG
160
13127
13146
56


 


407650
1
15436
15455
CCATGTGAGGGTTCATTTCA
160
13133
13152
57


 


407950
1
15441
15460
AGCTTCCATGTGAGGGTTCA
160
13138
13157
138


 


407951
1
15464
15483
AACAGCTTTTGTTTTTAAAA
160
13163
13182
139


 


407651
1
15482
15501
AGCCTCAAACATCTATCAAA
160
13181
13200
58


 


407652
1
15492
15511
TGGGAGCTACAGCCTCAAAC
160
13191
13210
59


 


407952
1
15498
15517
GGATCCTGGGAGCTACAGCC
160
13197
13216
140


 


407953
1
15513
15532
ACATCCAATTCCACAGGATC
160
13212
13231
141


 


407954
1
15523
15542
CAGGGAGAGAACATCCAATT
160
13222
13241
142


 


407653
1
15546
15565
AATATCATTGACAAGGGCTG
160
13245
13264
60


 


407955
1
15551
15570
TGTGAAATATCATTGACAAG
160
13250
13269
143


 


407957
1
15617
15636
GGCCTGGGAACTTGCATTTA
160
13312
13331
145


 


407654
1
15630
15649
CCACTGCAGCCAGGGCCTGG
160
13325
13344
61


 


407655
1
15653
15672
AGAGTGCAGCTTGCCAGGTC
160
13348
13367
62


 


407656
1
15658
15677
CAGCAAGAGTGCAGCTTGCC
160
13353
13372
63


 


407657
1
15664
15683
GGGACTCAGCAAGAGTGCAG
160
13359
13378
64


 


407658
1
15749
15768
GCTTGCCGGAGTCTGAGTGG
160
13444
13463
65


 


407958
1
15754
15773
GCCGTGCTTGCCGGAGTCTG
160
13449
13468
146


 


407659
1
15778
15797
GATGGCATCGAGTCCACTCT
160
13473
13492
66


 


407959
1
15783
15802
GCAGGGATGGCATCGAGTCC
160
13478
13497
147


 


407960
1
15800
15819
GTGCCCAGGACGGCCCTGCA
160
13495
13514
148


 


407961
1
15899
15918
AAGTAGATGATGCCTGAGTG
160
13564
13583
149


 


407660
1
15905
15924
GCTCTGAAGTAGATGATGCC
160
13570
13589
67


 


407962
1
15944
15963
TTGGAAGCAGCCCACGGCTG
160
13609
13628
150


 


407963
1
15957
15976
AGAGCAGGGTGCCTTGGAAG
160
13622
13641
151


 


407661
1
15963
15982
TTTACAAGAGCAGGGTGCCT
160
13628
13647
68


 


407604
2
297
316
CACACTGGTCCCCATCACTG
160
6730
6749
152


 


407620
2
652
671
AGGACCTGCCATGGACACTC
160
9906
9925
153


 


407621
2
657
676
ACAACAGGACCTGCCATGGA
161
723
742
154


 


407622
2
663
682
TCACCAACAACAGGACCTGC
160
10519
10538
155


 


407627
2
773
792
GCCCAGCACCGCGATCAGGT
160
10629
10648
156


 


407662
3
50
69
TCCTGCAGCCAGGCAGCCCT
160
632
651
159









Example 2
Dose-Dependent Antisense Inhibition of Human Factor 7 in HepB3 Cells
Several antisense oligonucleotides from Example 1 (see Table 1) exhibiting at least 80% in vitro inhibition of human Factor 7 were tested at various doses in HepB3 cells. Cells were plated at a density of 4,000 cells per well and treated with lipofectin reagent with 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM concentrations of antisense oligonucleotide, as indicated in Table 3. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Factor 7 mRNA levels were measured by real-time RT-PCR, as described herein. Human Factor 7 primer probe set RTS 2927 (forward sequence: GGGACCCTGATCAACACCAT, incorporated herein as SEQ ID NO: 164; reverse sequence: CCAGTTCTTGATTTTGTCGAAACA, incorporated herein as SEQ ID NO: 165; probe sequence: TGGGTGGTCTCCGCGGCCX, incorporated herein as SEQ ID NO: 166) was used to measure mRNA levels. Factor 7 mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented as percent inhibition of Factor 7, relative to untreated control cells. As illustrated in Table 3, Factor 7 mRNA levels were reduced in a dose-dependent manner.







TABLE 3







Dose-dependent antisense inhibition


of human Factor 7 in HepB3 cells














ISIS
3.125
6.25
12.5
25.0
50.0
100.0
SEQ ID


No.
nM
nM
nM
nM
nM
nM
No.

















407641
53
53
71
84
88
89
48


407606
20
0
49
55
83
84
17


407594
10
34
63
77
84
80
6


407662
0
35
58
74
81
83
166


407643
16
59
68
76
92
95
50


407935
57
76
78
89
90
89
123


407939
62
79
83
91
92
92
127


407900
52
58
80
87
94
91
88


407936
45
77
79
86
91
90
124


407910
31
44
69
68
82
89
98









Example 3
Antisense Inhibition of Human Factor 7 in HepB3 Cells
Antisense oligonucleotides targeted to a Factor 7 nucleic acid were designed and tested for their effects on Factor 7 mRNA in vitro. Certain antisense oligonucleotides from Table 3 were also retested for their effects on Factor 7 mRNA in vitro. Cultured HepB3 cells at a density of 4,000 cells per well were transfected using lipofectin reagent with 50 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Factor 7 mRNA levels were measured by real-time RT-PCR, as described herein. Human Factor 7 primer probe set RTS 2927 was used to measure mRNA levels. Factor 7 mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented as percent inhibition of Factor 7, relative to untreated control.
The chimeric antisense oligonucleotides in Table 4 were designed as 5-10-5 MOE gapmers. The gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of ten 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five nucleotides each. Each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. The first seven listed gapmers in Table 4 are from Table 3 and are designated by an asterisk (*). “Human Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the specified human gene sequence. “Human Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted in the specified human gene sequence. Each gapmer listed in Table 4 is targeted to SEQ ID NO: 1 (nucleotides 1255000 to 1273000 of GENBANK® Accession No. NT—027140.6), SEQ ID NO: 2 (GENBANK® Accession No. NM—019616.2), or SEQ ID NO: 167 (GENBANK® Accession No. NM—000131.3).








TABLE 4







Inhibition of human Factor 7 mRNA levels by chimeric



antisense oligonucleotides having 5-10-5 MOE wings


and deoxy gap targeted to SEQ ID NO: 1, SEQ ID NO: 2,


and SEQ ID NO: 167















Human
Human
Human







Target
Target
Target


SEQ



SEQ
Start
Stop

%
ID


ISIS No.
ID NO
Site
Site
Sequence (5′ to 3′)
Inhibition
No.

















*407606
1
9204
9223
CTGGTCCTTGCAGGAGCCCC
57
17



 


*407900
1
12141
12160
CCCCACAATTCGGCCTTGGG
78
88


 


*407910
1
14104
14123
AGTCCTTGCTGCCATCCGAG
72
98


 


*407641
1
14707
14726
CCTGAGGCCAGCAGATCACG
73
48


 


*407643
1
15098
15117
CACACATGGAGTCAGCATCG
80
50


 


*407935
1
15191
15210
ATGCATGGTGATGCTTCTGA
72
123


 


*407939
1
15255
15274
TGCAGCCCGGCACCCAGCGA
76
127


 


416492
1
616
635
GGATCATTCTGGCCCTGAGC
13
168


 


416493
1
738
757
TCTTGGGTGTGGATGTAAAT
0
169


 


416494
1
803
822
CAGATTTAAACTGCAGATGA
0
170


 


416495
1
838
857
TCTAGAATTCCAAACCCCTA
0
171


 


416496
1
855
874
CAACACTTCAAATACGATCT
0
172


 


416497
1
883
902
CGTGCAGGTGTTAAGGTGTG
0
173


 


416498
1
994
1013
GGCCAGTGGCCATGCATCCC
5
174


 


416499
1
1011
1030
GAGAGCTGCACCTGGCCGGC
7
175


 


416500
1
1026
1045
CTGAACACCCCAGCTGAGAG
1
176


 


416424
1
1151
1170
GAGACCATGATGAAATCTCT
27
177


 


416425
1
1182
1201
AAGCAGAAGGCAGAGGAGCC
13
178


 


416426
1
1187
1206
AGCCCAAGCAGAAGGCAGAG
34
179


 


416427
1
1193
1212
CCCTGAAGCCCAAGCAGAAG
5
180


 


416501
1
1251
1270
CTGCCCTTCCACCAAGTTTA
18
181


 


416502
1
1437
1456
GTTCTTTGAAAAATAATCCC
22
182


 


416423
1
2179
2198
GTGTTTCTCCTCCTGAGGCC
51
183


 


416503
1
2311
2330
TAGCCACCCCGCGGGCTGGC
0
184


 


416504
1
2484
2503
TCAGAAAAGCTCTCAAGAAC
0
185


 


416505
1
2495
2514
GCAGATTTGCATCAGAAAAG
45
186


 


416506
1
4766
4785
CTTTAAAATCAGTTTCACAC
18
187


 


416507
1
4847
4866
GGTTACTGAGCGCGGAAGAA
73
188


 


416508
1
4873
4892
CGAGTTCTGCAGGAGCGGCC
67
189


 


416509
1
4880
4899
AGGAGCCCGAGTTCTGCAGG
56
190


 


416510
1
4916
4935
GACGAGGCCTCAGGTGGACG
30
191


 


416511
1
4926
4945
TTGCTGGGAGGACGAGGCCT
35
192


 


416512
1
4934
4953
GACGACCTTTGCTGGGAGGA
43
193


 


416429
1
6022
6041
ACGCCGTGGGCTTCCTCCTG
47
194


 


416430
1
6066
6085
GCAGCTCCTCCAGGAACGCG
52
195


 


416431
1
6079
6098
AGGGAGCCCGGCCGCAGCTC
25
196


 


416432
1
6108
6127
AGCACTGCTCCTCCTTGCAC
44
197


 


416513
1
6399
6418
CTGATGTGAAAACCGGCATG
37
198


 


416514
1
6406
6425
GTATTTTCTGATGTGAAAAC
8
199


 


416515
1
8547
8566
TAGGCATGACCATCCTCAAT
39
200


 


416516
1
8599
8618
GTGAGAATACAACAGATGAG
24
201


 


416517
1
8708
8727
GGGTGCAGTAGCAGATGCAA
23
202


 


416518
1
8855
8874
GGGTGACCACACATTTCCTG
55
203


 


416434
1
9076
9095
CTGTAAGAAATCCAGAACAG
0
204


 


416491
1
9120
9139
GAGAAGGGTTGCAGGCAGGA
2
205


 


416438
1
9194
9213
CAGGAGCCCCCATTCTGGCA
63
206


 


416439
1
9201
9220
GTCCTTGCAGGAGCCCCCAT
56
207


 


416440
1
9213
9232
GGACTGGAGCTGGTCCTTGC
44
208


 


416441
1
9220
9239
AGATATAGGACTGGAGCTGG
32
209


 


416442
1
9223
9242
AGCAGATATAGGACTGGAGC
57
210


 


416443
1
9231
9250
GAGGCAGAAGCAGATATAGG
17
211


 


416444
1
9255
9274
ACAGTTCCGGCCCTCGAAGG
24
212


 


416519
1
9290
9309
AAATATGGGACCCAAAGTGG
9
213


 


416520
1
9298
9317
CCCTCTGCAAATATGGGACC
14
214


 


416521
1
9362
9381
CACCCCACCAGGTTGTGCAC
36
215


 


416522
1
9515
9534
GGCTGAGAATTGCCCAGGGC
25
216


 


416523
1
9522
9541
TCTCGAGGGCTGAGAATTGC
18
217


 


416524
1
9665
9684
TAATTTAATATTCAGATGGT
0
218


 


416525
1
9725
9744
TATGAGTCCTTCTAGTGAAT
5
219


 


416526
1
9848
9867
TGTCCACATGACCCCACAGG
19
220


 


416527
1
9912
9931
GAGCTTCCCAAGTTGGCAGT
49
221


 


416528
1
9942
9961
TGATAAAACCTCTGGACACC
10
222


 


416529
1
9999
10018
GGGCTGAGACTGAGGTCAGC
18
223


 


416530
1
10166
10185
AGGGTAGCCTTTGCCTTGGC
35
224


 


416531
1
10317
10336
AGATGACCAGCAGGAAGCCT
19
225


 


416532
1
10323
10342
GGACCCAGATGACCAGCAGG
12
226


 


416533
1
10330
10349
GCATTCTGGACCCAGATGAC
41
227


 


416534
1
10377
10396
ATGCACACCAGGGCTGCTGG
32
228


 


416535
1
10382
10401
GCAGGATGCACACCAGGGCT
47
229


 


416536
1
10398
10417
CGGGAAGGCCTGCCCTGCAG
23
230


 


416537
1
10677
10696
TGACCACTCTTCCGAGCAGC
55
231


 


416538
1
10807
10826
CGTGGACTGATCCAAAGGAC
46
232


 


416539
1
10837
10856
GACAGAGCCTGAGCTTGGCA
53
233


 


416445
1
11013
11032
TACTGCTCACAGCCGCCGTT
57
234


 


416446
1
11024
11043
GGTCACTGCAGTACTGCTCA
64
235


 


416540
1
11143
11162
GGACTGGTGTCATCTGGGAC
38
236


 


416541
1
11259
11278
CCACCCTTGGTGCCCAGATC
53
237


 


416542
1
11297
11316
CAGGGTGCCCATCCTAGTCA
56
238


 


416543
1
11395
11414
TCCTGCGAGTGGGAGTTGGA
0
239


 


416544
1
11499
11518
ATCCCATTTTCCCAGGAGCC
58
240


 


416545
1
11505
11524
AGAAACATCCCATTTTCCCA
8
241


 


416546
1
11519
11538
CCAGGCTGGTTTGGAGAAAC
21
242


 


416547
1
11729
11748
ATGAAATTCTACCTAAAGAT
0
243


 


416548
1
11735
11754
AGGTGAATGAAATTCTACCT
29
244


 


416549
1
11838
11857
GACAATGGTCAGGGCTGGTT
68
245


 


416550
1
11852
11871
TGGCTGGCTGAGGAGACAAT
55
246


 


416551
1
12000
12019
CAGAAACACCCATCCTCTGA
19
247


 


416448
1
12088
12107
TTTTTCCACATGGATATTCA
34
248


 


416449
1
12094
12113
TAGGTATTTTTCCACATGGA
68
249


 


416450
1
12122
12141
GGTTTGCTGGCATTTCTTTT
49
250


 


416451
1
12175
12194
GCCATGGACACTCCCCTTTG
75
251


 


416552
1
12398
12417
TCTGCACAGGGTAGACCCTC
46
252


 


416553
1
12403
12422
GGTTCTCTGCACAGGGTAGA
56
253


 


416554
1
12467
12486
AAAGATCCCACCTCAAAGAG
7
254


 


416555
1
12478
12497
AAAGATCAGGCAAAGATCCC
6
255


 


416556
1
12508
12527
ATAGCTTTGATCCAATGCTC
53
256


 


416557
1
12639
12658
TCCCAGGCAAAGCTGCTCAG
56
257


 


416453
1
12867
12886
GATTTTGTCGAAACAGTGGG
45
258


 


416558
1
13159
13178
TGACAGCACGAAGCCCAGAG
19
259


 


416559
1
13638
13657
GCCATTTCTAGGTCTGCAGG
25
260


 


416455
1
13760
13779
CGCCGGCTCTGCTCATCCCC
72
261


 


416456
1
13770
13789
CTGCGCCACCCGCCGGCTCT
58
262


 


416457
1
13780
13799
GGATGATGACCTGCGCCACC
48
263


 


416458
1
13831
13850
GGCGGAGCAGCGCGATGTCG
29
264


 


416459
1
13859
13878
TGGTCAGTGAGGACCACGGG
23
265


 


416460
1
13866
13885
CACCACATGGTCAGTGAGGA
49
266


 


416461
1
13923
13942
GAAGCGCACGAAGGCCAGCG
43
267


 


416462
1
14020
14039
GGCAGTCCTGGGTCATCAGC
60
268


 


416463
1
14027
14046
TGCTGCAGGCAGTCCTGGGT
39
269


 


416464
1
14072
14091
AACATGTACTCCGTGATATT
53
270


 


416465
1
14122
14141
CACTGTCCCCCTTGCAGGAG
51
271


 


416466
1
14132
14151
TGTGGGCCTCCACTGTCCCC
57
272


 


416467
1
14189
14208
CCCTGGCCCCAGCTGACGAT
55
273


 


416468
1
14234
14253
TGGGAGACCCTGGTGTACAC
46
274


 


416469
1
14251
14270
GCAGCCACTCGATGTACTGG
55
275


 


416470
1
14257
14276
GCTTTTGCAGCCACTCGATG
39
276


 


416471
1
14260
14279
TGAGCTTTTGCAGCCACTCG
62
277


 


416472
1
14348
14367
CAGCCTTGGCTTTCTCTCCA
76
278


 


416473
1
14613
14632
CCCTGCCCCTCTGTCCAGCG
61
279


 


416474
1
14642
14661
TGTCTGCCTCCAGGACAACC
40
280


 


416475
1
14653
14672
CTCAGCTGGGCTGTCTGCCT
70
281


 


416476
1
14686
14705
GCAGGTGGGCTTGGCTGAAG
58
282


 


416477
1
14710
14729
CAGCCTGAGGCCAGCAGATC
74
283


 


416478
1
14735
14754
GTCTCCAGCAATGAAGGCAG
44
284


 


416479
1
15103
15122
GACAGCACACATGGAGTCAG
56
285


 


416480
1
15132
15151
AAGTGAGAGCTAAACAACCG
25
286


 


416481
1
15157
15176
AGATGATAATGGATAAGAAC
15
287


 


416482
1
15188
15207
CATGGTGATGCTTCTGAATT
64
288


 


416483
1
15433
15452
TGTGAGGGTTCATTTCAGTG
22
289


 


416484
1
15485
15504
TACAGCCTCAAACATCTATC
0
290


 


416485
1
15489
15508
GAGCTACAGCCTCAAACATC
7
291


 


416486
1
15540
15559
ATTGACAAGGGCTGTGGCAG
12
292


 


416487
1
15571
15590
GCAGGTGCTCCCAGGGTCTC
39
293


 


416488
1
15639
15658
CAGGTCCTCCCACTGCAGCC
41
294


 


416489
1
15650
15669
GTGCAGCTTGCCAGGTCCTC
38
295


 


416490
1
15661
15680
ACTCAGCAAGAGTGCAGCTT
33
296


 


416560
1
15973
15992
TAAAACTTTATTTACAAGAG
0
297


 


416561
1
15985
16004
GGTGTGTTCCCATAAAACTT
16
298


 


416562
1
16185
16204
AAAGCAGAGCCAGCTCTGAC
12
299


 


416563
1
16596
16615
GCCTGCATTTCCCATTGGCA
0
300


 


416564
1
16738
16757
GCCACTCACAGAAAGCTGGA
0
301


 


416565
1
16872
16891
CAGGATGCTCATGGCAGACA
0
302


 


416566
1
16911
16930
GTTTGTATGGAGAGACCAAT
0
303


 


416567
1
16977
16996
TGGTGGCACCAGATGTCTGA
2
304


 


416568
1
17112
17131
CATTGTGCCTGGCACACAGG
14
305


 


416569
1
17136
17155
GCCTGGTGTGCACACATTGT
12
306


 


416428
2
110
129
CTGGGTTACGAAGACTGCAG
11
307


 


416422
167
107
126
AGCGACCCCGCCTGCAGCCA
8
308


 


416433
167
341
360
AGAAATCCAGAACAGCTTCG
0
309


 


416435
167
353
372
CCCATCACTGTAAGAAATCC
3
310


 


416436
167
360
379
ACTGGTCCCCATCACTGTAA
27
311


 


416437
167
366
385
AGGCACACTGGTCCCCATCA
52
312


 


416447
167
616
635
CATGGATATTCAACTGTGGG
17
313


 


416452
167
726
745
CCAACAACAGGACCTGCCAT
19
314


 


416454
167
846
865
CGTGCTCGCCCAGCACCGCG
56
315









Certain gapmers from Table 4 are 100% homologous to the rhesus monkey genomic sequence (nucleotides 691000 to 706000 of GENBANK Accession No. NW—00104507.1; incorporated herein as SEQ ID NO: 162) or the rhesus monkey mRNA sequence (GENKBANK Accession No. 3360—061_B; incorporated herein as SEQ ID NO: 163). Shown in Table 5 are the chimeric antisense oligonucleotides from Table 4, which are homologous with rhesus monkey.








TABLE 5







Human/rhesus monkey cross-reactive chimeric antisense



oligonucleotides having 5-10-5 MOE wings and deoxy gap






















Rhesus
Rhesus





Human
Human
Human

Rhesus
Monkey
Monkey



Target
Target
Target

Target
Target
Target
SEQ


ISIS
SEQ
Start
Stop

SEQ
Start
Stop
ID


No.
ID No.
Site
Site
Sequence (5′ to 3′)
ID No.
Site
Site
No.



















407606
1
9204
9223
CTGGTCCTTGCAGGAGCCCC
162
6771
6790
17



 


407900
1
12141
12160
CCCCACAATTCGGCCTTGGG
162
9865
9884
88


 


407910
1
14104
14123
AGTCCTTGCTGCCATCCGAG
162
11826
11845
98


 


407641
1
14707
14726
CCTGAGGCCAGCAGATCACG
162
12427
12446
48


 


407643
1
15098
15117
CACACATGGAGTCAGCATCG
162
12815
12834
50


 


407935
1
15191
15210
ATGCATGGTGATGCTTCTGA
162
12908
12927
123


 


416492
1
616
635
GGATCATTCTGGCCCTGAGC
162
24
43
168


 


416493
1
738
757
TCTTGGGTGTGGATGTAAAT
162
149
168
169


 


416494
1
803
822
CAGATTTAAACTGCAGATGA
162
214
233
170


 


416495
1
838
857
TCTAGAATTCCAAACCCCTA
162
259
278
171


 


416496
1
855
874
CAACACTTCAAATACGATCT
162
276
295
172


 


416497
1
883
902
CGTGCAGGTGTTAAGGTGTG
162
306
325
173


 


416498
1
994
1013
GGCCAGTGGCCATGCATCCC
162
417
436
174


 


416499
1
1011
1030
GAGAGCTGCACCTGGCCGGC
162
434
453
175


 


416500
1
1026
1045
CTGAACACCCCAGCTGAGAG
162
449
468
176


 


416424
1
1151
1170
GAGACCATGATGAAATCTCT
162
574
593
177


 


416425
1
1182
1201
AAGCAGAAGGCAGAGGAGCC
162
605
624
178


 


416426
1
1187
1206
AGCCCAAGCAGAAGGCAGAG
162
610
629
179


 


416427
1
1193
1212
CCCTGAAGCCCAAGCAGAAG
162
616
635
180


 


416501
1
1251
1270
CTGCCCTTCCACCAAGTTTA
162
674
693
181


 


416502
1
1437
1456
GTTCTTTGAAAAATAATCCC
162
858
877
182


 


416423
1
2179
2198
GTGTTTCTCCTCCTGAGGCC
162
1638
1657
183


 


416503
1
2311
2330
TAGCCACCCCGCGGGCTGGC
162
1771
1790
184


 


416504
1
2484
2503
TCAGAAAAGCTCTCAAGAAC
162
1944
1963
185


 


416505
1
2495
2514
GCAGATTTGCATCAGAAAAG
162
1955
1974
186


 


416506
1
4766
4785
CTTTAAAATCAGTTTCACAC
162
2556
2575
187


 


416507
1
4847
4866
GGTTACTGAGCGCGGAAGAA
162
2637
2656
188


 


416508
1
4873
4892
CGAGTTCTGCAGGAGCGGCC
162
2663
2682
189


 


416509
1
4880
4899
AGGAGCCCGAGTTCTGCAGG
162
2670
2689
190


 


416510
1
4916
4935
GACGAGGCCTCAGGTGGACG
162
2706
2725
191


 


416511
1
4926
4945
TTGCTGGGAGGACGAGGCCT
162
2716
2735
192


 


416512
1
4934
4953
GACGACCTTTGCTGGGAGGA
162
2724
2743
193


 


416513
1
6399
6418
CTGATGTGAAAACCGGCATG
162
4144
4163
198


 


416514
1
6406
6425
GTATTTTCTGATGTGAAAAC
162
4151
4170
199


 


416515
1
8547
8566
TAGGCATGACCATCCTCAAT
162
6104
6123
200


 


416516
1
8599
8618
GTGAGAATACAACAGATGAG
162
6156
6175
201


 


416517
1
8708
8727
GGGTGCAGTAGCAGATGCAA
162
6265
6284
202


 


416518
1
8855
8874
GGGTGACCACACATTTCCTG
162
6422
6441
203


 


416434
1
9076
9095
CTGTAAGAAATCCAGAACAG
162
6640
6659
204


 


416491
1
9120
9139
GAGAAGGGTTGCAGGCAGGA
162
6684
6703
205


 


416438
1
9194
9213
CAGGAGCCCCCATTCTGGCA
162
6761
6780
206


 


416439
1
9201
9220
GTCCTTGCAGGAGCCCCCAT
162
6768
6787
207


 


416440
1
9213
9232
GGACTGGAGCTGGTCCTTGC
162
6780
6799
208


 


416441
1
9220
9239
AGATATAGGACTGGAGCTGG
162
6787
6806
209


 


416442
1
9223
9242
AGCAGATATAGGACTGGAGC
162
6790
6809
210


 


416443
1
9231
9250
GAGGCAGAAGCAGATATAGG
162
6798
6817
211


 


416444
1
9255
9274
ACAGTTCCGGCCCTCGAAGG
162
6822
6841
212


 


416519
1
9290
9309
AAATATGGGACCCAAAGTGG
162
6857
6876
213


 


416520
1
9298
9317
CCCTCTGCAAATATGGGACC
162
6865
6884
214


 


416521
1
9362
9381
CACCCCACCAGGTTGTGCAC
162
6906
6925
215


 


416522
1
9515
9534
GGCTGAGAATTGCCCAGGGC
162
7059
7078
216


 


416523
1
9522
9541
TCTCGAGGGCTGAGAATTGC
162
7066
7085
217


 


416524
1
9665
9684
TAATTTAATATTCAGATGGT
162
7239
7258
218


 


416525
1
9725
9744
TATGAGTCCTTCTAGTGAAT
162
7299
7318
219


 


416526
1
9848
9867
TGTCCACATGACCCCACAGG
162
7422
7441
220


 


416527
1
9912
9931
GAGCTTCCCAAGTTGGCAGT
162
7479
7498
221


 


416528
1
9942
9961
TGATAAAACCTCTGGACACC
162
7509
7528
222


 


416529
1
9999
10018
GGGCTGAGACTGAGGTCAGC
162
7566
7585
223


 


416530
1
10166
10185
AGGGTAGCCTTTGCCTTGGC
162
7852
7871
224


 


416531
1
10317
10336
AGATGACCAGCAGGAAGCCT
162
8006
8025
225


 


416532
1
10323
10342
GGACCCAGATGACCAGCAGG
162
8012
8031
226


 


416533
1
10330
10349
GCATTCTGGACCCAGATGAC
162
8019
8038
227


 


416534
1
10377
10396
ATGCACACCAGGGCTGCTGG
162
8066
8085
228


 


416535
1
10382
10401
GCAGGATGCACACCAGGGCT
162
8071
8090
229


 


416536
1
10398
10417
CGGGAAGGCCTGCCCTGCAG
162
8087
8106
230


 


416537
1
10677
10696
TGACCACTCTTCCGAGCAGC
162
8376
8395
231


 


416538
1
10807
10826
CGTGGACTGATCCAAAGGAC
162
8505
8524
232


 


416539
1
10837
10856
GACAGAGCCTGAGCTTGGCA
162
8535
8554
233


 


416445
1
11013
11032
TACTGCTCACAGCCGCCGTT
162
8710
8729
234


 


416446
1
11024
11043
GGTCACTGCAGTACTGCTCA
162
8721
8740
235


 


416540
1
11143
11162
GGACTGGTGTCATCTGGGAC
162
8840
8859
236


 


416541
1
11259
11278
CCACCCTTGGTGCCCAGATC
162
8953
8972
237


 


416542
1
11297
11316
CAGGGTGCCCATCCTAGTCA
162
8991
9010
238


 


416543
1
11395
11414
TCCTGCGAGTGGGAGTTGGA
162
9089
9108
239


 


416544
1
11499
11518
ATCCCATTTTCCCAGGAGCC
162
9193
9212
240


 


416545
1
11505
11524
AGAAACATCCCATTTTCCCA
162
9199
9218
241


 


416546
1
11519
11538
CCAGGCTGGTTTGGAGAAAC
162
9213
9232
242


 


416547
1
11729
11748
ATGAAATTCTACCTAAAGAT
162
9434
9453
243


 


416548
1
11735
11754
AGGTGAATGAAATTCTACCT
162
9440
9459
244


 


416549
1
11838
11857
GACAATGGTCAGGGCTGGTT
162
9543
9562
245


 


416550
1
11852
11871
TGGCTGGCTGAGGAGACAAT
162
9557
9576
246


 


416551
1
12000
12019
CAGAAACACCCATCCTCTGA
162
9724
9743
247


 


416448
1
12088
12107
TTTTTCCACATGGATATTCA
162
9812
9831
248


 


416449
1
12094
12113
TAGGTATTTTTCCACATGGA
162
9818
9837
249


 


416450
1
12122
12141
GGTTTGCTGGCATTTCTTTT
162
9846
9865
250


 


416451
1
12175
12194
GCCATGGACACTCCCCTTTG
162
9899
9918
251


 


416552
1
12398
12417
TCTGCACAGGGTAGACCCTC
162
10127
10146
252


 


416553
1
12403
12422
GGTTCTCTGCACAGGGTAGA
162
10132
10151
253


 


416554
1
12467
12486
AAAGATCCCACCTCAAAGAG
162
10196
10215
254


 


416555
1
12478
12497
AAAGATCAGGCAAAGATCCC
162
10207
10226
255


 


416556
1
12508
12527
ATAGCTTTGATCCAATGCTC
162
10237
10256
256


 


416557
1
12639
12658
TCCCAGGCAAAGCTGCTCAG
162
10368
10387
257


 


416453
1
12867
12886
GATTTTGTCGAAACAGTGGG
162
10596
10615
258


 


416558
1
13159
13178
TGACAGCACGAAGCCCAGAG
162
10880
10899
259


 


416559
1
13638
13657
GCCATTTCTAGGTCTGCAGG
162
11360
11379
260


 


416455
1
13760
13779
CGCCGGCTCTGCTCATCCCC
162
11482
11501
261


 


416456
1
13770
13789
CTGCGCCACCCGCCGGCTCT
162
11492
11511
262


 


416457
1
13780
13799
GGATGATGACCTGCGCCACC
162
11502
11521
263


 


416458
1
13831
13850
GGCGGAGCAGCGCGATGTCG
162
11553
11572
264


 


416459
1
13859
13878
TGGTCAGTGAGGACCACGGG
162
11581
11600
265


 


416460
1
13866
13885
CACCACATGGTCAGTGAGGA
162
11588
11607
266


 


416461
1
13923
13942
GAAGCGCACGAAGGCCAGCG
162
11645
11664
267


 


416462
1
14020
14039
GGCAGTCCTGGGTCATCAGC
162
11742
11761
268


 


416463
1
14027
14046
TGCTGCAGGCAGTCCTGGGT
162
11749
11768
269


 


416464
1
14072
14091
AACATGTACTCCGTGATATT
162
11794
11813
270


 


416465
1
14122
14141
CACTGTCCCCCTTGCAGGAG
162
11844
11863
271


 


416466
1
14132
14151
TGTGGGCCTCCACTGTCCCC
162
11854
11873
272


 


416467
1
14189
14208
CCCTGGCCCCAGCTGACGAT
162
11911
11930
273


 


416468
1
14234
14253
TGGGAGACCCTGGTGTACAC
162
11956
11975
274


 


416469
1
14251
14270
GCAGCCACTCGATGTACTGG
162
11973
11992
275


 


416470
1
14257
14276
GCTTTTGCAGCCACTCGATG
162
11979
11998
276


 


416471
1
14260
14279
TGAGCTTTTGCAGCCACTCG
162
11982
12001
277


 


416472
1
14348
14367
CAGCCTTGGCTTTCTCTCCA
162
12070
12089
278


 


416473
1
14613
14632
CCCTGCCCCTCTGTCCAGCG
162
12333
125352
279


 


416474
1
14642
14661
TGTCTGCCTCCAGGACAACC
162
12362
12381
280


 


416475
1
14653
14672
CTCAGCTGGGCTGTCTGCCT
162
12373
12392
281


 


416476
1
14686
14705
GCAGGTGGGCTTGGCTGAAG
162
12406
12425
282


 


416477
1
14710
14729
CAGCCTGAGGCCAGCAGATC
162
12430
12449
283


 


416478
1
14735
14754
GTCTCCAGCAATGAAGGCAG
162
12455
12474
284


 


416479
1
15103
15122
GACAGCACACATGGAGTCAG
162
12820
12839
285


 


416480
1
15132
15151
AAGTGAGAGCTAAACAACCG
162
12849
12868
286


 


416481
1
15157
15176
AGATGATAATGGATAAGAAC
162
12874
12893
287


 


416482
1
15188
15207
CATGGTGATGCTTCTGAATT
162
12905
12924
288


 


416483
1
15433
15452
TGTGAGGGTTCATTTCAGTG
162
13130
13149
289


 


416484
1
15485
15504
TACAGCCTCAAACATCTATC
162
13184
13203
290


 


416485
1
15489
15508
GAGCTACAGCCTCAAACATC
162
13188
13207
291


 


416486
1
15540
15559
ATTGACAAGGGCTGTGGCAG
162
13239
13258
292


 


416487
1
15571
15590
GCAGGTGCTCCCAGGGTCTC
162
13270
13289
293


 


416488
1
15639
15658
CAGGTCCTCCCACTGCAGCC
162
13334
13353
294


 


416489
1
15650
15669
GTGCAGCTTGCCAGGTCCTC
162
13345
13364
295


 


416490
1
15661
15680
ACTCAGCAAGAGTGCAGCTT
162
13356
13375
296


 


416560
1
15973
15992
TAAAACTTTATTTACAAGAG
162
13638
13657
297


 


416561
1
15985
16004
GGTGTGTTCCCATAAAACTT
162
13650
13669
298


 


416562
1
16185
16204
AAAGCAGAGCCAGCTCTGAC
162
13849
13868
299


 


416563
1
16596
16615
GCCTGCATTTCCCATTGGCA
162
13931
13950
300


 


416564
1
16738
16757
GCCACTCACAGAAAGCTGGA
162
14071
14090
301


 


416565
1
16872
16891
CAGGATGCTCATGGCAGACA
162
14205
14224
302


 


416566
1
16911
16930
GTTTGTATGGAGAGACCAAT
162
14244
14263
303


 


416567
1
16977
16996
TGGTGGCACCAGATGTCTGA
162
14310
14329
304


 


416568
1
17112
17131
CATTGTGCCTGGCACACAGG
162
14445
14464
305


 


416569
1
17136
17155
GCCTGGTGTGCACACATTGT
162
14469
14488
306


 


416422
167
107
126
AGCGACCCCGCCTGCAGCCA
163
107
126
308


 


416433
167
341
360
AGAAATCCAGAACAGCTTCG
163
341
360
309


 


416435
167
353
372
CCCATCACTGTAAGAAATCC
163
353
372
310


 


416436
167
360
379
ACTGGTCCCCATCACTGTAA
163
360
379
311


 


416437
167
366
385
AGGCACACTGGTCCCCATCA
163
366
385
312


 


416447
167
616
635
CATGGATATTCAACTGTGGG
163
616
635
313


 


416452
167
726
745
CCAACAACAGGACCTGCCAT
163
726
745
314


 


416454
167
846
865
CGTGCTCGCCCAGCACCGCG
163
846
865
315









Example 4
Antisense Inhibition of Human Factor 7 in HepB3 Cells
Antisense oligonucleotides targeted to a Factor 7 nucleic acid were designed and tested for their effects on Factor 7 mRNA in vitro. Cultured HepB3 cells at a density of 4,000 cells per well were transfected using lipofectin reagent with 50 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Factor 7 mRNA levels were measured by real-time RT-PCR, as described herein. Human Factor 7 primer probe set RTS 2927 was used to measure mRNA levels. Factor 7 mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented as percent inhibition of Factor 7, relative to untreated control.
The chimeric antisense oligonucleotides in Table 6 were designed as 5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE gapmers. The 5-10-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of ten 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five nucleotides each. The 3-14-3 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of fourteen 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising three nucleotides each. The 2-13-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of thirteen 2′-deoxynucleotides. The central gap is flanked on the 5′ end with a wing comprising two nucleotides and on the 3′ end with a wing comprising five nucleotides. For each of the motifs (5-10-5, 3-14-3, and 2-13-5), each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. Each gapmer listed in Table 6 is targeted to SEQ ID NO: 1 (nucleotides 1255000 to 1273000 of GENBANK Accession No. NT—027140.6).








TABLE 6







Inhibition of human Factor 7 mRNA levels by chimeric



antisense oligonucleotides targeted to SEQ ID NO: 1















Target
Target



SEQ



ISIS
Start
Stop


%
ID


No.
Site
Site
Sequence (5′ to 3′)
Motif
Inhibition
No.

















422117
4834
4853
GGAAGAAGGGACGGAAGGTG
5-10-5
1
316



 


422186
4834
4853
GGAAGAAGGGACGGAAGGTG
3-14-3
14
316


 


422263
4834
4853
GGAAGAAGGGACGGAAGGTG
2-13-5
19
316


 


422118
4835
4854
CGGAAGAAGGGACGGAAGGT
5-10-5
15
317


 


422187
4835
4854
CGGAAGAAGGGACGGAAGGT
3-14-3
28
317


 


422264
4835
4854
CGGAAGAAGGGACGGAAGGT
2-13-5
24
317


 


422119
4836
4855
GCGGAAGAAGGGACGGAAGG
5-10-5
39
318


 


422188
4836
4855
GCGGAAGAAGGGACGGAAGG
3-14-3
37
318


 


422265
4836
4855
GCGGAAGAAGGGACGGAAGG
2-13-5
34
318


 


422120
4837
4856
CGCGGAAGAAGGGACGGAAG
5-10-5
40
319


 


422189
4837
4856
CGCGGAAGAAGGGACGGAAG
3-14-3
0
319


 


422266
4837
4856
CGCGGAAGAAGGGACGGAAG
2-13-5
13
319


 


422121
4838
4857
GCGCGGAAGAAGGGACGGAA
5-10-5
43
320


 


422190
4838
4857
GCGCGGAAGAAGGGACGGAA
3-14-3
12
320


 


422267
4838
4857
GCGCGGAAGAAGGGACGGAA
2-13-5
38
320


 


422122
4839
4858
AGCGCGGAAGAAGGGACGGA
5-10-5
24
321


 


422191
4839
4858
AGCGCGGAAGAAGGGACGGA
3-14-3
27
321


 


422268
4839
4858
AGCGCGGAAGAAGGGACGGA
2-13-5
63
321


 


422123
4840
4859
GAGCGCGGAAGAAGGGACGG
5-10-5
16
322


 


422192
4840
4859
GAGCGCGGAAGAAGGGACGG
3-14-3
27
322


 


422269
4840
4859
GAGCGCGGAAGAAGGGACGG
2-13-5
27
322


 


422124
4841
4860
TGAGCGCGGAAGAAGGGACG
5-10-5
0
323


 


422193
4841
4860
TGAGCGCGGAAGAAGGGACG
3-14-3
6
323


 


422270
4841
4860
TGAGCGCGGAAGAAGGGACG
2-13-5
15
323


 


422125
4842
4861
CTGAGCGCGGAAGAAGGGAC
5-10-5
8
324


 


422194
4842
4861
CTGAGCGCGGAAGAAGGGAC
3-14-3
11
324


 


422271
4842
4861
CTGAGCGCGGAAGAAGGGAC
2-13-5
32
324


 


422126
4843
4862
ACTGAGCGCGGAAGAAGGGA
5-10-5
22
325


 


422195
4843
4862
ACTGAGCGCGGAAGAAGGGA
3-14-3
37
325


 


422272
4843
4862
ACTGAGCGCGGAAGAAGGGA
2-13-5
12
325


 


422127
4844
4863
TACTGAGCGCGGAAGAAGGG
5-10-5
17
326


 


422196
4844
4863
TACTGAGCGCGGAAGAAGGG
3-14-3
2
326


 


422273
4844
4863
TACTGAGCGCGGAAGAAGGG
2-13-5
0
326


 


422128
4845
4864
TTACTGAGCGCGGAAGAAGG
5-10-5
27
327


 


422197
4845
4864
TTACTGAGCGCGGAAGAAGG
3-14-3
26
327


 


422274
4845
4864
TTACTGAGCGCGGAAGAAGG
2-13-5
24
327


 


422129
4846
4865
GTTACTGAGCGCGGAAGAAG
5-10-5
45
328


 


422198
4846
4865
GTTACTGAGCGCGGAAGAAG
3-14-3
50
328


 


422275
4846
4865
GTTACTGAGCGCGGAAGAAG
2-13-5
42
328


 


422199
4847
4866
GGTTACTGAGCGCGGAAGAA
3-14-3
65
188


 


422276
4847
4866
GGTTACTGAGCGCGGAAGAA
2-13-5
66
188









Example 5
Dose-Dependent Antisense Inhibition of Human Coagulation Factor 7 in HepB3 Cells
Gapmers (from Tables 1 through 6, above) exhibiting in vitro inhibition of Factor 7 were selected and tested at various doses in HepB3 cells. Cells were plated at a density of 4,000 cells per well and transfected using lipofectin reagent with 6.25 nM, 12.5 nM, 25.0 nM, 50.0 nM, and 100.0 nM concentrations of antisense oligonucleotide, as indicated in Table 7. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Factor 7 mRNA levels were measured by real-time RT-PCR, as described herein. Human Factor 7 primer probe set RTS 2927 was used to measure mRNA levels. Factor 7 mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented as percent inhibition of Factor 7, relative to untreated control cells. As illustrated in Table 7, Factor 7 mRNA levels were reduced in a dose-dependent manner.







TABLE 7







Dose-dependent antisense inhibition


of human Factor 7 in HepB3 cells













ISIS
6.25
12.5
25.0
50.0
100.0
SEQ ID


No.
nM
nM
nM
nM
nM
No.
















407643
36
49
70
83
93
50


407900
18
36
61
82
91
88


407935
38
53
70
82
86
123


407939
37
57
77
84
87
127


416438
11
33
56
75
82
206


416446
25
24
50
69
64
235


416449
19
33
45
65
85
249


416455
28
44
64
78
87
261


416472
16
44
64
80
88
278


416477
21
46
64
78
87
283


416507
30
53
70
77
72
188


416508
42
51
71
79
89
189


416549
38
44
63
73
78
245









Example 6
Antisense Inhibition of Human Factor 7 in HepB3 Cells by Oligonucleotides Designed by Microwalk
Additional gapmers were designed based on the gapmers presented in Table 7. These gapmers were designed by creating gapmers shifted slightly upstream and downstream (i.e. “microwalk”) of the original gapmers from Table 7. Gapmers were also created with various motifs, e.g. 5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE. These gapmers were tested in vitro. Cultured HepB3 cells at a density of 4,000 cells per well were transfected using lipofectin reagent with 50 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Factor 7 mRNA levels were measured by real-time RT-PCR. Factor 7 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Factor 7, relative to untreated control cells.
The in vitro inhibition data for the gapmers designed by microwalk was then compared with the in vitro inhibition data for the gapmers from Table 7, as indicated in Tables 8, 9, 10, 11, 12, and 13. The oligonucleotides are displayed according to the region on the human gene sequence to which they map.
The chimeric antisense oligonucleotides in Table 8 were designed as 5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE gapmers. The first listed gapmer in Table 8 is the original gapmer (see Table 7) from which the remaining gapmers were designed via microwalk and is designated by an asterisk (*). The 5-10-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of ten 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five nucleotides each. The 3-14-3 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of fourteen 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising three nucleotides each. The 2-13-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of thirteen 2′-deoxynucleotides. The central gap is flanked on the 5′ end with a wing comprising two nucleotides and on the 3′ end with a wing comprising five nucleotides. For each of the motifs (5-10-5, 3-14-3, and 2-13-5), each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. Each gapmer listed in Table 8 is targeted to SEQ ID NO: 1 (nucleotides 1255000 to 1273000 of GENBANK Accession No. NT—027140.6).
As shown in Table 8, all of the 5-10-5 MOE gapmers, 3-14-3 MOE gapmers, and 2-13-5 MOE gapmers targeted to the target region beginning at target start site 4868 and ending at the target stop site 4899 (i.e. nucleobases 4868-4899) of SEQ ID NO: 1 inhibited Factor 7 mRNA by at least 48%.
Certain gapmers within the target region (i.e. nucleobases 4868-4899) inhibited Factor 7 mRNA expression by at least 40%, for example, ISIS numbers 416508, 422138, 422213, 422290, 422139, 422214, 422291, 422140, 422215, 422292, 422141, 422216, 422293, 422142, 422217, 422294, 422218, 422295, 422143, 422219, 422296, 422144, 422220, 422297, 422145, 422221, 422298, 422146, 422222, 422299, 422147, 422223, 422300, 422148, 422224, 422301, 416509, 422225, and 422302.
Certain gapmers within the target region (i.e. nucleobases 4868-4899) inhibited Factor 7 mRNA expression by at least 50%, for example, ISIS numbers 416508, 422138, 422213, 422290, 422139, 422214, 422291, 422140, 422215, 422292, 422141, 422216, 422293, 422142, 422217, 422294, 422218, 422295, 422143, 422219, 422296, 422144, 422220, 422297, 422145, 422221, 422298, 422146, 422222, 422299, 422147, 422300, 422148, 422224, 422301, 416509, 422225, and 422302.
Certain gapmers within the target region (i.e. nucleobases 4868-4899) inhibited Factor 7 mRNA expression by at least 60%, for example, ISIS numbers 416508, 422138, 422213, 422139, 422140, 422215, 422292, 422141, 422216, 422293, 422142, 422217, 422294, 422218, 422295, 422143, 422219, 422296, 422297, 422298, 422299, 422147, 422300, 422224, 422301, 416509, and 422302.
Certain gapmers within the target region (i.e. nucleobases 4868-4899) inhibited Factor 7 mRNA expression by at least 70%, for example, ISIS numbers 422138, 422140, 422215, 422292, 422142, 422217, 422294, 422218, 422295, 422143, and 422296.








TABLE 8







Inhibition of human Factor 7 mRNA levels by chimeric



antisense oligonucleotides targeted to


nucleobases 4868 to 4899 of SEQ ID NO: 1















Target
Target








Start
Stop


%
SEQ ID


ISIS No.
Site
Site
Sequence (5′ to 3′)
Motif
Inhibition
NO

















*416508
4873
4892
CGAGTTCTGCAGGAGCGGCC
5-10-5
67
189



 


422138
4868
4887
TCTGCAGGAGCGGCCTAAAT
5-10-5
70
329


 


422213
4868
4887
TCTGCAGGAGCGGCCTAAAT
3-14-3
67
329


 


422290
4868
4887
TCTGCAGGAGCGGCCTAAAT
2-13-5
50
329


 


422139
4869
4888
TTCTGCAGGAGCGGCCTAAA
5-10-5
66
330


 


422214
4869
4888
TTCTGCAGGAGCGGCCTAAA
3-14-3
60
330


 


422291
4869
4888
TTCTGCAGGAGCGGCCTAAA
2-13-5
53
330


 


422140
4870
4889
GTTCTGCAGGAGCGGCCTAA
5-10-5
74
331


 


422215
4870
4889
GTTCTGCAGGAGCGGCCTAA
3-14-3
73
331


 


422292
4870
4889
GTTCTGCAGGAGCGGCCTAA
2-13-5
75
331


 


422141
4871
4890
AGTTCTGCAGGAGCGGCCTA
5-10-5
64
332


 


422216
4871
4890
AGTTCTGCAGGAGCGGCCTA
3-14-3
68
332


 


422293
4871
4890
AGTTCTGCAGGAGCGGCCTA
2-13-5
69
332


 


422142
4872
4891
GAGTTCTGCAGGAGCGGCCT
5-10-5
73
333


 


422217
4872
4891
GAGTTCTGCAGGAGCGGCCT
3-14-3
75
333


 


422294
4872
4891
GAGTTCTGCAGGAGCGGCCT
2-13-5
78
333


 


422218
4873
4892
CGAGTTCTGCAGGAGCGGCC
3-1-4-3
70
189


 


422295
4873
4892
CGAGTTCTGCAGGAGCGGCC
2-13-5
74
189


 


422143
4874
4893
CCGAGTTCTGCAGGAGCGGC
5-10-5
70
334


 


422219
4874
4893
CCGAGTTCTGCAGGAGCGGC
3-14-3
65
334


 


422296
4874
4893
CCGAGTTCTGCAGGAGCGGC
2-13-5
74
334


 


422144
4875
4894
CCCGAGTTCTGCAGGAGCGG
5-10-5
58
335


 


422220
4875
4894
CCCGAGTTCTGCAGGAGCGG
3-1-4-3
59
335


 


422297
4875
4894
CCCGAGTTCTGCAGGAGCGG
2-13-5
63
335


 


422145
4876
4895
GCCCGAGTTCTGCAGGAGCG
5-10-5
57
336


 


422221
4876
4895
GCCCGAGTTCTGCAGGAGCG
3-1-4-3
59
336


 


422298
4876
4895
GCCCGAGTTCTGCAGGAGCG
2-1-3-5
62
336


 


422146
4877
4896
AGCCCGAGTTCTGCAGGAGC
5-10-5
58
337


 


422222
4877
4896
AGCCCGAGTTCTGCAGGAGC
3-14-3
55
337


 


422299
4877
4896
AGCCCGAGTTCTGCAGGAGC
2-13-5
64
337


 


422147
4878
4897
GAGCCCGAGTTCTGCAGGAG
5-10-5
64
338


 


422223
4878
4897
GAGCCCGAGTTCTGCAGGAG
3-1-4-3
48
338


 


422300
4878
4897
GAGCCCGAGTTCTGCAGGAG
2-13-5
65
338


 


422148
4879
4898
GGAGCCCGAGTTCTGCAGGA
5-10-5
57
339


 


422224
4879
4898
GGAGCCCGAGTTCTGCAGGA
3-14-3
62
339


 


422301
4879
4898
GGAGCCCGAGTTCTGCAGGA
2-13-5
67
339


 


416509
4880
4899
AGGAGCCCGAGTTCTGCAGG
5-10-5
60
190


 


422225
4880
4899
AGGAGCCCGAGTTCTGCAGG
3-14-3
56
190


 


422302
4880
4899
AGGAGCCCGAGTTCTGCAGG
2-13-5
67
190









The chimeric antisense oligonucleotides in Table 9 were designed as 5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE gapmers. The first listed gapmer in Table 9 is the original gapmer (see Table 7) from which the remaining gapmers were designed via microwalk and is designated by an asterisk (*). The 5-10-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleotides each. The 3-14-3 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 14 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 3 nucleotides each. The 2-13-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 13 2′-deoxynucleotides. The central gap is flanked on the 5′ end with a wing comprising 2 nucleotides and on the 3′ end with a wing comprising 5 nucleotides. For each of the motifs (5-10-5, 3-14-3, and 2-13-5), each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. Each gapmer listed in Table 9 is targeted to SEQ ID NO: 1 (nucleotides 1255000 to 1273000 of GENBANK Accession No. NT—027140.6).
As shown in Table 9, most of the 5-10-5 MOE gapmers, 3-14-3 MOE gapmers, and 2-13-5 MOE gapmers targeted to the target region beginning at target start site 11830 and ending at the target stop site 11869 (i.e. nucleobases 11830-11869) of SEQ ID NO: 1 inhibited Factor 7 mRNA by at least 40%.
Certain gapmers within the target region (i.e. nucleobases 11830-11869) inhibited Factor 7 mRNA expression by at least 20%, for example, ISIS numbers 416549, 422154, 422231, 422089, 422155, 422232, 422090, 422156, 422233, 422091, 422157, 422234, 422092, 422158, 422235, 422093, 422159, 422236, 422094, 422160, 422237, 422095, 422161, 422238, 422162, 422239, 422096, 422163, 422240, 422097, 422164, 422241, 422098, 422165, 422242, 422099, 422166, 422243, 422100, 422167, 422244, 422101, 422168, 422245, 422102, 422169, 422246, 422103, 422170, 422247, 422104, 422171, 422248, 422105, 422172, 422249, 422106, 422173, 422250, 422107, 422174, and 422251.
Certain gapmers within the target region (i.e. nucleobases 11830-11869) inhibited Factor 7 mRNA expression by at least 30%, for example, ISIS numbers 416549, 422154, 422155, 422232, 422090, 422156, 422233, 422091, 422157, 422234, 422092, 422158, 422235, 422093, 422159, 422236, 422094, 422160, 422237, 422095, 422161, 422238, 422162, 422239, 422096, 422163, 422240, 422097, 422164, 422241, 422098, 422165, 422242, 422099, 422166, 422243, 422100, 422167, 422244, 422101, 422168, 422102, 422169, 422246, 422103, 422247, 422104, 422171, 422248, 422105, 422172, 422249, 422106, 422173, 422250, 422107, 422174, and 422251.
Certain gapmers within the target region (i.e. nucleobases 11830-11869) inhibited Factor 7 mRNA expression by at least 40%, for example, ISIS numbers 416549, 422232, 422090, 422233, 422091, 422157, 422234, 422158, 422235, 422093, 422159, 422236, 422094, 422160, 422237, 422095, 422161, 422238, 422162, 422239, 422096, 422163, 422240, 422097, 422164, 422241, 422098, 422165, 422242, 422099, 422166, 422243, 422100, 422167, 422244, 422101, 422102, 422169, 422246, 422104, 422171, 422248, 422105, 422249, 422173, 422250, and 422174.
Certain gapmers within the target region (i.e. nucleobases 11830-11869) inhibited Factor 7 mRNA expression by at least 50%, for example, ISIS numbers 416549, 422234, 422235, 422237, 422095, 422161, 422238, 422162, 422239, 422096, 422163, 422240, 422097, 422164, 422241, 422098, 422165, 422242, 422166, 422243, 422100, 422167, 422244, 422102, 422169, 422104, 422171, 422248, 422105, 422249, 422173, 422250, and 422174.
Certain gapmers within the target region (i.e. nucleobases 11830-11869) inhibited Factor 7 mRNA expression by at least 60%, for example, ISIS numbers 416549, 422234, 422095, 422238, 422239, 422096, 422240, 422164, 422241, 422242, 422166, 422243, 422102, 422171, 422248, and 422105.
Certain gapmers within the target region (i.e. nucleobases 11830-11869) inhibited Factor 7 mRNA expression by at least 70%, for example, ISIS number 422096.








TABLE 9







Inhibition of human Factor 7 mRNA levels by chimeric



antisense oligonucleotides targeted to nucleobases


11830 to 11869 of SEQ ID NO: 1















Target
Target








Start
Stop


%
SEQ ID


Oligo ID
Site
Site
Sequence (5′ to 3′)
Motif
Inhibition
NO

















*416549
11838
11857
GACAATGGTCAGGGCTGGTT
5-10-5
69
245



 


422088
11830
11849
TCAGGGCTGGTTTTGGAGGA
5-10-5
8
340


 


422154
11830
11849
TCAGGGCTGGTTTTGGAGGA
3-14-3
31
340


 


422231
11830
11849
TCAGGGCTGGTTTTGGAGGA
2-13-5
22
340


 


422089
11831
11850
GTCAGGGCTGGTTTTGGAGG
5-10-5
22
341


 


422155
11831
11850
GTCAGGGCTGGTTTTGGAGG
3-1-4-3
34
341


 


422232
11831
11850
GTCAGGGCTGGTTTTGGAGG
2-13-5
41
341


 


422090
11832
11851
GGTCAGGGCTGGTTTTGGAG
5-10-5
42
342


 


422156
11832
11851
GGTCAGGGCTGGTTTTGGAG
3-1-4-3
38
342


 


422233
11832
11851
GGTCAGGGCTGGTTTTGGAG
2-13-5
46
342


 


422091
11833
11852
TGGTCAGGGCTGGTTTTGGA
5-10-5
42
343


 


422157
11833
11852
TGGTCAGGGCTGGTTTTGGA
3-14-3
49
343


 


422234
11833
11852
TGGTCAGGGCTGGTTTTGGA
2-13-5
62
343


 


422092
11834
11853
ATGGTCAGGGCTGGTTTTGG
5-10-5
36
344


 


422158
11834
11853
ATGGTCAGGGCTGGTTTTGG
3-14-3
49
344


 


422235
11834
11853
ATGGTCAGGGCTGGTTTTGG
2-13-5
50
344


 


422093
11835
11854
AATGGTCAGGGCTGGTTTTG
5-10-5
42
345


 


422159
11835
11854
AATGGTCAGGGCTGGTTTTG
3-14-3
45
345


 


422236
11835
11854
AATGGTCAGGGCTGGTTTTG
2-1-3-5
44
345


 


422094
11836
11855
CAATGGTCAGGGCTGGTTTT
5-10-5
48
346


 


422160
11836
11855
CAATGGTCAGGGCTGGTTTT
3-14-3
42
346


 


422237
11836
11855
CAATGGTCAGGGCTGGTTTT
2-13-5
50
346


 


422095
11837
11856
ACAATGGTCAGGGCTGGTTT
5-10-5
60
347


 


422161
11837
11856
ACAATGGTCAGGGCTGGTTT
3-14-3
53
347


 


422238
11837
11856
ACAATGGTCAGGGCTGGTTT
2-13-5
64
347


 


422162
11838
11857
GACAATGGTCAGGGCTGGTT
3-14-3
59
245


 


422239
11838
11857
GACAATGGTCAGGGCTGGTT
2-13-5
67
245


 


422096
11839
11858
AGACAATGGTCAGGGCTGGT
5-10-5
76
348


 


422163
11839
11858
AGACAATGGTCAGGGCTGGT
3-14-3
56
348


 


422240
11839
11858
AGACAATGGTCAGGGCTGGT
2-13-5
66
348


 


422097
11840
11859
GAGACAATGGTCAGGGCTGG
5-10-5
59
349


 


422164
11840
11859
GAGACAATGGTCAGGGCTGG
3-14-3
64
349


 


422241
11840
11859
GAGACAATGGTCAGGGCTGG
2-13-5
61
349


 


422098
11841
11860
GGAGACAATGGTCAGGGCTG
5-10-5
53
350


 


422165
11841
11860
GGAGACAATGGTCAGGGCTG
3-14-3
57
350


 


422242
11841
11860
GGAGACAATGGTCAGGGCTG
2-13-5
64
350


 


422099
11842
11861
AGGAGACAATGGTCAGGGCT
5-10-5
48
351


 


422166
11842
11861
AGGAGACAATGGTCAGGGCT
3-14-3
63
351


 


422243
11842
11861
AGGAGACAATGGTCAGGGCT
2-13-5
62
351


 


422100
11843
11862
GAGGAGACAATGGTCAGGGC
5-10-5
59
352


 


422167
11843
11862
GAGGAGACAATGGTCAGGGC
3-14-3
53
352


 


422244
11843
11862
GAGGAGACAATGGTCAGGGC
2-13-5
55
352


 


422101
11844
11863
TGAGGAGACAATGGTCAGGG
5-10-5
42
353


 


422168
11844
11863
TGAGGAGACAATGGTCAGGG
3-1-4-3
30
353


 


422245
11844
11863
TGAGGAGACAATGGTCAGGG
2-13-5
24
353


 


422102
11845
11864
CTGAGGAGACAATGGTCAGG
5-10-5
62
354


 


422169
11845
11864
CTGAGGAGACAATGGTCAGG
3-14-3
56
354


 


422246
11845
11864
CTGAGGAGACAATGGTCAGG
2-13-5
46
354


 


422103
11846
11865
GCTGAGGAGACAATGGTCAG
5-10-5
38
355


 


422170
11846
11865
GCTGAGGAGACAATGGTCAG
3-14-3
28
355


 


422247
11846
11865
GCTGAGGAGACAATGGTCAG
2-13-5
36
355


 


422104
11847
11866
GGCTGAGGAGACAATGGTCA
5-10-5
59
356


 


422171
11847
11866
GGCTGAGGAGACAATGGTCA
3-14-3
61
356


 


422248
11847
11866
GGCTGAGGAGACAATGGTCA
2-13-5
60
356


 


422105
11848
11867
TGGCTGAGGAGACAATGGTC
5-10-5
60
357


 


422172
11848
11867
TGGCTGAGGAGACAATGGTC
3-14-3
39
357


 


422249
11848
11867
TGGCTGAGGAGACAATGGTC
2-13-5
52
357


 


422106
11849
11868
CTGGCTGAGGAGACAATGGT
5-10-5
32
358


 


422173
11849
11868
CTGGCTGAGGAGACAATGGT
3-14-3
51
358


 


422250
11849
11868
CTGGCTGAGGAGACAATGGT
2-1-3-5
54
358


 


422107
11850
11869
GCTGGCTGAGGAGACAATGG
5-10-5
36
359


 


422174
11850
11869
GCTGGCTGAGGAGACAATGG
3-14-3
55
359


 


422251
11850
11869
GCTGGCTGAGGAGACAATGG
2-13-5
36
359









The chimeric antisense oligonucleotides in Table 10 were designed as 5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE gapmers. The first listed gapmer in Table 10 is the original gapmer (see Table 7) from which the remaining gapmers were designed via microwalk and is designated by an asterisk (*). The 5-10-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleotides each. The 3-14-3 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 14 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 3 nucleotides each. The 2-13-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 13 2′-deoxynucleotides. The central gap is flanked on the 5′ end with a wing comprising 2 nucleotides and on the 3′ end with a wing comprising 5 nucleotides. For each of the motifs (5-10-5, 3-14-3, and 2-13-5), each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. Each gapmer listed in Table 10 is targeted to SEQ ID NO: 1 (nucleotides 1255000 to 1273000 of GENBANK Accession No. NT—027140.6).
As shown in Table 10, most of the 5-10-5 MOE gapmers, 3-14-3 MOE gapmers, and 2-13-5 MOE gapmers targeted to the target region beginning at target start site 13760 and ending at the target stop site 13789 (i.e. nucleobases 13760-13789) of SEQ ID NO: 1 inhibited Factor 7 mRNA by at least 30%.
Certain gapmers within the target region (i.e. nucleobases 13760-13789) inhibited Factor 7 mRNA expression by at least 20%, for example, ISIS numbers 416455, 422175, 422252, 422108, 422176, 422253, 422109, 422177, 422254, 422110, 422178, 422255, 422111, 422179, 422256, 422112, 422180, 422257, 422113, 422181, 422258, 422114, 422259, 422115, 422183, 422260, 422116, 422184, 422261, 416456, and 422185.
Certain gapmers within the target region (i.e. nucleobases 13760-13789) inhibited Factor 7 mRNA expression by at least 30%, for example, ISIS numbers 416455, 422175, 422252, 422108, 422176, 422253, 422109, 422177, 422254, 422110, 422178, 422255, 422111, 422179, 422112, 422180, 422257, 422113, 422181, 422258, 422114, 422259, 422115, 422183, 422260, 422116, 422184, 422261, 416456, and 422185.
Certain gapmers within the target region (i.e. nucleobases 13760-13789) inhibited Factor 7 mRNA expression by at least 40%, for example, ISIS numbers 416455, 422175, 422252, 422108, 422176, 422253, 422109, 422177, 422254, 422110, 422179, 422112, 422180, 422257, 422113, 422181, 422258, 422114, 422259, 422115, 422183, 422260, 422116, 422184, 422261, 416456, and 422185.
Certain gapmers within the target region (i.e. nucleobases 13760-13789) inhibited Factor 7 mRNA expression by at least 50%, for example, ISIS numbers 416455, 422175, 422252, 422108, 422176, 422253, 422109, 422177, 422110, 422112, 422180, 422257, 422113, 422181, 422258, 422114, 422259, 422115, 422183, 422260, 422116, 422184, 422261, and 416456.
Certain gapmers within the target region (i.e. nucleobases 13760-13789) inhibited Factor 7 mRNA expression by at least 60%, for example, ISIS numbers 422175, 422252, 422108, 422253, 422109, 422177, 422112, 422257, 422113, 422181, 422258, 422259, 422115, 422183, and 422261.
Certain gapmers within the target region (i.e. nucleobases 13760-13789) inhibited Factor 7 mRNA expression by at least 70%, for example, ISIS numbers 422252, 422177, 422183, and 422261.








TABLE 10







Inhibition of human Factor 7 mRNA levels by chimeric



antisense oligonucleotides targeted to nucleobases


13760 to 13789 of SEQ ID NO: 1















Target
Target








Start
Stop


%
SEQ ID


ISIS No.
Site
Site
Sequence (5′ to 3′)
Motif
Inhibition
NO

















*416455
13760
13779
CGCCGGCTCTGCTCATCCCC
5-10-5
51
261



 


422175
13760
13779
CGCCGGCTCTGCTCATCCCC
3-14-3
69
261


 


422252
13760
13779
CGCCGGCTCTGCTCATCCCC
2-13-5
70
261


 


422108
13761
13780
CCGCCGGCTCTGCTCATCCC
5-10-5
69
360


 


422176
13761
13780
CCGCCGGCTCTGCTCATCCC
3-14-3
52
360


 


422253
13761
13780
CCGCCGGCTCTGCTCATCCC
2-13-5
61
360


 


422109
13762
13781
CCCGCCGGCTCTGCTCATCC
5-10-5
68
361


 


422177
13762
13781
CCCGCCGGCTCTGCTCATCC
3-14-3
74
361


 


422254
13762
13781
CCCGCCGGCTCTGCTCATCC
2-13-5
42
361


 


422110
13763
13782
ACCCGCCGGCTCTGCTCATC
5-10-5
53
362


 


422178
13763
13782
ACCCGCCGGCTCTGCTCATC
3-14-3
37
362


 


422255
13763
13782
ACCCGCCGGCTCTGCTCATC
2-13-5
30
362


 


422111
13764
13783
CACCCGCCGGCTCTGCTCAT
5-10-5
37
363


 


422179
13764
13783
CACCCGCCGGCTCTGCTCAT
3-14-3
43
363


 


422256
13764
13783
CACCCGCCGGCTCTGCTCAT
2-13-5
29
363


 


422112
13765
13784
CCACCCGCCGGCTCTGCTCA
5-10-5
61
364


 


422180
13765
13784
CCACCCGCCGGCTCTGCTCA
3-14-3
54
364


 


422257
13765
13784
CCACCCGCCGGCTCTGCTCA
2-13-5
65
364


 


422113
13766
13785
GCCACCCGCCGGCTCTGCTC
5-10-5
68
365


 


422181
13766
13785
GCCACCCGCCGGCTCTGCTC
3-14-3
66
365


 


422258
13766
13785
GCCACCCGCCGGCTCTGCTC
2-13-5
66
365


 


422114
13767
13786
CGCCACCCGCCGGCTCTGCT
5-10-5
50
366


 


422182
13767
13786
CGCCACCCGCCGGCTCTGCT
3-14-3
11
366


 


422259
13767
13786
CGCCACCCGCCGGCTCTGCT
2-13-5
61
366


 


422115
13768
13787
GCGCCACCCGCCGGCTCTGC
5-10-5
66
367


 


422183
13768
13787
GCGCCACCCGCCGGCTCTGC
3-14-3
79
367


 


422260
13768
13787
GCGCCACCCGCCGGCTCTGC
2-13-5
56
367


 


422116
13769
13788
TGCGCCACCCGCCGGCTCTG
5-10-5
52
368


 


422184
13769
13788
TGCGCCACCCGCCGGCTCTG
3-14-3
55
368


 


422261
13769
13788
TGCGCCACCCGCCGGCTCTG
2-13-5
47
368


 


416456
13770
13789
CTGCGCCACCCGCCGGCTCT
5-10-5
50
262


 


422185
13770
13789
CTGCGCCACCCGCCGGCTCT
3-14-3
48
262


 


422262
13770
13789
CTGCGCCACCCGCCGGCTCT
2-13-5
0
262









The chimeric antisense oligonucleotides in Table 11 were designed as 5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE gapmers. The first listed gapmer in Table 11 is the original gapmer (see Table 7) from which the remaining gapmers were designed via microwalk and is designated by an asterisk (*). The 5-10-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleotides each. The 3-14-3 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 14 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 3 nucleotides each. The 2-13-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 13 2′-deoxynucleotides. The central gap is flanked on the 5′ end with a wing comprising 2 nucleotides and on the 3′ end with a wing comprising 5 nucleotides. For each of the motifs (5-10-5, 3-14-3, and 2-13-5), each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. Each gapmer listed in Table 11 is targeted to SEQ ID NO: 1 (nucleotides 1255000 to 1273000 of GENBANK Accession No. NT—027140.6).
As shown in Table 11, all of the 5-10-5 MOE gapmers, 3-14-3 MOE gapmers, and 2-13-5 MOE gapmers targeted to the target region beginning at target start site 14707 and ending at the target stop site 14732 (i.e. nucleobases 14707-14732) of SEQ ID NO: 1 inhibited Factor 7 mRNA by at least 48%.
Certain gapmers within the target region (i.e. nucleobases 14707-14732) inhibited Factor 7 mRNA expression by at least 40%, for example, ISIS numbers 416477, 407641, 422200, 422277, 422130, 422201, 422278, 422131, 422202, 422279, 422203, 422280, 422132, 422204, 422281, 422133, 422205, 422282, 407642, 422206, and 422283.
Certain gapmers within the target region (i.e. nucleobases 14707-14732) inhibited Factor 7 mRNA expression by at least 50%, for example, ISIS numbers 416477, 407641, 422200, 422277, 422130, 422201, 422278, 422131, 422279, 422203, 422280, 422132, 422204, 422281, 422133, 422205, 407642, 422206, and 422283.
Certain gapmers within the target region (i.e. nucleobases 14707-14732) inhibited Factor 7 mRNA expression by at least 60%, for example, ISIS numbers 416477, 407641, 422130, 422201, 422278, 422131, 422204, 422133, 422205, 407642, and 422206.
Certain gapmers within the target region (i.e. nucleobases 14707-14732) inhibited Factor 7 mRNA expression by at least 70%, for example, ISIS numbers 416477, 422130, 422201, and 422204.








TABLE 11







Inhibition of human Factor 7 mRNA levels by chimeric



antisense oligonucleotides targeted to nucleobases


14707 to 14732 of SEQ ID NO: 1















Target
Target



SEQ




Start
Stop


%
ID


ISIS No.
Site
Site
Sequence (5′ to 3′)
Motif
Inhibition
NO

















*416477
14710
14729
CAGCCTGAGGCCAGCAGATC
5-10-5
71
283



 


407641
14707
14726
CCTGAGGCCAGCAGATCACG
5-10-5
68
48


 


422200
14707
14726
CCTGAGGCCAGCAGATCACG
3-14-3
58
48


 


422277
14707
14726
CCTGAGGCCAGCAGATCACG
2-13-5
56
48


 


422130
14708
14727
GCCTGAGGCCAGCAGATCAC
5-10-5
79
369


 


422201
14708
14727
GCCTGAGGCCAGCAGATCAC
3-14-3
71
369


 


422278
14708
14727
GCCTGAGGCCAGCAGATCAC
2-13-5
64
369


 


422131
14709
14728
AGCCTGAGGCCAGCAGATCA
5-10-5
68
370


 


422202
14709
14728
AGCCTGAGGCCAGCAGATCA
3-14-3
49
370


 


422279
14709
14728
AGCCTGAGGCCAGCAGATCA
2-13-5
56
370


 


422203
14710
14729
CAGCCTGAGGCCAGCAGATC
3-14-3
52
283


 


422280
14710
14729
CAGCCTGAGGCCAGCAGATC
2-13-5
52
283


 


422132
14711
14730
GCAGCCTGAGGCCAGCAGAT
5-10-5
54
371


 


422204
14711
14730
GCAGCCTGAGGCCAGCAGAT
3-14-3
72
371


 


422281
14711
14730
GCAGCCTGAGGCCAGCAGAT
2-13-5
57
371


 


422133
14712
14731
AGCAGCCTGAGGCCAGCAGA
5-10-5
65
372


 


422205
14712
14731
AGCAGCCTGAGGCCAGCAGA
3-14-3
63
372


 


422282
14712
14731
AGCAGCCTGAGGCCAGCAGA
2-13-5
48
372


 


407642
14713
14732
CAGCAGCCTGAGGCCAGCAG
5-10-5
63
49


 


422206
14713
14732
CAGCAGCCTGAGGCCAGCAG
3-14-3
65
49


 


422283
14713
14732
CAGCAGCCTGAGGCCAGCAG
2-13-5
50
49









The chimeric antisense oligonucleotides in Table 12 were designed as 5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE gapmers. The first listed gapmer in Table 12 is the original gapmer (see Table 7) from which the remaining gapmers were designed via microwalk and is designated by an asterisk (*). The 5-10-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleotides each. The 3-14-3 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 14 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 3 nucleotides each. The 2-13-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 13 2′-deoxynucleotides. The central gap is flanked on the 5′ end with a wing comprising 2 nucleotides and on the 3′ end with a wing comprising 5 nucleotides. For each of the motifs (5-10-5, 3-14-3, and 2-13-5), each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. Each gapmer listed in Table 12 is targeted to SEQ ID NO: 1 (nucleotides 1255000 to 1273000 of GENBANK Accession No. NT—027140.6).
As shown in Table 12, all of the 5-10-5 MOE gapmers, 3-14-3 MOE gapmers, and 2-13-5 MOE gapmers targeted to the target region beginning at target start site 15098 and ending at the target stop site 15122 (i.e. nucleobases 15098-15122) of SEQ ID NO: 1 inhibited Factor 7 mRNA by at least 25%.
Certain gapmers within the target region (i.e. nucleobases 15098-15122) inhibited Factor 7 mRNA expression by at least 20%, for example, ISIS numbers 407643, 422207, 422284, 422134, 422208, 422285, 422135, 422209, 422286, 422136, 422210, 422287, 422137, 422211, 422288, 416479, 422212, and 422289.
Certain gapmers within the target region (i.e. nucleobases 15098-15122) inhibited Factor 7 mRNA expression by at least 30%, for example, ISIS numbers 407643, 422207, 422284, 422134, 422208, 422285, 422135, 422209, 422286, 422136, 422287, 422137, 422211, 422288, 416479, 422212, and 422289.
Certain gapmers within the target region (i.e. nucleobases 15098-15122) inhibited Factor 7 mRNA expression by at least 40%, for example, ISIS numbers 407643, 422207, 422284, 422134, 422208, 422135, 422209, 422286, 422136, 422287, 422137, 422211, 422288, and 416479.
Certain gapmers within the target region (i.e. nucleobases 15098-15122) inhibited Factor 7 mRNA expression by at least 50%, for example, ISIS numbers 407643, 422134, 422208, 422135, 422286, and 422136.
Certain gapmers within the target region (i.e. nucleobases 15098-15122) inhibited Factor 7 mRNA expression by at least 60%, for example, ISIS numbers 407643 and 422134.








TABLE 12







Inhibition of human Factor 7 mRNA levels by chimeric



antisense oligonucleotides targeted to nucleobases


15098 to 15122 of SEQ ID NO: 1















Target
Target








Start
Stop


%
SEQ ID


Oligo ID
Site
Site
Sequence (5′ to 3′)
Motif
Inhibition
NO

















*407643
15098
15117
CACACATGGAGTCAGCATCG
5-10-5
69
50



 


422207
15098
15117
CACACATGGAGTCAGCATCG
3-14-3
41
50


 


422284
15098
15117
CACACATGGAGTCAGCATCG
2-13-5
43
50


 


422134
15099
15118
GCACACATGGAGTCAGCATC
5-10-5
67
373


 


422208
15099
15118
GCACACATGGAGTCAGCATC
3-14-3
56
373


 


422285
15099
15118
GCACACATGGAGTCAGCATC
2-13-5
39
373


 


422135
15100
15119
AGCACACATGGAGTCAGCAT
5-10-5
53
374


 


422209
15100
15119
AGCACACATGGAGTCAGCAT
3-14-3
47
374


 


422286
15100
15119
AGCACACATGGAGTCAGCAT
2-13-5
53
374


 


422136
15101
15120
CAGCACACATGGAGTCAGCA
5-10-5
57
375


 


422210
15101
15120
CAGCACACATGGAGTCAGCA
3-14-3
25
375


 


422287
15101
15120
CAGCACACATGGAGTCAGCA
2-13-5
41
375


 


422137
15102
15121
ACAGCACACATGGAGTCAGC
5-10-5
45
376


 


422211
15102
15121
ACAGCACACATGGAGTCAGC
3-14-3
42
376


 


422288
15102
15121
ACAGCACACATGGAGTCAGC
2-13-5
44
376


 


416479
15103
15122
GACAGCACACATGGAGTCAG
5-10-5
47
285


 


422212
15103
15122
GACAGCACACATGGAGTCAG
3-14-3
30
285


 


422289
15103
15122
GACAGCACACATGGAGTCAG
2-13-5
34
285









The chimeric antisense oligonucleotides in Table 13 were designed as 5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE gapmers. The first listed gapmer in Table 13 is the original gapmer (see Table 7) from which the remaining gapmers were designed via microwalk and is designated by an asterisk (*). The 5-10-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleotides each. The 3-14-3 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 14 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 3 nucleotides each. The 2-13-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 13 2′-deoxynucleotides. The central gap is flanked on the 5′ end with a wing comprising 2 nucleotides and on the 3′ end with a wing comprising 5 nucleotides. For each of the motifs (5-10-5, 3-14-3, and 2-13-5), each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. Each gapmer listed in Table 13 is targeted to SEQ ID NO: 1 (nucleotides 1255000 to 1273000 of GENBANK Accession No. NT—027140.6).
As shown in Table 13, all of the 5-10-5 MOE gapmers, 3-14-3 MOE gapmers, and 2-13-5 MOE gapmers targeted to the target region beginning at target start site 15188 and ending at the target stop site 15211 (i.e. nucleobases 15188-15211) of SEQ ID NO: 1 inhibited Factor 7 mRNA by at least 41%.
Certain gapmers within the target region (i.e. nucleobases 15188-15211) inhibited Factor 7 mRNA expression by at least 40%, for example, ISIS numbers 407935, 416482, 422149, 422226, 422085, 422150, 422227, 422086, 422151, 422228, 422152, 422229, 422087, 422153, and 422230.
Certain gapmers within the target region (i.e. nucleobases 15188-15211) inhibited Factor 7 mRNA expression by at least 50%, for example, ISIS numbers 407935, 416482, 422149, 422085, 422150, 422227, 422086, 422151, 422228, 422152, 422229, 422087, 422153, and 422230.
Certain gapmers within the target region (i.e. nucleobases 15188-15211) inhibited Factor 7 mRNA expression by at least 60%, for example, ISIS numbers 407935, 416482, 422149, 422085, 422150, 422227, 422086, 422151, 422228, 422152, 422229, 422087, 422153, and 422230.
Certain gapmers within the target region (i.e. nucleobases 15188-15211) inhibited Factor 7 mRNA expression by at least 70%, for example, ISIS numbers 407935, 422085, 422150, 422086, 422228, 422152, 422229, and 422087.
Certain gapmers within the target region (i.e. nucleobases 15188-15211) inhibited Factor 7 mRNA expression by at least 80%, for example, ISIS numbers 422086 and 422087.








TABLE 13







Inhibition of human Factor 7 mRNA levels by chimeric



antisense oligonucleotides targeted to nucleobases


15188 to 15211 of SEQ ID NO: 1















Target
Target








Start
Stop


%
SEQ


Oligo ID
Site
Site
Sequence (5′ to 3′)
Motif
Inhibition
ID NO

















*407935
15191
15210
ATGCATGGTGATGCTTCTGA
5-10-5
79
123



 


416482
15188
15207
CATGGTGATGCTTCTGAATT
5-10-5
64
288


 


422149
15188
15207
CATGGTGATGCTTCTGAATT
3-14-3
61
288


 


422226
15188
15207
CATGGTGATGCTTCTGAATT
2-13-5
41
288


 


422085
15189
15208
GCATGGTGATGCTTCTGAAT
5-10-5
70
377


 


422150
15189
15208
GCATGGTGATGCTTCTGAAT
3-14-3
74
377


 


422227
15189
15208
GCATGGTGATGCTTCTGAAT
2-13-5
67
377


 


422086
15190
15209
TGCATGGTGATGCTTCTGAA
5-10-5
81
378


 


422151
15190
15209
TGCATGGTGATGCTTCTGAA
3-14-3
68
378


 


422228
15190
15209
TGCATGGTGATGCTTCTGAA
2-13-5
73
378


 


422152
15191
15210
ATGCATGGTGATGCTTCTGA
3-14-3
74
123


 


422229
15191
15210
ATGCATGGTGATGCTTCTGA
2-13-5
71
123


 


422087
15192
15211
CATGCATGGTGATGCTTCTG
5-10-5
83
379


 


422153
15192
15211
CATGCATGGTGATGCTTCTG
3-14-3
67
379


 


422230
15192
15211
CATGCATGGTGATGCTTCTG
2-13-5
65
379









Example 7
Dose Response Antisense Inhibition of Human Factor 7 in HepB3 Cells
Gapmers from Examples 5 and 6 (see Tables 7, 8, 9, 10, 11, 12, and 13), exhibiting in vitro inhibition of human Factor 7, were tested at various doses in HepB3 cells. Cells were plated at a density of 4,000 cells per well and transfected using lipofectin reagent with 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM concentrations of antisense oligonucleotide, as specified in Table 14. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Factor 7 mRNA levels were measured by quantitative real-time PCR. Human Factor 7 primer probe set RTS 2927 was used to measure mRNA levels. Factor 7 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Factor 7, relative to untreated control cells. As illustrated in Table 14, Factor 7 mRNA levels were reduced in a dose-dependent manner.







TABLE 14







Dose-dependent antisense inhibition of human Factor 7 in HepB3


cells via transfection of oligonucleotides with lipofectin














ISIS
3.125
6.25
12.5
25
50
100
SEQ ID


No.
nM
M
nM
nM
nM
nM
NO

















407935
19
39
58
70
85
87
123


407939
17
35
57
75
80
83
127


422086
8
26
46
65
83
90
378


422087
14
24
51
73
83
90
379


422096
17
23
43
60
68
82
348


422150
9
23
38
59
79
87
377


422152
18
29
47
67
83
89
123


422177
12
24
44
64
81
88
361


422183
28
37
56
78
84
88
367


422228
17
25
43
60
78
90
378


422229
21
32
53
72
86
92
123


422130
4
15
34
59
77
85
369


422140
18
35
49
64
74
74
331


422142
16
34
51
66
72
68
333


422215
13
30
54
65
84
82
331


422217
8
18
45
63
79
83
333


422252
18
17
45
61
77
87
261


422292
13
21
44
63
81
83
331


422294
9
21
32
61
77
84
333


422295
17
22
44
64
80
85
189


422296
21
27
39
67
78
82
334









The gapmers were also transfected via electroporation and their dose-dependent inhibition of human Factor 7 mRNA was measured. Cells were plated at a density of 20,000 cells per well and transfected via electroporation with 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM concentrations of antisense oligonucleotide, as specified in Table 15. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Factor 7 mRNA levels were measured by quantitative real-time PCR. Human Factor 7 primer probe set RTS 2927 was used to measure mRNA levels. Factor 7 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Factor 7, relative to untreated control cells. As illustrated in Table 15, Factor 7 mRNA levels were reduced in a dose-dependent manner.







TABLE 15







Dose-dependent antisense inhibition of human Factor 7 in HepB3


cells via transfection of oligonucleotides with electroporation














ISIS
3.125
6.25
12.5
25
50
100
SEQ ID


No.
μM
μM
μM
μM
μM
μM
No.

















407935
37
47
67
73
80
87
123


407939
18
28
42
56
68
82
127


422086
20
34
44
63
75
84
378


422087
22
26
48
57
72
80
379


422096
26
30
46
50
62
85
348


422150
17
20
40
54
68
83
377


422152
14
22
32
44
65
76
123


422177
3
10
29
21
31
63
361


422183
16
28
37
44
59
71
367


422228
18
33
40
47
71
83
378


422229
21
30
33
44
65
77
123


422130
28
29
59
61
73
86
369


422140
29
34
51
64
69
77
331


422142
26
47
63
67
77
78
333


422215
11
9
31
50
68
84
331


422217
15
19
29
55
61
83
333


422252
23
12
26
38
58
80
261


422292
36
25
47
59
75
86
331


422295
10
8
37
58
68
80
189


422296
62
40
56
56
82
84
334









Example 8
Selection and Confirmation of Effective Dose-Dependent Antisense Inhibition of Human Factor 7 in HepB3 Cells
Gapmers exhibiting in vitro inhibition of human Factor 7 in Example 7 were selected and tested at various doses in HepB3 cells. Cells were plated at a density of 20,000 cells per well and transfected via electroporation with 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM concentrations of antisense oligonucleotide, as specified in Table 16. After a treatment period of approximately 16 hours, RNA was isolated from the cells and human Factor 7 mRNA levels were measured by quantitative real-time PCR. Human Factor 7 primer probe set RTS 2927 was used to measure mRNA levels. Factor 7 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Factor 7, relative to untreated control cells. As illustrated in Table 16, Factor 7 mRNA levels were reduced in a dose-dependent manner.







TABLE 16







Dose-dependent antisense inhibition of human Factor 7 in HepB3


cells via transfection of oligonucleotides with electroporation














ISIS
3.125
6.25
12.5
25
50
100
SEQ ID


No.
μM
μM
μM
μM
μM
μM
NO:

















407935
63
79
84
90
90
91
123


407939
35
60
60
73
81
83
127


422086
25
48
74
82
86
90
378


422087
19
38
66
75
80
87
379


422130
14
24
61
68
82
88
369


422142
34
47
62
67
65
65
333


422150
0
31
53
67
77
86
377


422183
2
3
24
50
64
71
367


422229
30
45
67
79
90
92
123


422292
31
40
68
75
82
83
331


422296
47
44
70
78
80
82
334









Example 9
Antisense Inhibition of Human Factor 7 with Short (14-mer) Oligonucleotides
Short antisense oligonucleotides (shortmers) were designed to target a Factor 7 nucleic acid. The shortmers in Table 17 were designed as 2-10-2 MOE gapmers. The gapmers are 14 nucleotides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 2 nucleotides each. Each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines.
Shortmers were evaluated for their ability to reduce human Factor 7 mRNA in HepB3 cells and compared with one 5-10-5 chimeric oligonucleotide from Table 16, ISIS 407939. HepB3 cells at a density of 20,000 cells per well in a 96-well plate were transfected using electroporation with 1,000 nM of antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Factor 7 mRNA levels were measured by real-time RT-PCR, as described herein. Factor 7 mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Results are presented in Table 17 as percent inhibition of Factor 7 mRNA, relative to untreated control cells. ISIS 407939 is the first oligonucleotide in Table 17 to which the shortmers were compared, and is marked by an asterisk (*).
Each gapmer listed in Table 17 is targeted to human gene sequences, SEQ ID NO: 1 (nucleotides 1255000 to 1273000 of GENBANK Accession No. NT—027140.6) or SEQ ID NO: 2 (GENBANK Accession No. NM—019616.2). “Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the human gene sequence. “Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted in the human gene sequence.








TABLE 17







Inhibition of human Factor 7 mRNA levels by chimeric



antisense oligonucleotides having 2-10-2 MOE wings


and deoxy gap targeted to SEQ ID NO: 1 or SEQ ID NO: 2















Human
Human
Human







Target
Target
Target


ISIS
SEQ ID
Start
Stop


SEQ ID


No.
NO:
Site
Site
Sequence (5′ to 3′)
% inhibition
NO

















*407939
1
15255
15274
TGCAGCCCGGCACCCAGCGA
81
127



 


435758
1
610
623
CCCTGAGCTGGGCA
4
380


 


435759
1
657
670
GAGCCACAGAGCCT
11
381


 


435760
1
744
757
TCTTGGGTGTGGAT
17
382


 


435761
1
796
809
CAGATGAAAACTTG
5
383


 


435762
1
885
898
CAGGTGTTAAGGTG
0
384


 


435763
1
945
958
AGGAGAAAGGTCAG
2
385


 


435764
1
1018
1031
TGAGAGCTGCACCT
8
386


 


435765
1
1092
1105
CAAAGTTCTCTGCC
1
387


 


435634
1
1147
1160
TGAAATCTCTGCAG
5
388


 


435635
1
1152
1165
CATGATGAAATCTC
0
389


 


435636
1
1157
1170
GAGACCATGATGAA
13
390


 


435637
1
1196
1209
TGAAGCCCAAGCAG
11
391


 


435638
1
1201
1214
AGCCCTGAAGCCCA
55
392


 


435766
1
1217
1230
GCACCTGCAGCCAG
51
393


 


435767
1
1248
1261
CACCAAGTTTATGG
0
394


 


435768
1
1384
1397
GCCTGGATGCTGGT
75
395


 


435769
1
1442
1455
TTCTTTGAAAAATA
0
396


 


435770
1
1529
1542
CTCTGAGAGGCCCC
81
397


 


435771
1
2130
2143
GTCATAAGGCCATG
13
398


 


435772
1
2233
2246
GCAGTCCCTGCTCA
42
399


 


435773
1
2348
2361
CAGGGAACACCCTC
57
400


 


435774
1
2390
2403
GTCCCAGGGAAGGC
17
401


 


435775
1
2439
2452
TCTGGAAGGAACAG
25
402


 


435777
1
4713
4726
CTGAGGATGCAGGC
53
403


 


435778
1
4817
4830
GCTACTGGGCCACG
49
404


 


435779
1
4847
4860
TGAGCGCGGAAGAA
50
405


 


435780
1
4945
4958
GGAGGGACGACCTT
28
406


 


435781
1
5124
5137
ACCACGCGGTGGTG
0
407


 


435782
1
5209
5222
CGTGCGCTCAGCTC
36
408


 


435783
1
6396
6409
AAACCGGCATGCGC
49
409


 


435784
1
6433
6446
CCTAAGTGTGCTTT
17
410


 


435785
1
6540
6553
AGCTTCTCACCACG
46
411


 


435787
1
8481
8494
GAGTGTGAGGCTTC
34
412


 


435789
1
8518
8531
CATGTTAGCCTTTG
3
413


 


435790
1
8548
8561
ATGACCATCCTCAA
1
414


 


435791
1
8590
8603
ATGAGAAATGCTTT
0
415


 


435792
1
8651
8664
TGCTGAAGAAACTG
9
416


 


435793
1
8711
8724
TGCAGTAGCAGATG
27
417


 


435794
1
8776
8789
GAGGTACACAGGCT
26
418


 


435795
1
8828
8841
ACACCCGAGGTTCA
66
419


 


435796
1
8858
8871
TGACCACACATTTC
30
420


 


435797
1
8895
8908
AATCTTGGCCCCTC
16
421


 


435798
1
8933
8946
AGCCAGACTTTCCT
54
422


 


435639
1
9072
9085
TCCAGAACAGCTTC
31
423


 


435640
1
9081
9094
TGTAAGAAATCCAG
2
424


 


435799
1
9165
9178
CTGGTCCCCATCTG
33
425


 


435642
1
9168
9181
ACACTGGTCCCCAT
60
426


 


435478
1
9173
9186
GAGGCACACTGGTC
43
427


 


435643
1
9204
9217
CTTGCAGGAGCCCC
51
428


 


435644
1
9209
9222
TGGTCCTTGCAGGA
30
429


 


435645
1
9214
9227
GGAGCTGGTCCTTG
44
430


 


435646
1
9221
9234
TAGGACTGGAGCTG
13
431


 


435647
1
9226
9239
AGATATAGGACTGG
0
432


 


435648
1
9231
9244
GAAGCAGATATAGG
0
433


 


435649
1
9236
9249
AGGCAGAAGCAGAT
21
434


 


435650
1
9255
9268
CCGGCCCTCGAAGG
6
435


 


435651
1
9261
9274
ACAGTTCCGGCCCT
71
436


 


435800
1
9289
9302
GGACCCAAAGTGGG
0
437


 


435801
1
9339
9352
CGGTTGGCCAGGCC
38
438


 


435802
1
9420
9433
GTCACCTAGACCAA
61
439


 


435803
1
9517
9530
GAGAATTGCCCAGG
0
440


 


435804
1
9549
9562
CAGGGACTCTCAGC
26
441


 


435805
1
9716
9729
TGAATTACTGAACC
0
442


 


435806
1
9846
9859
TGACCCCACAGGGA
7
443


 


435807
1
9902
9915
CAGTACTTCCCACG
35
444


 


435808
1
9939
9952
CTCTGGACACCGGG
66
445


 


435809
1
10062
10075
CTGACAATGTGATG
5
446


 


435810
1
10114
10127
CTGAGGGAATGTTG
1
447


 


435811
1
10203
10216
GTGGTCAAGGATGA
27
448


 


435812
1
10355
10368
GCTGAGCAGAGATC
35
449


 


435813
1
10385
10398
GGATGCACACCAGG
55
450


 


435814
1
10584
10597
TGACGAGAGGACCA
31
451


 


435815
1
10677
10690
CTCTTCCGAGCAGC
59
452


 


435816
1
10803
10816
TCCAAAGGACAAGG
4
453


 


435817
1
10835
10848
CTGAGCTTGGCACC
39
454


 


435818
1
10978
10991
GTCATCCTTGTCTG
43
455


 


435652
1
10981
10994
CTGGTCATCCTTGT
48
456


 


435653
1
10986
10999
ATCAGCTGGTCATC
32
457


 


435654
1
11005
11018
GCCGTTCTCGTTCA
71
458


 


435655
1
11011
11024
ACAGCCGCCGTTCT
49
459


 


435656
1
11018
11031
ACTGCTCACAGCCG
58
460


 


435657
1
11023
11036
GCAGTACTGCTCAC
69
461


 


435658
1
11028
11041
TCACTGCAGTACTG
60
462


 


435659
1
11070
11083
TACCCCTCGTGGCA
18
463


 


435660
1
11088
11101
CCGTCTGCCAGCAG
58
464


 


435661
1
11095
11108
GGACACCCCGTCTG
18
465


 


435819
1
11181
11194
TTTGTCCAGTAAGA
1
466


 


435820
1
11294
11307
CATCCTAGTCACTG
19
467


 


435821
1
11454
11467
AGTCAGTACAGACA
0
468


 


435822
1
11494
11507
CCAGGAGCCTTTAC
37
469


 


435823
1
11622
11635
TGAGTCCCAGGCTG
41
470


 


435824
1
11724
11737
CCTAAAGATGAATC
21
471


 


435776
1
11732
11745
GGGACACTCACACT
51
472


 


435825
1
11830
11843
CTGGTTTTGGAGGA
0
473


 


435826
1
11861
11874
AAGTGGCTGGCTGA
27
474


 


435827
1
12057
12070
TCAGAAAGATGCAG
5
475


 


435662
1
12084
12097
TGGATATTCAACTG
19
476


 


435663
1
12089
12102
CCACATGGATATTC
50
477


 


435664
1
12094
12107
TTTTTCCACATGGA
4
478


 


435665
1
12099
12112
AGGTATTTTTCCAC
0
479


 


435666
1
12128
12141
GGTTTGCTGGCATT
71
480


 


435667
1
12141
12154
AATTCGGCCTTGGG
16
481


 


435479
1
12173
12186
CACTCCCCTTTGGG
9
482


 


435668
1
12182
12195
TGCCATGGACACTC
75
483


 


435828
1
12277
12290
GACCTGCCCATTTT
0
484


 


435829
1
12396
12409
GGGTAGACCCTCAG
18
485


 


435830
1
12493
12506
TGATTTTGCAAAGA
0
486


 


435831
1
12523
12536
GATCTTCACATAGC
51
487


 


435832
1
12632
12645
TGCTCAGACCTGGC
70
488


 


435671
1
12796
12809
TCACCAACAACAGG
28
489


 


435672
1
12842
12855
ACCCAGATGGTGTT
12
490


 


435673
1
12847
12860
AGACCACCCAGATG
34
491


 


435674
1
12863
12876
AAACAGTGGGCCGC
34
492


 


435675
1
12868
12881
TGTCGAAACAGTGG
24
493


 


435676
1
12873
12886
GATTTTGTCGAAAC
11
494


 


435833
1
13133
13146
AGACACTTGAGAGC
24
495


 


435834
1
13165
13178
TGACAGCACGAAGC
20
496


 


435835
1
13264
13277
TTATTTGGGCATGG
7
497


 


435836
1
13637
13650
CTAGGTCTGCAGGG
41
498


 


435837
1
13725
13738
CTCGCCTGGAAGGA
37
499


 


435678
1
13733
13746
AGGTCGTGCTCGCC
67
500


 


435679
1
13738
13751
CGCTGAGGTCGTGC
62
501


 


435680
1
13743
13756
GTGCTCGCTGAGGT
52
502


 


435681
1
13781
13794
ATGACCTGCGCCAC
40
503


 


435682
1
13786
13799
GGATGATGACCTGC
36
504


 


435683
1
13823
13836
ATGTCGTGGTTGGT
3
505


 


435684
1
13832
13845
AGCAGCGCGATGTC
56
506


 


435685
1
13856
13869
AGGACCACGGGCTG
40
507


 


435686
1
13861
13874
CAGTGAGGACCACG
31
508


 


435687
1
13866
13879
ATGGTCAGTGAGGA
7
509


 


435688
1
13871
13884
ACCACATGGTCAGT
0
510


 


435689
1
13890
13903
TTCGGGCAGGCAGA
23
511


 


435690
1
13895
13908
GTCCGTTCGGGCAG
55
512


 


435691
1
13946
13959
CAGCCGCTGACCAA
25
513


 


435692
1
13964
13977
CGGTCCAGCAGCTG
32
514


 


435480
1
14016
14029
GGTCATCAGCCGGG
53
515


 


435481
1
14017
14030
GGGTCATCAGCCGG
50
516


 


435482
1
14018
14031
TGGGTCATCAGCCG
62
517


 


435483
1
14019
14032
CTGGGTCATCAGCC
82
518


 


435484
1
14020
14033
CCTGGGTCATCAGC
45
519


 


435485
1
14021
14034
TCCTGGGTCATCAG
34
520


 


435486
1
14022
14035
GTCCTGGGTCATCA
64
521


 


435487
1
14023
14036
AGTCCTGGGTCATC
36
522


 


435488
1
14024
14037
CAGTCCTGGGTCAT
17
523


 


435693
1
14026
14039
GGCAGTCCTGGGTC
43
524


 


435694
1
14031
14044
CTGCAGGCAGTCCT
39
525


 


435695
1
14036
14049
GACTGCTGCAGGCA
48
526


 


435696
1
14081
14094
CAGAACATGTACTC
10
527


 


435697
1
14092
14105
AGTAGCCGGCACAG
53
528


 


435698
1
14119
14132
CCTTGCAGGAGTCC
61
529


 


435551
1
14144
14157
GTGGCATGTGGGCC

530


 


435699
1
14190
14203
GCCCCAGCTGACGA
48
531


 


435489
1
14231
14244
CTGGTGTACACCCC
72
532


 


435490
1
14232
14245
CCTGGTGTACACCC
49
533


 


435491
1
14233
14246
CCCTGGTGTACACC
61
534


 


435492
1
14234
14247
ACCCTGGTGTACAC
45
535


 


435493
1
14235
14248
GACCCTGGTGTACA
58
536


 


435494
1
14236
14249
AGACCCTGGTGTAC
47
537


 


435495
1
14237
14250
GAGACCCTGGTGTA
27
538


 


435496
1
14238
14251
GGAGACCCTGGTGT
47
539


 


435497
1
14239
14252
GGGAGACCCTGGTG
27
540


 


435498
1
14240
14253
TGGGAGACCCTGGT
37
541


 


435499
1
14241
14254
CTGGGAGACCCTGG
59
542


 


435500
1
14242
14255
ACTGGGAGACCCTG
49
543


 


435501
1
14243
14256
TACTGGGAGACCCT
51
544


 


435502
1
14244
14257
GTACTGGGAGACCC
69
545


 


435503
1
14245
14258
TGTACTGGGAGACC
17
546


 


435504
1
14246
14259
ATGTACTGGGAGAC
8
547


 


435714
1
14251
14264
ACTCGATGTACTGG
3
548


 


435715
1
14256
14269
CAGCCACTCGATGT
35
549


 


435716
1
14261
14274
TTTTGCAGCCACTC
22
550


 


435717
1
14266
14279
TGAGCTTTTGCAGC
0
551


 


435718
1
14303
14316
GCTCGCAGGAGGAC
26
552


 


435719
1
14354
14367
CAGCCTTGGCTTTC
50
553


 


435720
1
14662
14675
AGGCTCAGCTGGGC
16
554


 


435721
1
14667
14680
TAAGGAGGCTCAGC
12
555


 


435722
1
14687
14700
TGGGCTTGGCTGAA
50
556


 


435723
1
14707
14720
GCCAGCAGATCACG
68
557


 


435724
1
14712
14725
CTGAGGCCAGCAGA
52
558


 


435725
1
14717
14730
GCAGCCTGAGGCCA
79
559


 


435726
1
14734
14747
GCAATGAAGGCAGA
26
560


 


435727
1
15098
15111
TGGAGTCAGCATCG
73
561


 


435728
1
15103
15116
ACACATGGAGTCAG
16
562


 


435729
1
15108
15121
ACAGCACACATGGA
30
563


 


435730
1
15128
15141
TAAACAACCGCCTT
49
564


 


435731
1
15136
15149
GTGAGAGCTAAACA
11
565


 


435732
1
15141
15154
GAAAAGTGAGAGCT
0
566


 


435733
1
15181
15194
CTGAATTGTCTGAA
5
567


 


435734
1
15187
15200
ATGCTTCTGAATTG
16
568


 


435735
1
15192
15205
TGGTGATGCTTCTG
81
569


 


435736
1
15197
15210
ATGCATGGTGATGC
50
570


 


435737
1
15262
15275
GTGCAGCCCGGCAC
70
571


 


435738
1
15388
15401
CAGAGGATGAGCAC
16
572


 


435739
1
15393
15406
CATCTCAGAGGATG
5
573


 


435740
1
15430
15443
TCATTTCAGTGATG
0
574


 


435741
1
15435
15448
AGGGTTCATTTCAG
6
575


 


435742
1
15440
15453
ATGTGAGGGTTCAT
11
576


 


435743
1
15487
15500
GCCTCAAACATCTA
40
577


 


435744
1
15492
15505
CTACAGCCTCAAAC
19
578


 


435745
1
15497
15510
GGGAGCTACAGCCT
0
579


 


435746
1
15546
15559
ATTGACAAGGGCTG
32
580


 


435747
1
15551
15564
ATATCATTGACAAG
0
581


 


435748
1
15569
15582
TCCCAGGGTCTCTG
18
582


 


435749
1
15630
15643
CAGCCAGGGCCTGG
29
583


 


435750
1
15635
15648
CACTGCAGCCAGGG
14
584


 


435751
1
15650
15663
CTTGCCAGGTCCTC
28
585


 


435752
1
15655
15668
TGCAGCTTGCCAGG
12
586


 


435753
1
15660
15673
AAGAGTGCAGCTTG
9
587


 


435754
1
15665
15678
TCAGCAAGAGTGCA
16
588


 


435755
1
15670
15683
GGGACTCAGCAAGA
0
589


 


435756
1
15805
15818
TGCCCAGGACGGCC
20
590


 


435757
1
15895
15908
TGCCTGAGTGCCCC
51
591


 


435838
1
16009
16022
TCCATGGACACTAA
29
592


 


435839
1
16051
16064
GGTCAGCTGGTCTG
14
593


 


435840
1
16163
16176
GGGCCCGCCACTGG
0
594


 


435841
1
16205
16218
CTCGGACAAAAGAG
1
595


 


435842
1
16596
16609
ATTTCCCATTGGCA
0
596


 


435843
1
16730
16743
GCTGGAGCTGAGCC
0
597


 


435844
1
16773
16786
TGGCCAGTGGCCTC
30
598


 


435845
1
16872
16885
GCTCATGGCAGACA
21
599


 


435846
1
16910
16923
TGGAGAGACCAATG
7
600


 


435847
1
17139
17152
TGGTGTGCACACAT
36
601


 


435848
1
17207
17220
TGGCCACTGCCTCA
32
602


 


435849
1
17250
17263
TGCCGTAGTGGCCG
31
603


 


435850
1
17280
17293
CTTGGCCAGTGTGG
15
604


 


435851
1
17588
17601
ACAGGCCAGGCTGG
0
605


 


435786
1
48751
48764
AGGTGACCCGTGAG
68
606


 


435788
1
127793
127806
CCTGTGAGTGTGAG
20
607


 


435641
2
297
310
GGTCCCCATCACTG
51
608


 


435669
2
657
670
GGACCTGCCATGGA
36
609


 


435670
2
662
675
CAACAGGACCTGCC
47
610


 


435677
2
785
798
GTGCTCGCCCAGCA
60
611









Example 10
Antisense Inhibition of Murine Factor 7 In Vitro
Chimeric antisense oligonucleotides were designed as 5-10-5 MOE wings and deoxy gap were designed to target murine Factor 7 (nucleotides 10024000 to 10037000 of GENBANK Accession No. NT—039455.6; incorporated herein as SEQ ID NO: 160). The gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleotides each. Each nucleotide in each wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5′methylcytidines. The antisense oligonucleotides were evaluated for their ability to reduce murine Factor 7 mRNA in primary mouse hepatocytes. The antisense oligonucleotides were evaluated for their ability to reduce Factor 7 mRNA in primary mouse hepatocytes.
Primary mouse hepatocytes were treated with 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, and 200 nM of antisense oligonucleotides for a period of approximately 24 hours. RNA was isolated from the cells and Factor 7 mRNA levels were measured by quantitative real-time PCR, as described herein. Murine Factor 7 primer probe set RTS 2855 (forward sequence AATGAGGAACAGTGCTCCTTTGA, SEQ ID NO: 612; reverse sequence TGTAAACAATCCAGAACTGCTTGGT, SEQ ID NO: 613; probe sequence CCCGGGAGATCTTCAAGAGCCCX, SEQ ID NO: 614) was used to measure mRNA levels. Factor 7 mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN®. Certain murine antisense oligonucleotides reduced Factor 7 mRNA levels in a dose-dependent manner.
Example 11
Antisense Inhibition of Murine Factor 7 In Vivo
Four antisense oligonucleotides showing significant dose-dependent inhibition from the in vitro study (see Example 10) were evaluated for their ability to reduce Factor 7 mRNA in vivo. The antisense oligonucleotides are targeted to murine Factor 7 mRNA (nucleotides 10024000 to 10037000 of GENBANK Accession No. NT—039455.6; SEQ ID NO: 160). Target start sites for the four of the antisense oligonucleotides are as follows: 11326, 11336, 11613, and 11766.
Treatment
BALB/c mice were treated with ISIS 403102 (CCATAGAACAGCTTCACAGG, target site 11336, incorporated herein as SEQ ID NO: 161). BALB/c mice were injected subcutaneously with 5 mg/kg, 10 mg/kg, 25 mg/kg, or 50 mg/kg of ISIS 403102 twice a week for 3 weeks. A control group of mice was injected subcutaneously with PBS twice a week for 3 weeks. After the treatment period, whole liver was collected for RNA and protein analysis, and plasma was collected for clotting analysis (PT/aPTT).
RNA Analysis
RNA was extracted from liver tissue for real-time RT-PCR analysis of Factor 7. As shown in Table 18, ISIS 403102 achieved a dose-dependent reduction of murine Factor 7 over the PBS control. Results are presented as percent inhibition of Factor 7, relative to the control.







TABLE 18







Dose-dependent antisense inhibition of murine


Factor 7 mRNA in BALB/c mice










mg/kg
% inhibition














5
64



10
84



25
96



50
99










Protein Analysis

Plasma Factor 7 protein was measured using a Factor 7 chromogenic assay (Hyphen BioMed). As shown in Table 19, ISIS 403102 achieved a dose-dependent reduction of murine Factor 7 protein over the PBS control. Results are presented as percent inhibition of Factor 7, relative to control.







TABLE 19







Dose-dependent antisense inhibition of


murine Factor 7 protein in BALB/c mice










mg/kg
% inhibition














5
71



10
88



25
99



50
99










Clotting Analysis

Prothrombin Time (PT) and Activated Partial Thromboplastin Time (aPTT) were measured using platelet poor plasma (PPP) from mice treated with ISIS 403102. PT and aPTT values provided in Table 20 are reported as International Normalized Ratio (INR) values. INR values for PT and aPTT were determined by dividing the PT or aPTT value for the experimental group by the PT or aPTT for the PBS treated group. This ratio was then raised to the power of the International Sensitivity Index (ISI) of the tissue factor used. As shown in Table 20, PT was significantly prolonged in mice treated with ISIS 403102 compared to the control. aPTT was slightly prolonged in mice treated with ISIS 403102. These data suggest that ISIS 403102 has a greater affect on the extrinsic pathway of blood coagulation than the intrinsic pathway of blood coagulation.







TABLE 20







Effect of ISIS 403102 on PT and aPTT in BALB/c mice









mg/kg
PT INR
aPTT INR












5
1.09
1.05


10
1.33
1.04


25
2.33
1.09


50
4.37
1.16









Example 12
Single Dose Pharmacokinetic Assay of ISIS 403102
Treatment
The half-life and duration of action of ISIS 403102 in mice was evaluated. A group of 27 BALB/c mice was injected with 50 mg/kg of ISIS 403102. Three mice from the group were sacrificed at days 1, 2, 3, 4, 6, 8, 12, 24, and 56 after the single dose of ISIS 403102 was administered. A control group of 3 mice was injected with a single dose of PBS, and mice in this group were sacrificed on day 1. Mice in all groups were sacrificed by cervical dislocation following anesthesia with 150 mg/kg ketamine mixed with 10 mg/kg xylazine administered by intraperitoneal injection. Liver was harvested for RNA analysis and plasma was collected for clotting analysis (PT and aPTT).
RNA Analysis
RNA was extracted from liver tissue for real-time RT-PCR analysis of Factor 7. Results are presented as percent inhibition of Factor 7, relative to PBS control. As shown in Table 21, treatment with ISIS 403102 resulted in 92% inhibition of Factor 7 mRNA by day 4 after which the effect of ISIS 403102 gradually decreased and was reduced to 11% by day 24. By day 56, Factor 7 mRNA levels in ISIS 403102 treated mice are equal to that of the PBS control. Results are presented as percent inhibition of Factor 7, relative to control. These data show that the peak effect of a single dose of 50 mg/kg of ISIS 403102 occurs on about day 4 and duration of action lasts for at least 24 days.







TABLE 21







Antisense inhibition of murine Factor 7


mRNA in BALB/c mice in a single dose


pharmacokinetic study










Days
% Inhibition














1
61



2
87



3
92



4
92



6
86



8
80



12
72



24
11



56
0










PT and aPTT Assay

Prothrombin Time (PT) and Activated Partial Thromboplastin Time (aPTT) were measured using platelet poor plasma (PPP) from mice treated with ISIS 403102. PT and aPTT values provided in Table 22 are reported as International Normalized Ratio (INR) values. INR values for PT and aPTT were determined by dividing the PT or aPTT value for the experimental group (i.e. 50 mg/kg treatment with ISIS 403102) by the PT or aPTT for the PBS treated group. This ratio was then raised to the power of the International Sensitivity Index (ISI) of the tissue factor used.
As shown in Table 22, PT increased from 1.11 on day 1 to 1.97 on day 4. PT decreased gradually after day 4 until PT reached 1.10 on day 56. aPTT increased from 1.00 to 1.24 on day 4. aPTT decreased gradually after day 4 until aPTT reached 0.97 on day 56. Consistent with the mRNA expression data (above), these data show that the peak effect of a single dose of 50 mg/kg of ISIS 403102 occurs on about day 4 and duration of action lasts at least 24 days.







TABLE 22







PT and aPTT analysis in BALB/c mice in a


single dose pharmacokinetic study









Days
PT INR
aPTT INR












1
1.11
1.00


2
1.64
1.05


3
1.81
1.24


4
1.97
1.15


6
1.59
1.23


8
1.55
1.18


12
1.30
1.18


24
1.12
1.02


56
1.10
0.97









Example 13
Multiple Dose Pharmacokinetic Assay of ISIS 403102
Treatment
The duration of action of multiple doses of ISIS 403102 on antisense inhibition of murine Factor 7 and clotting time was evaluated. In a first group of mice, 25 mg/kg of ISIS 403102 was injected subcutaneously as a single dose. Mice from the first group were sacrificed on days 1 and 3 after the single dose. In a second group of mice, 25 mg/kg of ISIS 403102 was administered subcutaneously twice a week for 1 week. Mice from the second group were sacrificed on day 3 after the last dose of ISIS 403102 was administered. In a third group of mice, 25 mg/kg of ISIS 403102 was administered subcutaneously twice a week for 2 weeks. Mice from the third group were sacrificed on day 3 after the last dose of ISIS 403102 was administered. In a fourth group of mice, 25 mg/kg of ISIS 403102 was administered subcutaneously twice a week for 3 weeks. Mice from the fourth group were sacrificed on days 2, 7, 14, 28, 42, and 56 after the last dose of ISIS 403102 was administered. A control group of 3 mice was injected with PBS in a single dose. Mice from the control group were sacrificed 1 day later. Mice in all groups were sacrificed by cervical dislocation following anesthesia with 150 mg/kg ketamine mixed with 10 mg/kg xylazine administered by intraperitoneal injection. Liver was harvested for RNA analysis and plasma was collected for clotting analysis (PT and aPTT) for mice in all groups.
RNA Analysis
RNA was extracted from liver tissue for quantitative RT-PCR analysis of Factor 7. Results are presented as percent inhibition of Factor 7, relative to PBS control. As shown in Table 23, a single dose treatment of ISIS 403102 resulted in inhibition of Factor 7 as early as day 1. Inhibition increased through day 3 in the single dose treatment group. Two doses of ISIS 403102 resulted in increased inhibition on day 3 as compared to one dose of ISIS 403102. Inhibition increased through day 3 in the 2 dose treatment group. Four doses of ISIS 403102 resulted in increased inhibition in comparison to the 2 dose treatment group on day 3.
Six doses of ISIS 403102 resulted in increased inhibition on day 7 as compared to 6 doses of ISIS 403102 on day 2. In mice treated with 6 doses of ISIS 403102, Factor 7 inhibition declined progressively on days 14, 28, 42, and 56.







TABLE 23







Dose-dependent reduction of Factor 7 mRNA


in a multiple dose pharmacokinetic study









No. of
Days after
%


doses
last dose
inhibition












1
1
44


1
3
74


2
3
86


4
3
91


6
2
83


6
7
88


6
14
64


6
28
33


6
42
5


6
56
11









PT and aPTT Assay

Prothrombin Time (PT) and Activated Partial Thromboplastin Time (aPTT) were measured using platelet poor plasma (PPP) from mice treated with ISIS 403102. PT and aPTT values provided in Table 24 are reported as International Normalized Ratio (INR) values. INR values for PT and aPTT were determined by dividing the PT or aPTT value for the experimental group (i.e. 50 mg/kg treatment with ISIS 403102) by the PT or aPTT for the PBS treated group. This ratio was then raised to the power of the International Sensitivity Index (ISI) of the tissue factor used.
As shown in Table 24, PT was increased on day 3 in mice treated with a single dose of ISIS 403102 in comparison to mice treated with a single dose of ISIS 403102 on day 1. On day 3, PT increased in mice treated with 2 doses of ISIS 403102 over mice treated with a single dose of ISIS 403102. PT increased in mice treated with 4 doses of ISIS 403102 over those mice treated with 2 doses of ISIS 403102 on day 3. PT decreased in mice receiving 6 doses of ISIS 403102 from day 7 to day 56.
aPTT was slightly increased on day 3 in mice treated with a single dose of ISIS 403102 in comparison to mice treated with a single dose of ISIS 403102 on day 1. On day 3, aPTT increased in mice treated with 2 doses of ISIS 403102 over mice treated with a single dose of ISIS 403102. aPTT increased in mice treated with 4 doses of ISIS 403102 over those mice treated with 2 doses of ISIS 403102 on day 3. aPTT decreased in mice receiving 6 doses of ISIS 403102 from day 7 to day 56.







TABLE 24







PT and aPTT analysis in BALB/c mice in a


multiple dose pharmacokinetic study












No. of
Days after





doses
last dose
PT INR
aPTT INR
















1
1
1.04
1.08



1
3
1.20
1.10



2
3
1.43
1.29



4
3
2.13
1.53



6
2
1.64
1.62



6
7
2.08
1.67



6
14
1.37
1.25



6
28
0.96
0.90



6
42
1.00
1.00



6
56
0.99
0.98










Example 14
In Vivo Effect of Antisense Inhibition of Factor 7 with ISIS 403102 in the FeCl3 Induced Venous Thrombosis (VT) Model
Treatment
Three groups of BALB/c mice were injected with 25 mg/kg, 37.5 mg/kg, or 50 mg/kg of ISIS 403102, administered subcutaneously twice a week for 3 weeks. Two control groups of BALB/c mice were treated with PBS, administered subcutaneously twice a week for 3 weeks. Thrombus formation was induced with FeCl3 in half of the mice while the rest of the mice were assayed for tail bleeding. Two days after receiving the last dose of ISIS 403102 or PBS, mice were anesthetized with 150 mg/kg ketamine mixed with 10 mg/kg xylazine administered by intraperitoneal injection. Thrombus formation was induced with FeCl3 in all groups of the VT mice except the first control group.
In mice undergoing FeCl3 treatment, thrombus formation was induced by applying a piece of filter paper (2×4 mm) pre-saturated with 10% FeCl3 solution directly on the vena cava. After 3 minutes of exposure, the filter paper was removed. Thirty minutes after the filter paper application, a fixed length of the vein containing the thrombus was dissected out for platelet analysis. Liver was collected for RNA analysis.
RNA Analysis
RNA was extracted from liver tissue for real-time RT-PCR analysis of Factor 7. Results are presented as percent inhibition of Factor 7, relative to PBS control. As shown in Table 25, treatment with ISIS 403102 resulted in significant dose-dependent reduction of Factor 7 mRNA in comparison to the PBS control. These data show that antisense oligonucleotides can be used to inhibit expression of Factor 7.







TABLE 25







Dose-dependent reduction of Factor 7 mRNA


in the FeCl3 induced venous thrombosis model











%



mg/kg
inhibition














25
24



37.5
74



50
69










Quantification of Platelet Composition

Real-time RT-PCR quantification of platelet factor-4 (PF-4) was used to quantify platelets in the vena cava as a measure of thrombus formation. Results are presented as a percentage of PF-4 in ISIS 403102, as compared to the two PBS-treated control groups. As shown in Table 26, treatment with ISIS 403102 resulted in a reduction of PF-4 in comparison to the PBS control. Therefore, antisense oligonucleotides are useful for inhibiting thrombus and clot formation.







TABLE 26







Analysis of thrombus formation by real-time


RT-PCR quantification of PF-4 in the FeCl3


induced venous thrombosis model










mg/kg
PF-4















PBS − FeCl3

0



PBS + FeCl3

100



ISIS 403102
25
34




37.5
24




50
24










Tail Bleeding Assay

Mice not receiving treatment with FeCl3 solution were evaluated in a tail bleeding chamber. Mice were placed into the bleeding chamber two days after the final treatment of ISIS 403102 or PBS. Mice were anesthetized in the chamber with isofluorane and a small piece of tail (approximately 4 mm from the tip) was cut with sterile scissors. The cut tail was immediately placed in a 15 mL Falcon tube filled with approximately 10 mL of 0.9% NaCl buffer solution warmed to 37° C. The blood was collected over the course of 40 minutes. The saline filled tubes were weighed both before and after bleeding. The results are provided in Table 27.
Treatment with 25 mg/kg and 37.5 mg/kg ISIS 403102 caused a slight decrease in the amount of bleeding in comparison to PBS treated mice. Bleeding was the same in mice treated with 50 mg/kg ISIS 403102 and mice treated with PBS. These data suggest that treatment with ISIS 403102 does not increase hemorrhagic potential.







TABLE 27







Tail bleeding assay










Mg/kg
Blood (g)















PBS

0.10



403102
25
0.06




37.5
0.06




50
0.10










Example 15
In Vivo Antisense Inhibition of Murine Factor 7 by ISIS 403102 Compared to Warfarin
Treatment
ISIS 403102 and warfarin (Coumadin®) were evaluated in BALB/c mice. Four groups of BALB/c mice were treated with 5 mg/kg, 10 mg/kg, 20 mg/kg, or 40 mg/kg of ISIS 403102, administered subcutaneously twice a week for 3 weeks. Two days after receiving the last dose of ISIS 403102, mice were anesthetized with 150 mg/kg ketamine mixed with 10 mg/kg xylazine administered by intraperitoneal injection. A fifth group of BALB/c mice was treated with 3 mg/kg of warfarin, administered intraperioneally daily for 6 days. Four hours after the last dose of warfarin, mice were sacrificed. A control group of BALB/c mice were treated with PBS, administered subcutaneously twice a week for 3 weeks. Two days after the last dose of PBS, mice were anesthetized with 150 mg/kg ketamine mixed with 10 mg/kg xylazine administered by intraperitoneal injection. Thrombus formation was induced with FeCl3 in groups of mice except the first control group.
In mice undergoing FeCl3 treatment, thrombus formation was induced by applying a piece of filter paper (2×4 mm) pre-saturated with 10% FeCl3 solution directly on the vena cava. After 3 minutes of exposure, the filter paper was removed. Thirty minutes after the filter paper application, a fixed length of the vein containing the thrombus was dissected out for platelet analysis. Liver was collected for RNA analysis.
RNA Analysis
RNA was extracted from liver tissue for real-time RT-PCR analysis of Factor 7. Results are presented as percent inhibition of Factor 7, relative to PBS control. As shown in Table 28, treatment with ISIS 403102 resulted in significant dose-dependent reduction of Factor 7 mRNA in comparison to the PBS control. Conversely, treatment with warfarin did not result in significant reduction of Factor 7 as compared to the PBS control.







TABLE 28







Dose-dependent reduction of Factor 7 mRNA


in the FeCl3 induced venous thrombosis model











%



mg/kg
inhibition















PBS − FeCl3

0



PBS + FeCl3

0



Warfarin
3
10



ISIS 403102
5
59




10
84




20
95




40
99










Example 16
Effect of Dose-Dependent Antisense Inhibition of Murine Factor 7 on the FeCl3 Induced Venous Thrombosis (VT) Model Compared to Warfarin
Treatment
ISIS 403102 and warfarin (Coumadin®) were evaluated in BALB/c mice. Four groups of BALB/c mice were treated with 5 mg/kg, 10 mg/kg, 20 mg/kg, or 40 mg/kg of ISIS 403102, administered subcutaneously twice a week for 3 weeks. Two days after receiving the last dose of ISIS 403102, mice were anesthetized with 150 mg/kg ketamine mixed with 10 mg/kg xylazine administered by intraperitoneal injection. Six additional groups of BALB/c mice was treated with 0.5 mg/kg, 1 mg/kg, 2 mg·kg, 3 mg/kg, 4 mg/kg, or 5 mg/kg of warfarin, administered intraperioneally daily for 6 days. Four hours after the last dose of warfarin, mice were sacrificed. A control group of BALB/c mice were treated with PBS, administered subcutaneously twice a week for 3 weeks. Two days after the last dose of PBS, mice were anesthetized with 150 mg/kg ketamine mixed with 10 mg/kg xylazine administered by intraperitoneal injection. Thrombus formation was induced with FeCl3 in groups of mice except the first control group.
In mice undergoing FeCl3 treatment, thrombus formation was induced by applying a piece of filter paper (2×4 mm) pre-saturated with 10% FeCl3 solution directly on the vena cava. After 3 minutes of exposure, the filter paper was removed. Thirty minutes after the filter paper application, a fixed length of the vein containing the thrombus was dissected out for platelet analysis. Liver was collected for RNA analysis.
Quantification of Platelet Composition
Real-time RT-PCR quantification of platelet factor-4 (PF-4) was used to quantify platelets in the vena cava as a measure of thrombus formation. Results are presented as a percentage of PF-4 in ISIS 403102 or warfarin treated mice, as compared to the two PBS-treated control groups. As shown in Table 29, treatment with ISIS 403102 resulted in a dose-dependent reduction of PF-4 in comparison to the PBS control for dosages of 5 mg/kg and higher. Treatment with warfarin resulted in a reduction of PF-4 in comparison to the PBS control at a dose of 1 mg/kg and higher. Therefore, ISIS antisense oligonucleotides are useful for inhibiting thrombus and clot formation.







TABLE 29







Analysis of thrombus formation by real-time


RT-PCR quantification of PF-4 in the FeCl3


induced venous thrombosis model










mg/kg
PF-4















PBS − FeCl3

0



PBS + FeCl3

100



Warfarin
0.5
165




1
63




2
47




3
35




4
22




5
0



ISIS 403102
1
120




3
112




5
69




10
22




20
41




40
38










Example 17
Effect of Antisense Inhibition of Murine Factor 7 in a Tail Bleeding Assay Compared to Warfarin
Treatment
Tail-bleeding was measured to observe whether treatment with ISIS 403102 or warfarin causes internal hemorrhage in mice. ISIS 403102 and warfarin (Coumadin®) were evaluated in the tail bleeding assay. Six groups of BALB/c mice were treated with 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, or 40 mg/kg of ISIS 403102, administered subcutaneously twice a week for 3 weeks. An additional 6 groups of BALB/c mice were treated with 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, and 5 mg/kg of warfarin, administered intraperioneally daily for 6 days. A separate control group of BALB/c mice was treated with PBS, administered subcutaneously twice a week for 3 weeks.
Tail-Bleeding Assay
Two days after the final treatment of ISIS 403102, warfarin, or PBS, mice were placed in a tail bleeding chamber. Mice were anesthetized in the chamber with isofluorane and a small piece of tail (approximately 4 mm from the tip) was cut with sterile scissors. The cut tail was immediately placed in a 15 mL Falcon tube filled with approximately 10 mL of 0.9% NaCl buffer solution warmed to 37° C. The blood was collected over the course of 40 minutes. The saline filled tubes were weighed both before and after bleeding. The results are provided in Table 30.
Treatment with ISIS 403102 did not significantly affect bleeding as compared to PBS control mice. However, warfarin did increase bleeding in mice as compared to the PBS control mice. Increased doses of warfarin correlated positively with increased blood loss. These data suggest that the hemorrhagic potential of ISIS 403102 is low, especially in comparison to warfarin.







TABLE 30







Tail bleeding assay in the FeCl3 induced


venous thrombosis model












Dose in




Treatment
mg/kg
Blood (g)















PBS
0
0.06



Warfarin
0.5
0.16




1
0.28




2
0.58




3
0.78




4
0.66




5
0.93



ISIS 404071
1.25
0.03




2.5
0.03




5
0.03




10
0.09




20
0.09




40
0.09










Example 18
Effect of Antisense Inhibition of Murine Factor 7 in the Tail Bleeding Assay Compared to Apixaban
Treatment
ISIS 403102 and Apixaban were evaluated in BALB/c mice. In a first group of BALB/c mice, 40 mg/kg of ISIS 403102 was administered subcutaneously twice a week for 3 weeks. An additional 3 groups of BALB/c mice were treated with 5 mg/kg and 10 mg/kg of Apixaban, administered in a single intraperitoneal dose, and 10 mg/kg of Apixaban administered as a single subcutaneous dose. A control group of BALB/c mice was treated with PBS, administered subcutaneously twice a week for 3 weeks.
Tail-Bleeding Assay
Two days after the final treatment of ISIS 403102 or PBS, mice were placed in a tail bleeding chamber. Mice from the groups treated with Apixaban were analyzed 30 minutes after the single dose. Mice were anesthetized in the chamber with isofluorane and a small piece of tail (approximately 4 mm from the tip) was cut with sterile scissors. The cut tail was immediately placed in a 15 mL Falcon tube filled with approximately 10 mL of 0.9% NaCl buffer solution warmed to 37° C. The blood was collected over the course of 40 minutes. The saline filled tubes were weighed before and after bleeding. The results are provided in Table 31.
Mice treated with ISIS 403102 had less bleeding than PBS treated mice. Mice treated with 5 mg/kg of apixaban by intraperitoneal injection had the same amount of bleeding as PBS treated mice. Mice treated with 10 mg/kg of apixaban by intraperitoneal injection had increased bleeding as compared to the PBS treated mice. Mice treated with 10 mg/kg of apixaban by subcutaneous injection had less bleeding than PBS mice. These data suggest that the hemorrhagic potential of ISIS 403102 is low.







TABLE 31







Tail bleeding assay in BALB/c mice












mg/kg
Blood (g)
















PBS


0.22



ISIS 403102
40
(s.c.)
0.10



Apixaban
5
(i.p.)
0.22




10
(i.p.)
0.58




10
(s.c.)
0.04










Example 19
In Vivo Effect of Antisense Inhibition of Murine Factor 7 on Cancer Metastasis
The effect of inhibition of Factor 7 with ISIS 403102 on the formation of tissue factor-Factor 7 complex and its role in extravasation of cancer cells during metastasis will be evaluated. Two groups of severe combined immunodeficiency (SCID) mice will be treated with ISIS 403102, injected at a dose of 20 mg/kg twice a week for 3 weeks. A control group of mice will be injected with PBS twice a week for 3 weeks. Two days after the last dose of ISIS 403102 or PBS, one of the ISIS 403102 treated groups and the control group will be injected intravenously with 50×106 MDA-MB-231 breast carcinoma cells.
Two weeks after the injection with MDA-MB-231 breast carcinoma cells, mice will be sacrificed. The lungs will be harvested and real-time RT-PCR analysis of human GAPDH mRNA levels performed. The results will be normalized with mouse cyclophilin A mRNA levels. Human GAPDH levels will in the group treated with ISIS 403102 and MDA-MB-231 breast carcinoma cells group will be compared to human GAPDH levels in the other two groups of mice. This experiment is designed to assess the effect of inhibition of Factor 7 on the development of metastasis in the lungs.
Example 20
In Vivo Effect of Antisense Inhibition of Murine Factor 7 on Liver Fibrosis
The effect of inhibition of Factor 7 with ISIS 403102 on experimental liver fibrosis will be evaluated in the carbon tetrachloride liver injury model.
Treatment
In a first group of BALB/c mice, 20 mg/kg ISIS 403102 will be injected subcutaneously twice a week for 8 weeks. In a second group of mice, PBS will be injected subcutaneously twice a week for 8 weeks. Two weeks after the first treatment with ISIS 403102 or PBS, both groups of mice will be dosed intraperitoneally with 5 μl of carbon tetrachloride (CCl4) dissolved in 95 μl of mineral oil twice a week for 5 weeks. A third group of mice will be injected with 100 μl mineral oil alone. Mice will be sacrificed by cervical dislocation following anesthesia with isofluorane. Liver tissue will be harvested from all mice. Real-time RT-PCR will be used to determine the expression of fibrosis related genes, including, collagen type 1, α-smooth muscle actin, matrix metalloproteinase (MMP) 3, TGF-β, Timp1 and Timp2 (MMP inhibitors). The levels in the experimental group will be compared to the levels in the control mice to assess the effect of inhibition of Factor 7 on the development of liver fibrosis.
Example 21
In Vivo Effect of Antisense Inhibition of Murine Factor 7 on Collagen-Induced Arthritis
The effect of inhibition of Factor 7 with ISIS 403102 on the formation of tissue factor-Factor 7 complex and its role in fibrin accumulation in the joints leading to joint inflammation and rheumatoid arthritis will be evaluated in a collagen-induced arthritis model.
Treatment
In a first group of DBA/1J mice, 20 mg/kg of ISIS 403102 will be injected subcutaneously twice a week for 8 weeks. Two groups of mice will be injected with PBS twice a week for 8 weeks. Two weeks after the first treatment of ISIS 403102, type II bovine collagen (Chondrex) will be mixed with complete Freund's adjuvant, homogenized on ice and the emulsion, containing 100 μg of collagen, will be injected subcutaneously in the experimental group and the first control group. A booster injection containing 100 μg collagen type II in incomplete Freund's adjuvant will be injected subcutaneously 7 days after the first collagen injection in both these groups.
Mice in all groups will be examined each day from day 18 after the first collagen injection for the visual appearance of arthritis in peripheral joints. The clinical severity of arthritis will be scored as follows: 1 point for each swollen digit except the thumb (maximum, 4), 1 point for the tarsal or carpal joint, and 1 point for the metatarsal or metacarpal joint with a maximum score of 6 for a hindpaw and 5 for a forepaw. Each paw will be graded individually, the cumulative clinical arthritic score per mouse reaching a maximum of 22 points. Arthritis in the experimental groups will be compared to the control group to assess the effect of inhibition of Factor 7 on the development of arthritis in the joints.
Six weeks after the initial injection of collagen, the maximal level of arthritis will be induced. After mice are anesthetized with isofluorane and plasma is collected, the mice will be sacrificed by cervical dislocation. Livers will be harvested for RNA analysis of Factor 7 mRNA. Plasma collected from all three groups will be analyzed for clotting time (PT and aPTT). The measurement of thrombin-antithrombin (TAT) complexes in the plasma will also be performed by ELISA. The results in the experimental groups will be compared to the control group to assess the effect of inhibition of Factor 7 on the clotting time and formation of TAT complexes.

## Claims

1. A compound comprising a single-stranded modified antisense oligonucleotide consisting of 12 to 30 linked nucleosides targeted to a human Factor 7 nucleic acid, wherein the oligonucleotide has a sequence that is least 90% complementary to SEQ ID NO: 1 as measured over the entirety of said oligonucleotide, and wherein the oligonucleotide comprises:
a gap segment consisting of linked deoxynucleosides;
a 5′ wing segment consisting of linked nucleosides;
a 3′ wing segment consisting of linked nucleosides;
wherein the gap segment is positioned immediately adjacent to and between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.

2. The compound of claim 1, wherein the oligonucleotide comprises:
a gap segment consisting of ten linked deoxynucleosides;
a 5′ wing segment consisting of five linked nucleosides;
a 3′ wing segment consisting of five linked nucleosides;
wherein the gap segment is positioned immediately adjacent to and between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, wherein each cytosine in said oligonucleotide is a 5-methylcytosine, and wherein each internucleoside linkage of said oligonucleotide is a phosphorothioate linkage.

3. The compound of claim 2, wherein the oligonucleotide consists of 20 linked nucleosides.

4. A composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier or diluent.

5. The compound of claim 1, wherein the oligonucleotide has a sequence that is 100% complementary to SEQ ID NO: 1 as measured over the entirety of said oligonucleotide.

6. The compound of claim 1, wherein the oligonucleotide comprises at least one modified internucleoside linkage.

7. The compound of claim 6, wherein the modified internucleoside linkage is a phosphorothioate linkage.

8. The compound of claim 1, wherein the modified sugar is a 2′-O-methoxyethyl sugar or a bicyclic sugar.

9. The compound of claim 1, wherein the oligonucleotide comprises at least one modified nucleobase.

10. The compound of claim 9, wherein the modified nucleobase is a 5-methylcytosine.

11. A composition comprising a compound comprising a single-stranded modified antisense oligonucleotide consisting of 12 to 30 linked nucleosides targeted to a human Factor 7 nucleic acid, wherein the oligonucleotide has a sequence that is least 90% complementary to SEQ ID NO: 1 as measured over the entirety of said oligonucleotide, wherein the oligonucleotide comprises at least one modification selected from the group consisting of a modified internucleoside linkage, a modified sugar, and a modified nucleobase, a pharmaceutically acceptable carrier or diluent and a therapeutic agent selected from the group consisting of aspirin, clopidogrel, dipyridamole, heparin, lepirudin, ticlopidine, warfarin, apixaban, rivaroxaban, and lovenox.

12. A method comprising identifying an animal having a disease or condition associated with Factor 7 and administering to the animal a therapeutically effective amount of the compound comprising a single-stranded modified antisense oligonucleotide consisting of 12 to 30 linked nucleosides targeted to a human Factor 7 nucleic acid, wherein the oligonucleotide has a sequence that is least 90% complementary to SEQ ID NO: 1 as measured over the entirety of said oligonucleotide, wherein the oligonucleotide comprises at least one modification selected from the group consisting of a modified internucleoside linkage, a modified sugar, and a modified nucleobase such that expression of Factor 7 is inhibited.

13. The method of claim 12, wherein the disease or condition is selected from the group consisting of a thromboembolic complication, a hyperproliferative disorder, and an inflammatory condition.

14. The method of claim 13, wherein the thromboembolic complication is thrombosis, embolism, thromboembolism, deep vein thrombosis, pulmonary embolism, myocardial infarction, or stroke.

15. The method of claim 12, wherein the animal is a human.

16. The method of claim 12, comprising co-administering the compound and a therapeutic agent selected from the group consisting of aspirin, clopidogrel, dipyridamole, heparin, lepirudin, ticlopidine, warfarin, apixaban, rivaroxaban, and lovenox.

17. The method of claim 16, wherein the compound and the therapeutic agent are administered concomitantly.

18. The method of claim 12, wherein the oligonucleotide has a sequence that is 100% complementary to SEQ ID NO: 1 as measured over the entirety of said oligonucleotide.

19. The method of claim 12, wherein the oligonucleotide comprises at least one modified internucleoside linkage.

20. The method of claim 19, wherein the modified internucleoside linkage is a phosphorothioate linkage.

21. The method of claim 12, wherein the oligonucleotide comprises at least one modified sugar.

22. The method of claim 21, wherein the modified sugar is a 2′-O-methoxyethyl sugar or a bicyclic sugar.

23. The method of claim 12, wherein the oligonucleotide comprises at least one modified nucleobase.

24. The method of claim 23, wherein the modified nucleobase is a 5-methylcytosine.

25. The method of claim 12, wherein the oligonucleotide comprises:
a gap segment consisting of linked deoxynucleosides;
a 5′ wing segment consisting of linked nucleosides;
a 3′ wing segment consisting of linked nucleosides;
wherein the gap segment is positioned immediately adjacent to and between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.

26. The method of claim 12, wherein the oligonucleotide comprises:
a gap segment consisting of ten linked deoxynucleosides;
a 5′ wing segment consisting of five linked nucleosides;
a 3′ wing segment consisting of five linked nucleosides;
wherein the gap segment is positioned immediately adjacent to and between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, wherein each cytosine in said oligonucleotide is a 5-methylcytosine, and wherein each internucleoside linkage of said oligonucleotide is a phosphorothioate linkage.

27. The method of claim 26, wherein the oligonucleotide consists of 20 linked nucleosides.